PHOTOSYNTHESIS AND PHOTORESPIRATION IN THREE C …repositorio.ul.pt/bitstream/10451/1525/1/ulsd053692_td_tese.pdf · universidade de lisboa faculdade de ciÊncias departamento de

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA VEGETAL

PHOTOSYNTHESIS AND PHOTORESPIRATION IN THREE

C4 GRASSES OF DIFFERENT METABOLIC SUB-TYPES,

UNDER WATER STRESS

Ana Elizabete do Carmo Silva

DOUTORAMENTO EM BIOLOGIA

(Fisiologia e Bioquímica)

2008

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA VEGETAL

PHOTOSYNTHESIS AND PHOTORESPIRATION IN THREE

C4 GRASSES OF DIFFERENT METABOLIC SUB-TYPES,

UNDER WATER STRESS


DOUTORAMENTO EM BIOLOGIA

(Fisiologia e Bioquímica)

Tese orientada pela Professora Doutora Maria Celeste Arrabaça

2008

The experimental work leading to this PhD was done in collaboration

with

ROTHAMSTED RESEARCH

Department of Plant Sciences

Under the supervision of Professor Dr. Martin A. J. Parry

And close advice of Dr. Alfred J. Keys

In addition to the supervision and orientation of Professor Celeste Arrabaça, Professor Martin

Parry and Dr. Alfred Keys, the work here presented involved the collaboration with other

Professors and Researchers. The four chapters of results contained in the Thesis (II, III, IV and

V) correspond to the integral copy or extended versions of manuscripts submitted (or in final

phase of preparation to be submitted) for publication in peer-reviewed journals. The relative

contributions of each author apart from myself and the supervisors and advisors, who were

involved in the planning of the experiments, discussion of the results and elaboration of

manuscripts, are mentioned in the beginning of each chapter.

Para os devidos efeitos no n.º 2 do Art.8º do Decreto-Lei 388/70, o autor da tese declara que

participou na execução do trabalho experimental descrito, bem como na análise e interpretação

dos resultados e na redação dos textos e manuscritos submetidos para publicação.


Março de 2008

Acknowledgements / Agradecimentos

i

ACKNOWLEDGEMENTS / AGRADECIMENTOS

I am grateful to Fundação para a Ciência e a Tecnologia (Portugal) for financial support (PhD grant SFRH/BD/13730/2003), and to Faculdade de Ciências da Universidade de Lisboa (Portugal) and Rothamsted Research (UK) for receiving me and providing the conditions to do the work here presented. The grass seeds used were kindly provided by AgResearch, Margot Forde Forage Germplasm Centre, New Zealand and Alípio Dias & Irmão, Lda., Portugal.

Many people have somehow contributed to the successful realization of this PhD – many thanks to all!!! Here I can only express a special acknowledgement to a few of them!... Muitos foram aqueles que de alguma forma, directa ou indirectamente, contribuíram para a realização deste Doutoramento a bom termo – a todos, um grande bem-haja! Aqui, posso apenas agradecer a apenas algumas dessas pessoas!

Professora Doutora Maria Celeste Arrabaça pela sugestão do tema deste trabalho, pelo incentivo à sua execução e pelo acolhimento no Centro de Engenharia Biológica da FCUL. Agradeço-lhe a orientação e supervisão do trabalho, as demais lições científicas e afins, incluindo a da preserverança e a importância de manter a calma, e a disponibilidade que sempre mostrou para discutir os mais diversos aspectos que surgiram ao longo da sua realização. Agradeço-lhe também toda a amizade e o carinho.

Professor Dr. Martin Parry for receiving me at Rothamsted Research and showing me other faces of Science. I have learnt so many lessons while there, I have grown so much! I will always be thankful for the support, for the pushes applied when needed and for all the words and lessons – it did make a difference!

Dr. Alfred Keys, Alf… How can I find words that express the gratefulness for the many things you did for me? You were the main reason that made me to go to the UK and I will always owe you so much! The support, care and friendship, all the advices and the scientific or life-related discussions, the tears and the smiles… It is hard to imagine life far from you but (like with other parental relations) children must have wings and use them to fly around the world!

Todos os demais Professores e colegas do Departamento de Biologia Vegetal e do Centro de Engenharia Biológica da FCUL! Nomeadamente (e jamais exclusivamente!)... Prof. João Arrabaça pelo grande carinho (às vezes bem disfarçado!) com que me acolheu, pelo despertar do interesse pela cultura e pela transmissão de tão diversos e surpreendentes conhecimentos à “joven cientista”! Prof. Anabela Bernardes da Silva, Belucha, por me ter puxado para a Fisiologia e Bioquímca, pela amizade, pelos abraços e seus substitutos, pela transmissão de valores e conhecimentos tão importantes e pelas longas discussões - enzimáticas e afins! Prof. Jorge Marques da Silva, por todos os incentivos que transmitiu, directa ou indirectamente, pelos conhecimentos de stress hídrico e de fisiologia - alguns um tanto ou quanto fluorescentes! Dra. Ana Rita Matos pela lufada de ar fresco que trouxe ao CEB e pela capacidade de “apaziguação”. Ana Sofia Soares pelas aventuras e alegrias partilhadas no início de um percurso que acabou por tomar rumos diferentes! Prof. Cristina Cruz pela energia contagiante e pela recepção de braços abertos nas rápidas “visitas” ao laboratório da Ecologia! Prof. Cristina Máguas e Dr. Rodrigo Maia pelo apoio na determinação de composições isotópicas. Prof. Lia Ascensão pelo acolhimento no “cantinho da Microscopia” e pela transmissão de valores morais e científicos! Prof. Renata Meira (visitante do Brazil!) pela ajuda sempre pronta e pelo bom humor contagiante! Prof. Rui Malhó, Rui, pelo despertar de uma energia conquistadora e pela forma diferente de iniciar o dia, com sorrisos partilhados!! Manuela Lucas, Manelita, por ser uma segunda mãe, por tomar conta de mim, por todas as ajudas e por ser a maior mestra na “lei do desenrasque” – arranja sempre uma solução para o problema do próximo!

Acknowledgements / Agradecimentos

ii

All the researchers and colleagues at Rothamsted Research! John Andralojc for all the jokes and the help with Rubisco-related measurements and Pippa Madgwick for having a nice and comforting word always ready! Jeroni Galmés for sharing the hot lab, for the “paella” and for the company in beautiful Scotland! Marcela Baúdo, for being another Speedy Gonzalez (!!!) and for the deep breaths shared while planning drought experiments... Guy Kiddle for the advices on the HPLC, on the amino acid identification and also the ones on scientific life! Till Pellny for the technical, scientific and life advices and, most of all, for having fun teasing me!!! Simon Driscoll for the technical support on the gas-exchange measurements and for the knowledge transmitted on the subject. Also for sharing his life story and his music! Riekert van Heerden for the advices on proline estimation, on lab working and energy level maintenance!!! Richard Parkinson, Kate Rydlewski and Steve Harvey, who helped me to take care of my “little girls” in the greenhouse 28, compartment 106!!! Stephen Powers for all the statistical analysis and advices, the high-horses and the long (but productive) discussions, the support and comforting words and, especially, for Believing me as a scientist from the very first moment!

Amigos que conheci na FCUL! Entre os quais… André, pela loucura constante, Márcia pela boa disposição e energia positiva e Elsita pelos mimos tão gostosos! Cátia pela partilha de suspiros durante frases escritas e outros rabiscos que tal! Céu pelo melhor sorriso da manhã na FCUL!

All the good friends I met abroad! Namely… Rui for being my salvation at the Manor! Isabel for being a good listener and a good advisor, on PhDs and Life! Marta for sharing the experience on stress studies, Anneke for being a company in the late hours, Hiro for being pure, Francesca for being like a sister, Alexia for the joy! Maria Paz for being crazy, Nisreen for being a good friend, Ivânia and Luis for making part of the Portuguese mafia at RRes! Salvador for the sports company and for the friendship! Duncan for being such a good listener!!! My chocolate supplies will always be there… Eleonora for the smile and the NB copies! Tanya for the contagious energy!!!

Amigos em Casa... Sílvia Duarte, conselheira de saúde! Catarina Oliveira, assitente de imagem: a beleza é um conceito relativo! Bruno Serrão, assistente de informática – o que seria do meu portátil sem as tuas consultas terapêuticas!!! Ana Francisco, Anita, mesmo que distantes, não nos ausentamos de todo, estamos lá e sabemos disso! Foi um prazer enfrentar o microscópio contigo!!! Ana Catarino, Nocas, pela vontade contagiante de vencer nesta conquista por um lugar ao sol e de conhecer o mundo lá fora! Inês Bruno, por estar sempre a meu lado neste percurso que temos feito pela Vida - de alguma forma sei que vais estar sempre lá!

Friends at my second Home! Petra Bleeker, Peet, fofa, for being my older sister!!! Thanks for all the advices!!! Samuel Doufur, Sam, for all the Chemistry explanations, for the secret supplies… Also for the culture lessons and cooking sessions, for the constant company and support, even during the worst hours… and, most of all, for being such a nice friend!

Miguel pelos bons momentos que passámos juntos, o percurso de vida que caminhámos lado a lado, muitas vezes afastados... O apoio incontestável em todas as decisões difíceis, mesmo que muito discutidas e refutadas! A lição de vida que ficou e o carinho que permanece, sempre.

À minha família agradeço sobretudo a compeensão. Não é fácil colocar em palavras o quão importante foi para mim o apoio constante que recebi da vossa parte e a força que me dá, todos os dias, o Amor que nos une! A Cat e a Ju, pataca e pataquinha, que são crianças a sério, percebem o quanto as amo e aceitam que vá “lá para a Inglaterra”! O Daniel partilha uma certa estória e tem crescido muito rápido nesta Vida! Mãe, unidas pela distância e partilhando aquela forma especial de ver e de sentir! Pai… o que sou hoje resulta em grande parte daquilo que me ensinaste a Ser!

OBRIGADA!

iii

“Wisdom, Happiness and Courage Are not waiting somewhere (…) at the end of a straight line; They’re part of a continuous cycle that begins right here. They’re not only the ending, but the beginning as well. The more it snows, the more it goes on snowing.” “A Sabedoria, a Felicidade e a Coragem Não estão à espera algures (…) ao fundo de uma linha recta; São parte de um ciclo contínuo que começa aqui mesmo. Não são apenas o fim, mas também o princípio. Quanto mais neva, mais continua a nevar.” Benjamin Hoff in The Tao of Pooh

Contents

v

CONTENTS Summary ........................................................................................................................................ 1 Resumo .......................................................................................................................................... 3 Chapter I. General introduction and objectives ........................................................................... 7

C4 photosynthetic metabolism ................................................................................................. 17 Drought stress .......................................................................................................................... 23 Objectives of the Thesis ........................................................................................................... 31 References ................................................................................................................................ 32

Chapter II. Water relations and leaf anatomy of C4 grasses ...................................................... 47

Abstract .................................................................................................................................... 49 Introduction .............................................................................................................................. 50 Material and methods .............................................................................................................. 53 Results ...................................................................................................................................... 59 Discussion ................................................................................................................................ 71 References ................................................................................................................................ 79

Chapter III. Photorespiration and C4 photosynthesis under drought stress ............................... 85

Abstract .................................................................................................................................... 87 Introduction .............................................................................................................................. 88 Material and methods .............................................................................................................. 91 Results ...................................................................................................................................... 98 Discussion .............................................................................................................................. 107 References .............................................................................................................................. 115

Chapter IV. C4 enzymes in drought-stressed grasses................................................................ 121

Abstract .................................................................................................................................. 123 Introduction ............................................................................................................................ 124 Material and methods ............................................................................................................ 127 Results .................................................................................................................................... 131 Discussion .............................................................................................................................. 136 References .............................................................................................................................. 140

Chapter V. Rubisco from C4 grasses under drought stress .. .................................................... 145

Abstract .................................................................................................................................. 147 Introduction ............................................................................................................................ 148 Material and methods ............................................................................................................ 152 Results .................................................................................................................................... 159 Discussion .............................................................................................................................. 167 References .............................................................................................................................. 172

Chapter VI. General discussion and conclusions ..................................................................... 179

Concluding remarks ............................................................................................................... 192 Future perspectives ................................................................................................................ 193 References .............................................................................................................................. 194

List of Symbols and Abbreviations Used

vii

LIST OF SYMBOLS AND ABBREVIATIONS USED A net CO2 assimilation rate Ac net CO2 assimilation rate calculated from quadratic expression for enzyme-limited

photosynthesis An net CO2 assimilation rate calculated from the asymptotic exponential curve applied to the

variation of A with Ci ABA abscisic acid ACC 1-aminocyclopropane-1- carboxylic acid ADP adenosine 5’-diphosphate Ala alanine AlaAT alanine aminotransferase AMP adenosine monophosphate Asn asparagine Asp aspartate AspAT aspartate aminotransferase ATP adenosine 5’-triphosphate Bicine N,N-bis(2-hydroxy-ethyl)glycine BS bundle sheath Ca atmospheric CO2 concentration (in the gas phase) Ci CO2 concentration in the intercellular air-spaces Cm CO2 concentration in the mesophyll cells Cs CO2 concentration in the bundle sheath cells CA carbonic anhydrase CA1P 2-carboxyarabinitol-1-phosphate CABP 2-carboxyarabinitol-1,5-bisphosphate Chl chlorophyll δ isotope composition DTT 1,4-dithiothreitol DW dry weight EDTA ethylenediaminetetraacetic acid E4P erythrose-4-phosphate γ* half the reciprocal of Rubisco specificity gbs bundle sheath conductance to CO2

gi mesophyll conductance to CO2

gswa stomatal conductance to water vapour Gln glutamine Glu glutamate Gly glycine Hepes 4-(2-hydroxy-ethyl)-1-piperazine-ethanesulfonic acid HNV 5-hydroxy-L-norvaline (or 2-amino-5-hydroxypentanoic acid) FW fresh weight GOGAT ferredoxin (Fd-) or NADH-glutamate synthase Kc Michaellis-Menten constant of Rubisco for CO2


viii

Ko Michaellis-Menten constant of Rubisco for O2 Kp Michaellis-Menten constant of PEPC for CO2 KRuBP Michaellis-Menten constant of Rubisco for RuBP LN2 liquid nitrogen LSD least significant difference LWP leaf water potential M mesophyll MDH malate dehydrogenase MEA monoethanolamine Met methionine MS moderate drought stress NAD-ME NAD-malic enzyme NAD+ nicotinamide-adenine dinucleotide (oxidized) NADH nicotinamide-adenine dinucleotide (reduced) NADP-ME NADP- malic enzyme NADP+ nicotinamide-adenine dinucleotide phosphate (oxidized) NADPH nicotinamide-adenine dinucleotide phosphate (reduced) n.d. not determined O O2 partial pressure in the bundle sheath and mesophyll cells O2 oxygen OAA oxaloacetate P probability or level of significance PDBP D-glycero-2,3-diulose-1,5-bisphosphate PEG polyethylene glycol PEP phosphoenolpyruvate PEPC phosphoenolpyruvate carboxylase PEPCK phosphoenolpyruvate carboxykinase PG 2-phosphoglycolate PGA 3-phosphoglycerate Phe phenylalanine Pi orthophosphate PPdK pyruvate,orthophosphate dikinase PPFD photosynthetic photon flux density PPi pyrophosphate Pr rate of photorespiration PVP polyvinylpyrrolidone Rd leaf mitochondrial respiration Rm mesophyll mitochondrial respiration R2 percentage of variance accounted for by a model REML Residual Maximum Likelihood ROS reactive oxygen species Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Ru5P ribulose-5-phosphate RuBP ribulose-1,5-bisphosphate RWC leaf relative water content


ix

S non-watered or drought-stressed s2 residual mean square s.e. standard error of the mean SED standard error of differences Ser serine SLA specific leaf area SS severe drought stress SWC soil water content TCA tricarboxylic acid TFA trifluoroacetic acid TW turgid weight Vc rate of Rubisco carboxylation Vcmax maximum Rubisco carboxylation activity Vmax maximal activity Vo rate of Rubisco oxygenation Vomax maximum Rubisco oxygenation activity Vp rate of PEPC carboxylation Vpmax maximum PEPC carboxylation activity Vphysiol physiological activity WUE water use efficiency WWP water weight in pot

Summary

1

SUMMARY

Drought stress is one of the major constraints to plant photosynthetic performance. With

increasing water scarcity in many areas of the world the understanding of fundamental drought-

stress physiology and biochemistry is crucial to optimize the exploitation of plant responses and

improve water use efficiency. The CO2-concentrating mechanism present in C4 plants limits

photorespiration and increases their photosynthetic performance. The physiological and

biochemical responses to gradually-induced water deficit of three C4 grasses with different

decarboxylating mechanisms were compared. Paspalum dilatatum Poir. (NADP-ME) was less

resistant to drought than Cynodon dactylon Pers. (NAD-ME) and Zoysia japonica Steudel

(PEPCK). The faster leaf dehydration in the first species reflects the higher water requirement

associated with its high productivity. Leaf structure of all three species, especially C. dactylon

and Z. japonica, showed advantageous characteristics to cope with xeric environments. In all

three species photosynthesis and stomatal conductance decreased under water deficit but showed

continued CO2 assimilation even when leaf dehydration was severe. Photorespiration, as

evaluated by CO2 exchange rates at different concentrations of CO2 and O2, by mechanistic

modelling of C4 photosynthesis and by changes in amino acids in a 30 s dark period, remained

slow under drought conditions, supporting the photosynthetic efficiency of the CO2-

concentrating mechanism. Moderate drought stress did not affect dramatically the carboxylating

and decarboxylating enzyme activities but caused changes in the regulation of PEPC and

Rubisco. Considerable activity of PEPCK was found in all three species suggesting its

involvement in the C4 photosynthetic pathway, acting as a secondary decarboxylase in P.

dilatatum and C. dactylon, or in other non-photosynthetic processes occurring in the leaves of

the three C4 grasses. An unusual hydroxylated amino acid was drought-inducible and its

potential benefits for drought resistance should be further investigated.

KEYWORDS:

Water deficit, C4 photosynthesis, Photorespiration, Paspalum dilatatum, Cynodon dactylon,

Zoysia japonica

Resumo

3

RESUMO

O défice hídrico é um dos factores que mais limitam a produtividade vegetal. A escassez de água

é um problema que atinge proporções cada vez maiores em muitas áreas do planeta e tende a ser

exacerbado devido às reconhecidas alterações climáticas. A implementação de práticas de rega

mais correctas, envolvendo monitorização das condições hídricas do solo e/ou das plantas, bem

como a utilização de espécies e variedades de plantas melhor adaptadas a condições de baixa

disponibilidade de água poderão contribuir para uma mais eficiente gestão dos recursos hídricos.

O conhecimento da fisiologia e bioquímica em condições de stress são fundamentais para que as

respostas adaptativas das plantas a condições xéricas possam ser exploradas e a eficiência do uso

da água melhorada.

A pressão exercida sobre os diferentes utilizadores de água é assim cada vez maior e

afecta diversas áreas, incluindo a agricultura e o turismo. O crescente número de campos de

golfe em Portugal implica gastos hídricos adicionais que são muitas vezes criticados, sobretudo

nos verões em que a seca é mais severa e a necessidade de água para a produção agrícola torna o

desporto uma necessidade supérflua. A utilização de espécies e variedades de relvas mais

adequadas (que gastem menos água mantendo elevada performance) aliada a práticas de rega que

envolvam, por exemplo, reutilização de águas residuais irá contribuir para uma maior eficiência

do uso da água disponível.

O mecanismo de concentração de CO2 presente nas plantas com metabolismo

fotossintético em C4 torna-as menos sensíveis ao oxigénio e a resultante limitação da

fotorrespiração nestas plantas contribui para o aumento da eficiência do uso de água. As relvas

C4 estão, por esse motivo, geralmente associadas a uma maior resistência ao défice hídrico, mas

não há necessariamente uma relação directa entre eficiência de uso de água e resistência à seca.

A distribuição das espécies C3 e C4 sugere que a temperatura é o principal factor que se

correlaciona com a ocorrência de cada uma das variantes fotossintéticas. A precipitação, por

outro lado, parece estar relacionada coma distribuição diferencial dos vários subtipos de

fotossíntese em C4. As espécies do subtipo NADP-ME (enzima málico dependente do NADP)

são geralmente mais abundantes em áreas com maior precipitação e as espécies do subtipo NAD-

ME (enzima málico dependente do NAD) estão mais representadas em zonas mais secas,

enquanto que a distribuição das espécies do subtipo PEPCK (fosfoenolpiruvato carboxicinase)

em relação aos gradientes de precipitação apresenta um padrão mais incerto.

As respostas fisiológicas e bioquímicas ao défice hídrico foram estudadas em três

espécies de relvas C4 dos diferentes subtipos metabólicos: Paspalum dilatatum Poir. (NADP-

Resumo

4

ME), Cynodon dactylon Pers. (NAD-ME) e Zoysia japonica Steudel (PEPCK). As três espécies

foram comparadas em termos da presença de mecanismos de resistência ao stress envolvendo a

manutenção dos conteúdos hídricos da planta ou a tolerância a condições de desidratação foliar.

Uma abordagem inicial envolveu o estudo das relações hídricas e da estrutura foliar, a que se

seguiu o estudo das trocas gasosas, da fotorrespiração e de outros aspectos do metabolismo

fotossintético relativos aos processos de carboxilação e descarboxilação.

As plantas de P. dilatatum foram menos resistentes ao défice hídrico, observando-se uma

diminuição mais rápida do teor hídrico relativo das suas folhas que nas plantas de C. dactylon e

Z. japonica. As folhas das três espécies são caracterizadas por uma estrutura foliar especializada,

com anatomia foliar Kranz, que permite uma maior eficiência da utilização dos recursos hídricos.

A espécie Z. japonica, em particular, tem paredes celulares mais rígidas e possui maior

quantidade de esclerênquima, o que se traduz numa maior rigidez e dureza das folhas. A rigidez

celular permite a diminuição da pressão de turgescência quando o teor hídrico começa a diminuir,

promovendo o abaixamento do potencial hídrico das folhas, que foi mais acentuado em Z.

japonica e pode ser visto como uma estratégia que permite o aumento da tomada de água do solo

à medida que a sua disponibilidade diminui. A capacidade de acumulação de osmólitos,

incluindo a prolina e outros aminoácidos, permite aumentar a pressão osmótica e contribuem

assim para a diminuição do potencial hídrico. No entanto, o aumento de aminoácidos como a

prolina, metionina, valina, fenilalanina, isoleucina e leucina sugere também o seu envolvimento

directa ou indirectamente (através da produção de compostos do metabolismo secundário) em

mecanismos de defesa, nomeadamente protecção contra espécies reactivas de oxigénio e

utilização de poder redutor em excesso. Nas folhas de C. dactylon e Z. japonica foi identificado

um aminoácido hidroxilado não-proteico que não tinha sido previamente descrito como estando

presente em folhas e que apresenta características que devem ser exploradas. O aminoácido 5-

hidroxi-L-norvalina (HNV) ocorre em folhas de plantas de Z. japonica bem irrigadas e o seu

conteúdo aumenta com o défice hídrico. Em C. dactylon HNV não está presente nas folhas de

plantas bem irrigadas mas surge e o seu conteúdo aumenta acentuadamente com a desidratação

foliar. Em P. dilatatum o aminoácido não está presente e não é induzido pelo stress. Associando

este comportamento à elevada resistência à seca de C. dactylon e Z. japonica sugere-se que este

aminoácido poderá estar intimamente associado ao mecanismo de defesa destas plantas.

A anatomia foliar das plantas das três gramíneas não foi muito alterada em condições de

stress e sugere que as folhas destas espécies apresentam características intrínsecas que lhes

permitem uma melhor adaptação a condições de reduzida disponibilidade de água, conferindo-

lhes uma maior eficiência no uso de água. O aumento da proporção de células do mesófilo em

Resumo

5

relação às células da bainha perivascular em P. dilatatum e Z. japonica em condições de stress

sugere alguma capacidade de adaptação ao défice hídrico, mas a ausência de maiores alterações

morfológicas induzidas pelo stress reflecte a baixa plasticidade fenotípica das três espécies.

O mecanismo de concentração de CO2 característico do metabolismo fotossintético em

C4 limita a reacção oxigenativa da Rubisco e, consequentemente, a taxa de fotorrespiração. No

entanto, o fecho dos estomas em condições de défice hídrico pode levar à diminuição da

concentração intercelular de CO2 e ao aumento da fotorrespiração. Nas três gramíneas estudadas

houve uma diminuição da taxa de fotossíntese e da condutância estomática com a desidratação

foliar, mas as trocas gasosas continuaram mesmo quando o teor hídrico diminuiu para valores

inferiores a 80%, correspondente a cerca de 10 dias sem irrigação e um reduzido conteúdo de

água no solo. O decréscimo da fotossíntese com o aumento da concentração de oxigénio e a

diminuição do conteúdo em glicina ao fim de um período de 30 s em escuridão evidenciaram a

presença de taxas de fotorrespiração, embora muito mais lentas nestas três espécies C4 que em

plantas C3. A análise conjunta das trocas gasosas de CO2 a diferentes concentrações de CO2 e O2,

incluindo o ponto de compensação para o CO2 e sensibilidade da fotossíntese ao oxigénio e a

aplicação de um modelo de fotossíntese em C4 aos dados experimentais revelaram a manutenção

de taxas de fotorrespiração lentas em condições de défice hídrico. A variação do conteúdo em

aminoácidos, especialmente glicina, na fracção solúvel das folhas, suportou a capacidade das três

espécies estudadas para limitar a velocidade de oxigenação da RuBP mesmo em condições de

défice hídrico. O aumento do conteúdo em glicina, serina e etanolamina com a desidratação

foliar bem como os resultados comparativos da modelação das trocas gasosas em condições

controlo e de stress forneceram os únicos indícios para um ligeiro aumento do metabolismo

fotorrespiratório em relação à fotossíntese.

Para uma maior eficiência do metabolismo fotossintético em C4 é necessário haver uma

boa coordenação entre a carboxilação primária do CO2, levada a cabo pela PEPC no mesófilo, e

a subsequente descarboxilação dos ácidos C4 na bainha perivascular. As actividades da PEPC,

NADP-ME, NAD-ME e PEPCK não foram muito afectadas pelo défice hídrico e consideradas

no seu conjunto sugeriram uma maior eficiência fotossintética em C. dactylon que nas outras

duas espécies. O estado de fosforilação da PEPC aumentou, revelando uma potencial resposta

adaptativa das três gramíneas C4 ao stress no sentido de tornar a carboxilação primária do CO2

mais eficiente em condições que promovem o fecho dos estomas, reduzindo a disponibilidade de

CO2 a nível intercelular. Curiosamente, observaram-se consideráveis actividades da PEPCK nas

três espécies, pertencentes a cada um dos subtipos metabólicos de plantas C4, revelando que a

enzima poderá actuar como descarboxilase alternativa nas plantas de P. dilatatum e C. dactylon

Resumo

6

(subtipos NADP-ME e NAD-ME, respectivamente) ou desempenhar funções no metabolismo

não-fotossintético das três espécies. As potencialidades associadas à presença de elevadas

actividades da PEPCK em gramíneas C4 dos vários subtipos metabólicos devem ser investigadas

em maior detalhe no sentido de aumentar a compreensão do metabolismo fotossintético em C4.

Em condições de défice hídrico, a diminuição das taxas de assimilação de CO2 implica

que a actividade da Rubisco seja regulada de modo a manter o equilíbrio entre os diferentes

processos do metabolismo fotossintético. A diminuição do conteúdo em RuBP nas folhas

desidratadas das três espécies sugere que a regeneração deste substrato é afectada em condições

de défice hídrico, possivelmente devido a danos ao nível do ciclo de Calvin ou reduzida síntese

de ATP, limitando a assimilação de CO2. O aumento de inibidores nas mesmas folhas sugere que

estes se ligam à Rubisco em condições de stress protegendo a enzima contra o dano proteolítico,

não se observando alterações no conteúdo em Rubisco.

A aplicação de modelos de fotossíntese para simular os efeitos induzidos por alterações

ambientais tem sido comprometida pela falta de conhecimento de vários parâmetros

fundamentais e pela quantidade de pressupostos inerentes á sua utilização. As constantes

cinéticas para as actividades carboxilativa e oxigenativa da Rubisco parcialmente purificada de

cada uma das três gramíneas C4 estudadas irá permitir aumentar o rigor e a precisão com que se

fazem simulações baseadas nestes modelos. Em comparação com a espécie C3 utilizada como

modelo, o trigo, a Rubisco de P. dilatatum, C. dactylon e Z. japonica é caracterizada por factores

de especificidade mais baixos, e constantes de Michaelis-Menten para o CO2 e o O2 e

velocidades de carboxilação máxima mais elevadas.

As três espécies de gramíneas C4 estudadas apresentam elevada eficiência fotossintética,

sobretudo C. dactylon, e elevada resistência ao défice hídrico, sobretudo C. dactylon e Z.

japonica. O metabolismo fotossintético é regulado de modo adaptativo e a limitação da taxa de

fotorrespiração mantém-se em condições de stress. As potencialidades associadas ao aumento do

conteúdo em HNV com a desidratação foliar e à presença de elevadas actividades da PEPCK nas

três espécies pertencentes a diferentes subtipos metabólicos devem ser exploradas. As constantes

cinéticas da Rubisco determinadas para gramíneas C4 permitirão o rigor na simulação das

respostas do metabolismo fotossintético a diversas condições ambientais.

PALAVRAS-CHAVE:

Défice hídrico, Fotossíntese C4, Fotorrespiração Paspalum dilatatum, Cynodon dactylon, Zoysia

japonica

Chapter I.

GENERAL INTRODUCTION AND OBJECTIVES

General Introduction and Objectives

9

GENERAL INTRODUCTION AND OBJECTIVES

Climate change and water shortage – the problem!

Water is one of the most important factors limiting plant growth, development and survival. In

particular in Mediterranean climates, which are characterized by long hot and dry periods, water

availability is the major limitation to plant productivity (Turner 2004). The decrease of fresh

water availability is one of the most serious environmental problems of the planet, as recognized

by the United Nations Environment Programme (Global Environment Outlook: Environment for

Development, Geo-4 report 2007). The number of dry days per year is expected to raise in many

areas of the globe (Petit et al. 1999), exacerbating the problem.

The efficient management of water resources by agricultural and recreational systems is

therefore an outstanding priority in many regions of the world and there is increasing pressure on

the different irrigators to increase the efficiency of water use by crops and pastures or lawns.

Golf courses are probably the most criticized water-users, especially during summer-droughts, in

the USA and in some European countries, including Portugal. This is mostly because the game is

not considered a ‘primary need’. Even considering the enormous importance in terms of tourism

and development, serious concerns arise in terms of water consumption. The tourism sector has

been recognised as one of the key water users in Europe where there is big potential for water

savings, including the use of more efficient irrigation techniques and rain water harvesting in

golf courses (MAOTDR 2007b). In America great investments have been made in order to

promote increased water use efficiency at the level of the golf industry (Snow 2001), namely

through the use of varieties of turfgrass that require less water, better irrigation techniques and

management practices (e.g. monitoring the soil moisture for scheduling irrigation), and the use of

alternative water sources.

Deficit irrigation techniques have been successfully applied in the field to improve water

use efficiency by some crops and might have further application in water-limited environments

(Costa et al. 2007; Fereres & Soriano 2007). For instance, the strategy of partial rootzone drying,

which involves simultaneous exposition of the roots to wet and drying soil, allows the decrease

of stomatal conductance, decreasing water loss, without affecting plant performance (Davies et

al. 2002; Santos et al. 2003). The use of secondary, non-potable waters for the irrigation of

turfgrasses has been suggested as another possible alternative (Snow 2001; Marcum 2006) and

water re-utilization is already implemented in many golf courses in the South of Portugal

Chapter I.

10

(MAOTDR 2007a). Irrigation systems controlled by soil moisture sensors can also be used to

minimize water waste by matching turfgrass requirements (Pathan et al. 2007).

The use of plant species and cultivars better suited to the environment can also improve

water use efficiency and plant production and yields in water-limited regions (Turner 2004). As

recently proposed by Marcum (2006), turfgrasses with high salinity tolerance allow the use of

saline, non-potable waters for irrigation. Snow (2001) reported the use of stress-tolerant

bermudagrass (Cynoodn dactylon) cultivars with low water requirements yielding considerable

water savings in American golf courses, and also the successful irrigation of the extremely salt-

tolerant and highly productive seashore paspalum (Paspalum vaginatum) with salted waters. It is

also important to note that, in what concerns the golf courses in particular, there are different

areas associated with different game functions and the turfgrasses used in each of these should

correspond to the functional needs associated (Bernardes da Silva et al. 2008).

The grass family and the C4 photosynthetic pathway

The multifaceted contribution of the Poaceae to the world economy is the base for the superlative

importance of this plant family. The grass family includes the cereals (such as wheat, rice, maize

and sorghum), the most important source of sucrose in the world (sugarcane), and the forage and

turf grasses, which are the backbone of sustainable agriculture (Jones 1985). The paramount

importance of the forage and turf grasses arises from the economic benefits associated with its

use in golf courses and other recreational areas, the sustainability of live stocks and wild animals,

and from their contribution to the soil conservation and environment protection.

Grasses are commonly classified as cool-season and warm-season, reflecting low (ca.

15º-25ºC) or high (ca. 25º-35ºC) optimal daytime growth temperatures, respectively. The distinct

physiological characteristics between the two types of grasses result from the presence of the C4

photosynthetic pathway that elevates the CO2 concentration at the site of carboxylation in the

warm-season (C4) grasses (Furbank & Hatch 1987). C4 photosynthesis occurs in nearly half of

the species in the Poaceae (Hattersley 1988; Sage et al. 1999a) and some of the world’s most

important crop and turf species are C4 grasses (see Jones 1985; and Brown 1999). Warm-season

C4 grasses, by opposition to cool-season C3 species, are characterized by performing better at

high irradiance levels and high temperatures (Johnston 1996), showing higher rates of

photosynthesis than their C3 counterparts in full sunlight and at temperatures above 30ºC (Brown

1999). C4 grasses have generally higher water use efficiency (WUE) due to an efficient

assimilation of CO2 in combination with lower transpiration rates (Edwards et al. 1985). A


11

comparison by Gherbin et al. (2007) revealed that warm-season C4 grasses produce higher yields

than cold-season C3 grasses in warm dry summer conditions like those observed in the

Mediterranean climate and in some regions of the USA and Australia.

The distribution of C3 and C4 grasses in several regions of the world suggests that

temperature is the climatic variable best correlated with the relative occurrence of grass species

of the two photosynthetic pathways (Henderson et al. 1995; Sage et al. 1999b). As shown by

Cabido et al. (1997), C4 species tend to dominate in areas characterised by warmer temperatures

and their distribution is therefore dependent on a latitudinal and altitudinal gradient. Sage et al.

(1999b) noted that aridity is not a prerequisite for C4 dominance over C3 and the success of C4

species depends essentially on the presence of warm temperatures and high light intensities,

resulting in a high representation of this photosynthetic pathway in tropical and subtropical

regions, where more than two thirds of all grasses are C4.

The presence of a CO2-concentrating mechanism makes C4 photosynthesis more

competitive in conditions that promote photorespiration, like high temperatures and low

intercellular CO2 concentrations. Consequently, it is generally assumed that C4 species will

perform better in warm habitats and conditions that promote stomatal closure, including

decreased water availability. Importantly, Sage et al. (1999b) noted that the seasonality of

precipitation plays an important role and its occurrence during the warm season tends to favour

C4 dominance. Nevertheless, several reports suggest that C4 grasses may dominate in areas

where precipitation decreases in the warmer summer months (see Cabido et al. 2008) and their

exploitation can therefore bring advantage in terms of water savings.

The diversification of the anatomical-biochemical variants of C4 photosynthesis might be

related with natural selection pressures of changes in rainfall (Hattersley & Watson 1992), as

precipitation gradients seem to be the major determinant for the relative distribution of the three

classical variants of C4 photosynthesis. These are named after the main decarboxylating enzyme

in each pathway (Gutierrez et al. 1974; Hatch et al. 1975): NADP-malic enzyme (NADP-ME),

NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). The bio-

geographical distribution of C4 species with different decarboxylation mechanisms in several

regions of the world (e.g. Hattersley 1992; Cabido et al. 2008) shows that NADP-ME species are

relatively more abundant in areas with higher annual rainfall whereas NAD-ME species

predominate in arid zones, and PEPCK seem to have a less clear pattern of association with

precipitation gradients. Brown (1999) refers that most of the cultivated C4 species with

agronomic importance are NADP-ME, possibly as a consequence of their occurrence in wetter

areas, the first to be exploited during colonization. In the current climate conditions and

Chapter I.

12

assuming the future perspectives, the implementation of some potentially more drought-resistant

NAD-ME species might bring considerable advantage in terms of better use of water resources.

The positive and negative correlation of NAD-ME and NADP-ME species with aridity,

respectively, may be related with enhanced water use efficiency in the former species

(Ghannoum et al. 2002). However, it is unclear if this is due to the functional differences

between the two variants of C4 photosynthesis. Most grasses from the subfamily Chloridoideae

are C4 and belong to the subtypes NAD-ME or PEPCK, whereas the subfamily Panicoideae is

photosynthetically more variable, with representatives of C3, intermediate C3-C4 and C4

photosynthesis (Hattersley & Watson 1992). All three biochemical subtypes of C4 grasses occur

in the Panicoideae, but the great majority of C4 species in this subfamily are NADP-ME (Taub

2000). The distribution of the subfamilies Panicoideae and Chloridoideae is also strongly

correlated with the precipitation gradients (Taub 2000; Cabido et al. 2008), suggesting that

characteristics other than the biochemistry of photosynthesis may be responsible for the

geographical patterns observed, possibly reflecting some divergent patterns associated with the

multiple origins of C4 grasses (Kellogg 1999).

In addition to the specialised photosynthetic biochemistry, the leaves of most C4 grasses

show anatomical modifications associated with the functionality of the CO2-concentrating

mechanism. These characteristics are referred to as Kranz anatomy (see review by Dengler &

Nelson 1999). The term ‘Kranz’ refers to a wreath of cells surrounding the vascular tissues and

was first used by Haberlandt (1882) who initially referred to the presence of distinct leaf

anatomies and recognized that grasses could be divided into two groups and these were related

with their ecological adaptation. A suite of subtype-specific anatomical characteristics has been

further associated with each of the decarboxylation mechanisms (Prendergast & Hattersley 1987;

Dengler et al. 1994). Most C4 grasses belong to the ‘classical’ biochemical-anatomical subtypes

(Hattersley & Watson 1992), but some variations occur in nature (e.g. Prendergast et al. 1987).

As suggested by Hattersley (1992), differences in leaf structure could possibly be associated with

differential ability of different grass species to cope with decreased water availability.

C4 species and climate change

Although C4 plants represent less than 4% of the terrestrial plant species (Sage et al. 1999a) they

contribute about one-quarter of the primary productivity of the planet and a large fraction of the

primary production consumed by humans, directly or not, is derived from C4 crops and pastures

(Brown 1999). The specialized leaf structure, with a high density of vascular bundles, makes C4

grasses less digestible and tougher, and thus less appealing to herbivores, than C3 grasses


13

(Wilson & Hattersley 1989; Scheirs et al. 2001), but there is no consistent selection against C4

and in some areas C4 grasses are actually preferred in comparison to C3 (see Heckathorn et al.

1999). The increased interest in C4 grass pastures comes from the fact that they are more

productive and may be more drought resistant (Blaikie et al. 1988). The same reasons provide

evidence for their preferential use as turf in recreational areas. Additionally, the potential use of

C4 grasses as biofuels in energy production systems was recently outlined (Samson et al. 2005).

Climate change has remarkable consequences on biodiversity, species’ distribution and

their relative abundance. Water availability, in particular, is one of the most relevant

environmental factors affecting plant survival, productivity and distribution. The effects of

increasing atmospheric CO2 on growth and photosynthesis in C3 and C4 plants are still

controversial and associated with a great level of uncertainty (Chen et al. 1996; Campbell &

Smith 2000; Ainsworth & Long 2005; Korner 2006; Long et al. 2006; Soares et al. 2008), but

the predicted temperature rise is likely to favour C4 photosynthetic performance and

competitiveness and result in increased dominance of grasses with this photosynthetic pathway

(Henderson et al. 1995; Sage & Kubien 2003). Changes in rainfall scenarios are not as easy to

predict as those of temperature and involve more uncertainty but are likely to affect the relative

distribution of C4 grass species, with increased NAD-ME proportional occurrence in drier areas.

The comprehensive understanding of the C4 photosynthetic metabolism and the response

of plants with the C4 pathway to the environment are crucial if the best advantage is to be taken

from their potentialities. Recent progress has been made on the understanding of C4

photosynthesis in dicotyledon species e.g. (Voznesenskaya et al. 2007) and suggestion has been

made to adopt a species from the genus Cleome as a C4 model system (Brown et al. 2005). None

the less, there is great advantage on the use of C4 grass species. It is difficult to outcome the

economic, agronomic and ecological importance of the Poaceae. The C4 photosynthetic pathway

was first discovered in the grass family and photosynthetic variation within the Poaceae is

comprehensively understood (see review by Hattersley & Watson 1992). Most C4 grasses have

been biochemically typed in terms of photosynthetic variant and a checklist has been provided

by Hattersley some twenty years ago (Hattersley 1988). Therefore, grasses provide the ideal

system for the study of C4 photosynthesis under changing environments.

Chapter I.

14

The C4 grasses Paspalum dilatatum, Cynodon dactylon and Zoysia japonica

The three warm-season C4 grasses studied in the present work (Figure I.1) have been previously

classified as belonging to each of the different biochemical subtypes of C4 photosynthesis.

Paspalum dilatatum Poir. is a NADP-ME species (Usuda et al. 1984), Cynodon dactylon Pers. is

a NAD-ME species (Hatch & Kagawa 1974) and Zoysia japonica Steudel is a PEPCK species

(Gutierrez et al. 1974). P. dilatatum belongs to the subfamily Panicoideae, while C. dactylon and

Z. japonica belong to the subfamily Chloridoideae (Watson & Dallwitz 1992). The genus

Paspalum originated in South America, Cynodon in Africa and Zoysia in Southeastern Asia

(Brown 1999).

Paspalum dilatatum Cynodon dactylon Zoysia japonicaPaspalum dilatatum Cynodon dactylon Zoysia japonica Figure I.1. Plants of the three C4 grass species in the greenhouse during the early stages of development. Photograph was taken ca. two (P. dilatatum and C. dactylon) or four (Z. japonica) weeks after sowing.

Dallisgrass (P. dilatatum), bermudagrass (C. dactylon) and zoysiagrass (Z. japonica) are

warm-season perennial species used for turfgrass purposes throughout the world (Brown 1999).

Additionally, the first two species are important forage and cultivated pasture grasses and C.

dactylon is also one of the world’s most serious weeds (Jones 1985; Brown 1999).

The species P. dilatatum, native from South America, is an important forage grass in the

subtropical and warm regions of the world, mainly due to its high nutritive value (Venuto et al.

2003). Since long ago, dallisgrass has been a dominant pasture in Australia, especially during the

summer season (Stockdale 1983). Its great value is also derived from its cold tolerance and

ability to survive frosts in winter (Rowley 1976). Andrews & Crofts (1979a) evaluated the

possibility of replacing pastures of dallisgrass by bermudagrass in order to increase the growing


15

season recognizing the value of the latter in areas with low frost incidence. However, both the

highly productive, wide-temperature- and grazing-tolerant dallisgrass as well as the promising

bermudagrass were outyelded by an improved cultivar of Pennisetum clandestinum (Pearson et

al. 1985). None the less, dallisgrass is still an important pasture forage and progress has recently

been observed in the improvement of its forage yields (Venuto et al. 2007). In some modern

prairies, dallisgrass can become a weed to bermudagrass turf (see Henry et al. 2007).

The outstanding economic importance of C. dactylon results from the wide distribution

throughout tropical and subtropical areas and the enormous variability of the species (Taliaferro

1995). The recognition of ecotypes with different characteristics of establishment and persistence

(Andrews & Crofts 1979a) and of digestibility (Andrews & Crofts 1979b), suggested these could

be used most favourably to different purposes (e.g. control of soil erosion, feedstock, etc).

Bermudagrass, native from Africa, is the most widely used turfgrass (in lawns, golf courses and

other sports fields) in tropical and subtropical regions of the world (Brosnan & Deputy 2008) and

is used as forage for livestock (Starks et al. 2006). Additionally, C. dactylon can also be applied

in the stabilisation of soils (Vignolio et al. 2002; Moreno-Espindola et al. 2007) and has been

recently suggested as a potential energy crop for biofuel production (Boateng et al. 2007).

However, C. dactylon is sensitive to shading and requires full sun for best performance

(Guglielmini & Satorre 2002; Tegg & Lane 2004; Brosnan & Deputy 2008). The species is also

cold-sensitive, but different lines are now being developed with improved freeze tolerance

(Anderson et al. 2007). Bermudagrass is a target for genetic engineering for turf quality

improvement (Li et al. 2005; Wang & Ge 2005).

The species Z. japonica, native from Japan, is sometimes called Japanese or Korean lawn

grass (Duble 2002). Despite its slow growth rate, zoysiagrasses are shade tolerant and perform

well as lawns, being widely used in golf courses and other sports fields (Deputy & Hensley

1999). Zoysia japonica is widely used in Japan and other countries of Asia as a turfgrass for

sports fields and as a forage grass (see Cai et al. 2005 for references in Japanese!). Slow

establishment of lawns is one of the major barriers for the use of zoysiagrass, but it was recently

shown that different genotypes may establish faster (Patton et al. 2007). This grass species

requires watering during lawn establishment but afterwards it is one of the most drought- and

heat-resistant warm-season grasses (Deputy & Hensley 1999). Some contradictory references for

its cold resistance are related with differences in freeze tolerance among genotypes of

zoysiagrass (Patton & Reicher 2007) and the winter hardiness and high temperature tolerance

suggest that zoysiagrass can adapt to a wide range of environmental changes (see White et al.

2001). Zoysiagrass is characterized by very stiff leaf blades due to high content in silica (Duble

Chapter I.

16

2002) and is nearly as salt tolerant as bermudagrass due to the presence of salt secretion glands

in their leaves (Marcum 1999). Additionally, zoysiagrass seems to be less sensitive to low

nutrient supply than bermudagrass (Menzel & Broomhall 2006), with associated potential

savings in fertilizers. Its recognized turf value makes zoysiagrass subject of genetic

transformation (e.g. Ge et al. 2006).

The remarkable characteristics of P. dilatatum, C. dactylon and Z. japonica provide

evidence for the potentialities associated with the understanding of the responses of these grass

species to the environment, particularly to conditions of decreased water availability, and the

functional significance of their C4 photosynthetic pathways under these conditions.


17

C4 PHOTOSYNTHETIC METABOLISM

Biochemistry and anatomy of C4 photosynthesis

The C4 photosynthetic pathway, with specialised biochemical and anatomical characteristics,

results in elevated CO2 concentrations at the site of the carboxylating enzyme, ribulose-1,5-

bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), thereby increasing photosynthetic

efficiency in conditions promoting high rates of photorespiration. The main features associated

with C4 photosynthesis were identified in the two decades that followed the discovery of the C4

dicarboxylic acid pathway in the mid-1960’s (see historical overview by Hatch 1999).

In C4 grass leaves with Kranz anatomy, primary fixation of inorganic carbon by

phosphoenolpyruvate (PEP) carboxylase (PEPC; EC 4.1.1.31) occurs in the mesophyll (M) cells.

The C4 acids formed are then transported to the bundle sheath (BS) cells, where they undergo

decarboxylation and the released CO2 is subsequently assimilated by Rubisco in the C3 pathway

(Hatch 1987; Kanai & Edwards 1999). PEPC is confined to the cytosol of M cells and Rubisco is

confined to the chloroplasts of BS cells (Edwards et al. 2001). The specialized leaf structure (see

Dengler & Nelson 1999), namely the chemical modification and increased thickness of the BS

cell walls and the reduction of the exposure of BS surface area to intercellular spaces, decrease

leakage of CO2 back to M cells so that CO2 accumulates (Furbank et al. 1989; Brown & Byrd

1993; Evans & von Caemmerer 1996; Jenkins 1997; Kiirats et al. 2002). C4 photosynthesis

saturates at lower CO2 concentrations than in C3 plants, essentially because the affinity of PEPC

for HCO3- is much higher than the affinity of Rubisco for CO2 (Kanai & Edwards 1999). The use

of C4 photosynthesis mutants provided evidence that the flux through this pathway is controlled

by several enzymes, with special emphasis on Rubisco, PEPC pyruvate Pi dikinase (PPdK,

responsible for the regeneration of PEP in the M cells) (see review by Lea et al. 1999).

Three biochemical subtypes of the C4 photosynthetic pathway have been classically

defined according to the main enzymes responsible for the decarboxylation step (Gutierrez et al.

1974; Hatch et al. 1975): NADP-malic enzyme (NADP-ME, EC 1.1.1.40), NAD-malic enzyme

(NAD-ME, EC 1.1.1.39) and PEP carboxykinase (PEPCK, EC 4.1.1.49). The three mechanisms

of C4 photosynthesis were first described by Hatch (1987) and are summarised in Figure I.2.

These subtypes are distinguished by several aspects of leaf biochemistry and anatomy (see

reviews by Dengler & Nelson 1999; Kanai & Edwards 1999), which are discussed in further

detail in Chapter IV. The regulation of the C4 pathway, including the different subtypes, was

comprehensively revised by Leegood and Walker (1999) and details on the regulation of the

Chapter I.

18

CO2

CO2

NADP+

NADPH

Pyruvate

CHLOROPLAST

PEP

HCO3-

OAA OAA Malate

NADP+NADPH

CHLOROPLAST

PEP

ATP + Pi

AMP + PPi

Pyruvate

Malate

Calvin

Cycle

MESOPHYLL CELL BUNDLE SHEATH CELL

RuBP

MDH

PPdK

Rubisco

A NADP-ME

Pi

NADP-ME

PEPCCA

CO2

CO2

NADP+

NADPH

Pyruvate

CHLOROPLAST

PEP

HCO3-

OAA OAA Malate

NADP+NADPH

CHLOROPLAST

PEP

ATP + Pi

AMP + PPi

Pyruvate

Malate

Calvin

Cycle


RuBP

MDH

PPdK

Rubisco

A NADP-ME

Pi

NADP-ME

PEPCCA

NAD+

OAA

Calvin

Cycle

CO2

CO2

Pyruvate

CHLOROPLAST

PEP

HCO3-

OAA

CHLOROPLAST

PEP

ATP + Pi

AMP + PPi


RuBP

PPdK

Rubisco

B NAD-ME

Pi

Malate

Aspartate Aspartate

Alanine

NADH

PyruvatePyruvate

Pyruvate

Alanine(NH2)

MDH

NAD-ME

MITOCHONDRION

AspAT

AlaAT

(NH2)

AspAT

AlaATPEPCCA

NAD+

OAA

Calvin

Cycle

CO2

CO2

Pyruvate

CHLOROPLAST

PEP

HCO3-

OAA

CHLOROPLAST

PEP

ATP + Pi

AMP + PPi


RuBP

PPdK

Rubisco

B NAD-ME

Pi

Malate

Aspartate Aspartate

Alanine

NADH

PyruvatePyruvate

Pyruvate

Alanine(NH2)

MDH

NAD-ME

MITOCHONDRION

AspAT

AlaAT

(NH2)

AspAT

AlaATPEPCCA

Figure I.2. (See legend on next page.)


19

Calvin

Cycle

CO2

Pyruvate

CHLOROPLAST

PEP

HCO3-

OAA

CHLOROPLAST

PEP

ATP + Pi

AMP + PPi


RuBP

PPdK

Rubisco

C PEPCK

Pi

Aspartate

Pyruvate

Alanine

MITOCHONDRION

(NH2)

AspAT

AlaAT

PEPCCA

OAA Malate

NADP+NADPH

MDH

Aspartate

OAA PEPPEPCK

ADPATP

CO2

NAD+

NADH

Pyruvate

Malate

NAD-ME

H2O

O2

ATP

ADP

Alanine

Pyruvate

AlaAT

(NH2)

AspAT

CO2

Electron transport

Calvin

Cycle

CO2

Pyruvate

CHLOROPLAST

PEP

HCO3-

OAA

CHLOROPLAST

PEP

ATP + Pi

AMP + PPi


RuBP

PPdK

Rubisco

C PEPCK

Pi

Aspartate

Pyruvate

Alanine

MITOCHONDRION

(NH2)

AspAT

AlaAT

PEPCCA

OAA Malate

NADP+NADPH

MDH

Aspartate

OAA PEPPEPCK

ADPATP

CO2

NAD+

NADH

Pyruvate

Malate

NAD-ME

H2O

O2

ATP

ADP

Alanine

Pyruvate

AlaAT

(NH2)

AspAT

CO2

Electron transport

Figure I.2. Generalised scheme for the CO2-concentrating mechanisms of the three biochemical subtypes of C4 photosynthetic pathway (adapted from Kanai & Edwards 1999). In all subtypes, primary CO2 fixation by PEPC in the cytosol of the M cells results in the formation of C4 acids that are transported to the BS and decarboxylated increasing the CO2 concentration in these cells, where Rubisco and the C3 cycle is located. The C4 cycle is complete once PEP has been regenerated and becomes available for carboxylation in the mesophyll. In the NADP-ME subtype (A, previous page), malate is the C4 acid transported to the BS and decarboxylation occurs in the chloroplast of these cells; in the NAD-ME subtype (B, previous page), aspartate is the C4 acid transported to the BS where it is converted into malate that is decarboxylated in the mitochondria; in the PEPCK subtype (C, this page), aspartate is the main C4 acid transported to the BS where it is converted into oxaloacetate before decarboxylation in the cytosol but, concomitantly, malate is transported from the M chloroplasts to the BS mitochondria where its decarboxylation by NAD-ME provides the energy required for PEPCK (involving use of reducing power for the formation of ATP in the respiratory electron transport system) and contributes to enhance the CO2 concentration available for assimilation through the C3 cycle. Grey-dashed arrows indicate metabolite transport. Abbreviations used: AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; CA, carbonic anhydrase; MDH, malate dehydrogenase; NAD-ME, NAD-malic enzyme; NADP-ME, NADP-malic enzyme; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; PEPCK, PEP carboxykinase; Pi, orthophosphate; PPdK, pyruvate,orthophosphate dikinase; PPi, pyrophosphate; RuBP, ribulose-1,5-bisphosphate.

Chapter I.

20

enzymes involved in the carboxylating and decarboxylating reactions are given in Chapter IV

(PEPC and decarboxylating enzymes) and Chapter V (Rubisco).

The coordinate function between the M and BS cells (and thus the C3 and C4 cycles) is

crucial for C4 photosynthesis efficiency. Photosynthesis at high irradiance is limited by the

activities of PEPC and Rubisco (von Caemmerer & Furbank 1999) and a ratio of two between

PEPC and Rubisco carboxylation activities is enough to saturate CO2 assimilation. Reduction of

PEPC activity results in low assimilation rates and increased photorespiration rates at high light

(Maroco et al. 1998; Bailey et al. 2000), due to the decreased production of C4 acids and,

consequently, of CO2 released in the BS cells. Reduction of Rubisco activity leads to reduced

assimilation rates, increased CO2 concentration in BS cells and increased leakage from the BS

(Furbank et al. 1996; Siebke et al. 1997; von Caemmerer et al. 1997). Because the C4 pathway

has energy costs, associated with the regeneration of PEP (Hatch 1987; Kanai & Edwards 1999),

the leakage of CO2 back to the M cells increases the ATP used per mole of CO2 fixed and hence

decreases photosynthetic efficiency. At low irradiance, C4 photosynthesis is mostly limited by

the regeneration of RuBP and PEP, dependent on ATP and NADPH availability (von

Caemmerer & Furbank 1999).

The higher water use efficiency (WUE) of C4 plants relative to C3 results from the

combination of an efficient assimilation of CO2 through the C4 pathway, namely because the

CO2-concentrating mechanism limits photorespiration, with lower transpiration rates (Edwards et

al. 1985). As reported by Hatch (1987), the WUE in C4 plants corresponds to the double of that

observed in C3 plants and the disparity is likely to increase with temperature, essentially as result

of increased photorespiration in C3 plants.

Photorespiration in C4 plants

Rubisco properties limit the photosynthetic efficiency in C3 plants (Parry et al. 2007), essentially

because the enzyme acts both as a carboxylase and an oxygenase. When CO2 is used as a

substrate for the reaction with RuBP, the product 3-phosphoglycerate (PGA) is metabolised

through the Calvin cycle and carbon is assimilated into useful products. Conversely, reaction of

RuBP with O2 produces 2-phosphoglycolate (PG) and initiates the process of photorespiration,

which results in the loss of fixed carbon and consumption of energy (Kumarasinghe et al. 1977),

thereby decreasing the efficiency of CO2 assimilation. The photorespiratory carbon cycle (Figure

I.3) occurs in three subcellular compartments, the chloroplast, peroxisome and mitochondrion,

and results in the release of CO2 and NH3. The remaining carbon is recycled to PGA that re-

enters Calvin cycle and the ammonia released in the mitochondria must be re-assimilated in the


21

photorespiratory nitrogen cycle (Keys et al. 1978). The involvement of common intermediates in

these pathways reflects the great level of integration and interdependence of the photorespiratory

carbon oxidation cycle, the photorespiratory nitrogen cycle and the photosynthetic carbon

reduction cycle (Keys 1999). The biochemistry of photorespiration and its regulation have been

comprehensively reviewed by Leegood et al. (1995).

Glycolate oxidase

Amino-transferase

Calvin

Cycle

RuBP

PGA

Glycolate

PG

H2O

Pi

O2

Rubisco

P-glycolate oxidase

Glycolate

H2O2

O2

Glyoxylate Glycine

keto acidamino acid

Glycine (2)

CO2

NH3

Glycine decarboxylase&

Serine hydroxymethyltransferase

NAD+

NADH

SerineSerineGlycerate Glycerate

Hydroxypyruvate

CHLOROPLAST PEROXISOME MITOCHONDRION

Glyoxylate

GlycineAmino-

transferaseReductase

NAD+

NADH

ADP

Glycerate cinase

ATP

Glycolate oxidase

Amino-transferase

Calvin

Cycle

RuBP

PGA

Glycolate

PG

H2O

Pi

O2

Rubisco

P-glycolate oxidase

Glycolate

H2O2

O2

Glyoxylate Glycine

keto acidamino acid

Glycine (2)

CO2

NH3

Glycine decarboxylase&

Serine hydroxymethyltransferase

NAD+

NADH

SerineSerineGlycerate Glycerate

Hydroxypyruvate

CHLOROPLAST PEROXISOME MITOCHONDRION

Glyoxylate

GlycineAmino-

transferaseReductase

NAD+

NADH

ADP

Glycerate cinase

ATP

Figure I.3. Simplified scheme of the photorespiratory carbon cycle. The oxygenase reaction of Rubisco produces PG and PGA. The latter (not shown) is metabolised in the Calvin cycle and PG initiates the photorespiratory cycle. Glutamate and serine are the most common amino group donors for the amination of glyoxylate to glycine in the peroxisome. The subsequent conversion of two glycines into serine in the mitochondrion results in the release of both CO2 and NH3 and the remaining carbon is recycled to PGA that enters the Calvin cycle in the chloroplast. The regeneration of PGA from glycerate consumes ATP. Grey-dashed arrows indicate metabolite transport. Abbreviations used: PG, 2-phosphoglycolate; PGA, 3-phosphoglycerate; Pi, orthophosphate; RuBP, ribulose-1,5-bisphosphate.

The C4 photosynthetic pathway elevates the CO2 concentration in BS cells and the

resultant high CO2 to O2 ratio offsets the low affinity of Rubisco for CO2 and largely inhibits its

oxygenase activity and, hence, photorespiratory rates in air. Photorespiration rates correspond to

about one-quarter of CO2 assimilation rates in C3 species under normal chloroplast conditions at

25ºC (Keys 1986; Sharkey 1988), whilst in C4 plants photorespiration is likely to correspond to

less than 2% of the photosynthetic rate (Hatch 1987). Oxygen inhibits RuBP carboxylation but

Chapter I.

22

has no effect on PEP carboxylation in both maize and soybean leaf extracts (Bowes & Ogren

1972). Because CO2 and O2 are competing alternative substrates for reaction with RuBP

catalysed by Rubisco, the relative specificity of Rubisco and the concentrations of the gases will

determine the flow of carbon between the photorespiratory and the photosynthetic carbon cycles.

An increase in photorespiration is therefore expected when the intercellular CO2 decreases, as

may occur under stress conditions promoting stomatal closure.

The CO2-concentrating mechanism has energy costs (Hatch 1987) and the competitive

advantage of C4 plants in relation to C3 depends on the temperature, mostly because higher

temperatures result in increased photorespiration relative to photosynthesis in C3 plants due to

the enhanced ratio of O2 to CO2 in conditions that decrease the solubility of CO2 more than that

of O2 (see Edwards et al. 1985 and references therein).

The estimation of true rates of photosynthesis and photorespiration is difficult because of

the complexity of the CO2 and O2 exchanges involved (Sharkey 1988; Keys 1999). The re-

fixation of photorespired CO2 within the leaves (Loreto et al. 1999; Kiirats et al. 2002) further

compromises the estimation of photorespiration through CO2 exchange. Measurement of

photorespiration in C4 plants is especially complex given the level of biochemical and structural

specialization. The mathematical modelling of C4 photosynthesis by von Caemmerer and

Furbank (1999) uses basic equations to represent carbon fluxes in C4 plants. The model assumes

a steady-state balance between the release of CO2 in the BS through the C4 pathway, the

assimilation of CO2 by the C3 cycle and the leakage of CO2 from BS cells. At high irradiance,

CO2 fixation will be limited by PEPC and Rubisco activities and, the CO2 concentration in the

BS cells and, ultimately, the photorespiration rate can be estimated from the analysis of net

photosynthesis response to intercellular CO2. Additionally, amino acids can be used as

metabolite markers for the photorespiratory pathway (Foyer et al. 2003) and glycine and serine

contents were found to be well correlated with photorespiration (Novitskaya et al. 2002). Further

considerations on photorespiration rates in C4 plants are detailed in Chapter III.


23

DROUGHT STRESS

Drought and water deficit

Stress can be defined as “a deviation from the optimal condition of life” (Larcher 2003) and

elicits responses that may be adaptive or cause damage. Water stress occurs when water

availability is scarce (water deficit) or excessive (water logging) and becomes likely to affect

plant performance. Water deficit is often referred to as drought stress. Drought, in nature, is an

event of decreased water availability, resulting from low rainfalls, and is usually associated with

high temperatures and irradiance. In climates characterized by hot dry summers, like the

Mediterranean, plants are subjected to high temperatures and irradiance and may or not have

enough water, but are generally dependent on irrigation. An efficient management of water

resources is imperative in order to optimise the use of water in these regions.

Studies in the greenhouse generally attempt to promote conditions as close as possible to

those that plants experience in the field, controlling the imposition of stress conditions in order to

understand plant responses to the different variables that may affect their growth and

productivity. Drought stress, here used as a synonym to water deficit, is one of the major factors

limiting plant photosynthetic performance.

Plants may adopt a number of strategies to resist drought stress, including the

development of mechanisms of drought avoidance or drought tolerance. In the first case, the

hydration of the tissues is maintained through processes that minimize water loss or increase

water uptake from the soil, whilst in the latter one the physiological and biochemical parameters

are maintained when tissue dehydration occurs (Chaves 1991; Bray et al. 2000; Larcher 2003;

Blum 2005). Drought avoidance strategies are much dependent on the early signals triggered at

the root level in response to the drying soil (Chaves & Oliveira 2004). These signals warn the

plant to the decreased water status in the soil and allow the development of a stress response in

order to better cope with the unfavourable environment. The regulation of plant growth and

development in response to drying soil often involves chemical signalling that may

concomitantly result in stomatal closure to avoid excessive water loss from the tissues (Davies et

al. 2005). In fact, both stomatal closure (Schulze 1986) and shoot growth inhibition (Kramer

1983) occur at the earliest stages of water deficit and are likely to be induced by stress sensors or

through signal transduction pathways triggered in response to stress (Bartels & Sunkar 2005).

The long-distance signalling, involving the transit of molecules (like the hormone ABA and

other chemical regulators) from the root to the shoot and vice-versa, provide the plant with a

Chapter I.

24

means to measure water availability in the soil (Zhang & Davies 1989; Davies et al. 2005) and

quickly adjust to the environment. Plants with plastic genotypes may alter their growth and

development, and thereby optimize their response to the environment (Trewavas 1986; Sage &

McKown 2006).

The improvement of plant performance and the achievement of high stable yields under

limited water availability depend on the ecophysiological understanding of the interaction

between the plant and the growing environment in combination with the genetic background

behind it (Chaves & Oliveira 2004; Parry et al. 2005). Most of the drought-responsive genes that

have been identified and manipulated in order to increase water use efficiency under drought

conditions are involved in the protection of the cellular structure and function and, thus, the

primary metabolism, through the scavenging of reactive oxygen species (to prevent oxidative

damage) and the accumulation of osmolytes (to maintain water status) (Umezawa et al. 2006).

Many of the studies with these transgenic plants retrieved no concluding results or showed

compromised plant productivity in associations with drought resistance (Parry et al. 2005).

Considerable further research is required with rigorous evaluation of plant physiological

performance under field-like drought conditions (Chaves & Oliveira 2004).

Severe water deficit is an unlikely event in agriculture and recreational areas; it is more

likely to occur in natural ecosystems of arid, rain-fed areas, where plant survival becomes a

priority (e.g. Utrillas & Alegre 1997; Volaire et al. 1998). In the urbanized areas of the modern

world it is more important to understand plant physiological and biochemical responses to

moderate drought stress promoted by gradually-decreased water availability, including the

identification of traits associated with increased resilience to drought in better adapted species

more likely to cope with the expected climate changes, in order to improve plant performance

and water use efficiency.

Plant growth and development under conditions of decreased water availability

Plants can sense and respond to decreased water availability by regulating their growth and

development in order to cope better with the environment. In many plant species, the decrease in

biomass production is one of the earliest responses to decreased soil water availability (Kramer

1983). Hsiao & Xu (2000) showed that under drought stress conditions root elongation continues

but shoot growth is limited. Decreased shoot growth in response to decreased soil water potential

may result from changes in leaf cellular elongation and/or in protein metabolism and can occur

before alterations in leaf gas-exchanges (Lambers et al. 1998) or in leaf water potential (Saab &

Sharp 1989). Leaf elongation can be limited by the loss of cell turgor or cell wall stiffening (van


25

Volkenburgh & Boyer 1985) induced by chemical/hydraulic signals triggered in response to the

drying soil (Davies & Zhang 1991). However, despite initial suggestions of ABA accumulation

being the cause of growth inhibition, recent studies have shown that ABA restricts the

production of ethylene and functions to maintain rather than to inhibit plant growth (Sharp

2002). ABA seems to counteract completely the inhibition of root elongation by ethylene whilst

in the shoots the effect of ABA is not enough to avoid some growth limitation by ethylene and

therefore a reduction is observed in the aerial part of the plant but not in the root system. The

maintenance of root elongation at low soil water potentials is a strategy to improve water

absorption (Chaves et al. 2003) and allows plants to tolerate better, and in some cases survive,

water-limited conditions (Liu et al. 2005). On the other hand, the decreased shoot growth rate

and the diversion of the photoassimilates and energy into the synthesis of molecules involved in

plant defence can also be seen as an adaptive strategy essential for plant survival under severe

stress conditions (Chaves & Oliveira 2004).

Phenotypic plasticity or the ability of plants to adjust their development in response to the

environment may provide them with competitive advantage. For instance, plants native from

climates characterized by marked fluctuations have generally more plastic phenotypes and can

acclimate to the changing environment and cope better with those changes (Sage & McKown

2006). The leaf Kranz anatomy associated with C4 photosynthesis (Dengler et al. 1994) may

compromise the plasticity of C4 grasses, but both metabolic and/or structural adjustments have

been observed to occur in response to several environmental conditions and favour plant

performance (e.g. Cavaco et al. 2003; Soares et al. 2008). Moreover, the specialised leaf

structure of C4 grasses (discussed in further detail in Chapter III) is likely to be associated with

their increased drought resistance in comparison with C3 species.

Leaf water relations - diverse possibilities in response to water deficit

Water status can be defined in terms of the water chemical potential (adopting the Gibbs

concepts, see Kramer & Boyer 1995). The water potential, commonly used in ecophysiology,

depends on a number of components and, in the leaves, will essentially reflect pressure and

solute effects. Decreased leaf water potential (LWP) under water deficit conditions reflects

changes in the turgor pressure and in the osmotic potential. The turgor pressure depends on the

cell wall rigidity and has a determinant role in leaf expansion. The osmotic potential is a function

of the concentration of solutes in the cell: the accumulation of compatible compounds increases

the osmotic pressure and, as a consequence, the LWP becomes more negative, promoting the

water movement into the cell.

Chapter I.

26

Cell wall elasticity determines the extent to which LWP can decrease without

compromising leaf turgor. In general, species from drier climates have more elastic cell walls, a

drought-tolerance strategy that allows them to loose more water before reaching the turgor-loss

point (Lambers et al. 1998). On the other hand, the faster decrease of LWP in species with rigid

cell walls (see Kramer & Boyer 1995) increases the capacity of water absorption from the drying

soil, avoiding excessive leaf dehydration.

Osmotic adjustment is one of the most important mechanisms of adaptation to drought

stress (Bray 1993). Plants accumulate compatible solutes that reduce the osmotic potential of the

tissues, thereby minimizing water loss (Morgan 1984; Kramer & Boyer 1995). Examples of

substances with osmoregulation potential include amino acids, like proline; quaternary

ammonium compounds, like glycine-betaine and choline; carbohydrates, like sucrose and

trehalose; and other low molecular weight metabolites. As reviewed by Hare, Cress & van

Staden (1998), the advantage of increased contents in compatible solutes, or osmolytes, is

primarily, but not solely, related with turgor maintenance through increased osmotic pressure

and consequently decreased leaf water potential, which is a biophysical aspect, merely related

with cellular concentrations. The maintenance of the cellular water content maintains the

concentration of ions with regulatory properties unaffected, protecting the enzyme functioning

(Morgan 1984). As pointed by Hare et al. (1998), a number of other protective roles, possibly

with similar or even greater importance, have also been described for osmolytes and their

metabolism, including the scavenging of free radicals and the buffering of cellular redox

potential. Additionally, the same authors suggest that osmolyte accumulation might be a means

of storing energy and their degradation upon stress relief might provide the plant with reducing

power and sources of carbon and/or nitrogen for plant growth and recovery from stress injury.

Transgenic approaches to improve drought tolerance included the production of enzymes

involved in osmolyte biosynthesis and in the antioxidant defence system (e.g. Bajaj et al. 1999),

but most often giving no satisfactory results. Similarly, external application of proline to increase

plant resistance to drought stress has successfully increased crop yield in some, but not many,

plant species (Ashraf & Foolad 2007) and is likely to be of limited usefulness. Despite all the

putative benefits associated with osmolyte accumulation, which are likely to be of determinant

advantage for plant survival in natural ecosystems, osmoprotection of the above-ground plant

tissues seems to function mostly under severe dehydration levels. Osmolyte accumulation

usually occurs after gas-exchanges and leaf growth being affected and therefore is likely to have

little relevance for crop yield and production (Serraj & Sinclair 2002). However, osmolyte

accumulation at the below-ground level might give a positive and valuable contribution in the


27

maintenance of root growth as soil water availability decreases (Voetberg & Sharp 1991; Hsiao

& Xu 2000).

Protective roles associated with the accumulation of proline and other amino acids

In most plant species, including C4 grasses (Jones 1985), proline accumulates in response to

drought and other environmental stress conditions and it is likely that the unique characteristics

of its metabolism and the interaction with other intermediary pathways (see Hare & Cress 1997)

are in the basis of this widely distributed phenomenon. The protective roles of proline

accumulation under drought stress were recently reviewed (Ashraf & Foolad 2007) and include

the maintenance of cell turgor through osmotic adjustment, maintenance of protein function and

membrane integrity, scavenging of reactive oxygen species and buffer cellular redox potential.

As reviewed by Hare & Cress (1997) the involvement of proline metabolism in the

regulation of the cellular redox potential (and pH) results from the contribution of the

biosynthetic pathway to the oxidation of NADPH (and consumption of H+). The accumulation of

proline under stress is thought to result mostly from increased synthesis through the glutamate

pathway (Delauney & Verma 1993), which contributes twice as much to these processes than the

ornithine pathway. In fact, the shift in nitrogen metabolism associated with proline accumulation

is likely to be more associated with the reactions involved than with the product concentration

itself, most times insufficient to account for a significant contribution to the osmotic adjustment

and benefits associated with this biophysical aspect. This is supported by the observation that, in

most cases, external application of proline results in no benefit for plant drought resistance

(Ashraf & Foolad 2007). The increased activity of Δ1-pyrroline-5-carboxylate reductase,

involved in the final step of proline synthesis, is likely to be itself associated with the regulation

of intermediary metabolism through the maintenance of compatible redox potentials (Hare &

Cress 1997). Proline accumulation occurs especially under severe drought conditions (Jones

1985) and it is likely to have a particularly important role in plant survival where intensive

summer-droughts predominate (Utrillas & Alegre 1997; Volaire et al. 1998). As proposed by

Hare & Cress (1997), increased proline synthesis during the stress event results in increased

NADP+/NADPH ratios which promote increased activity of the oxidative pentose phosphate

pathway. This pathway provides precursors for the synthesis of secondary metabolites with

important roles in stress defence, and may protect the cells from photoinhibition when the use of

reducing power by the Calvin cycle is decreased by water deficit. The subsequent proline

degradation upon stress relief results in the formation of NADPH and increased ATP synthesis,

Chapter I.

28

which will benefit plant growth and recovery from stress. From this point of view, proline

accumulation can be seen as a means of energy storage (Hare & Cress 1997).

Hare & Cress (1997) discussed the tight link between proline biosynthesis and the

oxidative pentose phosphate pathway, which is regulated by the ratio NADP+/NADPH and will

be activated by NADPH oxidation. Both the oxidative and reductive pentose phosphate pathways

can provide carbon in the form of E4P, one of the precursors to the biosynthesis of phenylalanine

(together with PEP) by the shikimate pathway (Ireland 1997). Because photosynthesis (and the

Calvin cycle) becomes limited under stress conditions, E4P for the production of

phenylpropanoids and other aromatic secondary compounds potentially involved in stress

defence mechanisms, is likely to result essentially from the oxidative pentose phosphate pathway

(Dennis et al. 1997). The activity of this pathway is stimulated by the increased NADP+

production resulting from increased proline biosynthesis. Additionally, Hare & Cress (1997)

suggested the potential involvement of proline in stress metabolic signalling, as a secondary

messenger. This was supported by the observation of a sixty-fold increase in proline content in

the phloem sap of Medicago sativa possibly associated with the transport of the imino acid to

leaf meristematic tissues where it would promote growth maintenance (Girousse et al. 1996).

Methionine is a sulphur-containing amino acid with pivotal roles in the structure and

catalytic function of proteins. The methionine cycle leads to the production of S-adenosyl-L-

methionine (SAM), which can be used to the synthesis of ethylene, involving the intermediate 1-

aminocyclopropane-1-carboxylic acid (ACC), or to the synthesis of polyamines. Under stressful

conditions, including decreased water availability, methionine metabolism can be directed to an

enhanced production of ethylene (Wang et al. 2002) and polyamines (Groppa & Benavides

2008). Valine, leucine and isoleucine are precursors of secondary metabolites including

cyanogenic glycosides, glucosinolates and acyl-sugars (Coruzzi & Last 2000). Phenylalanine is

involved in the production of numerous secondary metabolites, including defensive phytoalexins,

bioactive alkaloids and structural lignin (Herrmann 1995). Lignin deposition reinforces vascular

tissues in grasses and its production involves the participation of free radicals. This structural

polymer and other phenolic compounds are often induced under stress conditions and can act in

the scavenging of reactive oxygen species (ROS), rendering the plant with increased antioxidant

capacity (Grace & Logan 2000).

The interactions between nitrogen and carbon metabolism and the metabolic shifts,

involving for instance proline metabolism and cycling with carbohydrates, SAM-dependent

methylation reactions and photorespiration, are likely to provide the plant with some flexibility

that enhances the capacity to cope with environmental stress conditions (Hare et al. 1998).


29

Stomatal closure in response to soil water deficit

The decrease of stomatal conductance is one of the first responses to water deficit (e.g. Flexas &

Medrano 2002; Chaves et al. 2003) and can occur due to leaf dehydration or due to signals

synthesized by the drying roots to warn the plant of water shortage and promote stomatal closure

before the leaf water status changes (Majumdar et al. 1991; Zhang et al. 2001). Chemical signals,

namely ABA synthesized in the roots when water availability is reduced (Davies & Zhang 1991;

Davies et al. 2005), control stomatal behaviour at moderate water deficits (Borel et al. 2001;

Sauter et al. 2001; Wilkinson & Davies 2002). Drought stress increased ABA concentrations and

decreased stomatal conductance in C3 (Zhang et al. 2001; Liu et al. 2003) and in C4 plants

(Stikic & Davies 2000). Hydraulic (Chazen & Neumann 1994; Comstock 2002) and electrical

(Wilkinson & Davies 1997; Fromm & Fei 1998) signals were also involved in stomatal closure.

As reviewed by Wilkinson & Davies (2002) chemical, hydraulic or electrical signals as well as

the interaction between them can mediate the regulation of stomata under drying soil conditions.

In the field, the control of stomatal conductance may be used in order to improve the water use

efficiency, which reflects the amount of carbon assimilated per amount of water lost (Chaves et

al. 2002). In some, but not other, grass species, stomata respond directly to changes in

evaporative demand, decreasing stomatal conductance in response to increased vapour pressure

deficit and thereby increasing water use efficiency and improving the capacity to resist drought

stress (Maroco et al. 1997).

Because decreased stomatal conductance is a widely generalised response of plants to

decreased water availability it has been suggested as a reference parameter to access the relative

importance and timing of different metabolic limitations to photosynthesis (Medrano et al. 2002;

Flexas et al. 2004).

Limitations to photosynthesis under drought stress

Water deficit may limit photosynthesis through stomatal closure and/or metabolic impairment

(see review by Chaves et al. 2003) and the timing and relative importance of these factors have

been much debated in the past (Flexas & Medrano 2002; Lawlor 2002; Lawlor & Cornic 2002).

The CO2 diffusion inside the leaf can also be limited and contribute to decreased assimilation

under drought conditions (see review by Flexas et al. 2008).

Both stomatal and non-stomatal factors were found to limit photosynthesis in drought-

stressed C4 plants and the relative importance of these factors may be different depending on the

rate of stress imposition (Saccardy et al. 1996; Marques da Silva & Arrabaça 2004a). In general,

Chapter I.

30

when water deficit is induced gradually, stomatal closure seems to be the major limitation to

photosynthesis under moderate stress with metabolic limitations becoming more relevant as

drought severity increases (e.g. Du et al. 1996). This may reflect a down-regulation of the

photosynthetic metabolism in order to match decreased availability of CO2 and growth rates

under drought (Chaves et al. 2003; Flexas et al. 2004).

The major metabolic processes limiting photosynthesis in conditions of decreased water

availability include photophosphorylation (Tezara et al. 1999), the capacity for regeneration of

RuBP (Giménez et al. 1992; Gunasekera & Berkowitz 1993) and the activity of Rubisco (Parry

et al. 2002). The importance of these and other metabolic factors will depend on the genotype

and plant developmental stage as well as on the rate, severity and duration of stress (Chaves et al.

2003). In C3 plants, decreased synthesis of ATP and RuBP are generally early events compared

to impairment of photochemical reaction and decreased Rubisco activity, which are mostly

observed only under severe stress conditions (Flexas & Medrano 2002).

In the field, plants are generally exposed to other stresses in addition to drought,

including high temperatures and irradiances, which ultimately result in oxidative stress. Powerful

plant antioxidant defence systems determine the resistance to stresses likely to cause oxidative

damage (Smirnoff 1998) by avoiding the irreversible impairment of the photosynthetic

machinery. Under conditions leading to decreased CO2 assimilation (e.g. water deficit) the

excess light may cause photoinhibition or promote the down-regulation of photosynthesis.

In plants with the C4 photosynthetic pathway, water deficit may affect the activities

and/or regulation of the enzymes involved in the C3 and C4 cycles and the regeneration of

substrates, thereby limiting or adjusting photosynthetic efficiency. A number of studies revealed

different possibilities of response to water deficit by the enzymes and substrates involved in the

mesophyll and bundle sheath reactions of C4 photosynthesis (Du et al. 1996; Lal & Edwards

1996; Saccardy et al. 1996; Foyer et al. 1998; Jagtap et al. 1998; Carmo-Silva et al. 2004;

Marques da Silva & Arrabaça 2004b; Carmo-Silva et al. 2007). Further detail on the activities

and regulation of the carboxylating and decarboxylating enzymes under drought conditions is

given in Chapters IV and V.

The understanding of the processes involved in the drought-induced limitation of

photosynthesis is fundamental to optimize plant performance and water use efficiency in the

present climate change scenario.


31

OBJECTIVES OF THE THESIS

The main objective of this work was the study and comparison of drought-stress physiology and

biochemistry of three C4 grasses from different subtypes. The grasses Paspalum dilatatum

(NADP-ME), Cynodon dactylon (NAD-ME) and Zoysia japonica (PEPCK) were chosen due to

their high photosynthetic performance and water use efficiency, which are duly associated with

their economic and agronomic importance. The target was to understand how these different

species respond to gradually-induced drought stress and what characteristics determine their

resistance to conditions of decreased water availability.

To achieve this goal, the water relations, relative growth and leaf anatomy of well-

watered and drought-stressed plants of the three species were first studied (Chapter II) in an

attempt to understand the factors determining the water loss in their leaves and their possible

association with plant resilience to water deficits.

The effects of water deficit on leaf gas-exchanges and variations in photorespiratory rates

were subsequently assessed (Chapter III) through the analysis of CO2 assimilation at several

concentrations of CO2 and O2, carbon and oxygen isotope compositions and amino acids content

in fully illuminated leaves and after a period of 30 s in darkness.

The activities of the enzymes involved in the primary carboxylation and subsequent

decarboxylation steps of C4 photosynthetic pathway were studied in leaves from well-watered

and drought-stressed plants of the three grasses (Chapter IV) including an assessment of C4

subtype biochemistry.

The amount, activities and regulation of Rubisco from the three C4 species, including the

analysis of RuBP and CA1P contents, were investigated under water deficit (Chapter V).

Additionally, the kinetic properties of Rubisco were determined in order to allow future studies

with a more realistic modelling of C4 photosynthesis and photorespiration under drought

conditions promoting stomatal closure and decreased CO2 availability.

All these approaches were conducted in order to attain the global target of improving the

understanding of photosynthesis and photorespiration in C4 grasses under water deficit.

Chapter I.

32

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Chapter II.

WATER RELATIONS AND LEAF ANATOMY OF C4

GRASSES

Chapter II.

48

A shorter version of this chapter will be shortly submitted to Annals of Botany:

Carmo-Silva A.E., Francisco A., Powers S.J., Keys A.J., Ascensão L., Parry M.A.J. & Arrabaça M.C. (To Submit) Drought resistance mechanisms are more effective in the C4 grasses Cynodon dactylon and Zoysia japonica than in Paspalum dilatatum.

Alfred J. Keys (Department of Plant Sciences, Rothamsted Research) was involved in the measurements of amino acids content and in the identification of HNV.

Ana Francisco and Lia Ascensão (Centro de Biotecnologia Vegetal and Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa) were involved in the studies of leaf anatomy, including the sample preparation, analysis and interpretation of the results obtained.

Stephen J. Powers (Department of Biomathematics and Bioinformatics, Rothamsted Research) was involved in the experimental design and in the statistical analysis of the data, especially the non-linear modelling and the analyses of variance.

Water Relations and Leaf Anatomy of C4 Grasses

49

DROUGHT RESISTANCE MECHANISMS ARE MORE EFFECTIVE IN THE C4 GRASSES Cynodon dactylon AND Zoysia japonica THAN IN Paspalum dilatatum

ABSTRACT

Plants respond to drought stress with a number of changes that may confer resistance to the

harmful environmental condition. The effects of gradually-induced water deficit were studied in

three grasses with different C4 photosynthetic mechanisms, characterized by different

biochemical and anatomical features: Paspalum dilatatum Poiret (NADP-ME), Cynodon

dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK). The leaf relative water

content decreased with drought in the three species but faster in P. dilatatum. The leaf water

potential decreased to a much greater extent in Z. japonica, the species that presented a higher

capacity to accumulate compatible solutes and has more rigid cell walls. All plants produced

fewer leaves and tillers in response to the drying soil. Increased leaf dry matter was observed in

all three species but leaf structure was not much altered by moderate drought stress. The

presence of cells with a specific role in leaf morphological responses that minimize water loss

was evident. Especially in C. dactylon and Z. japonica, drought stress caused increased contents

of amino acids in the soluble fraction of leaves. An unusual hydroxylated amino acid, not

previously reported in leaves, was present in Z. japonica and its synthesis was induced by water

deficit in C. dactylon.

KEYWORDS:

Water relations, shoot growth, leaf anatomy, amino acids, water deficit

Chapter II.

50

INTRODUCTION

Decreased water availability and the need to improve plant performance with increased water use

efficiency constitute one of the major challenges of the modern humanity, as recognised by the

Global Environment Outlook: Environment for Development (Geo-4), issued by the United

Nations Environment Programme in 2007. The use of species better adapted to conditions of

mild to moderate water deficit and the production of new varieties with increased drought

resistance, through plant breeding programmes and/or genetic engineering, are promising

approaches that depend on the understanding of plant responses under gradually-induced, field-

like drought conditions. A number of morphological, physiological, biochemical and molecular

changes might result in increased capacity of plants to resist unfavourable environments (Bray

1997; Chaves et al. 2003). Drought resistance strategies include mechanisms that minimize

tissue dehydration and mechanisms of dehydration tolerance, the latter allowing the maintenance

of active physiological and biochemical processes. Internal regulations including root-shoot

signals (Davies & Zhang 1991) and structural components in the leaves are likely to play a

determinant role in plant responses to drought stress (Lösch & Schulze 1995).

Some plants minimize water loss from the leaves through mechanisms that decrease

evapotranspiration, including stomatal closure and leaf rolling or folding, whilst others adopt

mechanisms that increase water absorption from the drying soil, for instance decreasing the leaf

water potential. The latter may involve changes in turgor pressure, which depends on cell wall

plasticity, or in the osmotic potential, which is a function of the concentration of solutes in the

cell (Kramer & Boyer 1995; Lambers et al. 1998). Osmotic adjustment, the ability of the plant to

decrease the osmotic potential through the accumulation of compatible, non-toxic solutes, and

hence decrease the water potential of the tissues, minimizing water loss (Morgan 1984; Kramer

& Boyer 1995), has long been recognized as one of the most important mechanisms of plant

adaptation to drought stress (Bray 1993). The capacity to adjust osmotically might be

determinant for survival under conditions of severe water deficit in regions where long summer-

droughts dominate (e.g. Utrillas & Alegre 1997; Volaire et al. 1998). A number of protective

roles have been ascribed to the presence of some osmolytes and their metabolism in the cells,

including the protection of membranes and proteins, the scavenging of free radicals and the

buffering of cellular redox potential (Hare et al. 1998). The beneficial roles of drought-induced

osmolyte accumulation for plant productivity and crop yield are still a matter of controversy

(Serraj & Sinclair 2002) and their correlation with plant drought tolerance is not always observed.

Nevertheless, the capacity for osmotic adjustment, involving either roles in osmoprotection or


51

the antioxidant defence system, has been associated with drought tolerance in maize (Nayyar

2003; Nayyar & Gupta 2006), sugarcane (Molinari et al. 2007) and pearl millet (Kusaka et al.

2005). Amino acids, and especially proline, can increase markedly in C4 grasses under water

deficit and are likely to play a role in their drought resistance capacity (Jones 1985). In addition

to the possible contribution to osmoregulation, increased contents of some amino acids such as

methionine and phenylalanine may be also associated with increased synthesis of compounds

involved in plant stress responses.

Decreased rates of shoot growth, mediated by ethylene (Hussain et al. 1999) are well

established effects of drought stress on C4 grasses and may involve decreased leaf expansion

(fewer or smaller cells), decreased rate of leaf appearance and/or leaf development (Jones 1985).

Inhibition of maize shoot growth in response to decreased water availability can occur before the

leaf water potential is decreased (Saab & Sharp 1989). Increased ABA production in response to

drying soil restricts ethylene production (Sharp 2002), thereby counteracting the inhibition of

root elongation and attenuating the inhibition of shoot growth. Root growth maintenance under

water deficit conditions is also an adaptive mechanism that allows water to be taken up from

deeper soil layers (Bartels & Sunkar 2005).

Water loss from the cells may cause leaf shrinkage. Severe dehydration often results in

impaired membranes and distorted organelles (Kramer & Boyer 1995) and, ultimately, folding of

the cell walls can be observed (e.g. Utrillas & Alegre 1997). Some plants may develop adaptive

strategies to resist cell shrinkage, for instance, through enhanced deposition of rigid cell wall

constituents (e.g. lignin). Controlled changes in leaf anatomy developed under gradually-induced

drought conditions may increase plant resistance by favouring the stability of macromolecules

and the maintenance of biochemical reactions. The leaf Kranz anatomy associated with the C4

photosynthetic pathway present in many grasses (see Dengler et al. 1994) may diminish their

potential for phenotypic plasticity and thus minimize their capacity for acclimation to the

environmental changes (Sage & McKown 2006).

Three classical biochemical subtypes of C4 photosynthesis have been identified based on

the major enzyme responsible for the decarboxylation of C4 acids, which are produced in the

mesophyll (M) cells, where primary assimilation of CO2 takes place, and then transported to the

bundle sheath (BS) cells, where carbon is assimilated through the C3 cycle. The decarboxylating

enzymes that give name to these subtypes are NADP-malic enzyme (NADP-ME subtype), NAD-

malic enzyme (NAD-ME subtype) and phosphoenolpyruvate carboxykinase (PEPCK subtype).

In addition to the biochemistry, there are differential anatomical features between these subtypes

(see Dengler & Nelson 1999 for review) and most C4 grasses fit into one of the three classical

Chapter II.

52

anatomical-biochemical subtypes (Hattersley & Watson 1992). Differences in leaf structure may

be related with differential water loss by the tissues and therefore it becomes important to

understand the possible contribution of leaf anatomy to plant responses under limited water

availability.

C4 plants are generally assumed to be better adapted to conditions of decreased water

availability than their C3 counterparts, mostly due to their greater water use efficiency (Long

1999). The use of warm-season (C4) turfgrasses in recreational areas might be a means to save

irrigation water. Several studies suggest an association between rainfall gradients and the

distribution of grass species from the different biochemical subtypes of C4 photosynthesis

(Henderson et al. 1995; Cabido et al. 2008), possibly reflecting different strategies or features

that allow some species to cope better with drought. In a previous study, different responses to

rapidly-induced water deficit conditions were observed in three warm-season turfgrasses of the

different C4 metabolic subtypes: Paspalum dilatatum Poir. (NADP-ME), Cynodon dactylon (L.)

Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK) (Carmo-Silva et al. 2007). However, it

was not possible to define whether those responses would reflect a better capacity of a certain

species to cope with conditions of decreased water supply in the field.

The aim of this work was to understand the primary responses to gradually-imposed

drought conditions in these three different C4 grasses. Water availability in the soil was

characterised and the corresponding water deficit in the plants measured, through the leaf

relative water content and water potential. The effects of drought stress on leaf and tiller

production, leaf dry matter and specific leaf area, leaf anatomy and soluble amino acids were

analysed in well-watered and non-watered plants of P. dilatatum (dallisgrass), C. dactylon

(bermudagrass) and Z. japonica (zoysiagrass).


53

MATERIAL AND METHODS

Plant material and drought stress induction

The C4 grasses Paspalum dilatatum Poir. cv. Raki (NADP-ME), Cynodon dactylon (L.) Pers var.

Shangri-Lá (NAD-ME) and Zoysia japonica Steudel ‘Jacklin Sunrise Brand’ (produced by

Jacklin Seed Company, USA) (PEPCK) were grown from seeds in pots with peat-free compost

(Petersfield Products, Leicester, UK) supplemented with a slow-release fertiliser (Hydro Agri

Ltd, Lincs, UK) in a glasshouse. Artificial light was provided whenever the natural light was

below a photosynthetic photon flux density (PPFD) of 500 μmol m-2 s-1 during a 16 h

photoperiod. Temperature was maintained at a minimum of 25ºC during the day and at 18ºC

during the night.

Seeds of each species were washed with 10% hypochlorite and soaked in water for 1 hour

before sowing. Water was supplied whenever needed during two weeks for P. dilatatum and C.

dactylon and four to five weeks for the slower growing Z. japonica. The seedlings were

transplanted to 1-L cylindrical pots containing equal amounts of soil. Five seedlings were used

per pot. All plants were well-watered until the beginning of the drought stress treatment. Pots

were placed according to a split-plot design, where each column of pots was a main plot of a

particular species and the sampling-days and the treatments (control vs. drought stress) were

randomised in the split-plots. Each pot corresponded to an independent sample. Water deficit

was imposed consecutively on the three species, separated by one day, with watering of the

‘stress’ pots being stopped first in C. dactylon, then in Z. japonica and at last in P. dilatatum in

order to get more similar degrees of leaf dehydration for the three species because the leaves

from P. dilatatum tended to dehydrate faster than those from Z. japonica and C. dactylon. The

‘control’ pots were watered once per day.

All three species were analysed simultaneously, for four consecutive days during the

drought period, starting when the weight of non-watered pots had been suitably decreased and

ending after a maximum of twelve days without watering. Five-week old plants of P. dilatatum

and C. dactylon and nine-week old plants of Z. japonica were analysed. The youngest fully

expanded leaf of each P. dilatatum plant and two young fully expanded leaves of each plant of C.

dactylon or Z. japonica were always used. Samples were collected in the growth environment

four or five hours after the beginning of the photoperiod. It was assumed that, within each pot,

all the leaves used were identical in terms of developmental stage, physiological and biochemical

properties, and would have experienced the same drought condition.

Chapter II.

54

Water relations and leaf parameters

Four control and eight non-watered pots were used per species per day during four consecutive

days making a total of 40 samples per species (16 control and 24 non-watered). A leaf sample

was collected from each pot, four hours after the beginning of the photoperiod, for determination

of the leaf relative water content (RWC). Taking into account the different leaf sizes, each

sample of P. dilatatum consisted of two leaves from two plants whereas each sample of C.

dactylon or Z. japonica consisted of five leaves from two plants. The fresh (FW), turgid (TW)

and dry (DW) weights were determined and used to calculate RWC by the equation RWC =

100((FW-DW)/(TW-DW)) (Catsky 1960). Leaf area was determined by scanning the turgid

leaves and analysing the image using the software Paint Shop Pro 9 (Jasc Software, Inc., USA)

and Image J 1.33u (National Institutes of Health, USA). The ratio between the dry and the turgid

leaf weight (DW/TW) was calculated as a percentage and the specific leaf area (SLA) was

calculated as the ratio between the leaf area and DW.

The leaf water potential (LWP) was measured on the middle part of young fully

expanded leaves taken from each pot five hours after the beginning of the photoperiod using a

pressure-chamber (Ritchie & Hinckley 1975).

The water weight in each pot (WWP) was determined as the weight of the pot at each

sampling time less 400 g (the mean weight of the pots with plants and totally dried soil was 404

± 16 g). The soil water content (SWC) was determined in three opposite locations in each pot

using an HH2 moisture meter with a Theta probe (type ML2x, AT Delta-t Devices Ltd.,

Cambridge, UK).

Growth parameters

Three control and three non-watered pots were used per species and all samples were analysed

on the same day. The mean numbers of leaves and tillers formed by the control and drought-

stressed plants in twelve days were determined by analysing all the five plants in each pot (15

plants per species per treatment, with a total of 90 plants) at the beginning of the experiment, the

last watering day for the first species, and at the end of the drought treatment, corresponding to

10, 11 and 12 days without watering the plants of P. dilatatum, Z. japonica and C. dactylon,

respectively.


55

Leaf anatomy

Leaf samples of three control and three non-watered pots of each species were taken for

anatomical observations at the end of the drought treatment (10-12 days without watering),

simultaneously for the three species. Because anatomical variation occurs along the leaf length

(e.g. Miranda et al. 1981), samples were taken from the middle portion of leaf blades, equidistant

from both ends. The leaf segments were fixed with 2.5% glutaraldehyde in 0.1 M sodium

phosphate buffer (pH 7.2), dehydrated in a graded ethanol series and then infiltrated with and

embedded in Leica Historesin (Leica Microsystems, Germany). Transverse sections (2 μm) were

cut using a rotatory microtome (RM2155, Leica Microsystems) and subsequently stained with

0.05% Toluidine Blue O (Gutmann 1995). Observations were carried out on a BX60F5 Olympus

light microscope (Olympus Optical Co., Ltd., Japan) and images were recorded digitally using a

DP50 Olympus camera and the Viewfinder Lite (Pixera Corporation, USA) software.

Quantitative anatomical data from the images of leaf transverse sections was gathered

using the Image-Pro Express 6.0 (Media Cybernetics Inc., USA) software. Three analytical

replicates (micro slides) were analysed for each biological sample (three controls and three

drought-stressed per species). The interveinal distance was measured for each pair of

longitudinal veins present on a half-leaf section. The leaf thickness was measured on the thickest

part of each vein of the same half-leaf section. The transverse half-leaf sections were divided

into four different zones, from the centre to the margin of the leaf, in order to have a

representative sampling of the leaf anatomy. These four zones corresponded to: (1) the central

longitudinal vein, (2) a large longitudinal vein or (3) two small longitudinal veins in between the

centre and the margin of the leaf, and (4) the two small longitudinal veins closest to the margin.

The cross-sectional areas of the bundle sheath cells, mesophyll cells, vascular tissues (including

mestome sheath if present), bulliform and colourless cells, sclerenchyma cells, intercellular

spaces, lower and upper epidermis (see diagram from Dengler et al. 1994 Figure 1), were

measured considering the four zones above.

Analysis of amino compounds by HPLC

Amino acids and other amino compounds contained in the leaves were determined by High-

Performance Liquid Chromatography (HPLC) of o-pthaldialdehyde (OPA) derivatives (Noctor

& Foyer 1998). Three control and five non-watered pots were used per species per day during

three consecutive days, making a total of 24 samples per species (nine control and fifteen non-

watered pots). The plants within each pot were used for the collection of a leaf sample that was

Chapter II.

56

immediately frozen in liquid N2 (LN2) in fully illuminated conditions (four hours after the

beginning of the photoperiod) and another leaf sample for the determination of RWC. Taking

into account the different leaf sizes, each frozen sample consisted of one leaf for P. dilatatum

and three leaves for C. dactylon and Z. japonica. Similarly, the corresponding RWC samples

consisted of two leaves for P. dilatatum and four leaves for C. dactylon and Z. japonica.

Reversed-phase HPLC was performed using a Waters Alliance 2695 Separation Module and a

474 Scanning Fluorescence Detector operated by the Millenium32 software (Waters, Milford,

USA) with a Waters Symetry C18 4.6×150 mm column (Part No. WAT 054278) protected with a

4×3 mm guard cartridge (Phenomenex, Torrance, USA). Since the fluorescent adducts formed by

reaction with OPA in the presence of 2-mercaptoethanol are unstable, the autosampler was set to

mix and pre-incubate 10 μL of each sample with 15 μL of OPA reagent for 2 min before

injecting the mixture onto the column. The eluent used for the amino compounds separation was

obtained by mixing solvents containing different proportions of methanol, sodium acetate pH 5.9

and tetrahydrofuran.

Amino compounds were extracted from the frozen leaf samples stored at -80ºC. Each

sample was ground in LN2 and then 1.4 mL of 0.1 M HCl was added to the fine powder. The

mixture was ground further during thawing and the homogenate was centrifuged for 10 min at

16000 ×g and 4ºC. Samples for HPLC were prepared by adding a sub-sample of each

supernatant to the internal standard and pure water, and these mixtures were stored at -20ºC. On

the following day, the mixtures were centrifuged for 40 min at 16000 ×g and 4ºC and then

filtered with syringe filters (0.2 μm) into HPLC autosampler vials. Standard solutions of α-

amino-n-butyric acid (internal standard), valine, methionine, phenylalanine, isoleucine, leucine,

ethanolamine and 5-hydroxy-norvaline were prepared in 0.1 M HCl and diluted to have

increasing concentrations for the calibration curves (0, 5, 10, 15, 20 and 25 μM).

Proline estimation

The proline content in the acid leaf extracts above was determined after reaction with ninhydrin

(Bates et al. 1973). Standard solutions of proline with concentrations rising from 0 to 100 μg /

mL in 0.1 M HCl were used for the calibration curve. According to Chinard (1952) a linear

relationship is obtained between the concentration of proline and the optical density in the range

0.02 mM and 0.1 mM. A volume of 0.25 mL was taken from each sample and standard solution

into a 15 mL centriuge tube and mixed with 0.75 mL of 3% sulfosalicylic acid, 1 mL of glacial

acetic acid and 1 mL of acid-ninhydrin reagent (140 mM ninhydrin in a solution 2:3 of glacial


57

acetic acid and 6 M orthophosphoric acid), consecutively. The tubes were thoroughly vortexed

and incubated at 100ºC for 1 hour. At a very low pH (~1.0) a red water-insoluble product is

formed in the reaction between proline and ninhydrin and no significant amounts of coloured

products seem to be formed by most of the other amino acids under these extremely acidic

conditions. The reaction was stopped by placing the tubes on ice for 5 min. Toluene (2 mL) was

added to each tube, and after mixing thoroughly, the phase separation occurred and the

absorbance of the upper phase at 520 nm was measured (CARY 300 Bio UV-Visible

Spectrophotometer, Varian Analytical Instruments, Varian Inc., Surrey, UK) using toluene as

background.

Statistical analysis

All the statistical analyses were made using GenStat® 9.2, 2005 (Lawes Agricultural Trust,

Rothamsted Research, UK). Regression analysis was applied to model the relationship between

WWP or SWC with days without watering, between SWC and WWP and between LWP or

DW/TW or SLA with SWC, taking into account the control vs. non-watered condition of the pots.

Regression analysis was also applied to model the variation of the amino acids content with

RWC, this time considering a squared term in this variable to check for non-linearity. The

variation of RWC with SWC and the relationship between LWP and RWC were assessed

through the use of non-linear regression (by Stephen J. Powers), fitting an asymptotic

exponential and a simple exponential model, respectively, and accounting for differences

between species by way of model parameters. Nested models were compared using F-tests and

then the non-significantly different (P > 0.05) parameters (t-tests) in the significant terms (P <

0.05) of each model were amalgamated in order to attain parsimony. The resulting best models

were plotted and the parameter estimates with their respective standard errors (s.e.), the

percentage of variance accounted for by the model (R2), the residual mean square (s2) and the

degrees of freedom (d.f.) are given with the plots. The residuals were checked and found to

generally conform to the assumptions of the analysis. All the absolute values and percentages

presented in the text were calculated in accordance with the regression analysis performed.

Analysis of variance (ANOVA) was applied (by Stephen J. Powers) to the growth

parameters (numbers of leaves and tillers) and leaf anatomy measurements (number of veins,

interveinal distance, leaf thickness, and the percentages of cellular areas) to check for statistically

significant (P < 0.05) differences between species, treatments (control vs. drought stress) and for

the interaction between these two factors. Prior to ANOVA, a square-root transformation was

Chapter II.

58

applied to the number of leaves and the number of tillers produced per plant. This transformation

ensured the assumptions of the analysis were not violated. For the analysis, the sets of five

individual plants in each pot were taken as analytical replicates, the variability within these sets

being output in the ANOVA. The numbers of leaves and tillers in the control and non-watered

plants at the beginning of the drought period were checked to be not significantly different

between the two treatments for each species (P > 0.05). The number of veins in each half-leaf

was analysed using a generalised linear mixed model (GLMM) by the fitting method of Schall

(1991) and assuming a Poisson distribution with a log link function for the model, which takes

account of the design structure of micro slides (analytical replicates) within samples. The

statistical significance of the different effects was assessed through an F-test (Welham &

Thompson 1997) on the appropriate degrees of freedom. The mean values of interveinal

distances, measured between each pair of consecutive veins, and the leaf thickness, measured at

the thickest part of each vein, per half-leaf were analysed using ANOVA, with a log

transformation of the data to ensure homogeneity of variance, and taking into account the design

structure of laminas within samples. For the different tissue areas, the grand total of area

measured per half-leaf was calculated as the sum of the areas of all four zones, and the

percentage of each type of tissue in relation to the grand total was determined. The values

obtained for each half-leaf of total area, tissue percentages and ratio M/BS were analysed using

ANOVA, again with a log transformation required. After all ANOVAs, following assessment of

the statistical significance of factors (species, treatments and their interaction), the Least

Significant Difference (LSD) at the 5% level of significance was used to test between relevant

means.


59

RESULTS

Amount of water in the soil

The amount of water in the soil of all pots was measured both as the water weight in pot (WWP)

and as the soil water content (SWC). The WWP and the SWC decreased linearly along the

sampling period, whilst the corresponding control pots used in each day showed no significant

variation in water availability (Figure II.1A-D). Parallel lines fitted the data from the three

species and hence the plants that were deprived of water in separate, consecutive days, had

reasonably the same amount of water in the soil on each sampling-day.

There was a very strong correlation (r = 0.996) between the SWC and the WWP (Figure

II.1E-F). The variation of SWC with WWP in the pots of C. dactylon and Z. japonica was not

significantly different (P > 0.05) and the two species were fitted with the same lines, whereas for

P. dilatatum separate, but parallel, lines were fitted, which is probably related with the plants of

this species being heavier than the former two species.

Leaf water status

The relative water content (RWC) in the leaves of P. dilatatum, C. dactylon and Z. japonica

started to decrease only when the amount of water in the soil decreased below a certain threshold,

so that the variation of RWC with the soil water content (SWC) was described by an asymptotic

exponential model (Figure II.2A). There was no significant difference (P > 0.05) between the

three species.

The leaf water potential (LWP) was lower in the non-watered plants compared to their

controls (Figure II.2B), decreasing linearly with SWC in the non-watered pots. In fully hydrated

leaves of P. dilatatum and C. dactylon the LWP was higher than in Z. japonica and drought-

stressed plants of the latter species showed a much steeper decrease of LWP with SWC.

As could be expected from the above, the relationship between the two variables related

with the leaf water status, RWC and LWP, was clearly different for Z. japonica than for the other

two species (Figure II.2C). An exponential model was used for the variation of LWP with the

RWC in P. dilatatum and C. dactylon. The LWP decreased from -0.6 MPa to a minimum of -1.3

MPa, as the RWC decreased from 99 to 93%, and then remained constant for further decreased

RWC. On the contrary, in Z. japonica the LWP decreased linearly with the RWC, from a value

Chapter II.

60

7 8 9 10 11 12

WW

P (g

)

0

100

200

300

400

500

7 8 9 10 11 12

0

50

100

150

200

Control pots Non-watered pots

Days without watering

7 8 9 10 11 12

SWC

(%)

0

10

20

30

40

Days without watering

7 8 9 10 11 12

0

2

4

6

8

10

12

A B

C D

WWP (g)

300 350 400 450 500 550

SWC

(%)

25

30

35

40

WWP (g)

0 50 100 150 200

0

2

4

6

8

10

12E F

y = 346 - 26.4 x (s.e. 24; 2.7)y = 382 - 26.4 x (s.e. 29; 2.7)y = 367 - 26.4 x (s.e. 26; 2.7)

y = 20.8 - 1.62 x (s.e. 1.5; 0.17)y = 23.6 - 1.62 x (s.e. 1.9; 0.17)y = 22.7 - 1.62 x (s.e. 1.7; 0.17)

y = 0.0585 x (s.e. 0.0011)y = 0.54 + 0.0585 x (s.e. 0.17; 0.0011)

y = 7.69 + 0.0585 x (s.e. 0.73; 0.0011)y = 8.82 + 0.0585 x (s.e. 8.82; 0.0011)

Strong correlation between SWC and WWP (r = 0.996)

Control pots Non-watered pots

Figure II.1. Amount of water in the soil in the control and non-watered pots with plants of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (white triangles). (A-D) Variation of the water weight in pot (WWP, g) and soil water content (SWC, %) during the sampling days, from the beginning of treatments when water was withheld from the stress pots of each species. WWP was calculated considering that pots with dry soil weighed 400g. (E-F) Relationship between SWC and WWP. Overall correlation coefficient, r = 0.996. In all cases, each data point corresponds to one sample (with 40 samples per species). The regression lines applied correspond to the best models statistically significant (P. dilatatum, solid lines; C. dactylon, dashed lines; Z. japonica, dotted lines; A-B, R2 = 97.7%, s2 = 665.9, d.f. = 113; C-D, R2 = 98.6%, s2 = 2.72, d.f. = 113; E-F, R2 = 99.7%, s2 = 0.469, d.f. = 116).


61

of -1.2 MPa, obtained in fully hydrated leaves, to -3.5 MPa in the most dehydrated leaves, with

ca. 77% RWC.

0 5 10 30 40

RW

C (%

)

70

75

80

85

90

95

100 A

SWC (%)

0 5 10 30 40

LWP

(MPa

)

-4

-3

-2

-1

0 B

RWC (%)

70 75 80 85 90 95 100

LWP

(MPa

)

-4

-3

-2

-1

0 C

y = 98.30 (1 - e - 0.4498 x ) (s.e. 0.31; 0.0100)

y = - 1.52 + 0.042 x (s.e. 0.10; 0.014)y = - 3.21 + 0.167 x (s.e. 0.17; 0.022)

y = - 1.347 + 3.47x10-17 e 0.3795 x (s.e. 0.052; 2.54x10-16; 0.0692)y = - 11.315 + 0.1021 x (s.e. 0.309; 0.0006)

Figure II.2. Water relations in the leaves of control and non-watered plants of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (white triangles). (A-B) Variation of the leaf relative water content (RWC, %) and leaf water potential (LWP, MPa) with the soil water content (SWC, %). (C) Relationship between LWP and RWC. Each data point corresponds to one sample (with 40 samples per species). The best models statistically significant were applied (P. dilatatum, solid lines; C. dactylon, dashed lines; Z. japonica, dotted lines; A, R2 = 81.5%, s2 = 6.387, d.f. = 118; B, R2 = 84.4%, s2 = 0.04003, d.f. = 114; C, R2 = 84.1%, s2 = 0.0408, d.f. = 115).

Chapter II.

62

Leaf parameters

The ratio between the dry and the turgid leaf weight (DW/TW) was much higher in Z. japonica

than in C. dactylon and was the smallest in P. dilatatum (Figure II.3A), revealing that the

percentage of dry matter in the leaves of the former species is greatest. An increase of the ratio

DW/TW with SWC was observed for the non-watered plants of the three species. The specific

leaf area (SLA) was higher in the control plants of P. dilatatum and C. dactylon than in Z.

japonica and decreased with SWC for non-watered plants of the first two grasses, but did not

change significantly (P > 0.05) in the drought-stressed leaves of Z. japonica (Figure II.3B).

0 5 10 30 40

DW

/TW

(%)

0

10

20

30

40

SWC (%)

0 5 10 30 40

SLA

(m2 K

g-1)

0

10

20

30

40

50

60

A

B

y = 17.96 - 0.3755 x (s.e. 0.74; 0.0690)y = 22.37 - 0.4109 x (s.e. 0.74; 0.0760)y = 33.93 - 0.5250 x (s.e. 0.74; 0.0713)

y = 41.09 + 1.046 x (s.e. 1.40; 0.188)y = 38.92 + 1.473 x (s.e. 1.53; 0.221)

Figure II.3. Leaf parameters of control and non-watered plants of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (white triangles). Variation of the ratio between the leaf dry weight and turgid weight (DW/TW, %) and the specific leaf area (SLA, m2 Kg-1) with the soil water content (SWC, %). Each data point corresponds to one sample (with 40 samples per species). The best models statistically significant were applied (P. dilatatum, solid lines; C. dactylon, dashed lines; Z. japonica, dotted lines; A, R2 = 97.4%, s2 = 1.131, d.f. = 113; B, R2 = 95.1%, s2 = 4.363, d.f. = 114).

Number of leaves and tillers

Although the plants were still not looking very stressed and no substantial decrease was observed

in their RWC by the end of the drought period, which last for a maximum of 12 days in this

experiment, there was a visible decrease in the number of leaves and tillers produced during the

drought period by the plants of all three species. Statistical analysis was applied to the square


63

root of the numbers measured. The graphs in Figure II.4 are presented in order to give a visual

impression of the variation of the raw data. There was a highly significant (P < 0.001) difference

between species and a strong effect (P ≤ 0.01) of the drought stress treatment on the square root

of the number of leaves and the square root of the number of tillers formed per plant during the

period of stress. However, there was no significant interaction between the species and treatment,

the stress effect being the same for the three species.

P. dilatatum C. dactylon Z. japonica

Num

ber o

f Lea

ves

(pla

nt-1

)

0

5

10

15

20 A


Num

ber o

f Tille

rs (p

lant

-1)

0

2

4

6

8 B

P. dilatatum C. dactylon Z. japonica Control Non-watered

Square root of N. of Leaves 2.54 3.95 3.95 3.64 3.32

SED 0.14 0.11

d.f. 12 12

LSD(5%) 0.29 0.24 Square root of

N. of Tillers 1.37 1.76 2.35 1.94 1.71

SED 0.09 0.07

d.f. 12 12

LSD(5%) 0.19 0.15

Figure II.4. Number of leaves and tillers produced (per plant) by the control (black bars) and non-watered (white bars) plants of P. dilatatum, C. dactylon and Z. japonica during the drought period (twelve days). Each value corresponds to the mean of fifteen plants (three control and three non-watered pots with five plants each were used per species) ± standard errors. Also presented are the means of the square root of the numbers of leaves and tillers produced by the plants of each species and by the control and non-watered plants, as given by the ANOVA, the standard error of the difference between means (SED), the degrees of freedom (d.f.) and least significant differences (LSD) at the 5% level for comparison between species and for comparison between tretaments.

Leaf anatomy

Leaf anatomical features of the C4 grasses, P. dilatatum, C. dactylon and Z. japonica, are shown

in Figures II.5 and II.6. Paspalum dilatatum is characterized by a proeminent midrib with

Chapter II.

64

B E

Bul

MMSBS

BS

M

Scl

C F

Bul M

MS

BS

M

BS

Scl

A D

M

BS BSM

Scl

B E

Bul

MMSBS

BS

M

Scl

C F

Bul M

MS

BS

M

BS

Scl

A D

M

BS BSM

Scl

Figure II.5. Transverse sections of young fully-expanded leaves of P. dilatatum (A, D), C. dactylon (B,

E) and Z. japonica (C, F) stained with Toluidine Blue O. The central longitudinal vein (A-C) and one

large longitudinal vein (D-F) of a half-leaf considered for the measurement of cellular areas are shown.

Abbreviations used: BS, bundle sheath; Bul, bulliform cells; M, mesophyll; MS, mestome sheath; Scl,

sclerenchyma. Scale bars = 30 μm.


65

A

B

C

D

F

E

Scl

Scl

ICS

A

B

C

D

F

E

Scl

Scl

ICS

Figure II.6. Transverse sections of young fully-expanded leaves of P. dilatatum (A, D), C. dactylon (B,

E) and Z. japonica (C, F) stained with Toluidine Blue O. Two small longitudinal veins between the centre

and the margin (A-C) or at the margin (D-F) of one half-leaf considered for the measurement of cellular

areas are shown. Abbreviations used: ICS, intercellular space; Scl, sclerenchyma. Scale bars = 60 μm.

numerous colourless cells and containing several veins, whilst in the other two species the midrib

is formed by one single vein. Bulliform cells are present in between consecutive veins in C.

dactylon and Z. japonica, but are absent in P. dilatatum (Figures II.5 and II.6). Sclerenchyma is

present in greatest proportion in Z. japonica and occurs at the leaf margins of both P. dilatatum

and Z. japonica, but not in C. dactylon.

Chapter II.

66

In the central longitudinal veins, the BS cells, filled with abundant chloroplasts, surround

the vascular tissues and are surrounded by the M cells, with less abundant and smaller

chloroplasts. In C. dactylon and Z. japonica, but not in P. dilatatum, a mestome sheath is present

between the BS cells and the vascular bundles. The chloroplasts of BS cells are oval and

distributed centrifugally or scattered around the cells in P. dilatatum and Z. japonica whilst in C.

dactylon the BS chloroplasts are elongated and have a centripetal position in the cells.

Quantitative data on leaf anatomy was obtained by measuring lengths and cellular areas

in the leaf transverse sections. A log transformation of each set of data presented in Figures II.7,

II.8 and II.9 was required in order to stabilise the variance across treatments and allowed the

ANOVA to be performed. Therefore, the graphs with the raw data are presented but all the

comparisons referred to were analysed on the log scale.

The leaves of P. dilatatum are wider and longer than those of the other two species,

which is associated with the greater number of veins in P. dilatatum than in Z. japonica and

lowest in C. dactylon (Figure II.7A). The analysis performed revealed no significant effect (P >

0.05) of drought stress on the vein number. Similarly, no significant differences (P > 0.05)

between control and drought-stressed plants were output by the analysis of the interveinal

distances (IVD) and leaf thickness (Figure II.7B,C). The IVD was much higher in C. dactylon

than in Z. japonica and was the lowest in P. dilatatum. The leaf thickness was lower in Z.

japonica than in the other two species.

Num

ber o

f vei

ns

0

10

20

30

A


IVD

(μm

)

0.00

0.05

0.10

0.15

B

Thic

knes

s (μ

m)

0.00

0.05

0.10

0.15

C

P. dilatatum C. dactylon Z. japonica P. dilatatum C. dactylon Z. japonica Figure II.7. Number of veins per half-leaf (A), interveinal distance (B, IVD) and leaf thickness (C) of control (black bars) and non-watered (white bars) plants of P. dilatatum, C. dactylon and Z. japonica at the end of the drought period (after twelve days without watering). Values are means ± standard errors of three leaf sections (analytical replicates) from each of three plant samples (biological replicates).


67

Pe

rcen

tage

tiss

ue (%

)

0

10

20

30

A


B C

P. dilatatum C. dactylon Z. japonica P. dilatatum C. dactylon Z. japonica P. dilatatum C. dactylon Z. japonica

DBundle Sheath

Mesophyll

Intercellularspaces

Bulliform cells

Perc

enta

ge ti

ssue

(%)

0

10

20

30

E


F G

P. dilatatum C. dactylon Z. japonica P. dilatatum C. dactylon Z. japonica P. dilatatum C. dactylon Z. japonica

H

Sclerechyma Vascular tissuesLower epidermis Upper epidermis

Figure II.8. Percentage of cellular area occupied by different tissues in the leaf transverse sections of control (black bars) and non-watered (white bars) plants of P. dilatatum, C. dactylon and Z. japonica at the end of the drought period (after twelve days without watering). Values correspond to the overall percentage of each tissue in relation to the total area of leaf measured considering the sums of all four zones (see Figures II.5 and II.6) and are means ± standard errors of three leaf sections (analytical replicates) from each of three plant samples (biological replicates).

Mes

ophy

ll / B

undl

e Sh

eath

0

1

2

3


M/BS

Figure II.9. Proportion of leaf transverse-sectional area occupied by mesophyll or bundle sheath cells in the control (black bars) and non-watered (white bars) plants of P. dilatatum, C. dactylon and Z. japonica at the end of the drought period (after twelve days without watering). Values correspond to the ratio between overall M or BS areas in a half-leaf considering the sums of all four zones measured (see Figures II.5 and II.6) and are means ± standard errors of three leaf sections from each of three plant samples.

Chapter II.

68

The cellular areas of half-leaf transverse sections were measured in four representative

zones, with two zones (Figure II.5) corresponding to one large (central or not) longitudinal vein

and two other (Figure II.6) corresponding to two small longitudinal veins each. Even though the

leaves of C. dactylon are smaller than those of Z. japonica and P. dilatatum, the total area

measured was higher in the first species (data not shown), due to its higher IVD (Figure II.7B).

The three species were characterised by different percentages of the total area measured being

occupied by the different types of tissues (Figure II.8). Hence, the percentage of BS was higher

in C. dactylon and Z. japonica than in P. dilatatum whilst M cells were proportionally less

abundant in Z. japonica than in the other two species. As a result, the ratio M/BS was much

greater in P. dilatatum than in C. dactylon and was lowest in Z. japonica (Figure II.9).

The percentage of ICS was substantially larger in P. dilatatum than in C. dactylon and

very low in Z. japonica (Figure II.8), whereas the percentage of leaf area occupied by bulliform

cells was considerable in C. dactylon and Z. japonica, but larger in the latter. No bulliform cells

were observed in P. dilatatum. The presence of sclerenchyma was more abundant in Z. japonica

than in the other two species (Figures II.5 and II.6) and the percentage of leaf area occupied by

sclerenchyma was highest in Z. japonica, lowest in C. dactylon and intermediate in P. dilatatum

(Figure II.8). The percentage of area occupied by vascular tissues was also highest in Z. japonica.

The epidermal cells were larger in P. dilatatum than in C. dactylon and Z. japonica (Figures II.5

and II.6), resulting in higher relative areas being occupied by the epidermis in P. dilatatum than

in the other two species (Figure II.8).

Gradually-imposed decrease in water availability did not cause many changes in the leaf

quantitative anatomy. Nevertheless, there was an overall marginal effect (P ≤ 0.1) of drought

stress on the percentage of area occupied by the M cells and on the ratio M/BS, with an increase

of the ratio M/BS in the leaves of the non-watered plants of P. dilatatum and Z. japonica, but not

of C. dactylon, compared to the controls (Figure II.9).

Amino acids

The proline content was much increased with drought stress in the leaves of three grasses under

study (Figure II.10A). A quadratic variation with RWC was found for this amino acid, the

increase being steeper as dehydration became more severe. Proline increased by about 2.5-fold in

the three species when the RWC decreased to 80% and the values rose abruptly below a certain

threshold of RWC (70%), reaching values of 8 mmol m-2 in the most dehydrated samples of Z.

japonica (40% RWC).


69

The content in methionine was higher in Z. japonica and C. dactylon than in P. dilatatum

and increased with the decrease of RWC in a non-linear way for the three species, more

accentuated as the level of dehydration increased (Figure II.10B). The amino acids phenylalanine,

valine, isoleucine and leucine were also much increased in the dehydrated leaves of Z. japonica

(Figure II.10C-F). In P. dilatatum and C. dactylon the content in leucine was negligible and the

other three amino acids increased with drought stress to a much lower extent than in Z. japonica.

Nevertheless, in C. dactylon valine increased three-fold when RWC decreased from 98 to 60%.

40 60 80 100

0.00

0.05

0.10

0.15

0.20

0.25

40 60 80 100

0.000

0.003

0.006

0.009

0.012

0.015

40 60 80 100

Amin

o Ac

ids

Con

tent

(mm

ol m

-2)

0

2

4

6

8

10

RWC (%)

40 60 80 100

0.00

0.03

0.06

0.09

0.12

0.15

Pro Met

RWC (%)

40 60 80 100

0.00

0.03

0.06

0.09

0.12

0.15

Phe

LeuIle

A B C

E F

y = 2106 - 42.22 x + 0.2124 x2 (s.e. 134; 3.72; 0.0248)

y = 3.81 - 0.0834 x + 0.000456 x2 (s.e. 0.24; 0.0066; 0.000044)

y = 3.90 - 0.0834 x + 0.000456 x2 (s.e. 0.24; 0.0066; 0.000044)

y = 40.14 - 0.937 x + 0.00543 x2 (s.e. 6.65; 0.152; 0.00089)y = 62.50 - 1.159 x + 0.00543 x2 (s.e. 4.35; 0.128; 0.00089)

y = 11.35 - 0.2290 x + 0.00118 x2 (s.e. 3.47; 0.0791; 0.00046)y = 27.17 - 0.3938 x + 0.00118 x2 (s.e. 2.27; 0.0667; 0.00046)

y = 19.14 - 0.1983 x + 0.000030 x2 (s.e. 0.72; 0.0093; 0.000015)

RWC (%)

40 60 80 100

0.0

0.1

0.2

0.3

0.4

0.5

Val

D y = 48.19 - 0.993 x + 0.00521 x2 (s.e. 7.90; 0.178; 0.00104)y = 49.73 - 0.993 x + 0.00521 x2 (s.e. 7.83; 0.178; 0.00104)y = 76.68 - 1.287 x + 0.00521 x2 (s.e. 5.10; 0.150; 0.00104)

Figure II.10. Variation of the amino acids content (mmol m-2) with the relative water content (RWC, %) in the leaves of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (open triangles): proline (Pro), valine (Val), methionine (Met), phenylalanine (Phe), isoleucine (Ile), leucine (Leu) (A-F, respectively). Each data point corresponds to one sample, with 24 samples per species. The regression lines and curves, when applied, correspond to the best models statistically significant (P. dilatatum, solid lines; C. dactylon, dashed lines; and Z. japonica, dotted lines; A, R2 = 90.5%, s2 = 3255, d.f. = 69; B, R2 = 94.3%, s2 = 3.792, d.f. = 66; C, R2 = 89.1%, s2 = 0.0102, d.f. = 68; D, R2 = 92.7%, s2 = 2.763, d.f. = 67; E, R2 = 93.7%, s2 = 0.7513, d.f. = 67; F, R2 = 91.2%, s2 = 0.8410, d.f. = 69).

Chapter II.

70

RWC (%)

40 60 80 100

MEA

(mm

ol m

-2)

0.00

0.02

0.04

0.06

0.08

0.10

RWC (%)

40 60 80 100

HN

V (m

mol

m-2

)

0.00

0.05

0.10

0.15

0.20

0.25

MEA HNV

A By = - 6.93 + 0.2503 x - 0.00167 x2 (s.e. 2.40; 0.0655; 0.00043)y = - 5.39 + 0.2503 x - 0.00167 x2 (s.e. 2.34; 0.0667; 0.00046)

y = 0.006967 x - 0.0000701 x2 (s.e. 0.000507; 0.0000055)y = 0.2330 - 0.001206 x (s.e. 0.0169; 0.000213)

Figure II.11. Variation of the content (mmol m-2) in mono-ethanolamine (A, MEA) and 5-hydroxy-L-

norvaline (B, HNV) with the relative water content (RWC, %) in the leaves of P. dilatatum (black

diamonds), C. dactylon (grey squares) and Z. japonica (open triangles). Each data point corresponds to

one sample, with 24 samples per species. The regression lines and curves, when applied, correspond to

the best models statistically significant (P. dilatatum, solid lines; C. dactylon, dashed lines; and Z.

japonica, dotted lines; A, R2 = 38.7%, s2 = 0.9491, d.f. = 67; B, R2 = 89.8%, s2 = 0.03151, d.f. = 68).

Higher values of mono-ethanolamine (MEA) were generally observed for Z. japonica

than for P. dilatatum or C. dactylon (Figure II.11A). Despite the high variability in the values

obtained, namely for Z. japonica, there was a slight increase of MEA in the earlier stages of

dehydration in all species, followed by a decrease in the most dehydrated samples. The non-

protein amino acid, 2-amino-5-hydroxypentanoic acid (or 5-hydroxy-L-norvaline, HNV) was

present in all leaves of Z. japonica and in leaves from drought-stressed plants of C. dactylon,

whereas no significant (P > 0.05) amounts were found to be present in P. dilatatum (Figure

II.11B). The content of HNV increased with leaf dehydration in both C. dactylon and Z. japonica,

but the increase was much steeper in the former species, attaining values similar to those found

in Z. japonica at ca. 65% RWC.


71

DISCUSSION

Soil and leaf water relations

Similar extents of decreased water availability in the soil of non-watered pots of each of the three

species were obtained with water withheld from the pots of C. dactylon first, Z. japonica next

and P. dilatatum last, in consecutive days, as given by the parallel lines of decreased water

availability through the experiment (Figure II.1), suggesting faster soil water absorption by P.

dilatatum. Withholding water from the pots of each species on consecutive, separate days was a

strategy adopted in order to obtain more similar levels of drought stress in the three species and

allow for a better comparison of their responses. Different rates of soil drying have been reported

for a C3 in comparison to a C4 monocot due to lower water consumption and increased water use

efficiency by the latter (Kalapos et al. 1996). The WWP and the SWC were highly correlated

(Figure II.1). The WWP corresponding to a certain SWC was greater in the pots of P. dilatatum

than in the pots of C. dactylon or Z. japonica, due to the greater biomass of the former species.

Accordingly, the drought-induced decrease of shoot growth in the three species (Figure II.4)

resulted in different, parallel lines applied to the control and non-watered pots (Figure II.1). The

soil water content, measured as the amount of water in relation to the amount of soil, might not

be a very useful tool because the soil water potential does not decrease linearly with the soil

water content (Kramer & Boyer 1995) and the water in the soil might not be all available to the

plant. The use of an appropriate pot to the plant size, allowing the roots to be homogeneously

distributed and occupying most of, but not exhaustively, the soil volume (results not shown), is

likely to have favoured the imposition of very similar levels of water deficit for the three species.

The leaf relative water content (RWC) decreased in the non-watered plants of the three

species (Figure II.2A), but only a few severely stressed samples were obtained. The sequence in

which watering was stopped for the plants of C. dactylon, Z. japonica and P. dilatatum in

consecutive days seemed adequate since similar RWC values were obtained for the samples of

the three species in this experiment. Occasionally, if the drying conditions were high, namely

through higher evaporative demand promoted by slightly increased temperatures in the first days

of the drought treatment (when water was being withheld from the pots of one species but not yet

from another), different levels of dehydration were attained in the leaves of each species, as

observed in the amino acid experiment. The faster absorption of water from the drying soil and

the faster water loss from the leaves of P. dilatatum than from the leaves of C. dactylon and Z.

japonica contrast with results previously obtained under rapidly-induced water deficit (Carmo-

Chapter II.

72

Silva et al. 2007) and are likely to be due to a dramatic response of C. dactylon to the artificial

PEG-induced decrease of water availability used in that study, promoting a fast dehydration in

the leaves of this species.

In P. dilatatum and C. dactylon, the LWP showed an initial decrease as the RWC started

to decrease but remained constant after reaching a minimum value of -1.4 MPa (Figure II.2).

This suggests a possible drought adaptation by these two species in the direction of producing

cells with increased plasticity, decreasing the turgor pressure and hence keeping the LWP

constant as RWC decreases (Lambers et al. 1998). LWP decreased to ca. -2.4 MPa in drought-

stressed NADP-ME and NAD-ME grasses (Ghannoum et al. 2002) and to a minimum of ca. -2.0

MPa in C. dactylon during a Mediterranean summer-drought (Utrillas & Alegre 1997).

Conversely, in Z. japonica the linear decrease of LWP with decreasing RWC suggests the

presence of rigid cell walls, associated with the hard, tough and stiff leaves in this species, and

reflected in the higher proportion of dry weight than in the other two species (Figure II.3). In fact,

Z. japonica is characterised by rigid cell walls that may become even more rigid upon exposure

to drought stress (White et al. 2001). The accumulation of compatible solutes, namely amino

acids like proline (Figure II.9), can also contribute to the osmotic adjustment of the tissues and to

the decreased leaf water potential, but the changes in turgor pressure are likely to have a

prevailing role in the drought-induced lowering of LWP (Kramer & Boyer 1995). The LWP at

zero turgor, reflecting changes in osmotic potential, decreased only slightly (less than 0.2 MPa)

in several Z. japonica genotypes (White et al. 2001), suggesting little contribution of compatible

solute accumulation to the decrease in LWP in this species (Figure II.2). The presence of rigid

cell walls promoting the decrease of LWP in response to the drying soil conditions can be seen

as a drought tolerance mechanism, by which water absorption from the soil is increased avoiding

excessive leaf dehydration. Both the capacity for osmotic adjustment and the cell wall rigidity

tend to be greater in C4 than in C3 grasses (Barker et al. 1993). However, these processes seem to

function essentially under severe drought conditions and might therefore have a pivotal role for

plant survival when severe summer-droughts are observed (Utrillas & Alegre 1997; Volaire et al.

1998) but may be of little significance when plant productivity is the target (Serraj & Sinclair

2002). Increased root depth, total root weight and/or root number might also result in increased

capacity of Z. japonica to keep absorbing water from the dry soil (Marcum et al. 1995). Carrow

(1996) associated increased drought resistance in bermudagrasses (C. dactylon) compared to

zoysiagrasses (Z. japonica) with deeper root systems of the former. Moreover, the presence of

salt glands in these two species (Oross & Thomson 1982; Marcum & Murdoch 1990) and the


73

active salt secretion from their leaves are associated with salt tolerance (Marcum 1999) and may

also benefit plant performance under drought conditions.

Plant growth and leaf structure

Plants of the three grass species produced fewer leaves and tillers in response to the drying soil

(Figure II.4). Decreased shoot growth is a well established effect of water deficit (Jones 1985;

Hsiao & Xu 2000) and seems to be an early event associated with signals produced in the roots

(Davies & Zhang 1991). Decreased shoot elongation rates were observed in several C4 grasses in

response to decreased water availability (Ghannoum et al. 2002) and decreased leaf production

during a Mediterranean summer was reported for two legume species under low rainfall field

conditions compared to irrigated conditions (Lefi et al. 2004). Decreased shoot growth may be

part of an adaptive response to drought stress with photoassimilates and energy being diverted to

the synthesis of molecules involved in plant defence (Chaves & Oliveira 2004).

The relative increase of leaf dry matter with water deficit (Figure II.3) might be

associated with the accumulation of starch or soluble compounds, including osmolytes with a

potentially protectant role against drought effects; or may reflect increased xeromorphy. For

instance, increased cell wall rigidity may result in increased dry matter due to changes in cell

wall thickness and constituents (Wilson et al. 1980). Increased proportion of leaf dry matter and

cell wall rigidity were observed in several Z. japonica genotypes exposed to drought conditions

(White et al. 2001). The increase in dry matter was not associated with increased leaf thickness

(Figure II.7), in agreement with previous observations for C. dactylon under field induced

drought conditions during a Mediterranean summer (Utrillas & Alegre 1997). The lower dry

matter and thicker leaves of P. dilatatum contrasted with the higher dry matter and thinner leaves

of Z. japonica. The decreased SLA in the non-watered plants of P. dilatatum and C. dactylon

may have resulted directly from the increased DW/TW (Figure II.3). On the other hand, the

unchanged SLA in drought-stressed compared to control plants of Z. japonica suggests increased

leaf area. No effects were observed on the number of veins or on IVD (Figure II.7), indicating

that leaf length rather than width might have changed.

Morphological changes including leaf ‘rolling’ and ‘folding’ are related with decreased

water content and leaf water potential and can play an important role in the response of grasses

to drought (O'Toole & Cruz 1980). These leaf movements can be delayed by osmotic adjustment

(Hsiao et al. 1984). The reduction of evaporative surface area contributes to minimize water loss

and the decreased light absorption minimizes photoinhibitory injury (Kramer 1983). In P.

dilatatum, C. dactylon and Z. japonica these morphological alterations were observed only at

Chapter II.

74

RWC values lower than ca. 80-75% (results not shown), which reflects the increasing

importance of these drought tolerance mechanisms as stress severity increases. Variations in leaf

morphological responses occur among C4 grasses under drought conditions (see Sanderson et al.

1997 for review). In the present work, the colourless and bulliform cells present in the leaves of

the three grasses caused different morphological responses. The leaves of P. dilatatum are

characterized by a large number of colourless cells in the midrib (Figure II.5) and the loss of

water from these cells results in the leaf folding. Conversely, in C. dactylon and Z. japonica the

midrib is formed solely by the central vascular bundle and bulliform cells are spread through the

leaf, in between each pair of consecutive vascular bundles. The differential arrangement of these

water-storing cells results in leaf shrinkage in C. dactylon and leaf curling in Z. japonica when

considerable dehydration occurs.

The anatomical characteristics of P. dilatatum, C. dactylon and Z. japonica (Figure II.5)

agree with the leaf anatomical features associated with each of the classical subtypes of C4

grasses (Dengler et al. 1994) and with previous descriptions for the three species (Watson &

Dallwitz 1992). Drought stress did not cause many changes in the leaf structure of the three

species but species-specific characteristics are likely to be associated with the differential

capacity to withstand low water availability. For instance, the higher percentage of leaf area

occupied by sclerenchyma in Z. japonica (Figure II.8) is likely to be associated with the leaf

stiffness and stronger xeromorphic characteristics of the species compared with the other two.

On the other hand, the higher percentage of area occupied by the vascular tissues in this species

may reflect improved water transport system. In several maize lines, drought resistance was

associated with larger leaf transverse sectional areas occupied by vascular tissues (Ristic & Cass

1991), possibly reflecting increased capacity for water uptake.

The ratio between the relative leaf areas occupied by mesophyll or bundle sheath cells

(M/BS) was greater in P. dilatatum than in the other two species (Figure II.9), which is in

agreement with previous descriptions for NADP-ME C4 grasses (Hattersley 1984; Dengler et al.

1994) and results essentially from the lower percentage of area occupied by BS cells (Figures

II.5, II.6 and II.8). The ratio M/BS increased in leaves of drought-stressed plants of P. dilatatum

and Z. japonica compared to well-watered plants, but was not changed in C. dactylon. The

ability of plants to alter their development and adjust, or acclimate, to environmental variations

may be of great importance to maintain photosynthetic performance. However, the structural

changes in leaves of C4 grasses must not disturb the functionality of the C3 and C4 cycles (Sage

& McKown 2006). Increased M area relative to BS area may result in increased CO2 being

delivered in the BS to be assimilated by the C3 cycle, which can be a strategy to overcome the


75

decreased CO2 concentrations resulting from stomatal closure. The high photosynthetic

performance of C. dactylon (Carmo-Silva et al. 2007; 2008) suggests no need for leaf structure

adjustments in moderate drought conditions, even though impairment of structural functionality

may be observed under severe drought stress (Utrillas & Alegre 1997).

Accumulation of soluble amino acids

The increased amino acids content in the drought-stressed leaves of the three species (Figure

II.10) is in accordance with previously reported data obtained for C4 grasses and other monocot

species (Barnett & Naylor 1966; Jones et al. 1980; Ford & Wilson 1981; Thakur & Rai 1982;

Kusaka et al. 2005; Simon-Sarkadi et al. 2006). Impaired protein metabolism, including both

decreased protein synthesis and increased hydrolysis, has been suggested to cause free amino

acids content to rise by several-fold (Barnett & Naylor 1966; Jones et al. 1980). However,

accumulation of amino acids is more likely to result from increased synthesis, possibly reflecting

the ability of plants to adjust osmotically in response to the drying soil or their enhanced

metabolism in the direction of synthesis of secondary metabolites with different roles in stress

defence. In Pennisetum glaucum (pearl millet, C4 NAD-ME), greater amino acids content were

observed in drought-stressed leaves of tolerant than sensitive accessions (Kusaka et al. 2005).

Amino acids accumulation is likely to result in increased osmotic potential and contribute

therefore to minimize water loss from the leaves and to stabilize membranes and proteins in the

cells. Moreover, their active biosynthesis of some amino acids is likely to be involved in the use

of excessive reducing power (in the event of drought-induced decreased photosynthetic

metabolism) as well as in the protection against reactive oxygen species.

Osmotic adjustment in response to drying soil has been shown to occur in C4 grasses to a

greater extent than in some of their C3 counterparts (Barker et al. 1993). Accumulation of

inorganic ions can play an important role in the osmotic adjustment of C4 grass species under

drought conditions (Ford & Wilson 1981; Utrillas et al. 1995) but soluble sugars seem to have a

relatively little contribution (e.g. Marques da Silva & Arrabaça 2004). However, in Sorghum

bicolor both increased sugars and inorganic ions accounted for most of the decrease in osmotic

potential under water deficit (Jones et al. 1980). Proline is one of the compatible solutes most

widely accumulated in response to hyperosmotic stresses (Delauney & Verma 1993) and, even

though its role in osmotic adjustment is a matter of controversy (see Hare & Cress 1997), the

capacity to accumulate this imino acid under drought conditions was related to the drought

resistance of different Sorghum bicolor varieties (Blum & Ebercon 1976). Moreover, increased

contents of proline (especially) and other amino acids, like valine, isoleucine and leucine, were

Chapter II.

76

observed in the phloem sap of Medicago sativa (drought-resistant C3 plant) in response to

progressively-induced drought stress (Girousse et al. 1996).

The proline content doubled when RWC decreased to nearly 80% in the leaves of the

three species studied (Figure II.10) but accumulation occurred only under severe leaf

dehydration, as described for other C4 grasses (Jones 1985). As recently reviewed by Ashraf &

Foolad (2007), the protective role of proline in the adaptation of plants to drought conditions is

likely to involve a number of functions other than osmoregulation. In Saccharum spp.

(sugarcane), for instance, the advantage of proline accumulation under water deficit was

associated with plant protection against oxidative stress, rather than osmoprotection (Molinari et

al. 2007). In several other C4 grass species, the contribution of proline accumulation to osmotic

adjustment was negligible, i.e. both occurred concomitantly in response to drought without one

being the cause of the other and the beneficial role of proline was more likely related with other

aspects of plant metabolism (Barker et al. 1993).

Increased content of methionine, especially in C. dactylon and Z. japonica, with leaf

dehydration (Figure II.9), is likely to reflect enhanced production of ethylene and/or polyamines

with drought. Ethylene is known to be involved in shoot growth restriction in response to the

drying soil (Hussain et al. 1999) whilst polyamines are thought to provide the plant with

increased drought tolerance, namely through antioxidant activity, although their mechanism of

action remains unclear (Groppa & Benavides 2008). Curiously, the content in methionine was

higher in C. dactylon and Z. japonica, the two C4 species having aspartate as the stable

photosynthetic product resulting from primary fixation of CO2 in the mesophyll cells, than in the

malate-producing species, P. dilatatum. However, the same pattern was not observed for

phenylalanine, also derived from aspartate. Greater increases in the contents of the amino acids

phenylalanine, valine, isoleucine and leucine were observed in Z. japonica than in the other two

species (Figure II.10), possibly contributing to the lower values of LWP (Figure II.2) and

eventually reflecting enhanced involvement of secondary metabolism. If the production of stress-

induced secondary metabolites is triggered, in order to allow the plant to cope with, and survive,

drought conditions, the metabolic fluxes will be diverted in that direction and increased contents

of the amino acids involved can be observed. For instance, induction of a protein involved in

lignin biosynthesis was observed in the roots of rice plants exposed to salt stress (Salekdeh et al.

2002). Increased lignin deposition might contribute to the cell wall strengthening under

conditions that favour water loss. Phenylalanine is involved in the synthesis of this polymer and

other aromatic secondary compounds that may play an important role in cellular protection

against reactive oxygen species (Grace & Logan 2000).


77

Mono-ethanolamine (MEA) was present in greater amount in Z. japonica than in C.

dactylon and P. dilatatum and increased slightly in the first stages of leaf dehydration (Figure

II.11). This amino alcohol is a precursor of phosphatidylethanolamine, one of the most abundant

phospholipids constituting cell membranes, and an intermediate in the biosynthetic pathway of

choline, a precursor of quaternary ammonium compounds known to confer protection under

stress conditions (Rhodes & Hanson 1993). Ethanolamine is the direct product of serine

decarboxylation and its increase in a C4 grass under moderate drought conditions, concomitant

with increased serine, suggested transiently enhanced photorespiration (Martinelli et al. 2007). In

the three C4 grasses here studied, a concomitant increase of both serine and ethanolamine was

observed when RWC decreased to ca. 80%, suggesting slightly increased photorespiratory

metabolic fluxes (see Chapter III, Carmo-Silva et al. 2008).

An unusual amino acid was also stress-responsive in the leaves of C. dactylon and Z.

japonica, but not of P. dilatatum (Figure II.11). To the best of our knowledge, 5-hydroxy-L-

norvaline has not been reported before to be present in plant leaves, although it is a well known

constituent of legume seeds (Thompson et al. 1964). The presence and drought-induced increase

of 5-hydroxy-L-norvaline in Z. japonica and it appearance in increasing concentrations in

dehydrated leaves of C. dactylon suggest that this non-protein amino acid might be involved in

the stress response and, possibly, playing a role in plant defence mechanisms.

CONCLUSIONS

Drought resistance mechanisms were present in the three grasses but were more effective in C.

dactylon (NAD-ME), and in Z. japonica (PEPCK) than in P. dilatatum (NADP-ME), with

greater water loss in the latter. The use of bermudagrass (C. dactylon) for recreational purposes,

including golf courses, seems to be a promising strategy to optimize water use. Zoysiagrass (Z.

japonica) can be used as an alternative and especially for zones where leaf hardness, toughness

and stiffness are not a concern. Dallisgrass (P. dilatatum) has a greater requirement for water and

is therefore less promising for the use as turfgrass. Nonetheless, its high productivity might be of

great advantage in its use as forage grass.

The different relationships between LWP and RWC in Z. japonica suggest increased cell

wall rigidity which might be involved in a strategy to improve water uptake from the soil. The

lack of considerable alterations in leaf anatomy under moderate drought stress in all three species

is probably associated with a low phenotypic plasticity and with the high leaf structure

specialization in these C4 grasses conferring considerable drought resistance. The increased

amino acids content are likely to be associated with enhanced synthesis of compounds involved

Chapter II.

78

in plant stress response and may additionally contribute to osmotic adjustment and, thus, to

minimize water loss. The presence and drought-induced increase of HNV on the leaves of the C4

grasses with greater drought resistance must be further exploited in order to understand the role

of this unusual amino acid and its possible association with stress defensive mechanisms.


79

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Serraj R. & Sinclair T.R. (2002) Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant Cell and Environment, 25, 333-341.

Sharp R.E. (2002) Interaction with ethylene: changing views on the role of abscisic acid in root and shoot growth responses to water stress. Plant Cell and Environment, 25, 211-222.

Simon-Sarkadi L., Kocsy G., Varhegyi A., Galiba G. & de Ronde J.A. (2006) Stress-induced changes in the free amino acid composition in transgenic soybean plants having increased proline content. Biologia Plantarum, 50, 793-796.

Thakur P.S. & Rai V.K. (1982) Dynamics of amino acid accumulation in two differentially drought resistant Zea mays cultivars in response to osmotic stress. Environmental and Experimental Botany, 22, 221-226.

Thompson J.F., Hunt G.E. & Morris C.J. (1964) The identification of L-α-amino-δ-hydroxynorvaleric acid and L-homoserine in Jack bean seeds (Canavalia ensiformis). Journal of Biological Chemistry, 239, 1122-1125.

Chapter II.

84


Utrillas M.J., Alegre L. & Simon E. (1995) Seasonal changes in production and nutrient content of Cynodon dactylon (L.) Pers. subjected to water deficits. Plant and Soil, 175, 153-157.

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Chapter III.

PHOTORESPIRATION AND C4

PHOTOSYNTHESIS UNDER DROUGHT STRESS

Chapter III.

86

An integral copy of this chapter is In Press in Plant, Cell and Environment:

Carmo-Silva A.E., Powers S.J., Keys A.J., Arrabaça M.C. & Parry M.A.J. (2008) Photorespiration in C4 grasses remains slow under drought conditions. Plant, Cell and Environment, x, xx-xx. (In Press)

Alfred J. Keys (Department of Plant Sciences, Rothamsted Research) was involved in the planning of the experiments, in the measurements of amino acids content and interpretation of the results.

Stephen J. Powers (Department of Biomathematics and Bioinformatics, Rothamsted Research) did the non-linear modelling and applied the equations of C4 photosynthesis mechanistic models.

Photorespiration and C4 Photosynthesis under Drought Stress

87

PHOTORESPIRATION IN C4 GRASSES REMAINS SLOW UNDER DROUGHT CONDITIONS

ABSTRACT

The CO2-concentrating mechanism present in C4 plants decreases the oxygenase activity of

Rubisco and, consequently, photorespiratory rates in air. Under drought conditions the

intercellular CO2 concentration may decrease and cause photorespiration to increase. The C4

grasses Paspalum dilatatum Poiret, Cynodon dactylon (L.) Pers and Zoysia japonica Steudel

were grown in soil and drought was imposed by ceasing to provide water. Net CO2 assimilation

(A) and stomatal conductance to water vapour decreased with leaf dehydration. Decreased

carbon and increased oxygen isotope composition were also observed under drought. The

response of A to CO2 suggested that the compensation point was zero in all species irrespective

of the extent of drought stress. A slight decrease of A as O2 concentration increased above 10%

provided evidence for slow photorespiratory gas-exchanges. Analysis of amino acids contained

in the leaves, particularly the decrease of glycine after 30 seconds in darkness, supported the

presence of slow photorespiration rates, but slightly faster in C. dactylon than in P. dilatatum and

Z. japonica. Although the contents of glycine and serine increased with dehydration and

mechanistic modelling of C4 photosynthesis suggested slightly increased photorespiration rates

in proportion to photosynthesis, the results provide evidence that photorespiration remained slow

under drought conditions.

KEYWORDS:

CO2- and O2-response curves, amino acids, isotope composition, modelling C4 photosynthesis

Chapter III.

88

INTRODUCTION

The main objective of this study was to investigate whether photorespiration was increased by

drought stress in three species of C4 grasses and could, as a consequence, contribute to metabolic

factors limiting net photosynthesis. Under drought conditions photosynthetic carbon assimilation

decreases in both C3 and C4 plants (e.g. Chaves et al. 2003). Closure of stomata is one of the

major causes of the decrease in photosynthesis but evidence has accumulated that metabolic

limitations also contribute (Du et al. 1996; Saccardy et al. 1996; Ghannoum et al. 2003; Marques

da Silva & Arrabaça 2004; Carmo-Silva et al. 2007). In C4 leaves with Kranz anatomy,

atmospheric CO2 is initially fixed by phosphoenolpyruvate (PEP) carboxylase (PEPC; EC

4.1.1.31) into C4 acids in the mesophyll (M) cells. The C4 acids are transported to the bundle

sheath (BS) cells where they undergo decarboxylation and the released CO2 enters the C3

pathway via ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco; EC 4.1.1.39).

Rubisco is confined to the BS cells and the specialized leaf anatomy decreases leakage of CO2

back to M cells so that CO2 accumulates.

Rubisco acts both as a carboxylase and an oxygenase. Molecules of CO2 and O2 are

competing alternative substrates for reaction with the enediol of RuBP catalysed by Rubisco

(Bowes & Ogren 1972; Laing et al. 1974) and, therefore, rates of photorespiration relative to

photosynthesis are determined by the relative concentrations of O2 and CO2 at the catalytic site

of Rubisco in the chloroplast stroma. In fully hydrated leaves, the CO2/O2 ratio in BS cells of C4

species is 3 to 6 times higher than in M cells under atmospheric levels of CO2 and O2 (Dai et al.

1993; Kiirats et al. 2002). Therefore the oxygenase activity of Rubisco, and consequently the

photorespiratory rate, is slow. The consequences are a low CO2 compensation point in C4 plants

(Forrester et al. 1966) and the absence of an enhancement of net photosynthesis when oxygen in

the gas-phase is decreased from 21 to 2% (Edwards et al. 1985). These observations were

initially interpreted as a lack of photorespiration in C4 plants. More recent studies with several C4

species, including the three main subtypes and both monocotyledons and dicotyledons (Dai et al.

1993; Maroco et al. 1997), have revealed that the maximum rate of net photosynthesis occurred

at O2 concentrations between 5 and 10%. The rise of C4 photosynthesis to a maximum at 5-10%

has been explained by an oxygen requirement for the production of extra ATP needed for the

CO2-concentrating mechanism (Maroco et al. 1997). The decrease in photosynthesis by C4

species when the O2 concentration is elevated above 10% depends on the CO2 concentration (Dai

et al. 1993; Maroco et al. 1997; 1998) and is assumed to be due to photorespiration. To the best


89

of our knowledge the effect of O2 on C4 photosynthesis by dehydrated leaves has not been

recorded.

Under water deficit conditions the CO2 concentration in the leaves may decrease because

of decreased stomatal conductance and should cause photorespiration to increase. Mechanistic

modelling of C4 photosynthesis is not used as frequently as that of C3 photosynthesis, mostly due

to the additional complexity resulting from the structural and biochemical specialization

characteristic of C4 plants (von Caemmerer 2000). The model of von Caemmerer & Furbank

(1999) uses basic equations to describe the carbon fluxes in C4 photosynthesis and, with careful

assumptions being made, can be used to estimate the CO2 concentration in the bundle sheath, the

rate of oxygenation of RuBP and hence photorespiration.

Photorespiratory metabolism requires the integration of the photorespiratory carbon

oxidation cycle, the photorespiratory nitrogen cycle (Keys et al. 1978) and photosynthetic carbon

assimilation (Keys 1999); consequently there is an interdependence of reactions in different parts

of the overall process. Since photorespiration is relatively little-influenced by metabolite signals,

amino acids can be used as metabolite markers for this pathway (Foyer et al. 2003). Although de

novo assimilation of nitrogen and recycling of ammonia during photorespiration interact (Stitt et

al. 2002), the ratio glycine/serine and both aspartate and alanine levels were strongly correlated

with photorespiration rates in C3 plants (Novitskaya et al. 2002). In the C4 dicotyledon

Amaranthus edulis, increased glycine content in the leaves with increasing O2 in the atmosphere

was taken as indicative of photorespiration (Maroco et al. 2000); although increased serine was

also observed in A. edulis, this amino acid decreased with increasing O2 in C3 plants and was

therefore discounted as an indicator of photorespiration. Post-illumination decreases in glycine

(Kumarasinghe et al. 1977; Rawsthorne & Hylton 1991) are mainly responsible for the post-

illumination burst of CO2 in C3 plants, a phenomenon leading to the early recognition of

photorespiration. Post-illumination CO2 bursts are generally not seen in C4 plants, mostly

because of the low conductance of BS cell walls to CO2, but the post-illumination decrease in

glycine should be observed if photorespiration is present.

Variations in carbon and oxygen isotope compositions (δ13C and δ18O) reflect the

influence of environmental factors on the kinetics of photosynthesis and the two isotopes are

conveniently measured on the same leaf samples. The CO2-concentrating mechanism present in

C4 plants results in lower discrimination against 13C than in C3 plants and hence an average δ13C

of -13.6‰ in C4 as opposed to -27.8‰ in C3 plants (Troughton 1979). Changes in δ13C under

stress conditions reflect mostly variations in the CO2 concentration at the carboxylation sites and

the coordination between the C3 and C4 cycles (Farquhar 1983). On the other hand, changes in

Chapter III.

90

δ18O are mainly the result of changes in evapotranspiration and reflect the isotope content of the

soil water as well as the fractionation during transpiration (Barbour 2007), which is likely to

change in conditions affecting the evaporative demand.

The experiments described aimed to detect photorespiration in three C4 grasses and to

determine whether the rate increased under drought conditions. The indicators of

photorespiration used were: the response of photosynthesis to CO2, inhibition of photosynthesis

by O2, content of amino acids, post-illumination changes in amino acids, and isotope

fractionation. The three C4 species studied have been reported to belong each to a different

biochemical subtype, according to the main enzyme responsible for the decarboxylation of C4

acids in the BS: Paspalum dilatatum Poiret, NADP-malic enzyme (NADP-ME), Cynodon

dactylon (L.) Pers, NAD-malic enzyme (NAD-ME) and Zoysia japonica Steudel, PEP

carboxykinase (PEPCK).


91





Jacklin Seed Company, USA) (PEPCK) were grown from seeds in pots with peat-free compost,

prepared to Rothamsted Research’s specification by Petersfield Products (Leicester, UK)

supplemented with a slow-release fertiliser (Hydro Agri Ltd, Lincs, UK) in a glasshouse.

Artificial light was provided whenever the natural light was below a photosynthetic photon flux

density (PPFD) of 500 μmol m-2 s-1 during a 16 h photoperiod. Temperature was maintained at a

minimum of 25ºC during the day and at 18ºC during the night.

Seeds of each species were washed with 10% hypochlorite and soaked in water for 1 hour

before sowing. Water was supplied whenever needed during two weeks for P. dilatatum and C.

dactylon and four to five weeks for the slower growing Z. japonica. The seedlings were

transplanted to 1-L cylindrical pots containing equal amounts of soil. Five seedlings were used

per pot. The dates of sowing and transplanting each species were adjusted and the timing of the

drought treatment was chosen in order to have plants at an adequate growth stage for taking

measurements or sampling all three grasses at the same time.

All plants were well-watered until the beginning of the drought stress treatment. Pots

were placed according to a split-plot design, where each column of pots was a main plot of a

particular species and the sampling-days and the treatments (control vs. drought stress) were

randomised in the split-plots. Each pot corresponded to an independent sample. All pots were

watered in the evening and weighed on the following morning in order to ensure that all of them

had similar amounts of water (mean overall weight of 800 ± 50 g). Water deficit was then

imposed on the ‘stress’ pots by ceasing to provide water. The ‘control’ pots were watered once

per day.

Five-week old plants of P. dilatatum and C. dactylon and nine-week old plants of Z.

japonica were analysed. Control and non-watered plants of the three species were either assayed

or harvested on consecutive days, starting when the weight of non-watered pots had been

suitably decreased, in order to obtain leaf samples with different levels of dehydration, and

ending after a maximum of nine to twelve days without watering. The water weight in each pot

(WWP, g) was determined as the weight of the pot at each sampling time less 400 g (the mean

weight of the pots with plants and totally dried soil was 404 ± 16 g).

Chapter III.

92

The youngest fully expanded leaf of each P. dilatatum plant and two young fully

expanded leaves of each plant of C. dactylon or Z. japonica were always analysed. Samples were

collected in the growth environment four hours after the beginning of the photoperiod. In vivo

measurements were made during the first half of the photoperiod. It was assumed that, within

each pot, all the leaves used were identical in terms of developmental stage, physiological and

biochemical properties, and would have experienced the same drought condition.

From each pot, a sample formed by similar leaves to those being used for gas-exchanges

or amino acid analysis was collected to determine the leaf relative water content (RWC). The

fresh (FW), turgid (TW) and dry (DW) weights were measured and used to calculate RWC by

the equation RWC = 100((FW-DW)/(TW-DW)) (Catsky 1960). Leaf area was determined by

scanning the turgid leaves and analysing the image using the software Paint Shop Pro 9 (Jasc

Software, Inc., Minneapolis, MN, USA) and the software Image J 1.33u (National Institutes of

Health, Bethesda, Maryland, USA).

In order to facilitate the analysis of the physiological responses to CO2 and O2, groups of

plants with different drought stress conditions were chosen for each species independently,

considering both the RWC as well as the response in terms of the measured gas-exchanges.

Therefore, control (C) corresponds to all well-watered plants; moderately-stressed (MS) to non-

watered plants with RWC values between 96-80% in P. dilatatum, 95-80% in C. dactylon and

95-85% in Z. japonica; and severely-stressed (SS) to non-watered plants with RWC values lower

than 80 or 85%. Unfortunately, no severely-stressed samples of C. dactylon were obtained in

these experiments as the leaves of this species did not dehydrate as much as the other two grasses.

Gas-exchange measurements (CO2- and O2-response curves)

Gas-exchanges of carbon dioxide and water vapour by attached leaves were measured by infra-

red gas analysis using a six-chamber system designed and developed at Rothamsted (Lawlor et

al. 1989). Experiments were conducted at 25 ± 2ºC, 35-40% relative humidity and a PPFD of

850 ± 50 μmol m-2 s-1, provided by overhead lamps. The air composition was controlled by a

gas-mixer supplying CO2 and O2, the balance being made up with N2. Two different experiments

were undertaken. In the first, CO2-response curves were determined at a constant O2

concentration (21%) and seven CO2 concentrations (~30, 100, 250, 360, 500, 750 and 1000 μmol

mol-1). In the second experiment, O2-response curves were determined at a constant CO2

concentration (360 μmol mol-1) and six O2 concentrations (2, 5, 10, 15, 21 and 30%).

Measurements on each plant were always taken with time intervals long enough for steady-state


93

CO2 uptake to be attained at each CO2 or O2 level. Ninety pots were used per experiment,

making a total of 30 samples per species (10 control and 20 non-watered) assayed over five

consecutive days during the drought period (two control and four non-watered per species per

day). Each sample was formed by the middle part of young fully expanded leaves of two plants

from one pot, but, due to their different size, a different number of leaves was used for the three

species: two leaves for P. dilatatum, four leaves for C. dactylon and three leaves for Z. japonica.

After each series of measurements, the area of leaf inside the chamber was determined. The net

CO2 assimilation rate (A) and the stomatal conductance to water vapour (gswa) by the control

and drought-stressed plants of each species were plotted against intercellular CO2 concentration

(Ci) or against the O2 concentration in the gas-phase.

Carbon and oxygen isotope compositions

A similar experimental design to that used to provide material for gas-exchange measurements

was used here. Three leaves of P. dilatatum and ten leaves of C. dactylon or Z. japonica of the

five plants in each pot were used for each sample. After determination of FW and TW, the leaf

samples were dried at 80ºC for more than 48h and weighed to allow calculation of RWC. Three

control and six drought-stressed samples from each species were selected according to their

RWC and sent to Unidade de Análise Instrumental, Faculdade de Ciências da Universidade de

Lisboa, Portugal, for the analyses of the carbon and oxygen isotope compositions (δ13C and

δ18O) on the leaf dry matter. Sub-samples of ground leaf tissue were analysed, to give three

technical replicates of each sample, using stable isotope ratio mass spectrometers (SIRA II, VG

Isogas Limited, Manchester, UK, for carbon, and IsoPrimeTM, Micromass UK Limited,

Manchester, UK, for oxygen, with automatic sample preparation systems EuroEA, EuroVector

S.p.A., Milan, Italy). The Pee Dee Belemnite (PDB) and the Vienne-Standard Mean Oceanic

Water (VSMOW) were used as standards for δ13C and δ18O calculations, respectively. The

results were expressed as parts per thousand deviation from the standards with an analytical

precision of ± 0.12‰.

Amino acid analysis

Amino acids contained in leaves were determined by High-Performance Liquid Chromatography

(HPLC) of o-pthaldialdehyde (OPA) derivatives (Noctor & Foyer 1998). Three control and five

non-watered pots were used per species per day during three consecutive days, making a total of

Chapter III.

94

24 samples per species (nine control and fifteen non-watered pots). The five plants within each

pot were used for the collection of three samples: one light-sample, immediately frozen with

liquid N2 (LN2) in fully illuminated conditions; one dark-sample, identical to the light-sample

but submitted to a period of 30 seconds in darkness before freezing in LN2; and a third sample

for the RWC determination. Taking into account the different leaf sizes, each light- or dark-

sample of P. dilatatum consisted of one leaf from one plant whereas each sample of C. dactylon

or Z. japonica consisted of three leaves from two plants.

Reversed-phase HPLC was performed using a Waters Alliance 2695 Separation Module

and a 474 Scanning Fluorescence Detector operated by the Millenium32 software (Waters,

Milford, USA) with a Waters Symetry C18 4.6×150 mm column (Part No. WAT 054278)

protected by a 4×3 mm guard cartridge (Phenomenex, Torrance, USA). Since the fluorescent

adducts formed by reaction with OPA in the presence of 2-mercaptoethanol are unstable, the

autosampler was set to mix and pre-incubate 10 μL of each sample with 15 μL of OPA reagent

for 2 min before injecting the mixture onto the column. The eluent used for the amino acids

separation was obtained by mixing solvents containing different proportions of methanol,

sodium acetate pH 5.9 and tetrahydrofuran.

Amino acids were extracted from the frozen leaf samples stored at -80ºC. Each sample

was ground in LN2 and then 1.4 mL of 0.1 M HCl was added to the fine powder. The mixture

was ground further during thawing and the homogenate was centrifuged for 10 min at 16000 ×g

and 4ºC. Samples for HPLC were prepared by adding a sub-sample of each supernatant to the

internal standard and pure water, and these mixtures were stored at -20ºC. On the following day,

the mixtures were centrifuged for 40 min at 16000 ×g and 4ºC and then filtered with syringe

filters (0.2 μm) into HPLC autosampler vials. Standard solutions of α-amino-n-butyric acid

(internal standard), serine, glycine, glutamate, glutamine, aspartate, asparagine, and alanine were

prepared in 0.1 M HCl. A stock solution with all the standards was prepared and then diluted in

order to have increasing concentrations for the calibration curves (0, 5, 10, 15, 20 and 25 μM).


All the analyses were made using GenStat® 8.2, 2005 (Lawes Agricultural Trust, Rothamsted

Research, UK). Non-linear modelling was used to fit an asymptotic exponential model to the

variation of the leaf relative water content (RWC) with the amount of water in the soil. Using F-

tests, non-significantly different (P > 0.05) parameters between species were amalgamated in

order to have a parsimonious model of the data. The responses of the net CO2 assimilation rate


95

(A) and the stomatal conductance to water vapour (gswa) to the intercellular CO2 concentration

(Ci) and to the given concentration of O2 were modelled similarly. Firstly, non-linear curves

were fitted to the data from each individual plant. Statistically, the best models were: an

asymptotic exponential for the variation of A with Ci, a modified logistic for the variation of

gswa with Ci and an ‘exponential plus linear’ for the variation of A with O2. The latter consisted

of an exponential-associated increase followed by a linear decrease effective after maximal A

was attained. Residual Maximum Likelihood (REML) analysis was then used to predict mean

values of estimated parameters for each species by stress level combination that would occur if

the number of plants in each group (C, MS and SS) was the same (Patterson & Thompson 1971).

Such means were compared using a t-test on the appropriate degrees of freedom from the REML

model and the standard errors of the difference (SED).

Regression analysis was applied to model the variation of the isotope compositions and

amino acids content with RWC, including a squared term in this variable to check for non-

linearity. Non-significantly different (P > 0.05) parameters (t-tests) in the significant (P < 0.05)

model terms were amalgamated in order to attain parsimony. The residuals were checked and

found to generally conform to the assumptions of the analysis. All the absolute values and

percentages presented in the text were calculated from the regression models pertaining to each

data set. The difference between the values obtained for the content of each amino acid in the

samples collected in the light and after 30 seconds in darkness was calculated (these being paired

samples). Regression analysis revealed no significant effect of RWC on this difference for any of

the amino acids studied, and therefore the REML method was used to output predicted mean

values for the difference (dark minus light) for each species. Significance from zero was assessed

through t-tests.

Modelling C4 photosynthesis

The mechanistic model of C4 photosynthesis of von Caemmerer & Furbank (1999), described in

detail by von Caemmerer (2000) and based on the models of Berry & Farquhar (1978) and

Peisker (1979), was applied to the data from the CO2-response curves measured at high

irradiance. A similar approach to that described by Massad et al. (2007) was used on a plant-by-

plant basis. Firstly, an asymptotic exponential model was found to provide the best description of

the variation of net CO2 assimilation rate (A) with the intercellular CO2 concentration (Ci) for

each plant. The equations for enzyme-limited photosynthesis (von Caemmerer 2000) were then

applied to the individual plants to estimate the maximum Rubisco carboxylation activity (Vcmax)

Chapter III.

96

and the maximum PEPC carboxylation activity (Vpmax), as well as the CO2 concentrations in the

bundle sheath (Cs) and in the mesophyll cells (Cm), for values of Ci from 0 to 560 µmol mol-1

using a step-size of 5 µmol mol-1. Applying the method, all four parameters were primarily

estimated and then, for each plant, Vpmax and Vcmax were fixed at the mean of estimated values

found between 75-175 µmol mol-1 and 300-400 µmol mol-1 Ci, respectively, to re-estimate Cs

and Cm (over Ci), this time free from the instability of the numerical calculation of all four

parameters simultaneously. The model parameters assumed as constant at 25°C (von Caemmerer

2000) are listed in Table III.1 and the equations applied were:

(1) ( )miin

CCgA −=

(2) d

s

ocs

csn

11

RC

O

KOKC

VCA max −

⎟⎟

⎠

⎞

⎜⎜

⎝

⎛−

⎟⎟⎠

⎞⎜⎜⎝

⎛++

=∗γ

(3) ( )mmsbs

pm

pm

nRCCg

KC

VCA max −−−

+=

In these equations, An represents the net CO2 assimilation rate calculated from the asymptotic

exponential curve. Mean values of Vpmax and Vcmax and mean curves for Cs and Cm versus Ci were

then calculated for the treatment structure of species by stress level over the plants.

Table III.1. Summary of C4 photosynthesis parameters assumed as constant at 25°C (von Caemmerer 2000) and used in the equations for enzyme-limited photosynthesis.

Parameter Value Description

Kc 650 µbar Michaelis-Menten constant of Rubisco for CO2

Ko 450 mbar Michaelis-Menten constant of Rubisco for O2

Kp 80 µbar Michaelis-Menten constant of PEPC for CO2

O 210 mbar O2 partial pressure in the bundle sheath and mesophyll cells

Rd 0.01 Vcmax Leaf mitochondrial respiration

Rm 0.5 Rd Mesophyll mitochondrial respiration

gbs 3 mmol m-2 s-1 Bundle sheath conductance to CO2

gi 2 mol m-2 s-1 Mesophyll conductance to CO2

γ* 0.000193 Half the reciprocal of Rubisco specificity


97

In Figure III.6, the rate of PEPC carboxylation, Vp, was calculated as

(4) pm

pm

p KC

VCV max

+= ,

and the net CO2 assimilation rate (Ac) was calculated as the solution to the quadratic expression

for enzyme-limited photosynthesis (von Caemmerer 2000), taking the fraction of O2 evolution in

the bundle sheath as zero:

0c

2

c=++ cbAaA ,

this being

(5) a

acbbA2

42

c

−−−= ,

with

1=a ;

( ) ( ) ( )( ){ }ocbsdcmbsmp

1 KOKgRVCgRVbmax

++−++−−= ;

( )( ) ( )( )ocbsdbscmbsmpdc

1 KOKgROgVCgRVRVcmaxmax

++−+−−= ∗γ .

In Figure III.7, a rate of photorespiration (Pr) was predicted for each species by stress level

combination using the equation below, which is derived from the equation of overall CO2

assimilation that describes Rubisco carboxylation in the bundle sheath (von Caemmerer, 2000):

(6) doc

0.5 RVVA −−= .

Considering that Pr will be half the rate of Rubisco oxygenation, Vo,

(7) ( )dco

0.5 RVAVPr +−−== ,

where A is the overall CO2 assimilation and the rate of Rubisco carboxylation, Vc, is calculated

as

⎟⎟⎠

⎞⎜⎜⎝

⎛++

=

ocs

csc

1 KOKC

VCV max .

Chapter III.

98

RESULTS

Water relations

The variation of the relative water content (RWC) in the leaves of P. dilatatum, C. dactylon and

Z. japonica with the water weight in pot (WWP) was described by an asymptotic exponential

model (Figure III.1). In the gas-exchange experiment, RWC decreased to lower values in P.

dilatatum (40%) than in C. dactylon and Z. japonica (75%). In the amino acids experiment,

watering of the pots of Z. japonica and C. dactylon was stopped respectively one or two days

prior to those of P. dilatatum. As a result, lower RWC values were observed for the most

stressed leaves of Z. japonica (40%) than for C. dactylon (60%) and P. dilatatum (80%).

Water Weight in Pot (g)

0 100 200 300 400 500

RW

C (%

)

40

50

60

70

80

90

100

Water Weight in Pot (g)

0 100 200 300 400 500

RW

C (%

)

40

50

60

70

80

90

100

Gas-exchangeexperiment

A B

y = 97.5 (1 - 2.78 e-0.0256 x) [s.e. 0.4; 0.33; 0.0015]

y = 97.5 (1 - e-0.0200 x) [s.e. 0.4; 0.0006]

Amino acidsexperiment

y = 98.9 (1 - e-0.0266 x) [s.e. 0.76; 0.0008]

Figure III.1. Leaf relative water content (RWC) as a function of the amount of water in the soil, measured as the water weight in pot (WWP), of the control and non-watered plants of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (open triangles). Each data point corresponds to one sample, with 30 (A) or 24 (B) samples per species. The asymptotic exponentials fitted correspond to the best models statistically significant (A, R2 = 89.6%, s2 = 11.9, d.f. = 176; B, R2 = 92.3%, s2 = 19.3, d.f. = 70).


99

CO2- and O2-response curves

The net CO2 assimilation rate (A) by the leaves of P. dilatatum, C. dactylon and Z. japonica

increased with the intercellular CO2 concentration (Ci), both in control and drought stress

conditions, as shown in Figure III.2 (A-C). The mean value for the maximal net CO2 assimilation

rate (Amax) in fully hydrated leaves of P. dilatatum and C. dactylon plants was higher than in Z.

japonica plants. In moderately-stressed (MS) leaves of the two former species Amax decreased in

relation to the control, whereas no significant difference (P > 0.05) between MS and control

leaves was observed in Z. japonica. The mean values of Amax obtained for the severely

dehydrated (SS) leaves of P. dilatatum and Z. japonica were slightly less than half of the values

observed for each species under control conditions.

A (μ

mol

m-2

s-1

)

0

5

10

15

20

25

30

Ci (μmol mol-1)

0 100 200 300 400 500

gsw

a (m

ol m

-2 s

-1)

0.0

0.1

0.2

0.3

0.4

0.5

Ci (μmol mol-1)

0 100 200 300 400 500

Ci (μmol mol-1)

0 100 200 300 400 500

A B C

D E F


Figure III.2. Mean values of net CO2 assimilation rate (A; A-C) and stomatal conductance to water vapour (gswa; D-F) in response to the intercellular CO2 concentration (Ci) in the control (black diamonds), moderately-stressed (grey squares) and severely-stressed (open triangles) plants of P. dilatatum, C. dactylon and Z. japonica. The bars correspond to the standard errors of each mean value. Measurements were taken at ambient O2 (21%), a PPFD of 850 μmol m-2 s-1 and 25ºC.

Chapter III.

100

The stomatal conductance to water vapour (gswa) decreased with increasing Ci for both

fully hydrated and drought-stressed plants of the three species (Figure III.2D-F). The mean

values of gswa in fully hydrated leaves of C. dactylon were generally higher than in the other

two species, especially at low CO2 concentrations, and were decreased by drought stress in all

species. In MS leaves of C. dactylon and in SS leaves of P. dilatatum and Z. japonica the

maximal value of gswa was lower than in control or less dehydrated leaves but no significant

differences (P > 0.05) were observed between the control and MS plants of P. dilatatum and Z.

japonica.

Photosynthesis by P. dilatatum, C. dactylon and Z. japonica plants under control or

drought stress conditions was not dramatically affected by the O2 concentration (Figure III.3A-

C). However, lower values of A were generally observed at 2% compared to higher O2

concentrations and an ‘exponential plus linear’ model was successfully applied to the variation

of A with O2 (Figure III.3D-F), showing that after the maximal value of net photosynthesis (Amax)

was attained, at O2 concentrations generally between 4 and 10% (O2*), a slight decrease of A

with increasing O2 tends to occur. Note that the predictions of the O2* are poor because there

was no clear definition of the point corresponding to Amax. Apart from an increase of O2* to

values closer to the atmospheric concentration in the dehydrated leaves of P. dilatatum (MS and

SS), no other significant differences (P > 0.05) were observed.

Carbon and oxygen isotope compositions

The carbon isotope composition (δ13C) in fully hydrated leaves of P. dilatatum (-15.2‰) was

less negative than in C. dactylon (-16.3‰) and Z. japonica (-16.6‰). Drought stress had an

identical effect on δ13C for the three species, δ13C being decreased by -0.5‰ when RWC

decreased to 60% (Figure III.4A). No significant differences in the oxygen isotope composition

(δ18O) were found between C. dactylon and Z. japonica (P > 0.05). Higher values were generally

observed for these two species than for P. dilatatum (Figure III.4B), and an increase of ca. 4‰

with dehydration (to 60% RWC) was observed for all species.


101

O2 (%)

0 10 20 30

O2 (%)

0 10 20 30

A (μ

mol

m-2

s-1

)

0

5

10

15

20

25

30

35

O2 (%)

0 10 20 30

O2 (%)

0 10 20 30

A (μ

mol

m-2

s-1

)

0

5

10

15

20

25

30

35

O2 (%)

0 10 20 30

O2 (%)

0 10 20 30

A B C

D E F



CMSSS

-0.0207 0.0062-0.0111

7.618.417.8

α* O2*C

MSSS

-0.0663-0.0934

---

6.04.3---

α* O2*C

MSSS

-0.0580-0.0816-0.0073

5.75.19.9

α* O2*

Figure III.3. (A-C) Mean values of net CO2 assimilation rate (A) at different O2 concentrations in the control (C, black diamonds), moderately-stressed (MS, grey squares) and severely-stressed (SS, open triangles) plants of P. dilatatum, C. dactylon and Z. japonica. The bars correspond to the standard errors of each mean value. Measurements were taken at ambient CO2 (360 μmol mol-1), a PPFD of 850 μmol m-

2 s-1 and 25ºC. (D-F) Representation of the ‘exponential plus linear’ model fitted to the variation of A with O2 in the C (solid lines), MS (long-dashed lines) and SS (short-dashed lines) plants of each species. The lines correspond to the curves obtained by plotting the best model statistically significant. Also shown are the mean values estimated for the slope α*, representing the linear decrease of A with O2 after Amax was attained, and the O2 concentration corresponding to Amax (O2*). The ‘average’ standard error of differences (SED) considering all data was 0.0683 for α* and 2.5 for O2* (with 55 d.f.).

Chapter III.

102

RWC (%)

60 70 80 90 100

δ 13

C (‰

)

-18

-17

-16

-15

RWC (%)

60 70 80 90 100

δ 18

O (‰

)

25

30

35

40

45A B

y = - 16.8 + 0.0157 x [s.e. 0.2; 0.0027]

y = - 17.7 + 0.0157 x [s.e. 0.2; 0.0027]y = - 18.1 + 0.0157 x [s.e. 0.2; 0.0027]

y = 42.7 - 0.1030 x [s.e. 2.1; 0.0242]

y = 47.0 - 0.1030 x [s.e. 2.1; 0.0242]

Figure III.4. Carbon (δ13C; A) and oxygen (δ18O; B) isotope compositions as a function of the relative water content (RWC) in the leaves of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (open triangles). Each data point corresponds to one sample, with 9 samples per species. The regression lines correspond to the best models statistically significant (P. dilatatum, solid lines; C. dactylon, dashed lines; and Z. japonica, dotted lines; A, R2 = 90.1%, s2 = 0.40, d.f. = 23; B, R2 = 65.3%, s2 = 3.23, d.f. = 24).

Amino acids analysis

The variation of the amino acids content in illuminated leaves of the three C4 grasses with leaf

dehydration is shown in Figure III.5. The fully hydrated leaves of P. dilatatum had generally

more serine (Ser) than the other two species. This amino acid increased steeply in all three

species when RWC decreased from 98 to 60% (C. dactylon and Z. japonica) or only down to

80% (P. dilatatum). Below 60% RWC a slight decrease was observed for the leaves of Z.

japonica. Glycine (Gly) had a quadratic variation with RWC. In C. dactylon there was only a

slight increase when RWC started to decrease and then glycine was kept nearly constant. Z.

japonica had less glycine than the other two species in fully hydrated conditions and the amount

increased as the RWC decreased from 98 to 60%, but was not so strongly affected by further

decreases in RWC. In P. dilatatum, although high variability was observed, the regression

showed increased glycine when RWC decreased from 99 to 80%. The ratio Gly/Ser was only

decreased with RWC in C. dactylon. Glutamate (Glu) decreased with decreasing RWC only in C.

dactylon and glutamine (Gln) was not significantly affected by leaf dehydration (P > 0.05), the

same being observed for the ratio Gln/Glu. Aspartate (Asp) decreased linearly with RWC in P.

dilatatum and Z. japonica, but no significant variation with RWC was observed for C. dactylon

(P > 0.05). Alanine (Ala) was not significantly affected by leaf dehydration (P > 0.05) in the


103

three species, showing high variability, especially in C. dactylon. Conversely, asparagine (Asn)

increased linearly with decreasing RWC, but only in C. dactylon and Z. japonica.

RWC (%)

40 60 80 1000.00

0.40

0.80

1.20

1.60

RWC (%)

40 60 80 1000.00

0.20

0.40

0.60

0.80

40 60 80 100

Amin

o Ac

ids

Con

tent

(mm

ol m

-2)

0.00

0.20

0.40

0.60

0.80

1.00

40 60 80 1000.00

0.20

0.40

0.60

0.80

40 60 80 1000.00

0.10

0.20

0.30

0.40

40 60 80 1000.00

0.05

0.10

0.15

0.20

40 60 80 1000.00

0.30

0.60

0.90

1.20

1.50

Ser Gly/SerGly

40 60 80 1000.00

0.10

0.20

0.30

0.40

0.50Glu Gln/GluGln

RWC (%)

40 60 80 1000.00

0.20

0.40

0.60

0.80Asp AsnAla

A B C

D E F

G H I

y = 0.0139 x - 0.000118 x2

[s.e. 0.0008; 0.000008]y = - 0.1187 + 0.0139 x - 0.000118 x2

[s.e. 0.015; 0.0008; 0.000008]

y = 0.256 - 0.000019 x2 [s.e. 0.021; 0.000002]y = 0.0024 x - 0.000019 x2 [s.e. 0.0002; 0.000002]

y = 0.0021 x - 0.000019 x2 [s.e. 0.0002; 0.000002]

y = 0.0044 x [s.e. 0.0003]

y = 0.0082 x [s.e. 0.0001]

y = - 0.560 + 0.0084 x [s.e. 0.354; 0.0037]

y = - 0.039 + 0.0046 x [s.e. 0.068; 0.0009]y = 1.876 - 0.0136 x [s.e. 0.170; 0.0019]y = 0.526 - 0.0046 x [s.e. 0.077; 0.0010]

Figure III.5. Variation of the amino acids content with the relative water content (RWC) in the leaves of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (open triangles) collected in the light: serine (Ser), glycine (Gly), Gly/Ser ratio, glutamate (Glu), glutamine (Gln), Gln/Glu ratio, aspartate (Asp), alanine (Ala), asparagine (Asn) (A-I, respectively). Each data point corresponds to one sample, with 24 samples per species. The regression lines and curves, when applied, correspond to the best models statistically significant (P. dilatatum, solid lines; C. dactylon, dashed lines; and Z. japonica, dotted lines; A, R2 = 59.8%, s2 = 0.297, d.f. = 69; B, R2 = 56.1%, s2 = 0.035, d.f. = 67; C, R2 = 40.5%, s2 = 0.012, d.f. = 68; D, R2 = 88.5%, s2 = 0.406, d.f. = 69; E, P > 0.05; F, P > 0.05; G., R2 = 56.1%, s2 = 0.035, d.f. = 67; H, P > 0.05; I, R2 = 56.1%, s2 = 0.035, d.f. = 67).

Chapter III.

104

The content of some amino acids in the leaves collected after a period of 30 seconds in

darkness changed significantly compared to corresponding leaves collected in the light (Table

III.2), but the difference ‘dark minus light’ was not affected by leaf dehydration (P > 0.05). In

the three species a decrease in glycine content after 30 seconds in darkness was observed. The

ratio between glycine and serine also decreased, mostly due to the decreased glycine, as no

significant differences were observed for serine. Both glycine and the ratio Gly/Ser showed a

larger decrease in C. dactylon than in P. dilatatum or Z. japonica. Glutamate decreased in the

dark- compared to the light-samples, but only for P. dilatatum and C. dactylon. Conversely,

glutamine content was not significantly changed (P > 0.05), and the ratio Gln/Glu increased only

in P. dilatatum, with no significant changes in C. dactylon or Z. japonica (P > 0.05). Aspartate

decreased after 30 seconds in darkness in C. dactylon and Z. japonica. Concomitantly, in these

two species a substantial increase in alanine was observed. The content in asparagine was not

significantly changed in 30 seconds of darkness (P > 0.05).

Table III.2. Estimated mean values of the difference between the amino acids content (mmol m-2) in the leaves of P. dilatatum, C. dactylon and Z. japonica collected in the light and after 30 seconds in darkness (‘dark minus light’) and respective standard errors of differences (in brackets). There was no significant variation with RWC (P > 0.05) and, therefore, the overall mean values obtained for the samples of each species (considering all control and non-watered plants, in a total of 24 samples per species) were analysed by the REML method. NS., not significantly different from zero (P > 0.05).

Species Ser Gly Gly/Ser Glu Gln Gln/Glu Asp .Ala Asn

P. dilatatum NS. -0.007 (0.004)

-0.025 (0.019)

-0.029 (0.020) NS. 0.125

(0.056) NS. NS. NS.

C. dactylon NS. -0.019 (0.004)

-0.145 (0.019)

-0.059 (0.020) NS. NS. -0.055

(0.019) 0.115

(0.023) NS.

Z. japonica NS. -0.007 (0.004)

-0.043 (0.019) NS. NS. NS. -0.031

(0.019) 0.085

(0.023) NS.

Mechanistic modelling of C4 photosynthesis

The variation of the enzyme-limited net CO2 assimilation rate (Ac) with the CO2 concentration in

the mesophyll cells (Cm) for each of the three C4 grasses was affected by drought stress as shown

in Figure III.6. The mean values estimated for the maximum PEPC carboxylation activity (Vpmax)

and maximum Rubisco carboxylation activity (Vcmax) were higher in C. dactylon than in P.


105

dilatatum and lower in Z. japonica and were significantly affected (P < 0.01) by the drought

stress level.

Cm (μmol mol-1)

0 100 200 300 400 500

Cm (μmol mol-1)

0 100 200 300 400 500

A c, V

p, V c

max

(μm

ol m

-2 s

-1)

0

5

10

15

20

25

30

35

Cm (μmol mol-1)

0 100 200 300 400 500

A C


B

CMSSS

33.629.916.6

Vcmax Vpmax64.357.227.7

CMSSS

34.330.5---

Vcmax Vpmax88.478.3---

CMSSS

30.826.417.5

Vcmax Vpmax51.848.432.6

Figure III.6. Enzyme-limited net CO2 assimilation rate (Ac) as a function of the mesophyll CO2 concentration (Cm) in the control (C, black lines), moderately-stressed (MS, dark-grey lines) and severely-stressed (SS, light-grey lines) plants of P. dilatatum, C. dactylon and Z. japonica. Also shown are the rates of PEP carboxylation (Vp, dashed lines) and the maximum Rubisco carboxylation activity (Vcmax, dotted lines). The mean values of Vcmax and maximum PEPC carboxylation activity (Vpmax) are presented below each graph. All parameters were calculated by applying a mechanistic model of C4 photosynthesis (von Caemmerer & Furbank, 1999). For simplification, assumption was made that the fraction of O2 evolution in the bundle sheath is null.

By application of the model equations, the concentration of CO2 in the bundle sheath (Cs)

is predicted to increase steeply with the increase in Ci, reaching values above 1000 μmol mol-1 at

low Ci (Figure III.7A-C). Accordingly, the rate of photorespiration (Pr) for each of the three C4

grasses at the different stress levels was estimated to be always lower than 1.5 μmol m-2 s-1 and

decreased with increasing Ci (Figure III.7D-F). The values predicted for Pr at ambient levels of

CO2 (360-390 μmol mol-1) were slightly higher for the MS plants of each species than in the

corresponding controls whereas slightly lower values were obtained for the SS relative to the MS

plants of P. dilatatum and Z. japonica.

Chapter III.

106

C

s (μ m

ol m

ol-1

)

0

2000

4000

6000

8000

10000

12000

14000 A C


B

Ci (μmol mol-1)

0 100 200 300 400 500

Ci (μmol mol-1)

0 100 200 300 400 500

Phot

ores

pira

tion

(μm

ol m

-2 s

-1)

0.00

0.25

0.50

0.75

1.00

1.25

1.50 D F

Ci (μmol mol-1)

0 100 200 300 400 500

E

CMSSS

175 85 ---

Ci Cs Ac Pr10250 5250

---

0.120.20---

3124---

CMSSS

155100110

Ci Cs Ac Pr450022502000

0.230.330.23

231912

CMSSS

145 80 85

Ci Cs Ac Pr430019501240

0.260.420.31

27209.5

Ca = 360 μmol mol-1

Figure III.7. Representation of the predicted CO2 concentration in the bundle sheath cells (Cs) and rate of photorespiration (Pr) as a function of the intercellular CO2 concentration (Ci) in the control (C, black lines), moderately-stressed (MS, dark-grey lines) and severely-stressed (SS, light-grey lines) plants of P. dilatatum, C. dactylon and Z. japonica. For the estimation of Cs and Pr, using the equations for enzyme-limited C4 photosynthesis (von Caemmerer & Furbank, 1999), Vcmax and Vpmax were fixed at the mean values obtained for each group of plants (see Figure III.6.) and other model parameters, namely the O2 concentration in the bundle sheath, were assumed to be constant at 25ºC. Also shown are the mean values of Ci, Cs, Ac and Pr, estimated through the modelling approach, at ambient concentrations of CO2 (360-390 μmol mol-1) and O2 (21%).


107

DISCUSSION

Drought stress condition and photosynthetic responses to CO2

The relative water content (RWC) in the leaves of P. dilatatum, C. dactylon and Z. japonica

decreased with the decrease in water available in the soil (Figure III.1) and was used as a

reference to analyse the effects of drought. The three species showed some differences in the

extent of leaf dehydration. In the gas-exchange experiment, P. dilatatum leaves were more

severely dehydrated than the other two species. In the amino acids experiment, the plants of C.

dactylon and Z. japonica were deprived of water before P. dilatatum in an attempt to get a

similar range of RWC values in the samples of the three species. However, C. dactylon and

(more dramatically) Z. japonica plants were more dehydrated in this experiment. The difficulty

in obtaining similar dehydration levels for the three species was a result of the faster loss of

water by the leaves of P. dilatatum than by C. dactylon and Z. japonica in response to the drying

soil. When water deficit was rapidly imposed in these C4 grasses by the addition of polyethylene

glycol to the nutrient solution (Carmo-Silva et al. 2007), C. dactylon showed a faster decrease in

RWC than P. dilatatum and Z. japonica. The differences are likely to result from the different

methods used for drought stress induction (Chaves et al. 2003).

Stomatal closure and decreased photosynthesis are generally accepted as early

consequences of leaf dehydration (e.g. Chaves et al. 2003) and this was observed among several

C4 grasses (Ghannoum et al. 2003; Marques da Silva & Arrabaça 2004; Carmo-Silva et al. 2007).

The net CO2 assimilation rate (A) decreased in the moderately-stressed (MS) leaves of P.

dilatatum and C. dactylon but not in Z. japonica (Figure III.2) compared to the controls,

suggesting that photosynthesis by the latter species might be more resistant to moderate drought

conditions. In severely-stressed (SS) leaves of P. dilatatum and Z. japonica, A decreased to about

half of the values observed in control conditions. Stomatal closure was also observed in response

to drought stress, as shown by the decrease in the stomatal conductance to water vapour (gswa)

in the MS and SS plants compared to the controls (Figure III.2). The maximal values of gswa in

the MS leaves of P. dilatatum and Z. japonica were not affected compared with the control,

which demonstrates that the effect of the low CO2 concentrations promoting the opening of

stomata was stronger than the drought-induced stomatal closure at that stage. However, for

higher CO2 concentrations, gswa was always lower in dehydrated leaves compared with the

control plants of each species, resulting in generally lower values of Ci for a given CO2

concentration. The decrease of Amax in the most dehydrated leaves of each grass species

Chapter III.

108

compared to their controls suggests that metabolic limitations of photosynthesis are also present.

The same conclusion has been reached for other C4 grasses (Ghannoum et al. 2003). Net CO2

assimilation consistently increased with increasing Ci and the CO2 concentration corresponding

to zero A (the CO2 compensation point) was not significantly different from zero (P > 0.05) for

either control or dehydrated leaves of any of the three species, suggesting little photorespiration.

O2-sensitivity of C4 photosynthesis

Net CO2 assimilation rates (A) at ambient CO2 (360 μmol mol-1) were little affected by the O2

concentration in the gas-phase but A was lowest at 2% O2 and decreased significantly with O2

after the maximal value (Amax) was attained (Figure III.3). Little sensitivity of C4 photosynthesis

to O2 was found in two other grasses, Panicum antidotale (NADP-ME) and Panicum coloratum

(NAD-ME) at low Ci (Ghannoum et al. 1998). The estimation of the O2 concentration

corresponding to Amax in the three C4 grasses of the different metabolic subtypes (O2*, Figure

III.3) agree with previously reported results (Maroco et al. 1997). The inhibition of A at low O2

concentrations is probably due to an extra energy requirement for the regeneration of PEP and

proper functioning of the C4 cycle (Ku et al. 1991; Dai et al. 1993; Maroco et al. 2000). A

reduced ATP production, by the O2-dependent photochemical reactions or even by mitochondrial

respiration (Maroco et al. 1997), might lead to a deficient function of the C4 pathway. The slight

decrease of A at O2 concentrations higher than 10% is likely to be due to the competing

oxygenation of RuBP that initiates the photorespiratory carbon oxidative cycle.

The rate of decreasing A with increasing O2 (Fig III.3D-F), partly due to photorespiration,

suggests that rates in ambient air would be in the range 0-2 μmol m-2 s-1 (representing 0-6% of

Amax) and slightly faster in C. dactylon than in the other two C4 grasses, but the variability is

large and there is no clear trend for its variation with leaf dehydration. Since A was affected by

drought (Figures III.2 and III.3), there is a tendency for increased photorespiration in proportion

to photosynthesis when leaf dehydration increases. Photorespiration in Amaranthus edulis (C4

dicotyledon), estimated by the release of NH3, represented 6% of the rate of CO2 assimilation

(Lacuesta et al. 1997). In two C4 grasses, a sharp increase of the CO2 compensation point when

the RWC fell below 60% compared with very low values in fully hydrated leaves, led to the

conclusion that photorespiration was greatly enhanced under drought conditions (Ghannoum et

al. 2003). The results presented here show that photorespiration by the three C4 grasses studied is

slow and not sufficient to explain the decrease observed in A under the drought conditions

attained (Figure III.1).


109

Effects of dehydration on carbon and oxygen isotope compositions

Drought stress affected leaf carbon and oxygen isotope compositions (δ13C and δ18O) similarly

in the three C4 grasses (Figure III.4), with a decrease of 0.5‰ in δ13C and an increase of 4‰ in

δ18O when the RWC decreased down to 60%. In cotton leaves (C3 plant), stomatal closure after

ABA treatment resulted in 13C and 18O enrichment (Barbour & Farquhar 2000). Conversely, a

study with several C4 grasses revealed a decrease of δ13C under drought but no consistent

variation in δ18O (Ghannoum et al. 2002). The less negative values of δ13C in P. dilatatum

(NADP-ME) than in C. dactylon (NAD-ME) and Z. japonica (PEPCK) agree with previously

reported differences among C4 grasses from the different subtypes (Hattersley 1982). On the

other hand, differences in the leaf length and interveinal distance among grasses (Helliker &

Ehleringer 2000) might explain the higher values of δ18O in C. dactylon and Z. japonica than in

P. dilatatum.

Variations in δ13C are mostly due to changes in the ratio of intercellular to atmospheric

CO2 concentrations (Ci/Ca) and/or changes in the fraction of CO2 fixed by PEPC that

subsequently leaks out from the BS without being assimilated by Rubisco (leakiness, φ)

(Farquhar 1983). Variation in φ can result either from alterations in the physical conductance of

BS cells to CO2 or alterations in the balance between PEPC and Rubisco activities (Peisker &

Henderson 1992). In Sorghum bicolor (Williams et al. 2001) and in sugarcane (Saliendra et al.

1996), both carbon isotope discrimination and φ increased under drought conditions, suggesting

that the coordination between the C4 and C3 cycles was affected. Conversely, Buchmann and co-

workers (1996) related the decrease of δ13C in several C4 grasses (including P. dilatatum, C.

dactylon and Z. japonica) under drought with decreased stomatal conductance, which affected

the intercellular CO2 concentration. Decreased gswa observed in the dehydrated leaves of each

species (Figure III.2) resulted in decreased Ci/Ca compared to the control plants of each species

(data not shown) and could contribute to the decrease in δ13C (Figure III.4A). However, the data

obtained do not exclude the possibility that impairment of photosynthetic metabolism increased

φ and contributed to the decrease in δ13C with drought in the three C4 grasses.

The increase of leaf δ18O with RWC (Figure III.4B) reflects the variation of δ18O in the

soil water, which becomes enriched in the heavy isotope under drought conditions due to

evaporation, and variation due to evaporative and diffusional effects during transpiration

(Barbour 2007). Decreased gswa in the dehydrated leaves (Figure III.2) probably contributed to

the leaf 18O enrichment because H218O diffuses more slowly and has lower vapour pressure than

H216O, causing the water in the leaf to become enriched in 18O during transpiration. Variations in

Chapter III.

110

evaporation during plant growth are integrated in the oxygen isotope composition of leaf

material (Barbour & Farquhar 2000). Although fractionation of oxygen isotopes occurs during

exchanges of CO2 and O2 with the atmosphere resulting from the sum of photosynthesis,

photorespiration and mitochondrial respiration, these effects are quickly buffered in the leaf due

to rapid isotopic exchange between the carbonyl oxygen in organic molecules and leaf water

(Barbour 2007).

Although some suggestions have been made that photorespiration should be considered

when interpreting δ13C (Gillon & Griffiths 1997) and δ18O (Farquhar et al. 1998) of leaf material,

the rate of photorespiration present in C4 plants is not likely to be of reasonable size to make

considerable contribution for the variations in isotope ratios of leaf dry matter. In the present

study, drought did not cause changes in δ13C in the same direction as observed for C3 species

(Cerling 1999), where photorespiration is rapid.

Effects of dehydration on steady-state contents of amino acids in illuminated leaves

The increased amounts of glycine and serine in the dehydrated leaves of all three species,

especially with decreases in RWC down to 80 and 60% (Figure III.5A,B), might be interpreted

as an increase in photorespiratory metabolism and flux of glyoxylate into the pathway because of

increased oxygenation of RuBP. Alternatively, these changes may reflect an increase in pool

sizes of glycine and serine because of slower transfer to the mitochondria and peroxisome.

Increased glycine and serine contents were also observed in droughted maize leaves (Foyer et al.

1998).

In leaves of C. dactylon, when RWC decreased from 98 to 60%, asparagine increased

from 0.5 to 1.1 mmol m-2 (Figure III.5I) and glutamate decreased from 0.8 to 0.5 mmol m-2

(Figure III.5D). An increase of 2- to 6-fold in the amount of asparagine was previously reported

in C. dactylon plants submitted to drought stress (Barnett & Naylor 1966) and the same authors

reported a concomitant decrease in glutamate and alanine. In Z. japonica the content of

asparagine increased by 4-fold and aspartate decreased when RWC decreased to 40% (Figure

III.5I,G) but glutamate amounts were not affected. No significant effect of RWC on the alanine

content was observed for any of the three C4 grasses (Figure III.5H). The contents in glutamate,

asparagine and alanine were generally higher in C. dactylon than in P. dilatatum and Z. japonica

whereas the content in glutamine was lower in the first species.


111

Amino acid changes in 30 seconds of darkness

In illuminated leaves, carboxylation and oxygenation of RuBP will produce P-glycerate and P-

glycolate. The later enters the photorespiratory carbon oxidative cycle and, as a result, glycine

will be converted into serine in the mitochondria. In darkness the regeneration of RuBP and the

production of both P-glycerate and P-glycolate will stop. The glycine pool will then decrease

because the amino acid is no longer being formed, but it is still converted into serine. Therefore,

the decrease in the amount of glycine in the leaves after 30 seconds in darkness compared to the

amounts in fully illuminated leaves during steady-state photosynthesis will be related to the rate

of photorespiratory production of P-glycolate through the oxygenation of RuBP. The pools of

glycine and serine in the leaves are thought to reflect the photorespiratory fixation of O2 quite

accurately, given that the synthesis of these two amino acids occurs mostly through this process

(Jolivet-Tournier & Gerster 1984). Earlier studies revealed that in C3 plants, steady-state

photosynthesis followed by one or two minutes in darkness results in considerable decrease in

glycine and increase in serine (Roberts et al. 1970, Kumarasinghe et al., 1977). The rate of

incorporation of 18O into glycolate and glycine in maize (C4 monocotyledon, NADP-ME) leaves

increased with increasing O2 concentrations, providing direct evidence for photorespiratory O2

uptake, albeit at a much lower rate than in wheat (de Veau & Burris 1989). In Amaranthus edulis

(C4 dicotyledon, NAD-ME), the decrease of both glycine and serine contents at very low O2 was

consistent with photorespiratory production of both amino acids at atmospheric O2 levels

(Maroco et al. 2000). In the C4 grasses P. dilatatum, C. dactylon and Z. japonica there was no

effect of decreased RWC (P > 0.05) on the difference between the content of amino acids in the

light- and the dark-samples and therefore the mean changes after 30 seconds in darkness for each

species, presented in Table III.2, were estimated considering all the data for the control and non-

watered plants together. A decrease in the glycine content was observed after 30 seconds in

darkness, but serine content was not affected in any of the three species. The changes in glycine

suggest that photorespiration was faster in C. dactylon (0.32 ± 0.07 μmol m-2 s-1) than in P.

dilatatum and Z. japonica (0.12 ± 0.07 μmol m-2 s-1). Maroco et al. (2000) proposed that changes

in the glycine pool would be a better indicator of the occurrence of photorespiration in maize

(C4) than changes in the serine pool. On the other hand, Novitskaya et al. (2002) demonstrated

that the ratio between the two amino acids is strongly correlated with the photorespiration in C3

plants. The decrease in Gly/Ser ratio in the dark was notably higher in C. dactylon than in P.

dilatatum and Z. japonica (Table III.2), suggesting that photorespiration in the NAD-ME grass

might be faster than in the other two species, although still much slower than the values reported

Chapter III.

112

for C3 species (Keys 1986; Novitskaya et al. 2002). Photorespiratory CO2 evolution rates at

atmospheric O2 (21%) and CO2 (350 μmol mol-1) for wheat (C3) and maize (C4) were estimated

to be 27 and 2%, respectively, of net photosynthetic CO2 assimilation (de Veau & Burris 1989).

More extensive studies on isotopic oxygen uptake in the light found slightly faster

photorespiration rates in NAD-ME compared to NADP-ME monocotyledon species (Furbank &

Badger 1982; Siebke et al. 2003).

Glutamine synthetase is responsible for the re-assimilation of NH3 produced in the

photorespiratory carbon oxidation cycle (Keys et al. 1978) and the glutamate produced by the

GOGAT complex during the photorespiratory nitrogen cycle acts as a donor of amino groups

needed for the photorespiratory carbon oxidation cycle (Keys 1999). The GOGAT reaction

would be expected to stop quickly in the dark, justifying the decrease in glutamate content in the

darkened leaves of P. dilatatum and C. dactylon, which was not evident in Z. japonica (Table

III.2). As suggested by Novitskaya et al. (2002), the trends in glutamate and glutamine might

well reflect processes linked to photosynthesis other than photorespiration, including primary N

assimilation, and can not be interpreted as a result of altered photorespiratory NH3 flux.

The decrease in aspartate, the primary C4 acid formed in the M cells of NAD-ME and

PEPCK species, in the darkened leaves of C. dactylon and Z. japonica (Table III.2) is a clear

consequence of stopping the primary fixation of CO2 by PEPC. Accordingly, the increase in

alanine in the same two species is consistent with continuing amination of pyruvate in the C4

cycle. In wheat and potato (C3 plants) decreased contents in both aspartate and alanine were

found under photorespiratory conditions (Novitskaya et al. 2002). However, in durum wheat a

period of 30 seconds in darkness induced no changes in aspartate or alanine although a clear

decrease in glycine and serine was observed (data not shown). Moreover, the post-illumination

changes in aspartate and alanine were restricted to C. dactylon and Z. japonica, with no changes

being observed in P. dilatatum (NADP-ME), suggesting their association with the C4

photosynthetic pathway rather than the photorespiratory metabolism.

Modelling the CO2-response of C4 photosynthesis under drought conditions

A mechanistic model of C4 photosynthesis (von Caemmerer & Furbank 1999) was applied to the

photosynthetic response of the three C4 species to the intercellular CO2 concentration at high

irradiance shown in Figure III.2. This approach allowed the simulation of the effect of moderate

and severe leaf dehydration on the rates of photosynthesis and photorespiration by the three

grasses. At high irradiance, photosynthesis is assumed to be enzyme-limited and mostly


113

determined by the rates of PEP and RuBP carboxylation by PEPC and Rubisco, respectively, and

by the regeneration of PEP (von Caemmerer 2000). The variation of the enzyme-limited net CO2

assimilation rate (Ac) with the CO2 concentration in the mesophyll cells (Cm) is represented in

Figure III.6. The values predicted seem to agree fairly well with the experimental results (Figure

III.2). However, there might be some overestimation of the modelled values of Ac for C. dactylon

and Z. japonica. For simplification, it was assumed that the fraction of O2 evolution in the BS is

null for all three species but this is not likely to be correct for the NAD-ME or PEPCK species,

in which PSII activity will probably increase the O2 concentration in the BS, decreasing the

actual value of photosynthesis. The mean values estimated for the maximum PEPC

carboxylation activity (Vpmax) and maximum Rubisco carboxylation activity (Vcmax) were higher

in C. dactylon than in P. dilatatum and lower in Z. japonica and decreased with leaf dehydration

(Figure III.6). These results suggest that both enzymes responsible for the carboxylation of PEP

and RuBP are down-regulated under drought conditions. The negative effect of drought on the

photosynthesis response to CO2 is more notable in the SS plants of P. dilatatum and Z. japonica,

but a clear effect of leaf dehydration on the two enzymes and on Ac was simulated for the MS

plants of each of the three species.

The application of the model equations to the experimental data from the CO2-response

curves allowed the simulation of a rate of photorespiration (Pr) for each of the three C4 grasses at

the different stress levels (Figure III.7). Even at the lowest Ci values, the predicted Pr was never

higher than 1.5 μmol m-2 s-1 and at ambient levels of CO2 (360-390 μmol mol-1) Pr was

estimated to be less than 3.5% of Ac for all species and stress levels. The modelled data suggest

that photorespiration is present at higher rates in the MS relative to the well-watered plants of

each species, especially in Z. japonica. The lower values of Pr estimated for the SS compared to

the MS plants of P. dilatatum and Z. japonica indicate that metabolic inhibition of

photosynthesis occurs at the level of Rubisco, affecting both the rates of photosynthesis and

photorespiration. Pr was estimated to be lower in C. dactylon than in the other two species.

However, higher values could have been predicted for either C. dactylon (NAD-ME) or Z.

japonica (PEPCK) if a more realistic O2 concentration in the bundle sheath, likely to be higher

than in P. dilatatum (NADP-ME), had been considered.

Stomatal closure causes decreased Ci under drought conditions (Figure III.2).

Concomitantly, lower values of Cs for a given CO2 concentration are estimated for the

dehydrated leaves compared with the controls (Figure III.7). The slower response of increasing

Cs with increasing Ci under drought stress suggest that metabolic impairment might be present,

affecting the effectiveness of the C4 cycle. As a result, Ac is also decreased and Pr increases, i.e.

Chapter III.

114

the predicted rates of photorespiration increase as a proportion to the modelled net

photosynthesis when leaf dehydration increases for all three species. Many assumptions were

made to allow the simulation of plant photosynthetic and photorespiratory responses by the

application of a mechanistic model and, consequently, much uncertainty is involved. It was

assumed that stomata apertures decreased uniformly and that gm and gbs were not changed with

stress. Furthermore, it was accepted that the estimated values of Vcmax and Vpmax changed with

stress. More accurate predictions will be possible when more measurements of the assumed

parameters are available as, for example, of the kinetic constants of Rubisco from each of the C4

species.

CONCLUSIONS

Drought stress induced decreased leaf water contents, stomatal closure and decreased net CO2

assimilation in the leaves of P. dilatatum, C. dactylon and Z. japonica. The response of net

photosynthesis to CO2 was typical of C4 leaves and extrapolation to zero A gave no evidence of a

significant CO2 compensation point even in droughted plants. Net CO2 assimilation was

decreased only slightly with increasing O2 concentrations above 10% in the atmosphere around

the leaves but this trend was not increased by drought stress. Leaves of both control and

droughted plants showed δ13C and δ18O typical of C4 plants and changes with RWC were not

indicative of changes in photorespiration. Increased contents of glycine and serine in the

dehydrated leaves of the three species provided the only suggestion for increased

photorespiratory metabolism by water deficit. Changes in amino acids content after 30 seconds

in darkness, especially the decrease in glycine, were consistent with slow photorespiration rates

in all three species, but slightly faster in C. dactylon (NAD-ME) than in P. dilatatum (NADP-

ME) and Z. japonica (PEPCK), and not changed by drought stress in either species. Mechanistic

modelling of CO2-response curves as well as the O2-sensitivity of C4 photosynthesis by the three

grasses suggested slightly increased photorespiration rates in proportion to photosynthesis.

However, the overall results presented here suggest that the C4 grasses of the three different

metabolic subtypes are able to maintain levels of CO2 at the Rubisco site sufficiently high to

limit oxygenase activity under drought stress.


115

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Chapter IV.

C4 ENZYMES IN DROUGHT-STRESSED GRASSES

Chapter IV.

122

An integral copy of this chapter has been submitted for publication in Photosynthesis Research:

Carmo-Silva A. E., Bernardes da Silva A., Keys A. J., Parry M. A. J. & Arrabaça M. C. (Submitted) The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes. Photosynthesis Research.

Anabela Bernardes da Silva (Centro de Engenharia Biológica, Faculdade de Ciências da Universidade de Lisboa) was involved in the planning of the experiment and interpretation of the results obtained.

C4 Enzymes in Drought-Stressed Grasses

123

THE ACTIVITIES OF PEP CARBOXYLASE AND THE C4 ACID DECARBOXYLASES ARE LITTLE CHANGED BY DROUGHT STRESS IN THREE C4 GRASSES OF DIFFERENT SUBTYPES

ABSTRACT

The efficiency of C4 photosynthesis depends on good coordination between the assimilation of

CO2 by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C4

acids, which might be affected under water deficit. The effects of gradually-induced drought

stress on the activities of PEPC and the decarboxylating enzymes in Paspalum dilatatum

(NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and

Zoysia japonica (PEP carboxykinase, PEPCK) were slight. Moderate leaf dehydration caused

increased physiological activity of PEPC in all three species, decreased activities of NADP-ME

in P. dilatatum and of NAD-ME and PEPCK in C. dactylon, but had no effect on the

decarboxylating enzymes in Z. japonica. Decreased inhibition of PEPC activity by L-malate

under drought suggested an increased PEPC phosphorylation state in all species. Considerable

PEPCK activity in all three species suggests its possible role as a supplementary decarboxylase

in P. dilatatum and C. dactylon or additional involvement in non-photosynthetic metabolism.

KEYWORDS:

NADP-ME, NAD-ME, PEPCK, P. dilatatum, C. dactylon, Z. japonica

Chapter IV.

124

INTRODUCTION

C4 photosynthesis is characterised by the presence of a CO2-concentrating mechanism, which

involves the initial fixation of CO2 by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in

the mesophyll (M) cells followed by the decarboxylation of the resulting C4 acids in the bundle

sheath (BS) cells, where the CO2 released is assimilated by ribulose-1,5-bisphosphate

carboxylase/oxygenase (Rubisco, EC 4.1.1.39). The increased CO2 concentration in the BS cells

results in low RuBP oxygenation and, consequently, low rates of photorespiration and increased

rates of photosynthesis by C4 plants (Kanai & Edwards 1999). In the present work, the relative

contribution of the three decarboxylases involved in C4 photosynthesis and the coordination

between the primary carboxylation and subsequent decarboxylation steps were evaluated in three

C4 grasses exposed to drought stress.

The three biochemical subtypes of C4 photosynthesis have been defined by the main

enzyme responsible for C4 acid decarboxylation (Gutierrez et al. 1974; Hatch et al. 1975; Hatch

1987): NADP-malic enzyme (NADP-ME, EC 1.1.1.40), NAD-malic enzyme (NAD-ME, EC

1.1.1.39) and PEP carboxykinase (PEPCK, EC 4.1.1.49). However, variations to these ‘classical’

mechanisms of C4 photosynthesis occur. PEPCK is present in various species of the NADP-ME

subtype (Walker et al. 1997; Voznesenskaya et al. 2006) and acts as a supplementary

decarboxylase in Zea mays (Wingler et al. 1999). A set of anatomical characteristics, including

the shape and position of the chloroplasts in the BS cells, is associated with each biochemical

subtype (Gutierrez et al. 1974; Prendergast et al. 1987; Dengler et al. 1994). Most C4 grasses fit

clearly into one of the three subtypes but Hattersley & Watson (1992) distinguished a total of ten

anatomical-biochemical variants of C4 plants.

The three C4 grasses studied in the present work were classified as belonging to each of

the different biochemical subtypes of C4 photosynthesis: Paspalum dilatatum as a NADP-ME

species (Usuda et al. 1984), Cynodon dactylon as a NAD-ME species (Hatch & Kagawa 1974)

and Zoysia japonica as a PEPCK species (Gutierrez et al. 1974). Dallisgrass (P. dilatatum),

bermudagrass (C. dactylon) and zoysiagrass (Z. japonica) are warm-season species used for

turfgrass purposes throughout the world (Brown 1999). Additionally, the first two species are

important forage and cultivated pasture grasses whilst C. dactylon is also one of the world’s most

serious weeds (Jones 1985).

The understanding of plant responses to drought and the identification of species better

adapted to the expected climate changes is crucial. Plant physiological and biochemical

responses to water deficit depend on the rate of induction and severity of the stress, the leaf age


125

and state of development, and the species studied (Chaves 1991). In a previous study on the

effects of rapidly-imposed drought conditions, C. dactylon leaves dehydrated faster but its

photosynthesis and instantaneous water use efficiency were more resistant to water deficit than

in Z. japonica and P. dilatatum (Carmo-Silva et al. 2007). Whether decreased CO2 assimilation

during drought stress can be partly attributed to metabolic limitation by the enzymes of the CO2-

concentrating mechanism in the three ‘classical’ biochemical subtypes of C4 photosynthesis is

still unclear.

The CO2-concentrating mechanism can be affected by water deficit either through effects

on the activities and/or regulation of the C4 cycle enzymes or through restriction of metabolite

transport between the M and BS cells. Studies with several C4 grass species of the NADP-ME

subtype, including Zea mays (Saccardy et al. 1996; Foyer et al. 1998), Sacharum sp. (Du et al.

1996) and Setaria sphacelata (Marques da Silva & Arrabaça 2004), showed contradictory

responses of the C4 enzymes involved in the primary carboxylation, PEPC, and in the

decarboxylation of malate, NADP-ME. Rapidly-induced leaf dehydration decreased PEPC

activity in Zoysia japonica, but did not affect the enzyme in Paspalum dilatatum and Cynodon

dactylon (Carmo-Silva et al. 2004; 2007). The decarboxylating enzymes considered to have an

important role in the decarboxylation of C4 acids by each of these grass species were not affected

to an extent that would limit photosynthesis under rapidly-induced water deficit conditions

(Carmo-Silva et al. 2004). The results obtained in that study suggested that the proportion of

decarboxylation accounted for by PEPCK and NAD-ME in Z. japonica could change with leaf

dehydration.

The regulation of the C4 enzymes involved in the carboxylation and decarboxylation

reactions was reviewed by Leegood and Walker (1999). PEPC in C4 plants is activated in the

light by reversible phosphorylation of a serine residue in the N-terminus and regulated

allosterically by metabolites (Chollet et al. 1996; Izui et al. 2004). The phosphorylation increases

the maximal activity and makes the enzyme less sensitive to inhibition by L-malate and more

sensitive to activation by glucose-6-phosphate (Huber & Sugiyama 1986). Jiao & Chollet (1989)

showed that PEPC sensitivity to L-malate could be used to assess the degree of phosphorylation

of the enzyme. Studies on drought-induced changes in the PEPC phosphorylation state, judged

by the inhibitory effect of L-malate on the enzyme activity, gave contradictory results in Zea

mays, with either decreased (Foyer et al. 1998) or unchanged (Saccardy et al. 1996) PEPC

sensitivity in dehydrated leaves. Information on the regulation of the decarboxylating enzymes

involved in C4 photosynthesis and on the effects that environmental factors exert on them is

sparse. The activity of NADP-ME in the light is modulated by changes in pH and by the

Chapter IV.

126

concentrations of L-malate and Mg2+ (Iglesias & Andreo 1990). NAD-ME activity is regulated

by adenylates and the ratio NADH/NAD+, requires Mn2+ and is stimulated in the presence of

fructose-1,6-bisphosphate and CoA (Murata et al. 1989). PEPCK has also an absolute

requirement for Mn2+, is regulated by the concentrations of metal ions and ATP (Walker et al.

1997), inhibited by a number of phosphorylated metabolites (Burnell 1986) and in some, but not

all, C4 plants is susceptible to phosphorylation (Walker & Leegood 1996). Adenylates can have

an important role in the coordination of NAD-ME and PEPCK activities (Walker et al. 1997).

The relative contribution of each of the three enzymes to C4 acid decarboxylation in different

species needed further research. The decarboxylation step in the C4 pathway is generally

assumed to be non-limiting for photosynthesis; however the coordination between the primary

carboxylation in the mesophyll and the further decarboxylation in the bundle sheath is crucial to

maximize the efficiency of the CO2-concentrating mechanism in C4 plants.

The aim of the present work was to characterise further three C4 grass species in relation

to the different photosynthetic mechanisms and to assess the response of the carboxylating and

decarboxylating enzymes to gradually-induced drought stress. The activities of PEPC, NADP-

ME, NAD-ME and PEPCK in the leaves of P. dilatatum, C. dactylon and Z. japonica were

studied under water deficit conditions. Evidence for drought-induced changes in the regulation of

the enzymes was obtained. The relative activities of the C4 acid decarboxylases under

physiological conditions suggest different contributions to the photosynthesis by the three

species.


127





Jacklin Seed Company, USA) (PEPCK) were grown from seeds in pots with peat-free compost

(Petersfield Products, Leicester, UK) supplemented with a slow-release fertiliser (Hydro Agri

Ltd, Lincs, UK) in a glasshouse. Artificial light was provided whenever the natural light was

below a photosynthetic photon flux density (PPFD) of 500 μmol m-2 s-1 during a 16 h


during the night. Each pot contained five plants and was well-watered until the beginning of the

drought stress treatment. Water deficit was then imposed on the ‘stress’ pots by ceasing to

provide water and the ‘control’ pots were watered once per day. Each pot corresponded to one

independent sample, with 12 control and 12 non-watered pots being used per species. The

treatments (control vs. drought stress) were randomised in a split-plot design with species as the

main plots (columns). From the full set, three control (C) and five non-watered (S) samples of

each species were selected according to their leaf dehydration level (in order to optimise the

range of drought stress intensities) and the corresponding frozen samples used for biochemical

measurements.

Leaf samples were collected in the growth environment four hours after the beginning of

the photoperiod, eight to ten days after drought stress induction: P. dilatatum was sampled first,

C. dactylon next and Z. japonica last. Five-week old plants of the two former species and nine-

week old plants of the slow-growing Z. japonica were analysed simultaneously. Taking into

account the different leaf sizes, each sample of P. dilatatum consisted of two leaves, while each

sample of the other two grasses consisted of five leaves, taken from the same pot. The youngest

fully expanded leaf of each plant of P. dilatatum and two young fully expanded leaves of each

plant of C. dactylon or Z. japonica were always used. It was assumed that, within each pot, all

the young fully expanded leaves were identical in terms of developmental stage, physiological

and biochemical properties, and would have experienced the same drought condition. Therefore,

leaf sub-samples were taken from each pot: the first was quickly frozen in liquid nitrogen (LN2)

and then stored at -80ºC for biochemical assays and the second was used to determine the leaf

relative water content (RWC). The fresh (FW), turgid (TW) and dry (DW) weights were

measured and used to calculate RWC by the equation RWC (%) = 100 × ((FW - DW) / (TW -

Chapter IV.

128

DW)) (Catsky 1960). Afterwards, another young fully expanded leaf was taken from each pot for

measurement of the leaf water potential (LWP) using a pressure-chamber (Ritchie & Hinckley,

1975). The soil water content (SWC, %, v/v) was determined in three opposite locations in each

pot using an HH2 moisture meter with a Theta probe (type ML2x, AT Delta-t Devices Ltd.,

Cambridge, UK).

Extraction of C4 enzymes

The frozen leaf samples (0.1 to 0.4 g FW) were ground in a cold mortar with quartz sand, 1%

(w/v) insoluble PVP and 10 (P. dilatatum and C. dactylon) or 15 volumes (Z. japonica) of ice-

cold extraction medium containing 50 mM Bicine-KOH pH 8.0, 1 mM EDTA, 5% (w/v)

PVP25000, 6% (w/v) PEG4000, 10 mM DTT, 50 mM 2-mercaptoethanol and 1% (v/v) protease

inhibitor cocktail (Sigma, St Louis, MO, USA). After grinding to produce a fine suspension,

aliquots were taken for total chlorophyll determination and the remaining homogenate was

centrifuged for 3 min at 14 000g and 4ºC. The supernatant was kept on ice while sub-samples

were taken for measuring each of the enzyme activities at 25ºC in continuous assays monitoring

absorbance at 340 nm (UV-500, Unicam Ltd., Cambridge, UK, with the software Vision 32).

The extraction medium and procedure was optimized in order to achieve the best recovery of the

four enzymes activities and to ensure that they remained stable for 1h. Each value presented is

the mean of at least two replicate measurements using the same leaf extract. The total

chlorophylls content in the leaf homogenates was determined after extraction in 96% ethanol

(Wintermans & de Mots 1965).

PEPC activities and sensitivity to effectors

PEPC activity was measured by coupling the carboxylase reaction with malate dehydrogenase

(MDH), essentially as described by Bakrim et al. (1992). The reaction mixture (1 mL) for the

determination of the maximal activity of the enzyme (Vmax), under optimal pH and substrate

conditions, consisted of 50 mM Hepes-KOH pH 8.0, 10 mM MgCl2, 10 mM NaHCO3, 0.2 mM

NADH (Sigma), 10 U MDH (Sigma), 10 mM PEP (Sigma) and 20 μL of crude extract. The

physiological activity (Vphysiol) was determined under similar conditions but at pH 7.3 and with

2.5 mM instead of 10 mM PEP. The reaction mixtures, with all the components except NADH,

were allowed to equilibrate at 25ºC for 1 min before starting the reaction. The activation state of

PEPC was calculated as the ratio Vphysiol / Vmax × 100.


129

One sub-sample of the leaf crude extracts was desalted by gel filtration (Sephadex G-25,

Pharmacia Biotech, Uppsala, Sweden, in PD-10 columns), to assess the effects of the inhibitor L-

malate and of the activator glucose-6P on PEPC physiological activity. The assays were

performed in the same conditions as described above for PEPC Vphysiol, using 40 μL of the

desalted leaf extract and measuring the activity in the absence and with increasing concentrations

of L-malate or glucose-6P. The inhibition or activation of the enzyme was assessed by

calculating the ratio of the activity at each concentration in relation to the activity in the absence

of the effector. Inhibition by aspartate was also tested in the leaf extracts of C. dactylon and Z.

japonica.

Activities of C4 acid decarboxylases

PEPCK was assayed in the carboxylation direction by coupling the reaction with malate

dehydrogenase (MDH) according to Walker et al. (2002) with minor modifications. The reaction

mixture (1 mL) for measurement of the enzyme activity under physiological concentrations of

divalent ions contained 100 mM Hepes-KOH pH 7.0, 100 mM KCl, 90 mM KHCO3, 4 mM

MgCl2, 10 μM MnCl2, 1 mM ADP (Sigma), 0.2 mM NADH, 12 U MDH (Sigma), 5 mM PEP

(Sigma) and the reaction was started by the addition of 40 μL of leaf crude extract. Maximal

activity of the enzyme from each of the three species was obtained under the same conditions but

in the presence of 5 mM instead of 4 mM MgCl2 and 2 mM instead of 10 μM MnCl2 and starting

the reaction by the addition of PEP after incubating the enzyme, contained in the leaf crude

extracts, at 25ºC with all the other components for 3 min. The activation state of PEPCK was

calculated as the ratio between the two activities multiplied by 100.

NADP-ME and NAD-ME activities under conditions that approximate the physiological

state were determined using the methods described by Ashton et al. (1990) with minor

modifications. For NADP-ME activity, the reaction mixture (1 mL) contained 50 mM Hepes-

KOH pH 8.0, 10 mM MgCl2, 0.5 mM NADP+ (Sigma), 5 mM L-malate and 40 μL of crude

extract. For NAD-ME activity, the reaction mixture (1 mL) contained 50 mM Hepes-KOH pH

7.2, 4 mM MnCl2, 0.1 mM CoA, 4 mM NAD+ (Sigma), 5 mM L-malate and 40 μL of crude

extract. In both cases, the enzymes contained in the leaf crude extracts were incubated at 25ºC

with all the components except the substrate for 3 min and the reactions were then started by the

addition of L-malate. This procedure was adopted after obtaining greater rates of NADP-ME

activity in P. dilatatum and NAD-ME activity in C. dactylon under these conditions.

Chapter IV.

130



Research, UK). Regression analysis was applied to model the variation of enzyme activities and

activation states with RWC. Non-significantly different (P > 0.05) parameters (t-tests) in the

significant model terms of the regression (F-tests, P < 0.05) were amalgamated in order to attain

parsimony. The resulting best models were plotted and the parameter estimates with their

respective standard errors (s.e.), the percentage of variance accounted for by the model (R2), the

residual mean square (s2) and the degrees of freedom (d.f.) are given with the plots. All the

absolute values and percentages presented in the text were calculated in accordance with the

regression analysis performed. Residual Maximum Likelihood (REML) analysis was used to

verify if there was a significant effect of each effector on the activity of PEPC. The ratio of the

enzyme activity with each concentration of effector in relation to the activity in its absence was

calculated for each sample. The significance of the treatments on this ratio was assessed through

the Wald test (Welham & Thompson 1997). Subsequently, mean values estimated for the control

and drought-stressed plants of each species at the different effector concentrations were

compared using t-tests on the appropriate degrees of freedom from the REML model and the

standard errors of differences (SED) for all possible comparisons. The least significant difference

at the 5% level (LSD(5%)) considering all data for each effector is given as a reference.


131

RESULTS

Drought stress induction

The soil water content (SWC), in the non-watered pots was four to five times lower than in the

control pots of each species (Table IV.1), resulting in lower values of leaf water potential (LWP)

and relative water content (RWC) in the drought-stressed samples relative to the controls.

Table IV.1. The soil water content (SWC), leaf relative water content (RWC) and leaf water potential (LWP) of well-watered (C) and drought-stressed (S) plants of P. dilatatum, C. dactylon and Z. japonica. The mean values and respective standard errors were calculated from measurements taken with three control and five drought-stressed samples of each species.

Species P. dilatatum C. dactylon Z. japonica

SWC C 36.7 ± 0.8 36.2 ± 0.9 39.3 ± 0.2 (%) S 6.4 ± 0.6 8.0 ± 1.0 7.7 ± 1.2

RWC C 98.1 ± 0.1 98.4 ± 0.2 98.1 ± 0.5 (%) S 87.1 ± 2.9 93.1 ± 1.5 93.4 ± 1.0

LWP C -0.93 ± 0.02 -0.82 ± 0.04 -0.95 ± 0.04 (MPa) S -1.21 ± 0.03 -1.16 ± 0.04 -1.65 ± 0.12

PEP carboxylase

PEPC activity was higher in C. dactylon than in the other two species (Figure IV.1). A very

slight, but significant (P ≤ 0.001), increase of Vphysiol was observed with decreasing RWC

whereas Vmax was not significantly affected by leaf dehydration in any of the three species (P >

0.05). PEPC activation state was lower in P. dilatatum (< 40%) than in the other two species (ca.

80-90%). The activation state of the enzyme increased by ca. 16% in P. dilatatum and 6% in C.

dactylon and Z. japonica when RWC decreased to 90%.

PEPC physiological activity in the leaves of P. dilatatum, C. dactylon and Z. japonica

was significantly affected (P < 0.001) by the concentration of L-malate (Figure IV.2). The

enzyme was less inhibited in the non-watered than in the control plants (P < 0.001) and the

extent of inhibition differed among the three species (P = 0.047), being greater in P. dilatatum

Chapter IV.

132

and Z. japonica than in C. dactylon. The inhibition of PEPC Vphysiol by aspartate in the two latter

species was less pronounced than by L-malate, but the enzyme was similarly more inhibited in Z.

japonica than in C. dactylon and the enzyme from control leaves of both species was more

sensitive to the inhibitor than from the dehydrated leaves. The effect of glucose-6P on PEPC

Vphysiol (P < 0.001) was significantly different among the three species (P < 0.001), with greatest

activation in P. dilatatum. The enzyme present in drought-stressed leaves of C. dactylon was

more strongly activated by glucose-6P than in the control leaves, but for the other two species

the activating effect was greater in the fully hydrated leaves. The overall difference between

control and non-watered samples was not significant (P > 0.05).


B C

PEPC

(μm

ol m

in-1

mg-1

Chl

)

0

5

10

15

20

25

30 A

y = 10.06 - 0.0553 x (s.e. 8.82; 0.0963) y = 26.15 - 0.0553 x (s.e. 9.19; 0.0963) y = 17.12 - 0.0553 x (s.e. 9.20; 0.0963)

RWC (%)

80 85 90 95 100

RWC (%)

80 85 90 95 100

E F

RWC (%)

80 85 90 95 100

Activ

atio

n st

ate

(%)

0

20

40

60

80

100 D

y = 85.6 - 0.576 x (s.e. 25.8; 0.282) y = 140.9 - 0.576 x (s.e. 26.9; 0.282) y = 140.9 - 0.576 x (s.e. 26.9; 0.282)

Figure IV.1. (A-C) PEPC activities (μmol min-1 mg-1 Chl) and (D-F) activation state (%) as a function of

the relative water content (RWC, %) in the leaves of P. dilatatum, C. dactylon and Z. japonica. Activity

was measured under physiological (Vphysiol; closed symbols) and optimal conditions (Vmax; open symbols)

and the activation state (D-F) was calculated as 100×Vphysiol/Vmax. Each data point corresponds to one

sample (with eight samples per species). Regression lines were fitted when the RWC effect was

significant (P < 0.05). Vphysiol, R2 = 91.0%, s2 = 4.325, d.f. = 20; activation state, R2 = 94.7%, s2 = 37.1, d.f.

= 21.


133

Inhibitor (mM)

0.00 0.25 0.50 0.75

PEPC

Act

ivity

(Rat

io)

0.0

0.2

0.4

0.6

0.8

1.0

Inhibitor (mM)

0.00 0.25 0.50 0.75

Inhibitor (mM)

0.00 0.25 0.50 0.75

Glucose-6P (mM)

0.00 1.25 2.50 3.75

PEPC

Act

ivity

(Rat

io)

0.0

1.0

2.0

3.0

4.0

Glucose-6P (mM)

0.00 1.25 2.50 3.75

Glucose-6P (mM)

0.00 1.25 2.50 3.75

Malate LSD(5%)

B CA

E FD

Malate LSD(5%)

Aspartate LSD(5%)

Malate LSD(5%)

Aspartate LSD(5%)

Glucose-6P LSD(5%) Glucose-6P LSD(5%) Glucose-6P LSD(5%)



Figure IV.2. Sensitivity of PEPC physiological activity (Vphysiol; at pH 7.3) to the concentration of the

inhibitor malate (A, B, C) and the activator glucose-6P (D, E, F) in the leaves from control (closed

symbols) and non-watered (open symbols) plants of P. dilatatum, C. dactylon and Z. japonica. The effect

of aspartate was also analysed on the two latter species. The ratio of the enzyme activity at each

concentration of malate (black triangles), aspartate (grey triangles) and glucose-6P (black squares)

relative to the activity in the absence of added effectors (closed circles) was calculated for each sample

and the mean values for each group of plants were analysed by the REML method. The overall least

significant difference at the 5% level (LSD(5%)) is 0.084 for malate (48 d.f.), 0.056 for aspartate (32 d.f.)

and 0.480 for glucose-6P (51 d.f.).

Chapter IV.

134

C4 acid decarboxylases

The activity of PEPCK at optimal concentrations of Mg2+ and Mn2+ was high in all three species

(Figure IV.3), compared with the other C4 acid decarboxylases. Conversely, the activity of the

enzyme in the presence of low but physiological concentrations of the divalent ions was low in P.

dilatatum, with an average activation state of 25.7 ± 2.7%, but high in C. dactylon and Z.

japonica, where the activation state was generally above 80%. PEPCK activity decreased with

leaf dehydration only in C. dactylon and at physiological concentrations of Mg2+ and Mn2+, with

no other significant changes being observed with decreased RWC (P > 0.05). The activation

state of the enzyme was differently affected in C. dactylon and Z. japonica, decreasing by 8.5%

in the former species and increasing by 24% in the latter when RWC decreased to 90%.

PEPC

K (μ

mol

min

-1 m

g-1 C

hl)

0

2

4

6

8

10


y = 0.0761 x (s.e. 0.0039)

CBA

RWC (%)

80 85 90 95 100

Activ

atio

n st

ate

(%)

0

20

40

60

80

100

RWC (%)

80 85 90 95 100

RWC (%)

80 85 90 95 100

FED

y = 0.9664 x (s.e. 0.0285) y = 322.9 - 2.44 x (s.e. 98.9; 1.04)

Figure IV.3. (A-C) PEPCK activities (μmol min-1 mg-1 Chl) and (D-F) activation state (%) as a function of the relative water content (RWC, %) in the leaves of P. dilatatum, C. dactylon and Z. japonica. Activity was measured under conditions assumed to represent the closest to the physiological (closed symbols) and at optimal concentrations of Mg2+ and Mn2+ (open symbols), and the activation state (D-F) was calculated as the ratio between the two multiplied by 100. Each data point corresponds to one sample (with eight samples per species). Regression lines were fitted when the RWC effect was significant (P < 0.05). ‘Physiological’, R2 = 82.4%, s2 = 23.67, d.f. = 21; activation state, R2 = 94.5%, s2 = 58.8, d.f. = 20.


135

In P. dilatatum NADP-ME activity decreased by 8% with decreasing RWC down to 90%

(Figure IV.4). The activity of this enzyme was very low in C. dactylon and Z. japonica (0.50 ±

0.09 μmol min-1 mg-1 Chl) and did not change with leaf dehydration (P > 0.05). On the other

hand, the activity of NAD-ME in fully hydrated leaves of C. dactylon (~3.8 μmol min-1 mg-1

Chl) was higher than in Z. japonica (1.59 ± 0.11 μmol min-1 mg-1 Chl) and very low in P.

dilatatum (0.62 ± 0.11 μmol min-1 mg-1 Chl) (Figure IV.4). In C. dactylon NAD-ME activity

decreased by 8% when RWC decreased to 90%, but no other significant variations with drought

stress were observed for this enzyme.

RWC (%)

80 85 90 95 100

NAD

-ME

(μm

ol m

in-1

mg-1

Chl

)

0

2

4

6

8

10

NAD

P-M

E (μ

mol

min

-1 m

g-1 C

hl)

0

2

4

6

8

10

RWC (%)

80 85 90 95 100

RWC (%)

80 85 90 95 100


A B C

D E F

y = 0.0688 x (s.e. 0.0014)

y = 0.0389 x (s.e. 0.0011)

Figure IV.4. Activities of (A-C) NADP-ME and (D-F) NAD-ME (μmol min-1 mg-1 Chl) as a function of

the relative water content (RWC, %) in the leaves of P. dilatatum, C. dactylon and Z. japonica. Activities

were measured under conditions assumed to represent the closest to the physiological. Each data point

corresponds to one sample (with eight samples per species). Regression lines were fitted when the RWC

effect was significant (P < 0.05). NADP-ME, R2 = 98.4%, s2 = 0.130, d.f. = 22; NAD-ME, R2 = 95.1%, s2

= 0.089, d.f. = 21.

Chapter IV.

136

DISCUSSION

Effects of leaf dehydration on C4 enzyme activities and regulation

Slowly-induced leaf dehydration had a similar effect on PEPC from P. dilatatum, C. dactylon

and Z. japonica. The physiological activity of the enzyme increased slightly with decreasing

RWC in all three species due to an increase in the activation state of the enzyme (Figure IV.1);

PEPC maximal activity was not affected by leaf dehydration. In Setaria sphacelata both

maximal and physiological activities and the activation state of PEPC increased with slowly-

induced drought stress (Marques da Silva & Arrabaça 2004). Conversely, in Saccharum sp. a

linear decrease of the enzyme activity with decreasing leaf water potential was observed (Du et

al. 1996). In Zea mays PEPC maximal activity was either little (Foyer et al. 1998) or not affected

(Saccardy et al. 1996) by gradually imposed water deficit. When leaf dehydration was rapidly

induced by the addition of polyethylene glycol 4000 to the nutrient solution, PEPC activities

were not affected in P. dilatatum (NADP-ME) and C. dactylon (NAD-ME) and decreased in Z.

japonica (PEPCK) only when RWC was below 70% (Carmo-Silva et al. 2004; 2007). The

different results are attributed to the different methods used for drought stress induction and are

species-dependent (Chaves 1991).

The drought-induced increase in PEPC physiological activity (Figure IV.1) is consistent

with increased phosphorylation of the enzyme as indicated by the decreased sensitivity to the

inhibitor L-malate in all three species (Figure IV.2). Saccardy et al (1996) found no drought-

induced changes on PEPC phosphorylation state, judged by the inhibitory effect of L-malate on

the enzyme from Z. mays, but Foyer et al. (1998) reported a decrease of PEPC sensitivity to the

inhibitor in dehydrated leaves of the same species. In fully hydrated leaves, PEPC from C.

dactylon was relatively less inhibited by L-malate than the enzyme from P. dilatatum and Z.

japonica, suggesting a higher phosphorylation state in the former species (Figure IV.2), whilst

the extent of inhibition of the enzyme from drought-stressed plants was similar for the three C4

grasses. Even though aspartate was a weaker inhibitor of PEPC than L-malate, similar relative

inhibition patterns were observed with the two C4 acids. The activation of PEPC by glucose-6P

was greater in the enzyme from P. dilatatum than from C. dactylon and Z. japonica. In vivo, it is

plausible that changes in the concentrations of metabolite effectors in the dehydrated leaves

might also regulate the activity of the enzyme. Decreases in L-malate and glucose-6P were

previously observed in severely dehydrated leaves of Saccharum sp. (Du et al. 1998).


137

PEPCK had appreciable activity in all three grass species (Figure IV.3), but the activation

state of the enzyme was much lower in P. dilatatum than in C. dactylon and Z. japonica. The

activity of PEPCK at optimal concentrations of Mg2+ and Mn2+ was not affected by drought

stress and the activity of the enzyme at physiological concentrations of the divalent ions changed

with leaf dehydration only in C. dactylon, decreasing by 8.5% when the RWC decreased to 90%.

The activation state of both PEPC and PEPCK was low in P. dilatatum and high in C. dactylon

and Z. japonica. In the latter species both increased with decreasing RWC, whilst in C. dactylon

an increase of PEPC activation state and a decrease of PEPCK activation state were observed

under the drought conditions attained. An effective coordination between the activation and

phosphorylation states of PEPC and PEPCK was observed in the PEPCK-type species Panicum

maximum (Bailey et al. 2007). In Z. japonica the increase of PEPCK activation state with leaf

dehydration could indicate that the enzyme from this species is also regulated by

phosphorylation/dephosphorylation, as in P. maximum and some other C4 grasses (Walker &

Leegood 1996), and therefore by the same type of covalent modification as PEPC, but activated

by dephosphorylation (Walker et al. 1997; Walker et al. 2002) under drought conditions.

Conversely, in C. dactylon a decrease of PEPCK activation state with decreasing RWC was

observed concomitantly with the increased PEPC activation state, suggesting that the enzyme

from this species might not be susceptible to modulation by phosphorylation but regulated by

other mechanism, possibly involving alterations in metabolite levels or ATP concentration

(Walker et al. 1997; Leegood & Walker 1999).

NADP-ME activity decreased with leaf dehydration only in P. dilatatum (Figure IV.4).

The enzyme had no appreciable activity in C. dactylon and Z. japonica, consistent with the view

that this enzyme has no important role in C4 acid decarboxylation in the BS cells of these species.

An abrupt decrease in the activity of NADP-ME in P. dilatatum was previously observed with

rapidly-induced leaf dehydration (Carmo-Silva et al. 2004), and may result from proteolysis or

down-regulation under stress conditions. NADP-ME activity also decreased in gradually

dehydrated leaves of S. sphacelata (Marques da Silva & Arrabaça 2004) and Saccharum sp. (Du

et al. 1996) but was not affected by water deficit in Z. mays (Saccardy et al. 1996).

The activity of NAD-ME was highest in C. dactylon, the species classified as belonging

to this subtype. The activity decreased slightly with leaf dehydration in C. dactylon (Figure IV.4)

but was not affected by decreasing RWC in the leaves of Z. japonica and had no appreciable

activity in P. dilatatum, where the major decarboxylating enzyme is NADP-ME. A decrease in

NAD-ME activity in C. dactylon, with no change in the enzyme activity in Z. japonica, was also

found when drought conditions were rapidly imposed (Carmo-Silva et al. 2004). The enzyme

Chapter IV.

138

from C. dactylon might be slightly affected by leaf dehydration, either through a direct effect

resulting in degradation or inactivation of the enzyme or through an indirect effect on the levels

of adenylates or on the NADH/NAD+ ratio (Leegood & Walker 1999). The NAD-ME from some

but not all species is activated by ATP, whereas ADP and AMP invariably inhibit the enzyme

(Furbank et al. 1991). Different regulation mechanisms of the enzyme are likely to be present, at

least under drought conditions, in C. dactylon and Z. japonica. NAD-ME activity in any species

with high PEPCK activities must be compatible with the high concentrations of ATP involved.

The regulation of both enzymes is likely to be much dependent on the levels of Mn2+ and ATP

and the allosteric interaction between them (Furbank et al. 1991).

C4 biochemical subtypes of the grass species P. dilatatum, C. dactylon and Z. japonica

In the classical definition of the biochemical subtypes of C4 photosynthesis (Gutierrez et al.

1974; Hatch et al. 1975), species belonging to the NADP-ME and NAD-ME subtypes were

assumed to have low activities of the other two C4 acid decarboxylases. Conversely, in the

PEPCK subtype the enzyme NAD-ME plays an important role, providing the required ATP and

contributing considerably to the release of CO2 in the BS cells (Kanai & Edwards 1999). More

recently, PEPCK was shown to be present in the BS of some NADP-ME type C4 species (Walker

et al. 1997; Voznesenskaya et al. 2006) and to contribute to the decarboxylation of C4 acids in

the BS of Zea mays (Wingler et al. 1999). Measuring simultaneously the activities of the three

C4 acid decarboxylases under physiological conditions (Figures IV.3 and IV.4), we observed the

presence of considerable activities of PEPCK in all three species, with the least in P. dilatatum.

Considerable activities of NAD-ME were observed in C. dactylon and Z. japonica and of

NADP-ME in P. dilatatum. To date, the activity of PEPCK has been found to be very low or

negligible in NAD-ME-type grasses. Edwards et al. (1971) reported very low activity of PEPCK

in C. dactylon, insufficient to account for C4 acid decarboxylation, and Hatch and Kagawa

(1974) and Prendergast et al. (1987) could not detect the presence of the enzyme in this species.

However, Figure IV.3 shows high activities of PEPCK in C. dactylon, well above the activity

found for NAD-ME. Prendergast et al. (1987) suggested that some species might be intermediate

biochemically, showing either predominance of NAD-ME or PEPCK activity. Studies in

progress showed that the appropriate leaf anatomical characteristics associated with each

biochemical subtype are present in P. dilatatum (NADP-ME), C. dactylon (NAD-ME) and Z.

japonica (PEPCK). In particular, the elongated shape and centripetal distribution of the BS

chloroplasts and the evenness of the BS outline in transverse leaf sections of C. dactylon are

consistent with previous descriptions for this species (Prendergast & Hattersley 1987; Watson &


139

Dallwitz 1992; Dengler et al. 1994) and support its classification as a ‘typical’ NAD-ME-type

species (Dengler & Nelson 1999).

The subtypes NAD-ME and PEPCK have many characteristics in common and it is

plausible that species belonging to the PEPCK subtype evolved from ancestral species with the

NAD-ME C4 photosynthetic pathway (Gutierrez et al. 1974; Hattersley & Watson 1992; Watson

& Dallwitz 1992) retaining considerable levels of decarboxylation in the mitochondria. The

decarboxylation by PEPCK can be viewed as a relatively simple addition to, or variant of, the

NAD-ME pathway (Kellogg 1999). The high activities of PEPCK in P. dilatatum and C.

dactylon, suggest that this enzyme might act as a supplementary decarboxylating enzyme to

NADP-ME and NAD-ME (Kellogg 1999; Lea et al. 2001) but it is also known to have non-

photosynthetic functions in amino acid, organic acid, sugar, lipid and secondary metabolism

(Leegood & Walker 2003).

CONCLUSIONS

The activities of PEP carboxylase and the C4 acid decarboxylases were only slightly changed by

gradually-induced drought stress. Decreased RWC affected the activity and regulation of PEPC

in a similar manner in the three different C4 grass species studied but had different effects on the

C4 acid decarboxylases, NADP-ME, NAD-ME and PEPCK. Appreciable activity of PEPCK was

observed in all three species, suggesting that this enzyme may act as a supplementary

decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.

The faster primary carboxylation by PEPC and the higher potential for C4 acid decarboxylation,

by both NAD-ME and PEPCK, suggest the presence of a highly effective CO2-concentrating

mechanism in the C4 grass C. dactylon.

Chapter IV.

140

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Bailey K.J., Gray J.E., Walker R.P. & Leegood R.C. (2007) Coordinate regulation of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase by light and CO2 during C4 photosynthesis. Plant Physiology, 144, 479-486.

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Chollet R., Vidal J. & Oleary M.H. (1996) Phosphoenolpyruvate carboxylase: A ubiquitous, highly regulated enzyme in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47, 273-298.




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Edwards G.E., Kanai R. & Black C.C. (1971) Phosphoenolpyruvate carboxykinase in leaves of certain plants which fix CO2 by the C4-dicarboxylic acid cycle of photosynthesis. Biochemical and Biophysical Research Communications, 45, 278-285.


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Izui K., Matsumura H., Furumoto T. & Kai Y. (2004) Phosphoenolpyruvate carboxylase: A new era of structural biology. Annual Review of Plant Biology, 55, 69-84.

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Marques da Silva J. & Arrabaça M.C. (2004) Photosynthetic enzymes of the C4 grass Setaria sphacelata under water stress: a comparison between rapidly and slowly imposed water deficit. Photosynthetica, 42, 43-47.

Murata T., Ohsugi R., Matsuoka M. & Nakamoto H. (1989) Purification and characterization of NAD malic enzyme from leaves of Eleusine coracana and Panicum dichotomiflorum. Plant Physiology, 89, 316-324.



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Voznesenskaya E.V., Franceschi V.R., Chuong S.D.X. & Edwards G.E. (2006) Functional characterization of phosphoenolpyruvate carboxykinase-type C4 leaf anatomy: Immuno-, cytochemical and ultrastructural analyses. Annals of Botany, 98, 77-91.

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Chapter V.

RUBISCO FROM C4 GRASSES UNDER DROUGHT

STRESS

Chapter V.

146

A manuscript using the data presented in this chapter is in preparation for submission to Plant Cell and Environment:

Carmo-Silva A.E., Keys A.J., Andralojc P.J., Powers S.J., Arrabaça M.C. & Parry M.A.J. (In Preparation) Rubisco properties and regulation in three different C4 grasses under drought stress.

Alfred J. Keys (Department of Plant Sciences, Rothamsted Research) was involved in the planning of the experiments, in the measurements of RuBP and Rubisco inhibitors and in the interpretation of the results.

P. John Andralojc (Department of Plant Sciences, Rothamsted Research) was involved in the determination of Rubisco kinetic parameters and did the Rubisco specificity factor measurements.

Stephen J. Powers (Department of Biomathematics and Bioinformatics, Rothamsted Research) was involved in the planning of the experiment and did the statistical analysis of the data.

Rubisco from C4 Grasses under Drought Stress

147

RUBISCO PROPERTIES AND REGULATION IN THREE DIFFERENT C4 GRASSES UNDER DROUGHT STRESS

ABSTRACT

Decreased water availability limits photosynthetic performance partly through impaired

metabolism. Under adverse environmental conditions, ribulose-1,5-bisphosphate

carboxylase/oxygenase (Rubisco) activity must be modulated to match the amounts of CO2 and

ribulose-1,5-bisphosphate (RuBP) available in the chloroplast stroma. The regulation of Rubisco

was studied in three C4 grasses of different metabolic subtypes, Paspalum dilatatum Poiret

(NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK)

exposed to drought stress. Decreased initial and total activities of Rubisco with unchanged

maximal activities or enzyme amounts suggested increased inhibition of the enzyme with leaf

dehydration. Decreased amounts of RuBP and increased amounts of an inhibitor, probably 2-

carboxyarabinitol-1-phosphate (CA1P), were observed in the leaves from drought-stressed plants.

The tight-binding inhibitor of Rubisco was present especially in the dark in Z. japonica. Rubisco

from each of the three species was partially purified and the kinetic constants for carboxylation

and oxygenation were determined. All three species had Rubiscos with smaller specificity factors,

larger Km values for CO2 (Kc) and O2 (Ko) and larger maximum carboxylation activities (Vc) than

observed for wheat Rubisco. The overall results suggest that Rubisco from the three C4 grasses

has evolved characteristics that optimise photosynthetic efficiency and down-regulation of

Rubisco occurs under drought stress, possibly by the binding of inhibitors that may confer

protection to the enzyme.

KEYWORDS:

Rubisco activities, RuBP amounts, inhibition, kinetic constants, P. dilatatum, C. dactylon, Z. japonica

Chapter V.

148

INTRODUCTION

Drought stress is one of the major constraints to plant productivity. There is an importance and

urgency to understand plant limitations to photosynthetic performance under drought conditions

so that plant productivity can be optimized for conditions of limited water availability that are

predicted to spread through several regions of the planet (Petit et al. 1999). Limitations to

photosynthesis under water deficit include both stomatal and non-stomatal factors (see

comprehensive review by Chaves et al. 2003). In most C3 species, stomatal closure, and

consequently decreased CO2 availability for carboxylation, seems to be the primary factor

limiting net assimilation under moderate drought stress, whilst metabolic impairment becomes

more important as stress severity increases (Flexas & Medrano 2002). Lawlor & Cornic (2002)

recognised two types of plant responses to drought, with metabolic limitations either becoming

important only under severe water deficit or contributing to decreased photosynthesis from the

earliest stages of water deficit. In both cases, impairment of metabolism is assumed to increase

as the leaf relative water content (RWC) decreases. Decreased capacity for RuBP regeneration is

probably a major metabolic limitation to photosynthesis under water deficit (Tezara et al. 1999;

Flexas & Medrano 2002) and may result from decreased ATP and/or NADPH synthesis or from

decreased turnover rates in the Calvin cycle due to impaired enzyme activities (Lawlor 2002).

The tissue content of RuBP in relation to the amount of Rubisco gives an indication of its

importance as a limitation to photosynthesis with progressive drought stress.

The presence of a CO2-concentrating mechanism that effectively limits photorespiration

(see Chapter III, Carmo-Silva et al. 2008) and the higher photosynthetic rates generally

associated with C4 plants are features of their improved water use efficiency compared to their

C3 counterparts (Long 1999) and contribute to a better performance of C4 species in areas with

increased aridity. Studies on the response of C4 species to water deficit (Du et al. 1996; Lal &

Edwards 1996; Ghannoum et al. 2003; Marques da Silva & Arrabaça 2004a; Carmo-Silva et al.

2007) showed that stomatal closure, with consequently decreased CO2 availability at the primary

carboxylation site, and non-stomatal factors, including decreased internal conductance to CO2

and metabolic impairment, may contribute to drought-decreased net photosynthesis. Specific

plant responses vary with the genotype and developmental stage, and depend on the rate,

duration and severity of the imposed stress (Chaves et al. 2003).

Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) is the main enzyme

ultimately responsible for CO2 assimilation in all plant species and one of the crucial points of

regulation of photosynthesis in fully hydrated leaves of the C4 species Flaveria bidentis (Furbank


149

et al. 1997). The importance of the most abundant leaf protein in the limitation of photosynthesis

under drought conditions is not well understood. Flexas & Medrano (2002) suggested that

Rubisco capacity was of little importance in drought-induced photosynthesis limitation. None the

less, decreased activity and/or amount of Rubisco were observed in many C3 and C4 species

exposed to water deficit (Majumdar et al. 1991; Parry et al. 1993; Du et al. 1996; Parry et al.

2002; Tezara et al. 2002; Bota et al. 2004; Marques da Silva & Arrabaça 2004b; Carmo-Silva et

al. 2007). As recently proposed by Flexas et al. (2006), effects of water deficit on Rubisco may

involve differential regulation of the enzyme during stress rather than a direct effect of leaf

dehydration. The factors behind this stress-induced regulation remain unclear. Decreased

Rubisco capacity may not be the cause of limited CO2 assimilation when water availability is

reduced but its down-regulation is likely to be a determinant of photosynthetic performance

under these conditions and may allow better recoveries upon stress release and better overall

plant productivities in environments where water scarcity is an increasingly important problem.

Rubisco activity is regulated by the extent of carbamylation of a lysyl residue within the

catalytic site (Lorimer & Miziorko 1980), by ATP-dependent Rubisco activase, which facilitate

carbamylation in vivo when the CO2 concentration is sub-optimal (Portis 1992; 2003), and by

interaction with various chloroplast metabolites (Hatch & Jensen 1980; Badger & Lorimer 1981;

Jordan et al. 1983), including some tight-binding inhibitors (Pearce & Andrews 2003; Kim &

Portis 2004). In some, but not all, plant species (Vu et al. 1984; Seemann et al. 1985; Servaites et

al. 1986; Holbrook et al. 1992; Sage & Seemann 1993), Rubisco is inhibited at night by a

specific tight-binding inhibitor, 2-carboxyarabinitol-1-phosphate (CA1P) (Gutteridge et al. 1986;

Berry et al. 1987; Moore et al. 1992). The regulation by carbamylation in the light is readily

investigated by comparing the activity in freshly made extracts, the initial activity, with activity

following incubation of extracts with an excess of CO2 and Mg2+ to fully carbamylate the

enzyme, the total activity. The effects of tight-binding inhibitors can be measured by pre-

incubating the extract with a high concentration of SO42- ions and subsequent desalting, which

removes tight-binding inhibitors, and then fully carbamylating the enzyme before measuring

again its activity, the maximal activity (Parry et al. 1997). The difference between maximal

activity and total activity gives an indication of the tight-binding inhibitors causing regulation in

the light. Knowing the amount of Rubisco in the tissue extracts, the number of catalytic sites

occupied by inhibitors can be calculated. Tight-binding inhibitors can also be measured by the

inhibition of the activity of a known amount of purified Rubisco caused by acid extracts of

leaves (Keys et al. 1995). This can be conveniently related to the number of catalytic sites by

comparison with inhibition caused by CA1P on the same amount of purified Rubisco. The

Chapter V.

150

inhibitor, CA1P, is not ubiquitous through the plant kingdom (Servaites et al. 1986; Sage &

Seemann 1993) but it inhibits Rubisco during the night in certain species and to varying extents.

The presence of inhibitors during the daytime (Keys et al. 1995) became associated with

misfire products of Rubisco catalysis (Pearce & Andrews 2003; Kim & Portis 2004). The main

contender for inhibition in the light (Kane et al. 1998), D-glycero-2,3-diulose-1,5-bisphosphate

(PDBP), is too labile for detailed study and its importance in regulation is not fully established.

Tight-binding inhibitors, such as CA1P, protect the Rubisco from proteolytic breakdown (Khan

et al. 1999) and may play an important role in the regulation of the enzyme under stress

conditions (Parry et al. 2008).

Photorespiration is initiated by the oxygenase activity of Rubisco and decreases both the

rate and efficiency of photosynthesis in C3 plants. Despite the use of genetic manipulation,

selection of many mutants and engineering of the Rubisco protein, no major improvements have

yet been made in decreasing oxygenase relative to carboxylase activity (Keys & Leegood 2002;

Parry et al. 2007). Some C3 plants have evolved Rubiscos that discriminate more strongly

against O2 (Galmés et al. 2005) and, although comparatively small, such changes point the way

to improve Rubisco specificity towards RuBP carboxylation (Parry et al. 2007). Rubiscos from

C4 plants tend to have higher sensitivity to O2 (von Caemmerer 2000) and higher maximal rates

of carboxylation (Sage 2002) than Rubiscos from C3 plants. The CO2-concentrating mechanism

present in C4 photosynthesis is one of the few successful strategies evolved in nature that

decreases oxygenation of RuBP and the proportion of photorespiration to photosynthesis. This is

because CO2 and O2 are competitive alternative substrates and thus competitive inhibitors each

to the reaction of the other (Bowes & Ogren 1972; Laing et al. 1974). The high CO2

concentration in the bundle sheath of C4 plants means that the oxygenase reaction of Rubisco is

much decreased in these plants. The CO2-concentrating mechanism may become less effective

under water deficit so that increased photorespiration would contribute to the limitation of

photosynthesis. From consideration of the kinetics of the carboxylation and oxygenation of

RuBP catalysed by Rubisco, a constant, often referred to as the specificity factor, has been

recognised to describe the relative reaction towards the two substrate gases when present

together (Laing et al. 1974). Thus the specificity factor (SF) is equal to VcKo/VoKc, where Vc and

Vo represent the maximum velocities of the carboxylase and oxygenase reactions respectively,

and Kc and Ko the Michaelis-Menten constants (Km) for CO2 and O2. As a consequence, at

concentrations of CO2 (C) and O2 (O) the relative rates of carboxylation and oxygenation can be

expressed as vc/vo = VcKoC/VoKcO. The individual kinetic constants are not known with accuracy

for most species and they become of increasing interest for mechanistic modelling of the


151

response of photosynthesis to environmental conditions (von Caemmerer & Furbank 1999; von

Caemmerer 2000). Photosynthesis mechanistic modelling can give valuable information in

predicting plant responses, such as the effects of decreasing water availability on photosynthetic

performance (e.g. Carmo-Silva et al. 2008; Chapter III). Currently, the assumptions that need to

be made in order to apply these models compromise the reliability and validity of the predictions

and would largely benefit from a deeper knowledge of C4 Rubisco properties. The Km for CO2

(Kc) and for RuBP (KRuBP) for Rubiscos from several C3 and C4 grasses have been reported

(Yeoh et al. 1980; 1981). Jordan and Ogren (1983) measured specificity factors and Kc values

for Rubisco from several species including C3, C4 and C3-C4 intermediates. More progress is

now being made to establish Rubisco kinetic parameters variation between the C3 and C4

photosynthetic pathways (Kubien et al. 2008), but the enzyme from C4 plants, especially

monocotyledons, needs to be better understood.

The objectives of this study were to investigate the effects of water deficit on the amount,

activities and regulation of Rubisco in three C4 grass species. This involved also a study of the

effects on the contents of RuBP and tight-binding inhibitors. The kinetic properties of Rubisco

present in the leaves of the three species were measured with a view to future mechanistic

modelling of the effects of water deficit on C4 photosynthesis.

Chapter V.

152


General methods

Rubisco was purified from young wheat leaves essentially as described by Keys & Parry (1990).

RuBP was prepared essentially as described by Wong et al. (1980) and purified by ion exchange

chromatography. CA1P was prepared from RuBP by reaction with cyanide, hydrolysis of the

cyanohydrins, separation of 2-carboxyarabinitol-1,5-bisphosphate (CABP), partial

dephosphorylation by alkaline phosphatase and purification by ion-exchange chromatography

(Gutteridge et al. 1989). Radioactivity of 14C labelled compounds was measured by liquid

scintillation spectroscopy, using a liquid scintillation analyser model 2100 TR and Ultima Gold

scintillation cocktail (Perkin-Elmer, Waltman, Massachussets, USA).

Specific mixtures of nitrogen and oxygen were prepared using a gas mixer (Model 820,

Signal Group, UK). Concentrations of O2 in solution were calculated by taking the solubility at

25ºC in water as 257.5 μM in a saturated atmosphere at 100% relative humidity and correcting

for the atmospheric pressure (P) during measurements [257.5 × (P – 11598) / (101325 – 11589),

where 11589 Pa is the saturated vapour pressure of water at 25ºC and 101325 Pa is the saturated

atmospheric pressure]. The concentration of CO2 in solution in equilibrium with HCO3- was

calculated assuming a pKa1 for carbonic acid at 25ºC of 6.11 and an accurate measure of the pH

of the buffer used.

Values of Michaelis-Menten constants and maximum carboxylation velocity were

estimated using the EnzFitter (Biosoft: Software for Science, Cambridge, UK) package for

curve-fitting.




Jacklin Seed Company, USA) (PEPCK) were grown from seeds in trays or pots using peat-free

compost (Petersfield Products, Leicester, UK) supplemented with a slow-release fertiliser

(Hydro Agri Ltd, Lincs, UK) in a glasshouse. Artificial light was provided whenever the natural

light was below a photosynthetic photon flux density (PPFD) of 500 μmol m-2 s-1 during a 16 h


during the night. Seedlings from the trays were transplanted five to a pot. All pots were well-


153

watered until the beginning of the drought stress treatment. Pots were then placed according to a

split-plot design, where each column of pots was a main plot of a particular species and the

sampling-days and the treatments (control vs. drought stress) were randomised in the split-plots.

Each pot corresponded to one independent sample, with eight control and twelve non-watered

pots being used per species. For the RuBP/CA1P experiment, the number of control and stress

samples was duplicated and organized in two blocks in order to allow the imposition of a

light/dark regime, by restricted randomization.

Water deficit was imposed on the ‘stress’ pots by ceasing to provide water consecutively,

with one-day intervals, to the plants of C. dactylon, then Z. japonica and at last P. dilatatum. The

‘control’ pots were watered once per day. Samples were taken from all three species

simultaneously for four consecutive days during the drought period. Leaf samples were collected

in the growth environment under fully illuminated conditions four hours after the beginning of

the photoperiod. Because the Rubisco tight-binding inhibitor CA1P is synthesized during the

night, leaf samples for the estimation of RuBP and CA1P were collected early in the morning

both under fully-illuminated conditions and after a period of 12 hours of darkness.

Five-week old plants of P. dilatatum and C. dactylon and nine-week old plants of the

slow-growing Z. japonica were analysed simultaneously. Taking into account the different leaf

sizes, each sample of P. dilatatum consisted of two leaves, while each sample of the other two

grasses consisted of five leaves, taken from the same pot. The youngest fully expanded leaf of

each plant of P. dilatatum and two young fully expanded leaves of each plant of C. dactylon or Z.

japonica were always used. It was assumed that, within each pot, all the young fully expanded

leaves were identical in terms of developmental stage, physiological and biochemical properties,

and would have experienced the same drought condition. Therefore, two leaf sub-samples were

taken from each pot: the first was quickly frozen in liquid nitrogen (LN2) and then stored at -

80ºC for biochemical assays and the second was used to determine the leaf relative water content

(RWC). The fresh (FW), turgid (TW) and dry (DW) weights were determined and used to

calculate RWC by the equation RWC (%) = 100 × ((FW - DW) / (TW - DW)) (Catsky 1960).

The soil water content (SWC, %, v/v) was determined in three opposite locations in each pot

using an HH2 moisture meter with a Theta probe (type ML2x, AT Delta-t Devices Ltd.,

Cambridge, UK).

Rubisco extraction and activities

Rubisco was extracted from the leaves by grinding the LN2 frozen samples (0.1 to 0.4 g FW) in a

cold mortar with quartz sand, 1% (w/v) insoluble PVP and 10 (P. dilatatum and C. dactylon) or

Chapter V.

154

15 volumes (Z. japonica) of ice-cold extraction medium containing 50 mM Bicine-KOH pH 8.0,

1 mM EDTA, 5% (w/v) PVP25000, 6% (w/v) PEG4000, 10 mM DTT, 50 mM 2-mercaptoethanol

and 1% (v/v) protease inhibitor cocktail (Sigma, St Louis, MO, USA). After grinding to produce

a fine suspension, aliquots were taken for total chlorophyll determination and the remaining

homogenate was centrifuged for 3 min at 16 000g and 4ºC. The supernatant was used for

measuring the activities and amounts of Rubisco, with two analytical replicates for each

measurement.

The activities of Rubisco in the leaf extracts were determined immediately after

extraction by incorporation of 14CO2 into phosphoglycerate at 25ºC (Parry et al. 1997, with some

modifications). The reaction mixture (0.5 mL) contained 100 mM Bicine-NaOH pH 8.2, 20 mM

MgCl2, 10 mM NaH14CO3 (0.5 μCi μmol-1) and 0.4 mM RuBP. Initial activity was determined

by adding 25 μL of crude extract to the mixture and quenching the reaction after 60 s with 0.2

mL of 10 M formic acid. Total activity was measured after incubating the same volume of

extract for 3 min with all the reaction mixture components except RuBP in order to allow

carbamylation of all the Rubisco available catalytic sites. The reaction was then started by

adding RuBP and stopped as above. Maximal activity was measured after removal of Rubisco

tight binding inhibitors. For this purpose, 250 μL of crude extract were incubated with (final

concentrations) 200 mM Na2SO4, 10 mM NaHCO3 and 20 mM MgCl2 for 30 min at 4ºC.

Rubisco protein was precipitated with 20% polyethylene glycol 4000 (PEG4000) and 20 mM

MgCl2 and the tight binding inhibitors were removed by washing the enzyme three times with

20% PEG4000, 100 mM Bicine-NaOH pH 8.2, 20 mM MgCl2, 10 mM NaHCO3 and 50 mM 2-

mercaptoethanol. Rubisco free from tight binding inhibitors was re-dissolved in the extraction

buffer before performing the assay as for total activity. The mixtures were completely dried at

100ºC and the residues re-hydrated in 0.4 mL H2O and mixed with 3.6 mL Ultima Gold

scintillation cocktail (Perkin-Elmer, Waltman, Massachussets, USA). Radioactivity due to 14C

incorporation in the acid-stable products was measured by scintillation counting (liquid

scintillation analyser model 2100TR, Perkin-Elmer).

Chlorophylls

The total chlorophyll content in the leaf homogenates was determined spectrophotometrically

(NOVASPEC 4049 Spectrophotometer, LKB Biochrom Ltd. Cambridge, England) after

extraction in 96% ethanol (Wintermans & de Mots 1965).


155

Rubisco amounts

The amount of Rubisco in the leaf crude or inhibitor-free extracts, used for the determination of

initial and total or for the maximal activities, respectively, was determined by the [14C]-CABP

binding assay (Parry et al. 1997). For this purpose, 100 μL of crude extract were incubated with

(final concentrations) 100 mM Bicine-NaOH pH 8.0, 20 mM MgCl2, 10 mM NaHCO3, 50 mM

2-mercaptoethanol, 100 mM Na2SO4 and 75 μM [14C]-CABP (1 μCi μmol-1) for 15 min at 4ºC.

The Rubisco with bound [14C]-CABP was precipitated with 20% PEG4000 and 20 mM MgCl2.

The precipitate was washed three times with 20% PEG4000, 100 mM Bicine-NaOH pH 8.2, 20

mM MgCl2, 10 mM NaHCO3 and 50 mM 2-mercaptoethanol, and then re-dissolved in 500 μL

1% (v/v) Triton X-100. A sub-sample (450 μL) of this solution was mixed with 3.6 mL Ultima

Gold scintillation cocktail and the radioactivity due to [14C]-CABP bound to Rubisco catalytic

sites was measured by scintillation counting.

RuBP and Rubisco tight-binding inhibitors

RuBP and Rubisco tight-binding inhibitors contained in the LN2 frozen leaves (0.1 to 0.4 g FW)

were extracted by grinding the samples to a fine powder in LN2 and then add 0.45 M

trifluoroacetic acid (TFA; 0.25 mL per 100 mg FW). The mixture was ground further during

thawing and then duplicate sub-samples of the homogenate (20 μl) were taken for chlorophyll

determination. The remaining homogenate was centrifuged for 5 min at 16 000g and 4ºC. For the

estimation of RuBP, 50 μL of the leaf extracts were taken into a glass vial and dehydrated in

high vacuum over CaCl2 and NaOH. The residues were re-dissolved in 50 μL H2O, dried down

again and re-dissolved in 50 μL H2O. The RuBP contained in each vial was converted into

phosphoglycerate by incubating at room temperature (ca. 25ºC) for 45 min in a reaction mixture

(0.5 mL) containing 100 mM Bicine-NaOH pH 8.2, 20 mM MgCl2, 8 mM NaH14CO3 (0.5 μCi

μmol-1) and 20 μg of activated Rubisco. The reaction was quenched with 0.1 mL of 10 M formic

acid.

Leaf extracts were frozen in LN2 prior to CA1P estimation, by reference to a calibration

curve obtained simultaneously with standard solutions containing increasing concentrations of

the inhibitor (in 0.45 M TFA). From each standard or sample, 20 μL were used and incubated for

5 min with 230 μL of (final concentrations) 100 mM Bicine-NaOH pH 8.2, 20 mM MgCl2, 10

mM NaH12CO3 and 10 μg of activated Rubisco. After precisely 5 min, the reactions were started

by adding 250 μL of (final concentrations) 100 mM Bicine-NaOH pH 8.2, 20 mM MgCl2, 10

Chapter V.

156

mM NaH14CO3 (0.5 μCi μmol-1) and 0.4 mM RuBP and quenched after another 2.5 min with 0.1

mL of 10 M formic acid.

The mixtures were completely dried at 100ºC and the cold residues re-hydrated in 0.4 mL

H2O and mixed with 3.6 mL scintillation cocktail. Radioactivity due to 14C incorporation in the

acid-stable products was measured by scintillation counting.

Rubisco kinetic constants

Plants of wheat (C3 species used as reference) and each of the three C4 grasses were grown in

trays for determination of Rubisco kinetic parameters using young and fresh leaf material. Leaf

samples (0.5 g FW) were quickly frozen into LN2 and used within one day. Rubisco was

extracted from the leaves by grinding the LN2 frozen samples in a cold mortar with quartz sand,

2.5% (w/v) insoluble PVP and 5 volumes (2.5 mL) of ice-cold extraction medium containing 100

mM Bicine-KOH pH 8.2, 0.1 mM EDTA, 6% (w/v) PEG4000, 10 mM DTT, 50 mM 2-

mercaptoethanol, 2 mM MgCl2, 10 mM NaHCO3, 1 mM benzamidine, 1 mM ε-aminocaproic

acid and 1% (v/v) protease inhibitor cocktail (Sigma). After grinding to produce a fine

suspension, the homogenate was centrifuged for 4 min at 16 000g and 4ºC. Low molecular

weight proteins and salts present in the leaf crude extracts were removed by passage of 1 mL of

the supernatant though a Sephadex G-200 (GE Healthcare Life Sciences) column (20 mL bed

volume, 1.5 × 11.5 cm) pre-equilibrated and developed using desalt buffer, containing 100 mM

Bicine-KOH pH 8.2, 0.5 mM EDTA, 1 mM KH2PO4, 20 mM MgCl2, 10 mM NaHCO3, 1 mM

benzamidine and 1 mM ε-aminocaproic acid. Fractions of 0.5 mL were collected and after

measuring the soluble protein content by the method of Bradford (1976), measuring the

absorbance at 595 nm (SpectraMax 340 PC, Molecular Devices Ltd., Workingham, UK, with the

software SoftMax Pro V5), the three protein-richest fractions were combined and supplemented

with 2.5% (v/v) protease inhibitor cocktail (Sigma), carefully mixed and divided into aliquots for

the analysis to be performed. Some of the aliquots were immediately frozen into LN2 (for later

measurement of Rubisco amount and for appropriate control assays).

All measurements for determination of kinetic parameters were conducted at 25ºC. The

Michalis-Menten constant for CO2 (Kc) was measured essentially as described by Bird et al.

(1982) and Ko was estimated by measuring Kc-apparent at several O2 concentrations (0, 21, 60 and

100%, balanced with N2). The carboxylation activity of Rubisco was determined at several

concentrations of CO2 for each gas-mixture. The assay buffer was pre-treated by bubbling

through a steady stream of the appropriate gas-mixture. Septum-sealed vials with stirring


157

magnets were connected in series through the septa using butyl rubber transfer tubes fitted at

each end with hypodermic needles and flushed with the appropriate CO2-free gas-mixtures at 20

μL min-1 for at least 30 min. Flushing of the vials was disconnected after addition of buffer but

before addition of the NaH14CO3. The reaction mixtures (1 mL) in the vials used for each gas-

mixture contained (final concentrations) 100 mM Bicine-NaOH pH 8.2, 20 mM MgCl2, 10 mg

mL-1 carbonic anhydrase (freshly dissolved), 0.4 mM RuBP and six different concentrations of

NaH14CO3 (0-10 mM; 1 μCi μmol-1). The reactions were started with 30 s intervals by the

addition of 20 μL of leaf extract previously activated by incubation with 10 mM NaH14CO3 (1

μCi μmol-1) for 30 min. The reactions were quenched after 2 min with 0.1 mL of 10 M formic

acid. Changes in the activity of Rubisco through the course of the assays were monitored by the

use of replicates of the same vial at staggered time points. The acidified mixtures were dried at

100ºC and the residues re-hydrated in 0.4 mL H2O and mixed with 3.6 mL scintillation cocktail.

Radioactivity due to 14C incorporation in the acid-stable products was measured by scintillation

counting.

Several control assays were performed in order to assure the presence of 14C

incorporation in the conditions of the assay at saturating CO2 and low O2 concentrations

(positive control) and the absence of 14C incorporation in the absence of the substrate RuBP,

when RuBP had been replaced by phosphoglycerate or when partially purified Rubisco had been

pre-incubated with CABP to block the catalytic sites (negative controls). The amount of Rubisco

in the partially purified extracts was determined essentially as described above.

Rubisco specificity factor

Rubisco was purified from fresh leaves of wheat and of each of the three C4 grasses grown in

trays and used to determine the specificity factor by total consumption of RuBP in the oxygen

electrode (Parry et al. 1989). The purification consisted of Rubisco precipitation from the leaf

extracts with 20% PEG4000, step elution from an anion exchange column and desalting (Haslam

et al. 2005).



Research, UK). Regression analysis was applied to model the variation of the different

measurements with RWC. Non-significantly different (P > 0.05) parameters (t-tests) in the

Chapter V.

158

significant model terms of the regression (F-tests, P < 0.05) were amalgamated in order to attain

parsimony. The resulting best models were plotted and the parameter estimates with their

respective standard errors (s.e.), the percentage of variance accounted for by the model (R2), the

residual mean square (s2) and the degrees of freedom (d.f.) are given with the plots. Additionally,

the method of Residual Maximum Likelihood (REML) was used to model the different

measurements and assess the statistical significance of species, treatment (control vs. drought

stress) and light/dark regime (when applicable). The significance of the different effects was

assessed through an F-test (Welham & Thompson 1997) on the appropriate degrees of freedom.

Mean values were estimated for the control and drought-stressed plants of each species. The least

significant differences at the 5% level (LSD(5%)) considering all data for each measurement are

given as a reference. The residuals from the various models generally conformed to the

assumptions of the analysis.


159

RESULTS

Drought stress induction

The leaf relative water content (RWC) decreased in response to decreased water availability in

the soil, as measured by the soil water content (SWC), in the non-watered pots of all three

species (Table V.1). However, RWC was less decreased in the drought-stressed samples

collected in the dark, at the end of the 12 hours night period, than in samples collected four hours

after the beginning of the photoperiod. The results obtained with the plants for measurement of

Rubisco activities and amounts (not shown) were similar to those obtained with the light samples

used for estimation of RuBP and CA1P contents.

Table V.1. The soil water content (SWC) and leaf relative water content (RWC) of control (C) and non-watered (S) plants of P. dilatatum, C. dactylon and Z. japonica used for the estimation of RuBP and CA1P contents, in the light or after a period of 12 hours in darkness overnight. The mean values and respective standard errors were calculated from measurements taken with eight control and twelve drought-stressed samples of each species.

Water relations P. dilatatum C. dactylon Z. japonica

SWC in the Light C 34.7 ± 0.6 37.1 ± 0.6 38.4 ± 0.4 (%) S 7.6 ± 0.8 9.0 ± 0.6 11.2 ± 0.6

SWC in the Dark C 36.1 ± 0.9 38.9 ± 0.4 38.5 ± 0.6 (%) S 8.1 ± 0.9 9.5 ± 0.7 11.4 ± 0.8

RWC in the Light C 98.2 ± 0.1 97.9 ± 0.1 97.4 ± 0.3 (%) S 86.8 ± 3.8 95.0 ± 0.5 94.1 ± 1.0

RWC in the Dark C 99.4 ± 0.1 99.3 ± 0.3 98.8 ± 0.1 (%) S 97.2 ± 0.6 98.4 ± 0.2 97.1 ± 0.3

Rubisco activities and amounts

The initial and total activities of Rubisco decreased with decreased RWC but maximal activities

of the enzyme were not significantly affected (P > 0.05) by leaf dehydration (Figure V.1). The

initial activity was higher in P. dilatatum than in C. dactylon and lowest in Z. japonica but no

significant differences (P > 0.05) were observed in the total or the maximal activities of the

enzyme from P. dilatatum and C. dactylon, which were higher than in Z. japonica.

Chapter V.

160

RWC (%)

75 80 85 90 95 100

Max

imal

s (μ

mol

min

-1 m

g-1 C

hl)

0.0

1.0

2.0

3.0

4.0

75 80 85 90 95 100

Tota

ls (μ

mol

min

-1 m

g-1 C

hl)

0.0

1.0

2.0

3.0 D

G

75 80 85 90 95 100

Initi

als

(μm

ol m

in-1

mg-1

Chl

)

0.0

0.5

1.0

1.5

2.0 A

y = 0.440 + 0.01067 x (s.e. 0.060; 0.00045)

y = 0.212 + 0.02120 x (s.e. 0.065; 0.00057)

2.57 0.06±

RWC (%)

75 80 85 90 95 100

75 80 85 90 95 100

75 80 85 90 95 100

RWC (%)

75 80 85 90 95 100

75 80 85 90 95 100

75 80 85 90 95 100

E

H

B

y = 0.165 + 0.01067 x (s.e. 0.059; 0.00045)

y = 0.212 + 0.02120 x (s.e. 0.065; 0.00057)

2.57 0.06±

F

I

C

y = 0.01067 x (s.e. 0.00045)

y = 0.02120 x (s.e. 0.00057)

2.17 0.08±


Figure V.1. Rubisco initial (A-C), total (D-F) and maximal (G-I) activities (μmol min-1 mg-1 Chl) as a function of the relative water content (RWC, %) in the leaves of P. dilatatum (A, D, G; black diamonds), C. dactylon (B, E, H; grey squares) and Z. japonica (C, F, I; white triangles). Initials were determined immediately after extraction, totals were determined after incubation in the presence of CO2 and Mg2+ and maximals were determined after removal of tight-binding inhibitors with sulphate. Each data point corresponds to one sample (with seven (P. dilatatum and Z. japonica) or eight (C. dactylon) control and twelve non-watered samples per species). The regression lines applied correspond to the best models statistically significant (Initials, R2 = 50.5%, s2 = 0.033, d.f. = 55, P < 0.001; Totals, R2 = 32.5%, s2 = 0.052, d.f. = 56, P < 0.001; Maximals, no regression on RWC, P > 0.05)


161

RWC (%)

75 80 85 90 95 100

RWC (%)

75 80 85 90 95 100

Activ

atio

n St

atus

(%)

0

20

40

60

80

RWC (%)

75 80 85 90 95 100

Inhi

bitio

n St

atus

(%)

0

10

20

30

40 y = 62.2 - 0.540 x (s.e. 18.7; 0.200)

67.3 1.5 51.2 1.0± ±

y = 62.2 - 0.540 x (s.e. 18.7; 0.200) y = 62.2 - 0.540 x (s.e. 18.7; 0.200)

51.2 1.0±

A B C


D E F

Figure V.2. Activation status (A-C, %) and inhibition status (D-F, %) as a function of the relative water content (RWC, %) in the leaves of P. dilatatum (A, D; black diamonds), C. dactylon (B, E; grey squares) and Z. japonica (C, F; white triangles). The activation and inhibition status were calculated as 100×Initial/Total and 100×Total/Maximal, respectively, using specific activities (μmol min-1 mg-1 Rubisco). Each data point corresponds to one sample (with seven (P. dilatatum and Z. japonica) or eight (C. dactylon) control and twelve non-watered samples per species). The regression line applied corresponds to the best model statistically significant (Activation status, no regression on RWC, P > 0.05; Inhibition status, R2 = 10.0%, s2 = 95.9, d.f. = 56; P = 0.009).

Rubisco activation status, calculated as a percentage of the initial to the total activity of

the enzyme, measured immediately upon extraction or after pre-incubation of the enzyme in the

presence of CO2 and Mg2+, respectively, was higher in P. dilatatum than in C. dactylon and Z.

japonica and was not significantly affected (P > 0.05) by leaf dehydration (Figure V.2A-C).

Conversely, the inhibition status, calculated as a percentage of the total to the maximal activity

of the enzyme, measured in the same conditions but with removal of inhibitors tightly-bound to

the enzyme before assaying maximal activity, was not significantly different (P > 0.05) among

the three species and was similarly increased with decreasing RWC (Figure V.2D-F).

Chapter V.

162

The amount of Rubisco was higher in P. dilatatum than in C. dactylon and lowest in Z.

japonica and remained unchanged (P > 0.05) with decreasing RWC (Figure V.3). However,

when the mean values obtained for control and drought-stressed plants were compared there was

a significant effect (P = 0.005) of drought on the Rubisco amount, with decreased contents

compared to the control (Table V.2). As a consequence, Rubisco initial and maximal, but not

total, specific activities were higher in drought-stressed plants relative to the control.

RWC (%)

75 80 85 90 95 100Rub

isco

cat

alyt

ic s

ites

(nm

ol m

g-1 C

hl)

0

10

20

30

26.6 0.8±

RWC (%)

75 80 85 90 95 100

RWC (%)

75 80 85 90 95 100

24.4 0.8 21.0 0.8± ±

A B C


Figure V.3. Rubisco catalytic sites (nmol mg-1 Chl), as a function of the relative water content (RWC, %) in the leaves of P. dilatatum (A, black diamonds), C. dactylon (B, grey squares) and Z. japonica (C, white triangles). Each data point corresponds to one sample (with seven or eight control and twelve non-watered samples per species). There was no significant regression on RWC (P > 0.05).

Table V.2. Rubisco initial (I), total (T) and maximal (M) specific activities and Rubisco amounts in the leaves from control and drought-stressed plants. Values are means of seven (P. dilatatum and Z. japonica) or eight (C. dactylon) control and twelve non-watered samples for each species, as given by the REML models. The analysis revealed significant effects of species on total (P = 0.010) and maximal activities (P = 0.001) and amount (P < 0.001) and significant effects of drought stress on initial (P = 0.045) and maximal activities (P = 0.030) and amount (P = 0.005). There were no other significant effects neither significant interactions (P > 0.05) between species and stress. The average standard errors of differences (SED) for each variable considering all data are presented as a reference.

P. dilatatum C. dactylon Z. japonica Rubisco CONTROL DROUGHT CONTROL DROUGHT CONTROL DROUGHT

SED (49 d.f.)

I 0.71 0.83 0.66 0.72 0.65 0.71 0.07

T 1.17 1.18 1.32 1.36 1.36 1.35 0.09 Specific activities

(μmol min-1 mg-1 Rubisco) M 1.30 1.39 1.45 1.60 1.46 1.50 0.07

Amount (mg mg-1 Chl) 1.93 1.83 1.83 1.63 1.63 1.38 0.11


163

RuBP and Rubisco tight-binding inhibitors

The RuBP content was greater in fully hydrated leaves of C. dactylon than in Z. japonica and

lowest in P. dilatatum (Figure V.4A). In all three species a marked decrease with RWC was

observed, but the decrease was much steeper in C. dactylon than in Z. japonica and very slight in

P. dilatatum, resulting in similar RuBP contents for an RWC of ca. 87%. The contents present in

the dark-samples were very low but still denoted a significant, very slight, decrease with RWC

RWC (%)

60 70 85 90 95 100

0

1

2

RuB

P (n

mol

mg-1

Chl

)

20

40

60

80

100

120

140 A

Dark

Light

y = -0.409 + 0.0064 x (s.e. 0.064; 0.0005)y = 0.0064 x (s.e. 0.0005)

y = 0.3285 x (s.e. 0.0358)y = -553 + 6.7 x (s.e. 164; 1.7)y = -277 + 3.5 x (s.e. 100; 1.1)

P. dilatatumC. dactylonZ. japonica

RuB

P (n

mol

mg-1

Chl

)

0

20

40

60

80

100

P. dilatatum C. dactylon Z. japonica0.0

0.5

B

Dark

LightLSD(5%)

Drought

Species

Figure V.4. (A) RuBP amounts (nmol mg-1 Chl), as a function of the relative water content (RWC, %) in the leaves of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (white triangles) collected in the light and after 12 hours in darkness. Each data point corresponds to one sample (with seven (P. dilatatum and Z. japonica) or eight (C. dactylon) control and twelve non-watered samples per species per light regime). The regression lines applied correspond to the best models statistically significant (P. dilatatum, solid lines; C. dactylon, dashed lines; Z. japonica, dotted lines; Light, R2 = 75.6%, s2 = 216.7, d.f. = 54; P < 0.001; Dark, R2 = 40.5%, s2 = 0.0556, d.f. = 58; P < 0.001). (B) RuBP amounts (nmol mg-1 Chl) in the leaves from control (black bars) and non-watered plants (white bars) of P. dilatatum, C. dactylon and Z. japonica collected in the light and after 12 hours in darkness. Values are means of all control or non-watered samples for each species and light regime. REML analysis revealed significant differences (P < 0.001) between species and between treatments (control vs. drought stress) with no significant interaction (P > 0.05) in the light, and no significant differences (P > 0.05) in the dark. The overall least significant difference at the 5% level (LSD(5%)) is 7.823 for species and 6.375 for treatment (104 d.f.).

Chapter V.

164

(P = 0.029). Analysis of the mean values obtained for the control and the drought-stressed

samples revealed lower RuBP contents under drought conditions in the light but no significant

differences (P > 0.05) in the dark (Figure V.4B).

The amount of CA1P present in the leaves collected after a period of 12 hours in

darkness was much greater in Z. japonica than in C. dactylon or P. dilatatum (Figure V.5A) and

increased with decreasing RWC, in a similar extent for the three species (with no significant

interaction (P > 0.05) between species and treatment). Much lower amounts of the nocturnal

inhibitor were found in the leaves collected under fully illuminated conditions (Figure V.5B),

with no significant differences (P > 0.05) between the three species and a very slight but

significant (P = 0.002) increase with decreasing RWC. Comparing the mean values of CA1P

present in the control and drought-stressed plants, no significant differences (P > 0.05) between

species or treatments were observed in the light. The inhibitor amount in the dark-samples was

much greater in Z. japonica than in C. dactylon and lowest in P. dilatatum, and was higher in the

leaves from drought-stressed than from well-watered plants (Figure V.5C).

In a preliminary assay, the total and maximal activities of Rubisco were determined for

one single sample of control or drought-stressed plants of each species collected in a similar

light/dark regime to that used for the CA1P experiment above. The results of the assay (Table

V.3) suggested a much greater difference in the inhibition status (determined as a percentage of

the total to maximal activity) between the dark and light samples in Z. japonica than in P.

dilatatum or C. dactylon. This suggests the presence of the nocturnal inhibitor in Z. japonica and,

additionally, the possibility for increased amounts under drought conditions.

Table V.3. Rubisco inhibition status (%) in leaves sampled after a period of 12 hours in darkness or in fully illuminated conditions. Results obtained for a preliminary assay using one single sample of control or non-watered plants of P. dilatatum, C. dactylon and Z. japonica grown and harvested in identical conditions to those used for CA1P quantification. The inhibition status was calculated as a percentage of the total to the maximal specific activities (μmol min-1 mg-1 Rubisco).

INHIBITION P. dilatatum C. dactylon Z. japonica (%) CONTROL DROUGHT CONTROL DROUGHT CONTROL DROUGHT

Dark 98 95 89 85 87 73

Light 109 95 94 79 115 113

Light - Dark 11 0 5 -6 28 40


165

RWC (%)

60 70 85 90 95 100

Inhi

bito

r (nm

ol m

g-1 C

hl)

0.0

0.5

1.0

1.5

2.0

94 96 98 100

CA1

P (n

mol

mg-1

Chl

)

0

2

4

6

8

10ADark

BLight

y = 36.0 - 0.359 x (s.e. 10.2; 0.104)y = 36.8 - 0.359 x (s.e. 10.3; 0.104)y = 40.4 - 0.359 x (s.e. 10.2; 0.104)

y = 2.27 - 0.0201 x(s.e. 0.57; 0.0060)

P. dilatatumC. dactylonZ. japonica

CA1

P (n

mol

mg-1

Chl

)

0

1

2

3

4

5

6

P. dilatatum C. dactylon Z. japonica0.0

0.5

C

Light

DarkLSD(5%)

Drought

Species

Figure V.5. (A-B) CA1P amounts (nmol mg-1 Chl), as a function of the relative water content (RWC, %) in the leaves of P. dilatatum (black diamonds), C. dactylon (grey squares) and Z. japonica (white triangles) collected after a period of 12 hours in darkness overnight (A) or in fully illuminated conditions (B). Each data point corresponds to one sample (with seven (P. dilatatum and Z. japonica) or eight (C. dactylon) control and twelve non-watered samples per species per light regime). The regression lines applied correspond to the best models statistically significant (P. dilatatum, solid lines; C. dactylon, dashed lines; Z. japonica, dotted lines; Dark, R2 = 79.1%, s2 = 1.138, d.f. = 56; P < 0.001; Light, R2 = 15.0%, s2 = 0.0805, d.f. = 56; P = 0.002). (C) CA1P amounts (nmol mg-1 Chl) in the leaves from control (black bars) and non-watered plants (white bars) of P. dilatatum, C. dactylon and Z. japonica collected in the light and after 12 hours in darkness. Values are means of all control or non-watered samples for each species and light regime. REML analysis revealed significant differences (P < 0.001) between species and between treatments (control vs. drought stress) with no significant interaction (P > 0.05) in the dark, and no significant differences (P > 0.05) in the light. The overall least significant difference at the 5% level (LSD(5%)) is 0.525 for species and 0.428 for treatment (103 d.f.).

Chapter V.

166

Rubisco kinetic parameters

Rubisco purified from each of the three C4 grasses was characterized by lower specificity factors

(SF) than in wheat (C3 species used as reference) (Table V.4). The Michaelis-Menten constants

of Rubisco for CO2 and O2 (Kc and Ko) estimated for each of the C4 species were in the same

range and were higher than the values estimated for wheat. The maximum Rubisco carboxylation

activity (Vc) was also higher in the C4 than in the C3 species and among the grasses was higher in

Z. japonica than in C. dactylon and lowest in P. dilatatum. Conversely, the maximum Rubisco

oxygenation activity (Vo) was not much different between wheat, P. dilatatum and C. dactylon

but was highest in Z. japonica. The replication was reasonably good, with low standard errors

associated with the mean values estimated for each of the kinetic parameters.

Table V.4. Kinetics parameters of Rubisco from wheat and from the three C4 grasses: P. dilatatum, C. dactylon and Z. japonica, at 25ºC. For the Michaelis-Menten constants of Rubisco for CO2 and O2 (Kc and Ko, μM) and the maximum Rubisco carboxylation and oxygenation activities (Vc and Vo, μmol min-1 mg-1 Rubisco) the mean values and respective standard errors were calculated from measurements taken with three biological and three analytical replicates. The specificity factor (SF) was determined in Rubisco purified from each species using a minimum of five analytical replicates.

SF Kc Ko Vc Vo Species (VcKo/VoKc) (μM) (μM) (μmol min-1 mg-1 Rubisco)

Wheat (C3) 100.0 ± 9.2 10.9 ± 0.9 341 ± 33 2.54 ± 0.07 0.79 ± 0.03

P. dilatatum 88.0 ± 7.1 19.9 ± 0.8 415 ± 5 3.11 ± 0.04 0.71 ± 0.03

C. dactylon 89.2 ± 9.0 21.0 ± 1.3 402 ± 27 3.41 ± 0.18 0.73 ± 0.06

Z. japonica 84.1 ± 7.7 18.5 ± 1.2 403 ± 27 3.78 ± 0.08 0.98 ± 0.08


167

DISCUSSION

Rubisco activities and substrate availability under drought stress

In all three C4 grasses, Rubisco initial activity showed down-regulation under drought stress with

decreasing activity as leaf dehydration increased (Figure V.1). The down-regulation of Rubisco

can be explained by decreased carbamylation or by the presence of competitive and tight-binding

inhibitors. Carbamylation may be impeded by negative effectors (Hatch & Jensen 1980; Badger

& Lorimer 1981; Jordan et al. 1983) or RuBP bound into the non-carbamylated sites (Brooks &

Portis 1988). Total activity was also decreased with leaf dehydration suggesting an increase in

tight-binding inhibitors. In fact, both initial and total activities decreased by ca. 20-25% when

the RWC decreased from control values to 75% in drought-stressed plants. Similar results were

obtained for C. dactylon and Z. japonica in a previous study on the effects of water deficit

rapidly-imposed by the addition of PEG4000 to the nutrient solution (Carmo-Silva et al. 2004;

2007). The discrepancy between the results reported here and the unchanged activities of

Rubisco in P. dilatatum in that previous study, when leaves were rapidly dehydrated, support the

view that plant responses to drought depend greatly on the rate of water deficit imposition

(Chaves et al. 2003; Flexas et al. 2004).

The initial activity of Rubisco was higher in P. dilatatum than in C. dactylon and Z.

japonica, reflecting both the higher activation state and higher amounts of the enzyme in the

former species (Figures V.2 and V.3). The lowest activities of Rubisco were found in Z. japonica

and this is consistent with the lower amounts of Rubisco protein in the leaves of this species

compared to the other two C4 grasses. As a consequence, the specific activities of the enzyme

were either lower in P. dilatatum than in C. dactylon and Z. japonica (total and maximal) or not

different between the three species (initial, due to higher activation state in P. dilatatum). The

maximal activities (Figure V.1) and enzyme amounts (Figure V.3) showed no statistically

significant trend as RWC decreased. However, in the comparison of mean values obtained for all

control with all drought-stressed plants, Rubisco amount was significantly affected (P = 0.005),

decreasing under drought, but the decrease was very slight. Marques da Silva & Arrabaça

(2004b) reported decreased Rubisco initial and total activities with unchanged enzyme amount in

slowly-dehydrated leaves of Setaria sphacelata, whilst Lal & Edwards (1996) found decreased

initial activities concomitant with decreased enzyme amounts in Amaranthus cruentus but not in

Zea mays. Overall, the results obtained for the Rubisco activities and amounts suggest that, in the

Chapter V.

168

three C4 grasses studied, the main effect of drought on the enzyme activity is a slightly increased

inhibition of the enzyme.

The decrease in RuBP content as leaf dehydration increased (Figure V.4) was large in C.

dactylon and Z. japonica but small in P. dilatatum. Whilst Tezara et al. (1999) observed a

dramatic drop in RuBP in the C3 plant, Helianthus annuus and Bota et al. (2004) found

decreased RuBP contents in several C3 species under severe drought conditions, Lal & Edwards

(1996) reported a slight increase of RuBP in Zea mays as water deficit was established. Although

the drought-induced decrease in ATP was less dramatic than the decrease in RuBP in H. annuus,

it has been concluded that decreased ATP synthesis in response to stress conditions caused

decreased RuBP regeneration (Tezara et al. 1999; Lawlor 2002). The mean nmol of catalytic

sites (Et) of Rubisco in P. dilatatum per mg of chlorophyll was 27 (Figure V.3) whilst the

amount of RuBP as nmol per mg chlorophyll (Rt) was only marginally above this at ca. 34 even

in well watered plants (Figure V.4). Even taking into account that only some 60-70% (Figure

V.2) of the catalytic sites were active the ratio Rt/Et is less than 2 and so approaching the range

where RuBP can be considered limiting (Lawlor 2002). The RuBP content in C. dactylon and Z.

japonica was much higher in control plants, giving Rt/Et ratios of ca. 3.5 and 2.9, respectively,

but the values fell steeply as RWC decreased, to give ratios similar to those for P. dilatatum at

85% RWC. These results support the conclusion that the amounts of RuBP in the leaves of all

three species at the lowest values of RWC (Figure V.4) are close to the critical value for RuBP-

dependent limitation of photosynthesis (Lawlor 2002).

Rubisco regulation by tight-binding inhibitors

Inhibition of Rubisco activity in Figure V.5 is interpreted as due to CA1P but could be in part

due to inhibition by PDBP, especially in the light (Parry et al. 2002). The drought-induced

increase in the amounts of tight-binding inhibitors, estimated by inhibition of purified Rubisco,

agrees fairly well with the increased inhibition of Rubisco from each species, as estimated from

the percentage of total to maximal activities in a preliminary experiment (Table V.3). The

inhibitor in the acid extracts of leaves collected in the dark is most likely to be CA1P, with most

in Z. japonica and least in P. dilatatum, but the identity needs to be confirmed. Sage & Seemann

(1993) have previously reported regulation of Rubisco at low irradiance in Z. japonica that may

have been due to accumulation of CA1P. No indications were then obtained for the presence of

CA1P in P. dilatatum or C. dactylon.

The leaf relative water content is re-established during the night, through decreased

evaporation from the leaves and continued water absorption from the soil. As a result, samples


169

collected from non-watered pots at the end of the 12 h dark period had higher RWC values than

samples collected in the light period (Table V.1), but generally greater Rubisco inhibition.

Therefore, the increased inhibitor content in the non-watered plants (Figure V.5) is more likely

to reflect down-regulation of leaf metabolism under drought conditions than a direct effect of

leaf dehydration. Increased synthesis of tight-binding inhibitors may protect Rubisco from

proteolysis in the stressed leaves (Khan et al. 1999). The extent of inhibition in the dark samples

measured in vitro, compared with the number of Rubisco catalytic sites present in the leaves in

vivo, predicts inhibition to be of the order of 25%, 5% and 2.5% respectively for Z. japonica, C.

dactylon and P. dilatatum (Figures V5.A and V.3) and to increase in all species with drought

stress. A single preliminary assay (Table V.3) confirms a much higher inhibition in dark leaf

samples of Z. japonica (above 25% and increasing with drought) than in P. dilatatum and C.

dactylon (inconclusive), as estimated from the difference between total and maximal activities. It

is most likely that inhibition in these samples is due to CA1P although to the best of our

knowledge the presence of CA1P in Zoysia species has not been unequivocally demonstrated.

There was a slight increase in estimated tight-binding inhibitors in the light with

decreasing RWC but this was mostly caused by the high values obtained for a few samples with

lower RWC values. The predicted amounts based on the CA1P standard were less than 1.5 nmol

per mg chlorophyll (Figure V.5B) and are therefore sufficient to account for less than 6%

inhibition of Rubisco while the inhibition suggested by the difference between total and maximal

activity appears to be larger than this (Figure V.2.B). Rubisco total activity decreased in wheat

and tobacco under drought stress (Parry et al. 1993; 2002) and this was attributed partly to

increased amounts of the daytime inhibitor (PDBP), rather than CA1P (Parry et al. 2002). The

extent of inhibition of Rubisco in vivo by PDBP is very difficult to assess accurately because of

its extreme lability. Varying rates of Rubisco degradation, and turnover, in different species

under normal conditions (Esquível et al. 1998) may be associated with different inhibitor

contents in the leaves, and the presence of these inhibitors may confer competitive advantage

under unfavourable environmental conditions (Parry et al. 2008).

The modulation of Rubisco activity in vivo, both through carbamylation and the binding

of inhibitors, is affected by Rubisco activase, the enzyme that promotes Rubisco activation

(Spreitzer & Salvucci 2002; Portis 2003). Rubisco activase plays a central role in photosynthesis

limitation under heat stress (Crafts-Brandner & Salvucci 2000; Salvucci & Crafts-Brandner

2004) due to its low thermal stability (Salvucci et al. 2001). To the best of our knowledge, the

effects of drought stress on Rubisco activase have not been reported. Due to the ATP

requirement for Rubisco activation through Rubisco activase (Portis et al. 2008), it is likely that

Chapter V.

170

the event of impaired ATP synthesis by water deficit (Lawlor 2002) and the resulting high

ADP/ATP ratio, would decrease the activity of Rubisco activase, thereby affecting Rubisco

activation. The results presented here support the view that in the three C4 grasses studied

Rubisco modulation occurs in order to match the capacity for RuBP regeneration with the

capacity for RuBP utilization. This modulation is essentially due to Rubisco inhibition as the

enzyme amount is not affected and the activation status remains unchanged.

Kinetic properties of Rubisco from the C4 grasses

The data presented in Table V.4 support the view that Rubisco from C4 plants is less specific for

CO2 but has the potential for faster rates of carboxylation than the enzyme from C3 species (von

Caemmerer 2000; Kubien et al. 2008). Using appropriate controls, it was possible to estimate Kc,

Ko and Vc using partially purified extracts of Rubisco from the leaves of wheat and each of the

three C4 grasses. Attempts were made to measure Vo by an HPLC method based on that of

Yaguchi et al. (1996) to measure the amounts of 3-phosphoglycerate and 2-phosphoglycolate

produced by the oxygenation of RuBP at increasing concentrations of O2, using similar enzyme

extracts. However, these attempts were unsuccessful. As a consequence, Vo was estimated

indirectly by measuring the specificity factor and solving the equation SF = VcKo/VoKc. The

errors on the estimates of SF were too high to allow much precision to be credited to the

estimates of Vo. The Ko of Rubisco is the kinetic parameter for which there is the greatest

variation reported in the literature (see von Caemmerer 2000), with estimates generally in the

range of 200-650 μM being obtained for C3 species. The values obtained in the present study for

the three C4 grasses (ca. 400-420 μM) were higher than the value obtained for wheat, supporting

the conclusion that Rubisco from C4 species may have higher Ko, as well as higher Kc, than

Rubisco from C3 species (von Caemmerer 2000). In view of the effects of drought on the

amounts of RuBP in the leaves, a measure of Km for RuBP (KRuBP) would be a necessary

addition to the kinetic constants to be considered in future studies. Values for KRuBP for many C4

plants have been published (Yeoh et al. 1981) showing a wide range, which suggests that

measurements are needed for any species where the objective is the mechanistic modelling of

photosynthesis.


171

CONCLUSIONS

Rubisco activity was down-regulated in all three C4 grasses and the extent of down-regulation

was increased under drought stress conditions. A decreased capacity for RuBP regeneration with

drought is likely to be a major factor controlling the photosynthetic rate, at least in C. dactylon

and Z. japonica. Evidence was found for the presence of tight-binding inhibitors in the leaves of

the C4 grasses analysed, but the values measured were insufficient to account for the difference

between total and maximal activities of Rubisco in plants in the light. The nature of the inhibitors

that play a role in the inhibition of Rubisco in the light in the three species needs further

investigation. In Z. japonica there was strong evidence for a tight-binding inhibitor in the dark,

possibly CA1P. The kinetic properties of the Rubiscos from the three C4 grasses show a lower

affinity for CO2 and a higher Vc than the enzyme from C3 plants. These characteristics fit the

enzymes for high carboxylation capacity in the environment of the bundle sheath with its high

CO2 concentration.

Chapter V.

172

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Chapter VI.

GENERAL DISCUSSION AND CONCLUSIONS

General Discussion and Conclusions

181

GENERAL DISCUSSION AND CONCLUSIONS

Drought resistance in C4 grasses

The use of plant species and varieties better adapted to conditions of low water availability and

the production of new varieties with improved performance by traditional or molecular breeding

will improve the use of water available. The C4 grasses Paspalum dilatatum, Cynodon dactylon

and Zoysia japonica and their adaptive response to water deficit were studied in order to

understand the mechanisms underlaying their photosynthetic performance under conditions of

decreased water availability. The three species showed some differences in their drought-stress

physiology and biochemistry but also some responses to gradually induced drought stress

conditions that are common to many plant species (see comprehensive review by Chaves et al.

2003). The decreased shoot growth and photosynthesis are an adaptive response to decreased

water availability and allow energy to be diverted to the production of molecules involved in

stress defence mechanisms (Chaves & Oliveira 2004). Plants adopt diverse strategies to maintain

water status under water deficit (Schulze 1986). In all three C4 grasses, stomatal closure was an

early event in the response to water deficit and decreased stomatal conductance contributed to

the maintenance of control RWC values in the earlier stages of water deficit. Decreased LWP

also contributed to minimize water loss. Additionally, in Z. japonica more severe dehydration is

impeded by leaf curling simultaneously with continuous lowering of the LWP, which increases

the capacity for water absorption from the soil. Deep root systems have been observed in C.

dactylon and Z. japonica (Marcum et al. 1995; Carrow 1996) and we may suggest that increased

root/shoot ratio through sustained root growth concomitantly with decreased shoot growth rates

may help to retrieve more water from the soil. Decreased shoot growth and stomatal conductance

are among the primary effects of decreased water availability in the soil that commonly occur in

many plant species (Chaves et al. 2003) and were observed in the three C4 grasses.

The leaf structure of Z. japonica and the response of this species to water deficit in terms

of leaf water relations (Chapter II) together with the presence of rigid cells walls in association

with its drought resistance (White et al. 2001) support the view that this species is well adapted

to xeric environments. The faster decrease in water availability in the soil observed with P.

dilatatum reflects the high productivity of these plants at the expense of higher water

consumption. This is also a consequence of the different phenotype, with bigger and fewer

leaves, and the lower ratio of dry weight to turgid weight than in the other two C4 grasses.

Chapter VI.

182

The lower drought resistance of P. dilatatum (NADP-ME, subfamily Panicoideae)

compared to C. dactylon (NAD-ME, subfamily Chloridoideae) and Z. japonica (PEPCK,

subfamily Chloridoideae) agree to some extent with several reports on C4 grass distributions

suggesting that dominance of NADP-ME species in comparison to the other C4 subtypes is

positively correlated with precipitation gradients (see Cabido et al. 2008). It has been suggested

that the C4 subtype of photosynthetic pathway may be relevant for the ecology and management

of desertified grasslands, with persistent and drought resistant NAD-ME grasses potentially more

fitted to arid regions (Hattersley 1992). However, the lower resistance of P. dilatatum is also in

agreement with the high correlation of the grass subfamily Panicoideae distribution with higher

precipitation levels compared to the subfamily Chloridoideae (Taub 2000). In C3- and C4-like

subspecies of Alloteropsis semialata (Ueno & Sentoku 2006) responses to drought were

contradictory to the paradigm of higher drought resistance in C4 relative to C3 species, with C4-

like individuals coping poorly with decreased water availability compared to the C3-like

individuals (Ripley et al. 2007). Therefore, the intrinsic characteristics of each species determine

its resistance to drought more than the photosynthetic pathway or taxonomic background.

The accumulation of Proline (Chapter II) may be involved in plant resistance to drought

conditions. Besides the possible roles of this amino acid in osmoregulation and in sustaining

homeostasis, it has been suggested to act as an energy storage compound (Hare & Cress 1997).

The synthesis of Proline involves the consumption of NAD(P)H and the degradation of the

amino acid upon stress release supplies energy to re-establish growth and allow plants to recover

from the harmful effects. Increased content of other amino acids, like methionine and

phenylalanine, with leaf dehydration may be related to increased synthesis of secondary

metabolites with a role in plant stress defence mechanisms. Additionally, in C. dactylon and Z.

japonica an unusual amino acid, 5-hydroxy-L-norvaline (HNV) was induced by water deficit. As

far as we are aware, this amino acid has not been previously reported to be present in plant

leaves and its biosynthetic pathway in plants is unclear. The presence of HNV in well watered

plants of Z. japonica and its increased content in this species and in C. dactylon with water

deficit suggest that this amino acid may have an important role in C4 grass resistance to drought.

The properties of this amino acid suggest its possible involvement in plant defence mechanisms

that may be triggered under abiotic stress. This hypothesis needs further investigation and may

prove beneficial for plant engineering towards increased drought resistance.


183

Rapid vs. slow water deficit

In the present work, drought stress was slowly induced by ceasing to provide water to the plants

of the three C4 grass species (Chapter II). As a consequence, water availability in the soil

decreased gradually through the experiment, affecting plant growth, leaf water relations, leaf

gas-exchanges and photosynthetic metabolism. Several differences were identified in the

different aspects of plant responses to water deficit in comparison with a previous study, where

leaf dehydration was imposed rapidly, by the addition of polyethylene glycol 4000 (PEG4000) to

the nutrient solution used to water the plants (Carmo-Silva et al. 2004; 2007). It is known that

plant responses can be markedly different depending on the rate of leaf dehydration (Chaves et al.

2003; Flexas et al. 2004).

The most striking difference observed in the three C4 grasses studied was the dramatic

decrease of RWC in C. dactylon in response to the artificial PEG-induced water deficit (Carmo-

Silva et al. 2007). The addition of solutes to the nutrient solution allows the decrease of its water

potential and consequent decrease in water availability to be taken up by the plant. Due to its

high molecular weight, PEG will not penetrate the root system and its use as an osmolite for

water deficit induction has been validated by several authors (Zhang et al. 2001; Ober & Sharp

2003). Moreover, PEG-induced water deficit did not cause dramatic effects on photosynthesis,

stomatal conductance, photochemistry or enzyme activities in C. dactylon (Carmo-Silva et al.

2004; 2007), suggesting that a toxicity effect of the osmolyte on the plants of this species should

be disregarded. The lack of a faster response in terms of stomatal closure may be the explanation

for the dramatic drop in RWC of C. dactylon observed under rapidly-induced water deficit. As

referred above, the present results suggest that decreased stomatal conductance may be the major

factor controlling water loss in order to minimize leaf dehydration in C. dactylon.

The leaf relative water content (RWC) was not much decreased in the samples taken for

biochemical analysis. This was partly due to collection early in the day. Tissue water status

recovered during the night because of stomatal closure and hence decreased evaporation from the

leaves but continued water absorption from the soil. Because samples were taken four hours after

the beginning of the photoperiod, assuming that steady-state photosynthesis would then have

been attained and plant responses would reflect differences in water availability, the RWC was

not as much decreased as if it would have been in the middle of the day. In fact, the drought

samples taken at the end of a 12-hour-nocturnal period (Chapter V) had RWC values that were

not as much decreased as those taken from plants that had been exposed to four hours in the light

(Table V.1). None the less, the samples taken from non-watered pots, eight to twelve days after

Chapter VI.

184

stress imposition, were effectively experiencing stress conditions, as revealed for instance by the

decreased leaf water potentials (Figure II.2).

The photosynthetic responses observed under rapidly-induced water deficit in the three

C4 grasses are likely to result from direct effects of decreased water content in the leaves whilst

the responses observed under slowly-induced water deficit include the effects of adaptive

strategies adopted by the plants to cope with decreased water availability in the soil. The

disparity of results between the two water deficit induction systems adopted provides evidence

for the need to promote conditions more accurately representing those observed under natural

conditions. The gradual decrease in water availability in the soil promoted by ceasing to provide

water to the plants of the three species is therefore a more useful method to understand the

drought stress physiology and biochemistry of these grass species and provide information to the

exploitation of their capacities under water-limited conditions.

Stomatal and metabolic limitations to photosynthesis in C4 grasses

Net CO2 assimilation rates under water deficit may be limited by stomatal closure, metabolic

factors or a combination of both. The relative importance and the timing of the different effects

caused by water deficit that will have a negative impact on photosynthesis have been the target

of much debate some years ago (Flexas & Medrano 2002; Lawlor & Cornic 2002). In P.

dilatatum, C. dactylon and Z. japonica decreased water availability in the soil caused decreased

net CO2 assimilation rate and decreased stomatal conductance to CO2 (Figure III.2). The faster

decrease observed in stomatal conductance than in photosynthesis reveals that stomatal closure is

effectively induced by water deficit, minimizing water loss and slightly enhancing water use

efficiency. Decreased photosynthesis in several C4 grasses has previously been attributed to

stomatal closure (Du et al. 1996; Lal & Edwards 1996; Marques da Silva & Arrabaça 2004;

Carmo-Silva et al. 2007).

The decrease of the maximal rate of net CO2 assimilation attained at saturating CO2

concentrations in moderately and severely-stressed plants suggests that metabolic limitations to

photosynthesis are also present in the three C4 grasses. Moreover, these are likely to become

more important with increased severity of drought stress. An increased relative importance of

metabolic to stomatal limitations to photosynthesis was observed in other C4 grasses (Du et al.

1996; Marques da Silva & Arrabaça 2004) and suggests down-regulation of the photosynthetic

metabolism when CO2 availability is diminished (Chaves et al. 2003; Flexas et al. 2004).

Decreased capacity for RuBP regeneration, possibly as a result of impaired ATP

synthesis, has been considered as one of the major metabolic limitations to photosynthesis in C3


185

plants (Tezara et al. 1999; Flexas & Medrano 2002). An effective decrease of RuBP content was

observed with water deficit in the leaves of the three C4 grasses (Figure V.4), suggesting limited

capacity for the regeneration of this substrate, possibly as a consequence of decreased energy

levels. Concomitantly, decreased Rubisco initial and total activities were also observed and were

associated with increased contents of Rubisco tight-binding inhibitors in the drought-stressed

plants (Chapter V). The increased inhibition of Rubisco supports the hypothesis of ATP being a

limiting factor in the leaves of plants exposed to drought stress. The ATP-dependent Rubisco

activase facilitates the activation of Rubisco both by promoting the carbamylation of the enzyme

and by removing the tight-binding inhibitors (Portis 2003). Thus, if ATP decreases, Rubisco

activase will be less active and more inhibitors may remain bound to Rubisco. The binding of

inhibitors to Rubisco may protect the enzyme against proteolytic breakdown as may occur under

stress conditions (Khan et al. 1999).

The activation state of Rubisco was not affected by water deficit, suggesting that the CO2

concentration at the Rubisco site is not decreased to an extent that will affect substantially the

enzyme carbamylation. The CO2 concentration available for carboxylation by PEPC will be

decreased due to decreased stomatal conductance. However, the physiological activity of PEPC

increased slightly with water deficit, as a result of an increased phosphorylation state of the

enzyme (Figures IV.1 and IV.2). The increased PEPC activity in the mesophyll may be a

strategy to maximize the primary fixation of the less abundant CO2 in the mesophyll.

Accordingly, the activities of the C4 acid decarboxylases in each species were always

considerably higher (at least 4-fold) than Rubisco activity (Figures IV.3, IV.4 and V.1). Thus,

Rubisco is supplied with CO2 concentrations high enough to avoid a considerable increase in

photorespiration under water deficit (Chapter III).

Taken together, the results present here support the view that C4 photosynthesis in the

three grasses studied is primarily limited by decreased CO2 availability resulting from stomatal

closure. Biochemical adjustments are also observed. Decreased ATP synthesis may occur and

contribute to limit photosynthesis, but this hypothesis needs further investigation. RuBP

regeneration is considerably affected and the substrate becomes limiting for CO2 assimilation.

The photosynthetic metabolism is down-regulated in order to avoid severe impairment to occur

in response to the unfavourable conditions. The general effects of water deficit observed on the

photosynthetic metabolism involving the C4 and the C3 cycles may be summarized as:

→ increased PEPC physiological activity (higher activation state),

→ decreased Rubisco initial and total activity (increased enzyme inhibition),

→ and decreased RuBP contents.

Chapter VI.

186

Pyruvate,orthophosphate dikinase (PPdK), the enzyme responsible for the production of

PEP from pyruvate, is one of the control points of C4 photosynthesis (Furbank et al. 1997) and

was reported to limit photosynthesis in drought-stressed leaves of Saccharum sp. (Du et al.

1996). Therefore, both the activity of PPdK and the PEP content should be further investigated

together with determination of the ATP content under drought stress conditions. These studies

should provide the necessary additional information to allow a better understanding of the

drought-stress response of the C4 photosynthetic pathway in these three grasses.

Photorespiration and photosynthetic performance of the C4 pathway

The CO2-concentrating mechanism provides C4 plants with a more efficient photosynthetic

pathway, especially at high irradiance and temperatures. The oxygenase activity of Rubisco is

limited due to the high CO2 environment in the BS cells, resulting in higher CO2 assimilation

rates, which are associated with increased water use efficiencies (Edwards et al. 1985; Long

1999). Stomatal closure is one of the earliest and most common responses to water deficit and

results in decreased water loss at the expense of decreased CO2 availability for assimilation.

Under these conditions, photorespiration may increase and decrease the efficiency of C4

photosynthesis.

Photorespiration consumes ATP and reducing power and has been suggested as an

efficient process conferring protection under stress conditions against imbalanced redox

potentials by maintaining electron flow (Keys & Leegood 2002). The limitation of CO2

assimilation results in lower energy requirements for the Calvin cycle and this has been

suggested in the past to result in photoinhibition. However, most studies reveal that

photoinhibition is an unlikely event that seems to occur only in some plant species and under

very severe drought stress conditions (see Flexas & Medrano 2002). Photorespiration remained

slow under drought conditions, increasing only slightly in proportion to photosynthesis in the

three C4 grass species (Chapter III).

The photosynthetic metabolism of all three C4 grasses seems to adjust to the

environmental conditions in order to maximize efficiency and minimize damage under

unfavourable conditions. Cynodon dactylon tends to present higher photosynthetic rates of CO2

assimilation (Chapter III) and higher activities of the carboxylating and decarboxylating enzymes

(Chapter IV), suggesting that this species may have a more efficient photosynthetic performance.

Additionally, the highest content of RuBP (Chapter V) agrees with the presence of a highly

effective photosynthetic metabolism in C. dactylon. The hypothesis that metabolic impairment

may occur under severe drought stress conditions can not be disregarded, but the data here


187

presented suggest that photosynthesis slows down but considerable photosynthetic activity is

maintained under moderate water deficit.

The three biochemical subtypes of C4 photosynthesis in the grasses studied

The three ‘classical’ mechanisms of C4 photosynthesis (Hatch 1987) occur among the Poaceae

(Hattersley & Watson 1992) and species may be classified into one of these biochemical

subtypes by the relative activities of the three C4 acid decarboxylases: NADP-ME, NAD-ME and

PEPCK (Hattersley 1988). Species belonging to the ‘classical’ NADP-ME and NAD-ME

biochemical subtypes (Gutierrez et al. 1974; Hatch et al. 1975) have low activities of the other

two C4 acid decarboxylases. Conversely, in the PEPCK subtype the enzyme NAD-ME

contributes considerably to the release of CO2 in the BS cells and provides the required ATP for

PEPCK activity (Kanai & Edwards 1999). By measuring simultaneously, in the same leaf

extracts, the activities of the three C4 acid decarboxylases under conditions that were assumed to

be close to the physiological state, we observed the presence of PEPCK activity in all three

species, whereas NAD-ME activity was present in C. dactylon and Z. japonica but was very low

in P. dilatatum, and NADP-ME was present in P. dilatatum but was very low in the other two

grasses (Figures IV.3 and IV.4).

The high activity of NADP-ME found in P. dilatatum agrees with previous description of

this species as belonging to this biochemical subtype (Usuda et al. 1984). Accordingly, the

activity of NAD-ME was very low in the leaves of this species but, curiously, PEPCK had

considerable activity at physiological concentrations and high activity at optimal concentrations

of Mg2+ and Mn2+ (ca. 2.3 and 8.9 μmol min-1 mg-1 Chl, respectively). PEPCK has been

previously shown to be present in some NADP-ME type C4 species, such as Zea mays and

Paspalum notatum (Walker et al. 1997). Subsequent studies in Zea mays revealed that PEPCK

decarboxylates oxaloacetate derived from aspartate whilst NADP-ME is responsible for the

decarboxylation of malate, with both enzymes contributing to the release of CO2 in the BS cells

of this species (Wingler et al. 1999).

High values for the activity (generally ≥ 6 μmol min-1 mg-1 Chl) and the activation state

(generally ≥ 80%) of PEPCK were observed in both C. dactylon and Z. japonica (Figure IV.3).

Considerable activities of NAD-ME were also present in the leaves of the two species (Figure

IV.4), but this enzyme was not as active as PEPCK. These results agree with the typing of Z.

japonica as a PEPCK species (Gutierrez et al. 1974), retaining considerable decarboxylation of

malate in the BS mitochondria by NAD-ME. However, questions arise on what concerns the

Chapter VI.

188

previous characterization of C. dactylon as a NAD-ME species (Hatch & Kagawa 1974). As

referred above, the presence of PEPCK was previously shown in several NADP-ME type

monocots (Walker et al. 1997; Wingler et al. 1999; Voznesenskaya et al. 2006). However, to the

best of our knowledge, this is the first time the presence of high activities of PEPCK has been

found in a NAD-ME-type grass species, C. dactylon.

Edwards and co-workers (1971) were first to report the presence of high PEPCK levels in

several C4 grasses. In that study, very low levels of PEPCK, insufficient to account for C4 acid

decarboxylation, were found in C. dactylon. Subsequently, Hatch & Kagawa (1974) found high

NAD-ME activities in several species, including C. dactylon, and were only able to detect the

presence of PEPCK in species with significantly lower NAD-ME activity. These results led to

the typing of C. dactylon as a NAD-ME species. However, the value obtained for the activity of

this enzyme in C. dactylon (1.4 μmol min-1 mg-1 Chl) was less than half the activity present in

the other species classified as belonging to the same subtype (≥ 3 μmol min-1 mg-1 Chl).

Prendergast et al. (1987) have also reported the absence of PEPCK activity and the presence of

high activities of NAD-ME in this grass species (2.0-3.3 μmol min-1 mg-1 Chl, depending on

plant growth conditions). High activity of NAD-ME in C. dactylon was similarly found in the

present work (≥ 3 μmol min-1 mg-1 Chl), but PEPCK was more active in the same leaves (≥ 6

μmol min-1 mg-1 Chl). C4 grasses with relative activities of the two enzymes as close as 2 NAD-

ME to 3 PEPCK have been classified as belonging to the PEPCK subtype (Gutierrez et al. 1974).

Prendergast et al. (1987) suggested that some species might be intermediate biochemically,

showing either predominance of NAD-ME or PEPCK activity. The same authors found masked

activity of PEPCK in the C4 grass Tridens brasiliensis, suggesting the presence of some

compound in the leaves of this species that interfered with PEPCK detection and measurement

by the methods then used. Therefore, special care is taken to introduce the most suitable

components to the extraction medium to maximize the extraction and recovery of the enzyme

activity. The methods adopted were similar to those recently reported by other authors (Bailey et

al. 2007; Marshall et al. 2007; Sudderth et al. 2007) and the values obtained for the activities of

the three C4 acid decarboxylases are in the same range as previously reported data for grass

species (Gutierrez et al. 1974; Hatch et al. 1975; Hatch et al. 1982; Ueno & Sentoku 2006).

The high capacity of PEPCK found in all three C4 grasses, previously reported as

belonging to each of the different biochemical subtypes, suggests the possible role of the enzyme

as a secondary decarboxylase to NADP-ME and NAD-ME (Kellogg 1999; Lea et al. 2001).

However, the hypothesis that the high activity of PEPCK may be related to other aspects of plant

metabolism must not be disregarded. Leegood & Walker (2003) described roles in the cytosol


189

metabolism and in the interface between diverse metabolic pathways in the leaves, such as those

involving amino acids, organic acids, sugars, lipids and some secondary metabolites. Further

investigation is therefore required to clarify the functional significance of the high PEPCK

activities found in the three C4 grass species.

A set of anatomical characteristics is generally associated with the biochemical

mechanism present in a particular species. One of the distinctive characteristics among the three

‘classical’ subtypes is the position of the BS chloroplasts: centrifugal in NADP-ME, centripetal

in NAD-ME and centrifugal or even in PEPCK (Dengler & Nelson 1999). Optical microscopy of

transverse leaf sections stained with Toluidine Blue O revealed the presence of centrifugal or

scattered chloroplasts in the BS cells of P. dilatatum and Z. japonica and centripetal chloroplasts

in the BS cells of C. dactylon (Figure II.5), supporting previous descriptions for the species

(Prendergast & Hattersley 1987; Watson & Dallwitz 1992; Dengler et al. 1994). Moreover, the

elongated shape of the chloroplasts and the evenness of the BS outline in cross section in C.

dactylon support the anatomical classification of this species as a ‘typical’ NAD-ME species

(Dengler et al. 1994; Dengler & Nelson 1999). Exceptions to the characteristics associated with

the different anatomical-biochemical subtypes may occur. As proposed by Gutierrez et al. (1974)

a possible evolutionary pattern in C4 grasses could be from NAD-ME species with centripetal

chloroplasts to PEPCK species, with even or centrifugal chloroplasts and some activity of NAD-

ME being retained, to NADP-ME species, with centrifugal chloroplasts (to optimise the

exchange of metabolites between M and BS cells through plasmodesmata) and varying degrees

of grana reduction in the BS chloroplast. However, the three C4 subtypes do not seem to

represent a sequence of progressive biochemical modifications (Hattersley 1988). None the less,

the ‘classical’ subtypes NAD-ME and PEPCK have many characteristics in common in what

concerns their leaf anatomy and it is plausible to consider that species belonging to the PEPCK

subtype evolved from ancestral species with the NAD-ME C4 photosynthetic pathway

(Hattersley & Watson 1992; Watson & Dallwitz 1992) retaining considerable levels of

decarboxylation in the mitochondria. As proposed by Kellogg (1999), the decarboxylation by

PEPCK can be viewed as a relatively simple addition to, or variant of, the NAD-ME pathway.

The carbon isotope composition of leaf dry matter shown in Figure III.4 was different for

each of the three grass species. The anatomical variations among the C4 subtypes, and especially

the presence or absence of a suberin lamella in the BS cells, may be associated with their

conductance properties. The carbon isotope composition tends to be more negative in NAD-ME

than in NADP-ME grass species, with PEPCK species showing intermediate values (Hattersley

1982). This is thought to reflect a faster rate of leakage of CO2 from the BS in the former species,

Chapter VI.

190

which lack a suberin lamella in the BS cells (Hattersley & Browning 1981; Prendergast &

Hattersley 1987; Dengler et al. 1994). The fraction of CO2 fixed by PEPC that subsequently

leaks out of the BS without being assimilated by Rubisco, estimated from short-term carbon

isotope discrimination measured simultaneously with gas-exchanges (Henderson et al. 1992),

was higher for a NAD-ME-type monocot (0.30), than for other monocots belonging to the

NADP-ME or PEPCK subtypes (≤ 0.25). The carbon isotope composition of leaf dry matter was

lower in C. dactylon (-16.3‰) than in P. dilatatum (NADP-ME; -15.2‰) but slightly higher

than in Z. japonica (PEPCK; -16.6‰) (Figure III.4; Carmo-Silva et al. 2008), suggesting that the

former species might have evolved compensatory features that avoid considerable leakage of

CO2 from the BS (Hattersley & Browning 1981) and optimize the photosynthetic performance of

the species.

Heterogeneity in biochemistry and/or leaf structure can be the result of natural

hybridization between photosynthetically different individuals or derive from active evolution of

the photosynthetic pathway taking place. These were suggested as possible explanations to the

variability of C3 and C4 mechanisms observed among different individuals of Alloteropsis

semialata (Hattersley & Watson 1992). This hypothesis was supported by a recent study

showing that plants of A. semialata were not typically C3 or C4 but C3-like or C4-like (Ueno &

Sentoku 2006), showing considerable variability in the expression of the C4 syndrome.

Nowadays, the production of grass seeds and development of new varieties through breeding

techniques might result in some heterogeneity among different individuals of the same species

and explain the results obtained with C. dactylon.

Paspalum dilatatum, Cynodon dactylon or Zoysia japonica?

In practical terms, the goal beyond the fundamental research presented here was to show which

of the three grass species is the best in terms of water economy. The “best” grass will obviously

depend on the perspective and situation. The results obtained support the hypothesis that both C.

dactylon (bermudagrass) and Z. japonica (zoysiagrass) are very resistant, more than P. dilatatum

(dallisgrass), to environmental factors causing plant stress, namely decreased water availability.

Both C. dactylon and Z. japonica could therefore be viewed as better alternatives for the use as

turfgrass in golf courses. However, even though C. dactylon performed very well under

moderate drought conditions, the plants of this species can be severely damaged upon intense

desiccation (Utrillas & Alegre 1997). Zoysia japonica has characteristics that make it more fit to

xeric conditions, notably the higher relative leaf dry matter, superior compaction of leaf tissues


191

and more rigid cell walls that allow lower leaf water potentials to be reached. As a consequence,

zoysiagrass will probably be able to cope better and survive under conditions of severe drought,

as may be observed in future! However, “there is no rose without thorns” and these structural

features result in the harder and stiffer leaves of Z. japonica. Due to its coarse texture, this

species can not be used for the greens (on golf courses), where a great smoothness is needed. Its

higher content in fibers will also compromise its use as forage.

The “best” grass among the three studied when the high productivity is the target for use

as forage or pasture grass when water is not too restricted is Paspalum dilatatum. The potential

benefits associated with the use of mixtures of C3 and C4 forage and turf grasses (Johnston 1996)

should be further investigated as this may provide a better option.

In terms of drought resilience, when the intention is to have a green lawn, looking good

for most winter and summer seasons, and surviving severe summer droughts, the best option will

most likely be the use of Zoysia japonica, but this conclusion should be investigated further in

relation to severe drought conditions.

Overall the best grass for the use as turf and forage grass seems to be Cynodon dactylon,

for its great photosynthetic performance both under well-watered conditions and when water

availability is decreased and for the presence of good strategies to avoid excessive water loss. It

will perform very well under moderate levels of drought stress but will most likely be more

affected than Z. japonica in the case of extreme droughts as observed by Utrillas & Alegre

(1997). Nonetheless, given its overall characteristics, including the fine texture, there is great

advantage in its use for golf courses in Portugal in the present and near future.

Chapter VI.

192

CONCLUDING REMARKS

The three C4 grasses of the different metabolic subtypes showed some differences in their

responses to gradually induced water deficit, even though a generalized pattern could be

observed. The presence of drought resistance mechanisms that minimize water loss was more

effective in Cynodon dactylon and Zoysia japonica than in Paspalum dilatatum. The leaf

structure of Z. japonica makes this species more fit to cope with severe drought conditions. An

unusual amino acid (5-hydroxy-L-norvaline) was drought-responsive in both C. dactylon and Z.

japonica and is likely to be involved in stress defence mechanisms.

Photosynthesis was affected in all three C4 species under water deficit. Stomatal closure

and decreased RuBP contents were the major factors affected by drought stress that may

contribute to limit photosynthesis. Analysis of the CO2-response of photosynthesis and changes

in amino acids content suggested slightly increased photorespiratory rates as a proportion of

photosynthetic rates under water deficit. Nevertheless, the results provided evidence that

photorespiration remains slow in the three C4 grasses under water deficit conditions.

The carboxylating enzymes were similarly affected by water deficit in the three species,

with slightly increased PEPC physiological activity and decreased initial activity of Rubisco.

Down-regulation of Rubisco is likely to be advantageous in terms of protection of the enzyme

when its natural substrate is present at lower concentrations. The activities of the C4 acid

decarboxylases, NADP-ME, NAD-ME and PEPCK, changed only slightly with water deficit and

were more variable and species specific. High activities of PEPCK are present in the three

species, from each of the C4 biochemical subtypes.

Overall, the results provide evidence that an efficient photosynthetic metabolism is

present in the three C4 grasses. Water deficit decreased plant growth and photosynthesis and both

physiological and biochemical adjustments were observed. These responses allow the plants of

each species to perform well under adverse conditions and cope with moderate gradually induced

drought conditions. The plants of the three species and of C. dactylon in particular maintain

considerably high photosynthetic activity at moderate levels of drought stress.


193

FUTURE PERSPECTIVES

Given the broad field of plant responses to drought stress, it would not be possible to cover all

the aspects that needed investigation in the scope of the work here presented. The results

obtained suggest that the immediate next target should be to investigate the effects of water

deficit on photophosphorylation and ATP synthesis.

The applicability of the results obtained should be further investigated in the field,

through the imposition of different irrigation strategies in established lawns of each of the three

species. Special emphasis should however be given to C. dactylon and Z. japonica. Additionally,

other grass species and/or varieties should be included to broaden the screening of drought

resistance in grass species.

Some interesting details arose from the studies performed and two in particular must be

further investigated. The high PEPCK activity found in all three species belonging to each of the

classical biochemical subtypes of C4 photosynthesis should be complemented with studies of

cDNA sequencing, SDS-PAGE and immunoblotting to characterize and quantify the enzyme

type present in each of the three species. It is known (essentially from studies done by Leegood

and co-workers) that structural properties of PEPCK are related to the regulation of the enzyme

by (de)phosphorylation and to its coordination with PEPC. The functional significance of a high

activity of PEPCK in the leaves of the three species studied and in other C4 grasses must be

clarified.

The characterization of the stress-inducible amino acid 5-hydroxy-L-norvaline and its

metabolic pathway must be elucidated in order to exploit the potentialities associated with its

presence and enhancement in response to water deficit conditions.

Chapter VI.

194

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