Bastiaan Brouwer

Umeå Plant Science CentreFysiologisk BotanikUmeå 2012

Shedding Lighton

Shade- and Dark-InducedLeaf Senescence

Bastiaan Brouwer

Akademisk avhandling

Som med vederbörligt tillstånd av Rektor vid Umeå universitet för avläggande av filosofie doktorsexamen i Växters cell- och

molekylärbiology, framläggs till offentligt försvar i KB3A9, KBC-huset, Fredagen den 25 Maj, kl. 13:00.

Avhandlingen kommer att försvaras på engelska.

Fakultetsopponent: Professor Karin Krupinska, Botanisches Institute und Botanischer Garten, Christian-Albrechts-Universität zu Kiel,

Germany.

Umeå Plant Science Centre, Department of Plant PhysiologyUmeå UniversityUmeå, Sweden, 2012

Organization Document type Date of publicationUmeå University Doctoral thesis 04th of May 2012Fysiologisk Botanik

AuthorBastiaan Brouwer

TitleShedding Light on Shade- and Dark-Induced Leaf Senescence

AbstractLeaf senescence is the final stage of leaf development, during which the leaf relocates most of its

valuable nutrients to developing or storing parts of the plant. As this process progresses, leaves lose

their green color and their capacity to perform photosynthesis. Shade and darkness are well-known

as factors inducing leaf senescence and it has been proposed that senescence can be initiated by

reductions in photosynthesis, photomorphogenesis and transpiration. However, despite the fact that

the signaling mechanisms regulating each of these processes have been extensively described,

particularly in seedlings, their contribution to the initiation of senescence in mature leaves still

remains unclear. Furthermore, the use of different experimental systems to study shade-induced

leaf senescence has yielded several divergent results, which altogether complicate the overall

understanding of leaf senescence.

To address this, darkened plants and individually darkened leaves, which show different rates of leaf

senescence, were studied. Comparing the transcriptome and metabolome of these two dark-

treatments revealed that they differed distinctly with regard to their metabolic strategies. Whole

darkened plants were severely carbohydrate-starved, accumulated amino acids and slowed down

their metabolism. In contrast, individually darkened leaves showed continued active metabolism

coupled to senescence-associated degradation and relocation of amino acids.

This knowledge was used to set up a new system to study how shade affects leaf senescence in the

model plant Arabidopsis thaliana. Use of this system revealed that different senescence-associated

hallmarks appeared in response to different intensities of shade. Some of these hallmarks were

further shown to be part of both leaf senescence and photosynthetic acclimation to low light.

Finally, using this system on phytochrome mutants revealed that loss of phytochrome A increased

the loss of chlorophyll under shade, without increasing the expression of senescence-associated

genes.

Together, these findings suggest that shade-induced leaf senescence, which is generally perceived as

a single process, is actually an intricate network of different processes that work together to

maintain an optimal distribution of nutrients within the plant.

KeywordsArabidopsis, darkness, light, photosynthesis, phytochrome, shade, senescence

Language ISBN Number of pagesEnglish 978-91-7459-437-9 52 + 3 papers

Shedding Light on

Shade- and Dark-Induced

Leaf Senescence

Bastiaan Brouwer

Umeå Plant Science Centre

Fysiologisk Botanik

Umeå 2012

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

ISBN: 978-91-7459-437-9

Front cover by: Bastiaan Brouwer

Electronic version available at http://umu.diva-portal.org/

Printed by: KBC, Umeå University

Umeå, Sweden 2012

iii

Abstract

Leaf senescence is the final stage of leaf development, during which the

leaf relocates most of its valuable nutrients to developing or storing parts of

the plant. As this process progresses, leaves lose their green color and their

capacity to perform photosynthesis. Shade and darkness are well-known as

factors inducing leaf senescence and it has been proposed that senescence

can be initiated by reductions in photosynthesis, photomorphogenesis and

transpiration. However, despite the fact that the signaling mechanisms

regulating each of these processes have been extensively described,

particularly in seedlings, their contribution to the initiation of senescence in

mature leaves still remains unclear. Furthermore, the use of different

experimental systems to study shade-induced leaf senescence has yielded

several divergent results, which altogether complicate the overall

understanding of leaf senescence.

To address this, darkened plants and individually darkened leaves, which

show different rates of leaf senescence, were studied. Comparing the

transcriptome and metabolome of these two dark-treatments revealed that

they differed distinctly with regard to their metabolic strategies. Whole

darkened plants were severely carbohydrate-starved, accumulated amino

acids and slowed down their metabolism. In contrast, individually darkened

leaves showed continued active metabolism coupled to senescence-

associated degradation and relocation of amino acids.

This knowledge was used to set up a new system to study how shade

affects leaf senescence in the model plant Arabidopsis thaliana. Use of this

system revealed that different senescence-associated hallmarks appeared in

response to different intensities of shade. Some of these hallmarks were

further shown to be part of both leaf senescence and photosynthetic

acclimation to low light.

Finally, using this system on phytochrome mutants revealed that loss of

phytochrome A increased the loss of chlorophyll under shade, without

increasing the expression of senescence-associated genes.

Together, these findings suggest that shade-induced leaf senescence,

which is generally perceived as a single process, is actually an intricate

network of different processes that work together to maintain an optimal

distribution of nutrients within the plant.

iv

Sammanfattning

Senescens är det sista steget i ett blads livscykel och leder till att

värdefulla näringsämnen överförs till växande eller lagrande delar av växten.

Allt eftersom denna process fortskrider, förlorar bladet sin gröna färg samt

sin förmåga att fotosyntetisera. Skuggning och mörkläggning är välkända

faktorer som initierar bladsenescens och detta kan triggas genom

långsammare fotosyntes, fotomorfogenes och transpiration. Trots att

signaleringen bakom dessa processer är utförligt studerade och beskrivna i

groddplantor, finns många oklarheter om hur detta leder till initiering av

senescensen hos fullt utvecklade blad. Att olika experimentella system

använts för att studera skugg-inducerad bladsenescens har dessutom lett till

varierande resultat, vilket försvårar en övergripande förståelse av processen.

För att utreda detta vidare studerades hela växter i mörker vilka

jämfördes med enskilt mörklagda blad, behandlingar som uppvisar olika

hastigheter av senescens. Genom att jämföra transkriptom och metabolom

under sådana mörkerexperiment visades att de metaboliska strategierna klart

skilde sig åt mellan behandlingarna. När hela växten mörklades uppvisade

bladen klara tecken på kolhydratsvält och ackumulering av aminosyror

samtidigt som metabolismen gick på sparlåga. I individuellt mörklagda blad

fortsatte däremot en aktiv metabolism kopplad till degradering och

borttransport av aminosyror.

Denna information användes sedan för att sätta upp ett nytt system för

att studera hur skuggning av blad påverkar bladsenescens i modellväxten

Arabidopsis thaliana. Resultaten från detta visade på många typiska

kännetecken för bladscenescens som en följd av olika grader av skuggning.

Några av dessa faktorer var gemensamma för senescens och acklimering av

fotosyntesen till lågt ljus.

Användandet av fytokrommutanter i detta experimentella system visade

dessutom att signallering via fytokrom kan bidra till processen. I en mutant

utan fytokrom A fördröjdes skugg-inducerad minskning av klorofyll men

utan att uttrycket av senesces-associerade gener ökade.

Sammantaget tyder dessa upptäckter på att bladsenescens som initieras

av beskuggning, vilket oftast betraktas som en enhetlig process, i

verkligheten är ett komplicerat nätverk av olika processer som opererar

tillsammans för att upprätthålla en optimal fördelning av näringsämnen inom

växten.

v

List of Papers

I. Abdul Ahad*, Olivier Keech*, Andreas Sjödin, Pernilla Lindén,

Bastiaan Brouwer , H Stenlund, Thomas Moritz, Stefan Jansson

and Per Gardeström

Comparisons between leaves from darkened plants and individually

darkened leaves reveal differential metabolic strategies in response

to darkness

Manuscript

II. Bastiaan Brouwer, Agnieszka Ziolkowska, Matthieu Bagard,

Olivier Keech and Per Gardeström

The impact of light intensity on shade-induced leaf senescence

Plant, Cell & Environment, 2012, DOI: 10.1111/j.1365-

3040.2011.02474.x

III. Bastiaan Brouwer, Per Gardeström and Olivier Keech

Far-red light reduces senescence-associated chlorophyll loss under

low light via a Phytochrome A-mediated Far-red High Irradiance

Response

Manuscript

* These authors contributed equally

Paper II has been reproduced with kind permission of the publisher.

The papers will be referred to by their Roman numbers in the text.

vi

vii

Table of Contents

Abstract iii

Sammanfattning iv

List of Papers v

Preface ix

Abbreviations x

INTRODUCTION 1

A Leaf's Life 1

Leaf Senescence 1

Why study leaf senescence? 1

The process of leaf senescence 2

Chlorophyll degradation 2

Protein degradation 4

Nitrogen relocation 6

Lipid degradation 8

Mitochondria and loss of cellular organization 9

What induces and inhibits leaf senescence? 10

Shade- and Dark-induced leaf senescence 12

Shade and Darkness 12

Photosynthesis 12

Photomorphogenesis 14

Phytochromes 15

Transpiration 17

SUMMARY 18

AIM 19

RESULTS AND DISCUSSION 20

Different experimental systems 20

Darkened plants versus individually darkened leaves 21

Darkened leaves are sugar-starved 22

Darkened plants differ from individually darkened leaves 23

Shade-induced leaf senescence depends on the light intensity 25

Different light intensities induce different hallmarks of senescence 26

viii

PRP as part of the senescence process 27

Leaf senescence overlaps with PA 28

The LCP as a threshold to enhance expression of SAGs 28

Shade-induced chlorophyll loss depends on phytochromes 29

FR does not enhance chlorophyll loss under low light 29 PHYA inhibits chlorophyll loss 29

PHYA inhibits chlorophyll loss via the FR-HIR 30

PHYA does not directly inhibit the expression of SAGs 31

Could PHYA inhibit leaf senescence via chlorophyll biosynthesis? 32

Shedding light on leaf senescence 34

Summary 37

CONCLUSIONS AND FUTURE PERSPECTIVES 39

ACKNOWLEDGEMENTS 40

REFERENCES 42

ix

Preface

If you have plants in your house, you have undoubtedly observed the

occasional yellowing leaf. Most people remove such leaves and provide the

plant with some extra fertilizer to prevent further yellowing. Some of you

will have observed that such leaves most often form on the side of the plant

that receives the least light. The process causing such yellowing of leaves is

called shade-induced leaf senescence. Leaf senescence is a process that the

plant uses to redirect limiting nutrients (often nitrogen) from older and

shaded leaves to younger and growing leaves or storage-tissues. Because of

this feature, understanding how shade and light initiate and postpone leaf

senescence, respectively, could lead to increased crop productivity and

product shelf-life. However, this understanding is still fragmented and the

grand aim of this thesis therefore is to connect these fragments and shed

more light on the process of shade-induced leaf senescence.

Bastiaan Brouwer, 2012

x

Abbreviations

Abbreviations are explained when they first appear in the text.

1

INTRODUCTION

A Leaf's Life

When the temperature is high enough, light stimulates imbibed plant

seeds to germinate and grow upwards. Upon reaching an illuminated surface,

the light will promote seedlings to undergo photomorphogenesis, during

which they develop functional chloroplasts and become green

photoautotrophic organisms. Photoautotrophic organisms use light to

assimilate carbon dioxide (CO2) into sugars and other carbohydrates via the

process of photosynthesis. Within a plant canopy, plants compete for light by

elongating their stems and developing new leaves. These new leaves often

overshadow the older ones, which as a result experience continuous changes

in their light environment. In response to these changes, shaded leaves

acclimate their photosynthetic machinery to optimize their function. This

acclimation occurs on two levels: within leaves and between leaves. Within

leaves, the ratios between the light harvesting antennae and the photosystems

are adjusted through a process termed Photosynthetic Acclimation (PA).

Between leaves, whole-plant photosynthesis is optimized by re-allocating

limiting nutrients like nitrogen from older and shaded leaves to younger and

well-illuminated plant parts through a process termed Photosynthetic

Resource Partitioning (PRP). Leaves that cannot acclimate sufficiently or

that receive cues signaling the end of plant development undergo leaf

senescence. During this final process, the structure of the leaf is slowly

degraded, while limiting nutrients, such as nitrogen, sulfur and phosphate,

are relocated to developing and storing organs such as new leaves, roots and

seeds.

Leaf Senescence

Why study leaf senescence?

Derived from the latin verb 'senescere', which means 'to grow old', leaf

senescence is generally defined as the developmental stage that follows leaf

maturation and has leaf death as the inevitable outcome (Leopold, 1961;

Smart, 1994; Gan & Amasino, 1997; van Doorn & Woltering, 2004). During

this process, leaves turn yellow as the photosynthetic machinery is degraded

and the resulting nutrients are transported to other parts of the plant

(Gregersen et al., 2008; Masclaux-Daubresse et al., 2008).

While leaf senescence is best known for causing the idyllic scenery in

deciduous forests during autumn, it actually constitutes a major part of our

everyday lives. Stress-induced senescence reduces both the yield

(Hörtensteiner, 2009) and the shelf-life of green plant produce (King &

Morris, 1994). Additionally, major food-crops such as wheat and barley

2

employ leaf senescence to remobilize most of their leaf nutrients during seed

filling (Gregersen et al., 2008). During these processes, the rate of

senescence influences the nutrient-composition of both leaves and seeds and

thereby affects the desired quality of the produce (Gregersen et al., 2008). A

better understanding of the senescence process would therefore allow us to

modulate both plants and growing conditions to selectively postpone or

induce leaf senescence, depending on our needs with respect to growth

season, seed filling, nutrient composition or resistance to different stresses.

The process of leaf senescence

Leaf senescence is a genetically controlled process (Yoshida, 1963),

during which chloroplasts, which contain the photosynthetic machinery, are

converted into gerontoplasts (Thomas et al., 2003). This conversion is

realized by the degradation of chlorophyll and chloroplastic protein

(Krupinska, 2006) and ultrastructural changes such as the loss of chloroplast

grana and thylakoids, increased formation of plastoglobuli and decreases in

both the number and the size of the plastids (Zavaleta-Mancera et al., 1999;

Keech et al., 2007; Wada et al., 2009). Alongside these plastidial changes,

mitochondria increase in size and show a relative increase in respiration

activity, most likely to support the active metabolism accompanying leaf

senescence (Keech et al., 2007). Meanwhile, enzymes involved in protein

degradation and the synthesis of transport amino acids become relatively

abundant (Masclaux-Daubresse et al., 2008). Finally, as these processes are

completed, cells lose their structure and collapse.

Chlorophyll degradation

The most commonly observed aspect of leaf senescence is the loss of

chlorophyll, which as such is considered to be a good marker to assess the

progression of leaf senescence (Leopold, 1961; Ougham et al., 2008).

Senescence-associated degradation of chlorophyll is under nuclear control

(Thomas & Stoddart, 1980) and has been intensively studied over the last

decade (Ougham et al., 2008; Schelbert et al., 2009; Sakuraba et al., 2012).

While chlorophyll was initially thought to be degraded solely via

chlorophyllase (CLH; Tsuchiya et al., 1999), this view has recently been

questioned as mutants in the CLH-genes are still able to degrade chlorophyll

during leaf senescence (Schenk et al., 2007) and expression of CLH1 and

CLH2 is positively regulated by light (Banas et al., 2011). Instead, the

degradation of chlorophyll during leaf senescence occurs mainly via the

enzyme pheophytinase (PPH; Schelbert et al., 2009). In both degradation

pathways, chlorophyll b is converted to chlorophyll a in two steps (Figure 1).

Step one is catalyzed by a chlorophyll b reductase complex that contains

Non-Yellow Coloring1 (NYC1) and NYC1-like (NOL) as essential

components (Kusaba et al., 2007; Sato et al., 2009). Step two is catalyzed by

3

7-HydroxyMethyl Chlorophyll a Reductase (HMCR) and yields chlorophyll

a (Meguro et al. 2011). In case of CLH-based degradation (Figure 1, blue

arrows), chlorophyll a is converted to chlorophyllide a by CLH and

subsequently stripped of its Mg by a metal chelating substance (MCS;

Suzuki & Chioi, 2002). In PPH-based degradation (Figure 1; pink arrows),

chlorophyll a is stripped of its Mg by MCS and the resulting pheophytin a

(phein a) converted to pheophorbide a (pheide a) by PPH (Schelbert et al.,

2009). After the formation of pheide a, the two pathways converge and the

risk for photoxic reaction products is removed by the concerted action of

Pheide A Oxygenase (PAO) and Red Chlorophyll Catabolite Reductase

(RCCR; Rodoni et al., 1997; Wüthrich et al., 2000; Pružinská et al., 2003).

The resulting primary fluorescent Chl catabolyte (pFCC) is modified in the

cytosol and transported to the vacuole, where it is converted to and stored as

non-fluorescing chlorophyll catabolites (NCCs; Oberhuber et al., 2003).

Figure 1: Pathways describing chlorophyll (Chl) degradation during leaf

development (via CLH; blue arrows) and leaf senescence (via PPH; pink Arrows).

Chl biosynthesis during leaf development is represented by its final steps (CS and

CAO). Conversions of Chlide a between and Chlide b occur by the same enzymes

mediating the conversions between Chl a and Chl b. Arrow thickness indicates either

minor (thin) or major (thick) contribution to chlorophyll degradation associated to

leaf senescence. Abbreviations: CAO, Chl a oxygenase; Chlide, chlorophyllide; CS,

Chl synthase; RCC, red Chl catabolite. Other abbreviations are mentioned in the

text. Modified after Schelbert et al., (2009) and Sakuraba et al., (2012).

Recently, it was discovered that most of the chloroplastic enzymes

related to the PPH-pathway interact with the protein Stay-GReen (SGR;

Ougham et al., 2008; Figure 1), which is thought to act as a scaffold to

optimize the concerted action of these enzymes (Sakuraba et al., 2012).

4

Disconcerted action of these enzymes causes two opposing effects:

accelerated cell death and cosmetic stay-green phenotypes. Accelerated cell

death occurs when the function of HCMR, PAO or RCCR is impaired and

photoxic intermediates such as pheide a or RCC accumulate. In contrast,

lack of SGR, NYC1, NOL or PPH results in a cosmetic stay-green

phenotype, in which the light harvesting complexes are retained as well

(reviewed in Ougham et al., 2008). These observations suggest that

chlorophyll degradation is related to its stabilizing influence on light

harvesting complex (LHC) protein (Apel & Kloppstech, 1980) and that

removal of chlorophyll allows proteases to access this significant amount

(20%) of cellular protein (Hörtensteiner, 2006).

Protein degradation

While loss of chlorophyll is the most visible aspect of leaf senescence,

its onset is preceded by that of protein loss (Hensel et al., 1993; Keskitalo et

al., 2005). As much as 70% of the leaf protein is located within the

chloroplast (Gan & Amasino, 1997; Hörtensteiner & Feller, 2002), most of

which is in the form of proteins such as Rubisco and LHC-protein (50 and

20% of the leaf protein, respectively; Mae et al., 1983; Hörtensteiner, 2006).

To degrade these proteins, chloroplasts contain a number of proteases such

as Clp, FtsH, DegP and Lon (Adam & Clarke, 2002; Figure 2; chloroplast).

Many of these proteases and their subunits, e.g. the Clp protease system and

FtsH proteases, are constitutively expressed during leaf development and

serve in the turnover and repair of the photosynthetic machinery. Some of

the genes associated to these proteases show increased expression during

leaf senescence (Lin & Wu, 2004), suggesting that the corresponding

proteases contribute to this final developmental stage (Martínez et al.,

2008a). However, isolated chloroplasts from senescing leaves accumulate

specific Rubisco protein fragments (Kokubun et al., 2002), suggesting that

senescence-associated protein degradation is not completed within the

chloroplasts, but relies on the export of cleaved protein fragments to other

cellular compartments for further degradation (Martínez et al., 2008a).

Over the last decade, two types of senescence-associated bodies that

contain partially degraded plastid protein have been identified: Rubisco-

containing bodies (RCBs; Chiba et al., 2003) and Senescence-associated

vacuoles (SAVs; Otegui et al., 2005). The first type, RCBs, are small

spherical bodies (0.4–1.2 µm in diameter) that are found in both the

cytoplasm and the vacuole during the early phases of natural leaf senescence

(Chiba et al., 2003). These bodies contain stromal proteins such as Rubisco

and chloroplastic glutamine synthetase (GS2) and their double membrane, in

addition to their content, suggests that they originate from the chloroplast

(Figure 2, RCB pathway). Additional membrane and tubular structures

surrounding these bodies (Chiba et al., 2003) and the lack of RCBs in

5

AuTophaGy (ATG) mutants such as atg4 or atg5 indicate that the autophagic

system is involved (Ishida et al., 2008; Wada et al., 2009; Figure 2, bottom

pathway). While the ATG-dependent autophagy is required to reduce the

chloroplast size, which remains constant in atg4a4b-1 mutant plants (Wada

et al., 2009), it is not the only system able to reduce stromal chloroplast

protein, as exemplified by similar declines in the Rubisco content in wild-

type and atg4a4b-1 mutant plants. Furthermore, disabling the autophagic

system causes enhanced leaf senescence under conditions of limiting

nutrients, when nutrient relocation is promoted (Doelling et al., 2002;

Hanaoka et al., 2002; Thompson et al., 2005; Phillips et al., 2008). This

implies that although autophagy seems to be involved during regular leaf

senescence, it can be replaced or supplemented by other, more aggressive,

degradation processes (Martínez et al., 2008a).

Figure 2: Representation of protein degradation pathways during leaf senescence, in

particular those involving Rubisco containing bodies (RCB) and senescence

associated vacuoles (SAV). Other abbreviations are explained in the text. Modified

after Gregersen et al., (2008).

A strong candidate for such process is the senescence-specific formation

of the second type of bodies; SAVs, which are small (0.55–0.70 µm) lytic

vacuoles with a low pH (5.2) that do not depend on a functional autophagic

system (Otegui et al., 2005; Figure 2, SAV pathway). Besides partially

degraded chloroplast stromal protein, they may contain chlorophyll a, but

lack thylakoid proteins such as photosystem II reaction center protein D1 or

light harvesting complex protein II (Martínez et al., 2008b). In contrast to

isolated chloroplasts, isolated SAVs continue to degrade their protein via

proteases such as Senescence-Associated Gene 12 (SAG12; Otegui et al.,

2005; Martínez et al., 2008b), the gene of which is expressed late during leaf

senescence (Weaver et al., 1998). Both RCBs and SAVs are believed to

6

deliver their contents to the central vacuole for storage and further

degradation (Martínez et al., 2008a; Wada et al., 2009), as a number of genes

associated to vacuolar protease activity, such as SAG2, RD21 and γVPE,

show enhanced expression during leaf senescence (Kinoshita et al., 1999;

Buchanan-Wollaston et al., 2003; Gepstein et al., 2003).

In addition to chloroplast and vacuole-based protease systems, the

cytosolic/nuclear ubiquitin proteosome pathway appears to be enhanced

during leaf senescence (Park et al., 1998; Masclaux-Daubresse et al., 2008).

Important demonstrations of the involvement of this pathway involve

mutations of genes such as ORESARA 9 (ORE9) and Nitrogen Limitation

Adaptation (NLA). The ORE9 protein contains an F-box domain and has

been shown to be able to form a ubiquitin E3 ligase complex, suggesting that

it can ubiquitinate specific substrates to target them for degradation by the

26S proteasome and the ore9-1 mutant shows delayed leaf senescence (Woo

et al., 2001). The NLA protein interacts with an ubiquitin conjugase in the

nucleus and mutation of the NLA gene leads to enhanced senescence under

nitrogen-limiting conditions (Peng et al., 2007).

Altogether, senescence-associated protein degradation is carried out in

different cellular compartments by the concerted action of several protease-

systems, some of which are constitutive and some of which are enhanced

during leaf senescence.

Nitrogen relocation

One of the main functions attributed to leaf senescence is the relocation

of nitrogen from senescing to developing leaves (Smart, 1994; Lim et al.,

2007). To this end, the breakdown products of the degraded proteins are

incorporated into different amino acids for transport through the phloem

(Masclaux-Daubresse et al., 2008; figure 3, upper part). Transcript and

metabolite profile studies have shown that during leaf senescence, a number

of enzymes involved in the biosynthesis and transamination of amino acids

are enhanced consistently in different plant species (Masclaux-Daubresse et

al., 2008). Among these, two enzymes have been extensively studied:

Glutamate DeHydrogenase (GDH) and cytosolic Glutamine Synthase (GS1).

Within the mitochondria, GDH converts glutamate, originating from

transamination reactions, to α-ketoglutarate and ammonium. While α-

ketoglutarate can be used as a respiratory substrate via the tricarboxylic acid

(TCA) cycle, ammonium is exported to the cytoplasm, where it can be used

by GS1 to convert glutamate into glutamine (Masclaux-Daubresse et al.,

2008). In turn, glutamine can be used to convert aspartate into asparagine by

Asparagine Synthetase (AS), whose transcripts increase during leaf

senescence in both sunflower (Herrera-Rodriguez et al., 2006) and

Arabidopsis (Lin & Wu, 2004). Lin & Wu (2004) further proposed, based on

7

Figure 3: The concerted action of nitrogen remobilization (upper part),

galactolipid mobilization (insert) and lipid degradation (lower part). Abbreviations:

ACX, acyl CoA oxidase; α-gal, α-galactosidase; α-KG, α-ketoglutarate; AS,

asparagine synthase; Asn, asparagine; Asp, aspartate; Asp AT, aspartate

aimontransferase; AT, amino transferases; β-gal, β-galactosidase; Chl, chlorphyll;

CoA, coenzyme A; DAG, diacylglycerol; DGAT, DAG acyltransferase; GDH,

glutamate dehydrogenase; g-lip, galactolipase; Gln, glutamine; Glu, glutamate; GS1,

cytosolic glutamine synthase; ICL, isocitrate lyase; LACS, long-chain acyl CoA

lyase; Mal, malate; MDH, malate dehydrogenase; MS, malate synthase; OAA,

oxaloacetate; PEP, phosphoenol pyruvate; PEPCK, PEP carboxykinase; PPDK, PEP

dikinase; SAVs, senescence-associated vacuoles; Suc, succinate; TAG,

triacylglycerol; TCA cycle, tricarboxylic acid cycle. Modified after Thompson et al.,

(1998), Masclaux-Daubresse et al., (2008) and Dörmann (2010).

8

the senescence-associated increase in several gene transcripts in darkened

plants, that the aspartate required for asparagine synthesis is derived from

pyruvate through the combined action of Phosphoenol Pyruvate DiKinase

(PPDK), PhosphoEnol Pyruvate CarboxyKinase (PEPCK) and aspartate

aminotransferase. Additionally, PEPCK increases in response to increased

ammonium and is present specifically in phloem companion cells (Chen et

al., 2004), further supporting a role for this enzyme during the generation

and export of nitrogen-rich amino acids from senescing leaves.

Lipid degradation

As chloroplasts are converted into gerontoplasts, thylakoid lipid

degradation becomes a prominent feature (figure 3, lower part), providing

both carbon backbones for the formation of amino acids as well as

respiratory carbon (Buchanan-Wollaston, 1997; Krupinska, 2006). When

thylakoid membranes are dismantled, their breakdown products are stored in

plastoglobuli, which increase in both number and size (Krupinska, 2006).

However, in contrast to other membranes, thylakoid membranes contain a

large fraction of galactolipids (Lee, 2000), which are not directly stored in

the plastoglobuli (Tevini & Steinmüller, 1985). Instead, galactolipids are first

degraded inside the chloroplasts via a pathway involving α-galactosidases, β-

galactosidases and galactolipases (Krupinska, 2006; Dörmann, 2010) (figure

3, insert). These enzymes are upregulated during leaf senescence in various

plant species (Thompson et al., 1998; Lee et al., 2004; Krupinska, 2006) and

increased activity of an α-galactosidase in rice was recently shown to

directly influence the abundance of thylakoid membranes (Lee et al., 2009).

The resulting diacylglycerols (DAGs) are subsequently converted into

triacylglycerols (TAGs) by DAG acyltransferase (DGAT), which is strongly

increased during leaf senescence (Kaup et al., 2002). Meanwhile, free fatty

acids originating from galactolipase action and phytol, a byproduct from

chlorophyll degradation, are converted into fatty acid phytyl esters and

together with TAGs sequestered in the plastoglobuli (Kaup et al., 2002;

Dörmann, 2010). Plastoglobuli are subsequently extruded from the

chloroplast into the cytoplasm, through which they gain access to

glyoxysomes (Guiamet et al., 1999) (Figure 3, lower part). Glyoxysomes are

peroxisomes (Pracharoenwattana & Smith, 2008) that specialize in the β-

oxidation of TAG-derived fatty acids into acetyl coenzyme A to form

succinate via the glyoxylate cycle (Thompson et al., 1998). During leaf

senescence, various genes in these pathways, such as isocitrate lyase, malate

synthase, long-chain acyl CoA lyases and acyl CoA oxidases are upregulated

(Yang & Ohlrogge, 2009). Succinate is subsequently exported to the

mitochondria, where it enters the Krebs cycle (Thompson et al., 1998). From

this cycle, malate is exported into the cytosol and by NAD+-dependent

malate dehydrogenase converted into oxaloacetate, which can be used either

9

as an amino acid backbone (Lin & Wu, 2004) or via PhosphoEnolPyruvate

(PEP) converted into sugars for export or ATP-production (Buchanan-

Wollaston, 1997; Thompson et al., 1998).

As senescence progresses, non-thylakoid membranes are also degraded

through the concerted action of phospholipase D, phosphatidic acid

phosphatase, lytic acyl hydrolase and lipoxygenase (Thompson et al., 1998).

It appears that this is facilitated by the action of an acyl hydrolase encoded

by senescence-associated gene SAG101, as inhibition of this gene delays

leaf senescence somewhat and its overexpression enhances it (He & Gan,

2002).

Mitochondria and loss of cellular organization

As previously mentioned, mitochondria have a central role in the

remobilization of nutrients and providing the energy to establish this

(Buchanan-Wollaston, 1997). As senescence progresses, mitochondria

undergo distinct morphological changes as they increase in size, become

rounder in shape and decrease in number (Keech et al., 2007). Despite these

alterations and a considerable loss of their protein, mitochondria maintain a

relatively high activity, likely to sustain energy levels and carbohydrate

backbones for the relocation of nutrients (Thompson et al., 1998, Keech et

al., 2007; 2010). In addition, despite the early disruption of the microtubule

network (Keech et al., 2010), mitochondria retain a residual mobility

(Keech, 2011). This mobility indicates that cytoplasmic streaming is at least

partially retained until the end of leaf senescence via the conservation of

actin filaments (Keech, 2011).

When the above-described processes have progressed towards a critical

state, tonoplast rupture and the spill of lytic vacuolar content into the

cytoplasm cause a rapid loss of the remaining cellular structure and

inevitably lead to cell death (Hörtensteiner & Feller, 2002; van Doorn &

Woltering, 2004).

10

What induces and inhibits leaf senescence?

There are various types of plant senescence, each induced by specific

factors. An early classification of the process (Leopold, 1961) described

senescence to be either somatic (whole plant), top (stem and leaves),

deciduous (leaves only) or progressive (part by part). Nowadays, with the

increasing knowledge of different factors that can induce leaf senescence, it

is more common to specify leaf senescence based on its inducing factor. The

most recognized factor in this respect is autumn (Keskitalo et al., 2005),

which has recently been shown to induce senescence via the accompanied

reduction in daylength (Fracheboud et al., 2009). Other factors include a

number of biotic and abiotic factors, such as pathogens (Smart et al., 1994),

drought (Rivero et al., 2007), ozone (Miller et al., 1999), shade (Rousseaux

et al., 1996) and darkness (Weaver & Amasino, 2001). In addition, leaf

senescence is also modulated by plant hormones such as ethylene, abscissic

acid and cytokinins (Smart, 1994).

Ethylene is well known as an enhancer of various forms of senescence

(Abeles et al., 1988; Grbic & Bleecker,1995; Weaver et al., 1998) and the

genes related to its biosynthesis, such as 1-aminocyclopropane-1-carboxylic

acid (ACC) synthase or ACC oxidase are upregulated in senescing leaves

(van der Graaff et al., 2006; Lim et al., 2007). Inhibition of ethylene

biosynthesis correspondingly results in enhanced leaf longevity and

continued production of chlorophyll in tomato (John et al., 1995). In

Arabidopsis, leaf senescence is also delayed in ethylene perception mutants

such as ethylene resistant 1 (etr1; Grbic & Bleecker, 1995), and ethylene

signal transduction mutants such as ethylene insensitive 2 (ein2; Oh et al.,

1997). However, plants that are grown continuously under high ethylene or

mutants that have a constitutive ethylene signal transduction (ctr1) show no

premature leaf senescence (Kieber et al., 1993). The senescence-associated

response to ethylene appears to be related to leaf age, since mutants that

exhibit a different onset of leaf death (old) show altered responses to

ethylene (Jing et al., 2002; 2005).

Abscissic acid is well known for causing stomatal closure and enhancing

leaf abscission and senescence (Weaver et al., 1998; Dodd, 2003; Lee et al.,

2011). During leaf senescence, the level of ABA increases (Gepstein &

Thimann, 1980) and an ABA-inducible Receptor-like Protein Kinase, RPK1,

has recently been identified as a positive regulator of age-dependent leaf

senescence (Lee et al., 2011).

Cytokinins are well known inhibitors of leaf senescence (Mothes, 1960;

Zavaleta-Mancera et al., 1999). Presence of cytokinin causes relocation of

amino acids towards the application site (Mothes, 1960) and enhances

transcription of factors that are important in nitrate metabolism, carbon

metabolism and protein synthesis (Sakakibara et al., 2006). Additionally, in

Chenopodium rubrum L., cytokinins promote the expression of extracellular

11

invertase CIN1 and hexokinase transporters CST2 and 3, which correlated

with an increased uptake of sugars (Ehneß & Roitsch, 1997). Further

research in tobacco has shown that the inhibition of leaf senescence by

cytokinins actually depends on the enhanced expression of extracellular

invertase and the unloading of sugars from the phloem (Balibrea-Lara et al.,

2004).

Besides hormonal influences, the availability of additional nutrients such

as nitrogen often delays or even reverses leaf senescence (Mothes, 1960;

Ono & Watanabe, 1997; Schildhauer et al., 2008). Some species, which

maintain a symbiosis with nitrogen-fixing bacteria, show altered leaf

senescence. Black alder trees, for example, have a high nitrogen content and

show delayed autumn leaf senescence (Côtté & Dawson, 1986). These

observations suggest that, as long as leaves have access to sufficient

metabolizable nitrogen, leaf senescence and the accompanying nitrogen

relocation can be postponed.

On the other hand, the influence of sugars (glucose, fructose and

sucrose) on leaf senescence is a more complicated story, as sugars have been

shown to both inhibit and induce leaf senescence, depending on the

conditions of the senescing material (van Doorn, 2008). For example, while

in detached senescing leaves, addition of sugars can reduce the senescence-

specific expression of SAG12 (Noh & Amasino, 1999), accumulation of

sugars by means of disrupting phloem export via steam-girdling is

accompanied by leaf senescence (Parrot et al., 2005). Leaf senescence is also

induced when plants are grown under low nitrogen conditions and either

high light (Ono et al., 1996) or in the presence of additional sugars (Pourtau

et al., 2004). Interestingly, both the enhanced sugar levels and the leaf

senescence are reduced by addition of nitrogen to the system (Ono and

Watanabe, 1997). Finally, in senescing leaves of whole plant systems, sugars

increase around the same time as chlorophyll decreases (Masclaux et al.,

2000; Wingler et al., 2006), after a considerable decrease in levels of both

protein and amino acids (Masclaux et al., 2000). Together, these

observations suggest that the ability of sugars to enhance leaf senescence is

tightly connected to the reduced availability of nitrogen.

Over the years, this large variety of factors that influence leaf senescence

has been studied using many different experimental systems. Among these

systems, darkening plants or leaves has proven to be a convenient method to

induce a leaf senescence that exhibits similar features as natural senescence

(Buchanan-Wollaston et al., 2005, van der Graaff et al., 2006, Keech et al.,

2010). Additionally, inhibition of dark-induced leaf senescence by light has

been used extensively to draw conclusions with regard to shade-induced leaf

senescence (for review, see Biswal & Biswal, 1984).

12

Shade- and Dark-induced leaf senescence

As mentioned earlier, both shade and darkness can induce leaf

senescence. However, the rate at which these types of leaf senescence

progress depends on whether the plant is completely or partially shaded or

darkened. Completely shaded or darkened plants show a reduced rate of leaf

senescence (Mae et al., 1993; Weaver & Amasino, 2001, Keech et al., 2007),

whereas partial darkening induces a high rate of leaf senescence in the

darkened parts (Weaver & Amasino, 2001; Keech et al., 2007). This

difference in rates appears to be caused by the reduced metabolism of

completely shaded or darkened plants (Ono et al., 1996; Keech et al., 2007).

Shade and Darkness

While complete darkness occurs rarely in natural plant habitats, shade is

very common as plants overshadow each other. Leaf chlorophyll absorbs red

(R) and blue light and both transmits and reflects the photosynthetically

inactive green and far-red light (FR). As a result, leaf shade consists of

decreases in both the light intensity and the ratio between red and far-red

light (R/FR ratio). These decreases are detected by plants through three light-

dependent mechanisms, each of which has been connected to leaf

senescence: photosynthesis, photomorphogenesis and transpiration.

Photosynthesis

Photosynthesis is the process in which light is absorbed by the plant and

converted to chemical energy in the form of carbohydrates. The

photosynthetic process can be divided into two parts: a) light harvesting and

electron transport in the chloroplast thylakoids, which yield ATP and

NADPH and b) the Calvin cycle in the chloroplast stroma, which uses the

ATP and NADPH to fix CO2 into carbohydrates (Figure 4a).

Light harvesting occurs when light induces charge-separation in

chlorophyll molecules of the light harvesting antennae, followed by electron

transport that results in the generation of ATP and NADPH. To optimize this

process, chlorophyll is organized into light harvesting antennae that funnel

the electrons via other chlorophyll molecules into the reactions centers of

two different photosystems. Photosystem I (PSI) absorbs light of 700nm

(far-red light) and is located in the thylakoid lamellae exposed to the

chloroplast stroma, while photosystem II (PSII) absorbs light of 680nm (red

light) and is located in the thylakoid grana stacks (Anderson et al., 1988,

Wagner et al., 2008, Figure 4b). Concerted action of both PSI and PSII is

necessary to establish an electron transfer chain that splits water into oxygen,

protons and electrons. While oxygen is a side product, the electrons are used

to increase the proton gradient and eventually reduce NADP+ to NADPH,

while the proton gradient is used to generate ATP (Figure 4a).

The products of light harvesting, ATP and NADPH, are subsequently

13

used in the Calvin cycle, in which RibUlose-1,5-BISphosphate Carboxylase

Oxygenase (Rubisco) and other enzymes work together to assimilate CO2

into carbohydrates. Most of these carbohydrates are stored as starch in the

chloroplast or exported to form sucrose in the cytosol. Besides sucrose and

starch, a fraction of carbohydrates is also used to generate cellular building

blocks such as fatty acids or to assimilate nitrate into amino acids. During

the night, when photosynthesis does not occur, starch is used for respiration

or reconverted into sucrose and exported via the phloem.

Figure 4: a) Schematic representation of the process of photosynthesis, divided in

light reactions (left) and Calvin cycle (right). b) Chloroplast organization and

location of PSI and PSII within thylakoid lamellae and grana stacks, respectively

(insert).

However, before a net carbohydrate production can be established,

carbohydrate production by photosynthesis needs to exceed its consumption

by respiration. The light intensity where the two processes are in balance is

called the light compensation point (LCP). To maintain a positive

carbohydrate production in response to shade, plants can optimize their

photosynthesis through different acclimation strategies: Photosynthetic

Acclimation (PA) and Photosynthetic Resource Partitioning (PRP; Evans,

1993). Photosynthetic acclimation is induced by reductions in light intensity

to increase the size of the light harvesting antennae relative to the

photosystem cores (Anderson et al., 1995; Walters, 2005). Furthermore,

reductions in the R/FR ratio cause PA to adjust the relative abundances of

PSI and PSII so that the altered activity of PSI balances that of PSII

(Anderson et al., 1995, Wagner et al., 2008). Whereas shading of whole

plants induces only PA, shading parts of plants will additionally induce PRP,

which relocates nutrients from shaded to young, non-shaded plant parts

(Evans, 1993; Hikosaka et al., 2005). Although PRP decreases the

photosynthetic capacity of the shaded leaves, it effectively increases the

photosynthetic capacity of the whole plant (Evans, 1993).

14

Over the past decades, photosynthesis has been mentioned intermittently

as a process by which light could inhibit leaf senescence and a number of

observations suggest that a functional photosynthesis can inhibit leaf

senescence. First, inhibition of electron transport by 3-(3,4-

DiChlorophenyl)-1,1-diMethylUrea (DCMU) or 2,5-DiBromo-3-Methyl-6-

Isopropyl-p-Benzoquinone (DBMIB) tends to induce senescence in

illuminated leaves (Goldwaithe & Laetsch, 1967; Thimann et al., 1977;

Okada & Katoh, 1998). Although contradicting results have been published

(Haber et al., 1969, Ono & Watanabe, 1997), both the induction (Thimann et

al., 1977) and inhibition (Ono & Watanabe., 1997) of leaf senescence in

response to DCMU were accompanied by a decrease in leaf carbohydrate

content, which is well known to influence leaf senescence in different ways

(van Doorn, 2008). Second, light requires CO2 (Satler & Thimann, 1977)

and light intensities above the LCP to inhibit leaf senescence (Veierskov,

1987; Boonman et al., 2006). Finally, photosynthetic protein and activity

show a marked decline prior to the enhanced expression of senescence-

associated genes SAG2 and SAG4, suggesting the decline in photosynthesis

to precede leaf senescence (Hensel et al., 1993).

Regarding the acclimation responses, leaf senescence and PA have been

shown to occur alongside each other and PA has been discussed to interfere

with senescence by reducing the degradation of both light harvesting

complex proteins and chlorophyll (Mae et al., 1993). Degradation of

chlorophyll and protein occurs as part of both leaf senescence and PRP.

Nevertheless, PRP has been argued to differ from leaf senescence on the

bases of the chloroplast-specific degradation during PRP (Hikosaka et al.,

2005) and the specific initiation of leaf senescence by FR (Pons & de Jong

van Berkel, 2004).

Photomorphogenesis

Photomorphogenesis refers to the collection of processes regulating

plant development in response to light signals (Franklin & Quail, 1020).

Photomorphogenic processes are best known for their role during seedling

development and in establishing the photosynthetic machinery (Franklin &

Quail, 2010; Shin et al., 2009). In plants, there are several known types of

light receptors: phytochromes, which absorb red and far-red light (Franklin

& Quail, 2010), cryptochromes and phototropins, which absorb blue light,

UVR8, which absorbs UV-B (Brown and Jenkins, 2008) and an unknown

receptor that absorbs green light (Zhang et al., 2011). Of these light

receptors, phytochromes have been studied most extensively with regard to

leaf senescence.

15

Phytochromes

Phytochromes (PHY) are a class of light receptors that mainly absorb R

and FR light (Franklin & Quail, 2010). They are synthesized in an inactive

Pr form and upon absorption of R undergo photoconversion into their active

Pfr form (Figure 5a). This active form can absorb FR, which converts them

back into their inactive Pr form, something that also occurs passively in

darkness through the process of dark reversion (Rausenberger et al., 2010).

While in their active Pfr form, most phytochromes, like PHYB in figure 5a,

expose a nuclear localization sequence that allows the Pfr to be translocated

into the nucleus (Chen et al., 2005). In the nucleus, Pfr can either bind to

phytochrome interaction factors (PIFs) and initiate their degradation

(Franklin & Quail, 2010) or repress the COnstitutive Photomorphogenic/

De-ETiolated/ FUSca (COP/DET/FUS) system (Lau & Deng, 2010) (Figure

5b). Both PIFs and the COP/DET/FUS system negatively influence

photomorphogenesis; PIFs by being transcription factors that repress

photomorphogenesis-related genes and the COP/DET/FUS system by

mediating the degradation of transcription factors, such as HYpocotyl

elongated 5 (HY5), that promote photomorphogenesis (Saijo et al., 2003,

Lau & Deng, 2010). Thus, by reducing the levels of PIFs and promoting

those of HY5, phytochromes indirectly promote photomorphogenesis.

Figure 5: Light signaling by phytochromes. a) Activation of light-stable

(represented by PHYB) and light labile (PHYA) phytochromes and the mechanisms

that translocate them to the nucleus. Modified after Rausenberg et al., (2011). b)

Simplified phytochrome signaling pathways within the nucleus, involving different

photomorphogenesis-related transcription factors. Modified after Lau & Deng

(2010).

16

Plants contain multiple phytochromes that can be subdivided into two

types: light-stable and light-labile phytochromes (Franklin & Quail, 2010).

In light-grown plants, the main type is light-stable phytochrome, which in

Arabidopsis consists of four members: PHYB, C, D and E. Of these

members, PHYB is the most abundant (Sharrock & Clack, 2002) and shows

redundant functions with PHYD and PHYE (Franklin & Quail, 2010). Light-

stable phytochromes mediate Low Fluence Responses (LFR), the strength of

which depends on the relative amount of active phytochrome. Short periods

of light, even pulses, can be sufficient to induce a response. For instance, a

pulse of R can induce de-etiolation in a dark-grown plant, whereas a

subsequent FR pulse can inhibit this signal. Examples of photoreversible

responses regulated by light-stable phytochromes are de-etiolation and

shade-avoidance (Franklin & Quail, 2010).

The other type, light-labile phytochrome, consists solely of PHYA and

while signal transduction of light-stable phytochromes is relatively

straightforward, that of PHYA is more complex. First of all, while PHYA is

very abundant in seeds and dark-grown seedlings, it is rapidly degraded

under light conditions (Clough & Vierstra, 1997). However, despite this

degradation, a small quantity of PHYA forms a small, but stable pool that

contributes to a variety of functions such as shade avoidance, photoperiod

detection and internode elongation (Franklin et al., 2007; Franklin & Quail,

2010). Second, in contrast to light-stable phytochromes, PHYA lacks a

localization sequence. Instead, activated PHYA depends on binding to Far-

red elongated HYpocotyl 1 (FHY1) or FHY1-Like (FHL) for transport to the

nucleus (Rausenberger et al., 2011) (Figure 5a). Once inside the nucleus,

PHYA requires inactivation by FR to be released from these shuttling

proteins and reactivation by R before light signals can be transduced. These

unique properties cause PHYA-signaling to be stronger under low light

conditions that are enriched in FR (Rausenberger et al., 2011) and thereby

reduce the inhibiting effect of FR on the other phytochromes (Yanovsky et

al., 1995; Smith et al., 1997). Third, PHYA mediates two distinct response

modes: the Very Low Fluence Response (VLFR) and the High Irradiance

Response (HIR) (Casal et al., 1998; Zhou et al., 2002). Of these modes, the

VLFR is extremely sensitive and can be triggered by pulses of either R or

FR at extremely low light fluences, while the HIR requires continuous light.

Phytochromes have been implicated with leaf senescence based on

observations that R inhibits loss of chlorophyll and protein and that

subsequent FR can negate this inhibition (Sugiura, 1963, Tucker, 1981,

Biswal & Biswal 1984). Since effective inhibition of leaf senescence by R is

accomplished by R pulses of a mere few minutes long, it is unlikely that the

inhibition is caused by photosynthesis (de Greef & Fredericq, 1971; Okada

& Katoh, 1998). Recent observations in phytochrome quintuple mutants,

17

which develop green leaves under white or blue light and turn yellow after

transfer to red light, indicate that the combination of an established

photosynthetic machinery and photosynthetic light irradiance is not

sufficient to inhibit leaf yellowing (Strasser et al., 2010). Furthermore,

prolonged addition of FR to white light has been reported to induce leaf

senescence (Rousseaux et al 1996, 1997), whereas plants that overexpress

either PHYA or PHYB exhibit delayed leaf senescence (Cherry et al 1991,

Rousseaux et al 1997, Thiele et al 1999).

However, phytochrome signaling is well-known to affect expression of

photosynthesis-related genes (Kaufman et al., 1984; Shin et al., 2009).

Concomitantly, plants overexpressing either PHYA or PHYB contain

enhanced levels of chlorophyll and a number of photosynthetic proteins

(Cherry et al., 1991, Sharkey et al., 1992, Rousseaux et al., 1997, Thiele et

al., 1999). Moreover, the loss of chlorophyll in PHYB-overexpressing potato

starts at the same time as the wild type (Thiele et al., 1999). These

observations suggest that the enhanced levels of chlorophyll and protein may

not be due to enhanced leaf senescence, but are rather a consequence of the

phytochrome overexpression phenotype that interferes with the markers for

visual leaf senescence, namely chlorophyll and protein. Altogether, while

phytochromes have been shown to be important for retaining the

photosynthetic machinery and delaying leaf senescence, little information is

available regarding the link between the phytochrome signaling and its

inhibiting effect on leaf senescence.

Transpiration

The third light-dependent mechanism by which plants can detect

changes in both light quality and light intensity is transpiration (Pieruschka

et al., 2010). Most transpiration occurs when water in the leaf apoplast

evaporates after absorbing light energy. The rate of evaporation by light

depends on both the intensity and the wavelength of the light; since higher

light intensities and lower wavelengths contain more energy and thus

evaporate more water than lower light intensities and higher wavelengths

(Pieruschka et al., 2010). The evaporated water is evacuated from the leaf

through open stomata, thus reducing leaf temperature. Besides regulating

leaf temperature, evaporation also drives xylem-mediated transport of water,

mineral nutrients and root-synthesized cytokinins (Sakakibara, 2006).

Cytokinins can stimulate transpiration by promoting the opening of

stomata (Dodd, 2003). Open stomata, besides stimulating transpiration, have

further been suggested to delay leaf senescence in two other ways; by

enhancing the CO2-concentration within the leaf and thereby photosynthesis

(Biswal & Biswal, 1984) and by preventing accumulation of senescence-

stimulating hormone ABA, which occurs after stomatal closure (Gepstein &

Thimann, 1980).

18

Reducing transpiration by increasing the relative humidity around

individual leaves has been shown to reduce the amount of cytokinins and

cytokinin-responsive factors in the leaf and to result in enhanced leaf

senescence (Boonman et al., 2007). However, contradictory results were

obtained when reducing transpiration by application of petroleum-ether on

both sides of attached leaves, which did not particularly induce leaf

senescence (Weaver & Amasino, 2001).

SUMMARY

Understanding the process of shade-induced leaf senescence is important

with regard to improve the cultivation of crops. When cultivating crops at

high density, crop yields may be reduced by senescence of the lower leaves.

Such leaf senescence is induced by relative changes in the light conditions

lower in the leaf canopy, which are caused by shade from the upper leaves.

As mentioned previously, research regarding shade-induced leaf senescence

has been conducted using a variety of experimental setups, ranging from

detached leaves to whole plants at different developmental stages.

Physiologically different systems have shown to cause considerable

differences in the rate of leaf senescence, as exemplified in darkened plants

and individually darkened leaves. Therefore, comparing observations

between different experimental systems should be done with care, taking

into account the limitations of the experimental systems, as well as the

consequential implications with regard to leaf physiology. As previously

mentioned, light has been shown to inhibit leaf senescence through different

processes such as photosynthesis, photomorphogenesis and transpiration.

However, the signaling and molecular mechanisms by which these processes

can inhibit leaf senescence are still unclear. In order to address these

questions, it would be convenient to use an experimental system based on

the model plant Arabidopsis thaliana that resembles the physiology of a

plant participating in a crop-canopy.

19

AIM

With regard to the above, the aim of this project was to establish an

experimental system in Arabidopsis to study shade-induced leaf senescence,

similar to that in canopies, and to use this system to increase the

understanding of how changes in light affect leaf senescence. To achieve this

aim, the following questions were asked:

1) How does the type of darkening, whole plants or individual leaves, affect

the senescence process?

2) How do changes in light intensity and R/FR ratio affect leaf senescence in

individually shaded leaves?

3) To what extent do phytochromes contribute to shade-induced leaf

senescence?

20

RESULTS AND DISCUSSION

Integrating the three mechanisms by which changes in the light

environment control shade-induced leaf senescence, photosynthesis,

photomorphogenesis and transpiration, is difficult due to their simultaneous

occurrence. Additionally, the use of different experimental systems, growth

conditions and methods to assess the progression of leaf senescence may

have affected one or more components of these mechanisms and thereby

affected the senescence-process. I will therefore start this discussion by

comparing different experimental systems that have been used to study

shade- and dark-induced leaf senescence. Then I will relate the basics of the

experimental system and how it was used to study how changes in light

intensity and light quality affect leaf physiology and senescence. After a

discussion on the overlap between leaf senescence and photosynthetic

acclimation responses, the focus will shift to the role that phytochrome A

plays under shade. Finally, I will discuss how these results integrate in the

present knowledge and how this knowledge suggests that photosynthesis,

photomorphogenesis and transpiration work together to regulate shade-

induced leaf senescence.

Different experimental systems

Shade-induced leaf senescence naturally occurs in plant canopies, where,

from top to bottom, light intensity and R/FR ratio decrease and relative

humidity (RH) and leaf age increase. These parameters differ per canopy,

depending on variables such as growth conditions, plant species, and density

of the plant populations within that canopy. To reduce the number of

variables and to identify important components of the senescence-process,

shade-induced leaf senescence has been studied using different experimental

setups:

Earlier studies on shade- and dark-induced leaf senescence often used

detached leaves or leaf segments floating in aqueous solutions (Figure 6a),

which allowed studies regarding the effects of light (Sugiura, 1963), air-

composition (Satler & Thimann, 1983) and exogenous application of

chemical substances (Goldthwaite & Laetsch, 1967; Gepstein & Thimann,

1980) on the senescence-process. While such a system is relatively simple to

handle, detached leaves lack the physiological interactions with the rest of

the plant, such as regulated supplies of water, nitrogen and cytokinins via the

xylem and sugars and other nutrients via the phloem. Physiologically

regulated transport of these nutrients to the apoplast can to some extent be

bypassed by adding supplements to the aqueous solution, which through the

stomata can directly access the apoplast (van Doorn, 2005; van Doorn,

2008).

21

Other studies resorted to shading or darkening whole plants (Mae et al.,

1993; Ono et al., 1996, Weaver & Amasino, 2001; Figure 6b).

Physiologically, this type of shading mimics plants that spend part or all of

their development underneath a closed canopy. The advantage of darkening

plants instead of detached leaves is its convenience and the fact that the

communication between leaves and plant is kept intact. However, during

shade-induced leaf senescence in natural conditions, such a system does not

subject a plant to a light-gradient such as in a crop-canopy. In crops, the light

gradient regulates relocation of nitrogen from shaded to non-shaded leaves

(Hikosaka et al., 2005). Furthermore, the rate of chlorophyll degradation in

this system is reduced compared to that of detached leaves (Ono et al., 1996;

Weaver et al., 1998), suggesting that these systems differ in leaf senescence

(Weaver et al., 1998).

Figure 6: Three experimental systems to study shade- and dark-induced leaf

senescence; detached leaves floating on an aqueous solution (a); darkened plants:

the cover is shown semitransparent to reveal the setup underneath, which is

ventilated through holes in the floor (b); individually darkened leaves: darkening

envelopes reduce both light and heat, while the rest of the plant remains under

growth light conditions (c).

A more recent experimental system that better approaches the partial

plant shading in a crop canopy involves darkening only a few leaves per

plant (Figure 6c). This system allows the rest of the plant to produce

carbohydrates and to act as a sink for senescence-associated relocation of

nitrogen (Weaver & Amasino 2001). As a result, the darkened leaves in this

system senesce at a faster rate than leaves of darkened plants and this has

been connected to a reduced metabolic status in shaded and darkened plants

(Ono et al., 1996, Keech et al., 2007).

Darkened plants versus individually darkened leaves

To improve our understanding of why darkened plants show a reduced

rate of leaf senescence compared to individually darkened leaves, we

22

investigated the metabolomes and the transcriptomes of the darkened leaves.

Profiling these metabolomes and transcriptomes using principal component

analysis showed that leaves from the two dark treatments differed

considerably from illuminated leaves after up to 6 days of treatment (Paper I,

Figure 1a,c). Further analysis of the metabolomes, using supervised

Orthogonal Projection to Latent Structures - Discriminant Analysis (OPLS-

DA; Wiklund et al., 2008), revealed that leaves of darkened plants became

increasingly different from individually darkened leaves as the dark-

treatments progressed (Paper I, Figure 1b). Much like the metabolome, the

transcriptome also showed the two dark-treatments to be different (Paper I,

Figure 1c). However, in contrast to the metabolomes of the darkened leaves,

their transcriptomes did not diverge as the treatment progressed. Because of

these characteristics, further transcript analyzes were based on the average of

the different time points.

Darkened leaves are sugar-starved

Comparing the metabolomes of leaves from both dark-treatments to

those of illuminated leaves was done using a Shared and Unique Structure

(SUS) plot (Paper I, Figure 2a). This comparison revealed that, compared to

illuminated leaves, darkened leaves contained less metabolites related to

photosynthesis and photorespiration, such as sucrose, fructose and glycine.

Meanwhile, darkened leaves, in particular those from darkened plants,

showed increased levels of amino acids (Table 2). Additional comparison of

the transcriptomes of the respective treatments revealed that both dark-

treatments caused a reduced expression of genes related to photosynthesis

and photorespiration, while expression of genes related to cell wall

degradation, gluconeogenesis and biosynthesis of nitrogen-rich amino acids

was increased. These changes in the metabolomes and transcriptomes of the

dark-treatments indicated that the darkened leaves were carbon-starved. To

investigate this further, the transcript profiles of both dark-treatments were

compared to those of sucrose-starved cell suspension cultures (Contento et

al., 2004). In individually darkened leaves, 28% of the up- and 59% of the

down-regulated genes behaved similar to those in the cell suspension

cultures. In darkened plants, these values were 75% and 65%, respectively,

indicating that at least the darkened plants exhibited sucrose starvation.

Sucrose starvation has been described to relieve the inhibition of protein

KINases KIN10 and KIN11 (Baena-Gonzales et al., 2007). These kinases are

both members of the Starvation-induced sucrose Non-fermenting-1-Related

protein Kinase-1 (SnRK1) family and promote a variety of processes related

to catabolism (Baena-Gonzales et al., 2007, Baena-Gonzales & Sheen 2008).

To assess whether KIN10 played a role during the starvation response in

darkened plants, the transcript profile of darkened plants was compared to a

list of genes that were specifically up- or down-regulated in protoplasts

23

overexpressing KIN10. Many of the KIN10-related changes in transcription

matched the direction of expression in darkened plants. Changes that did not

match, involved genes related to DNA-synthesis and photosynthesis and are

likely caused by the KIN10-overexpressing material being a cell suspension

culture grown under light. Interestingly, the transcript profile of individually

darkened leaves showed the same directional changes in KIN10-regulated

genes, albeit at a reduced amplitude. These parallels suggest that both

darkened plants and individually darkened leaves are starving for sugars,

even though the starvation in darkened plants appears to be more severe.

Darkened plants differ from individually darkened leaves

Even though both darkened plants and individually darkened leaves

showed comparable changes in their metabolomes and transcriptomes

compared to illuminated leaves, they showed considerable differences when

compared to each other. As mentioned earlier, the metabolome of darkened

plants became increasingly different from that of individually darkened

leaves as the dark-treatments progressed (Paper I, Figure 1b). Compared to

darkened plants, individually darkened leaves contained more sugars,

galactosyl glycerol and phytol, while the darkened plants contained more

nitrogen-rich amino acids, glycerol and xylose (Paper I, Table 1). While

these differences were relatively small after the first day, they became

increasingly pronounced as the dark-treatments progressed (Paper I, Table

S2). Metabolically, the most obvious difference between the two dark-

treatments was the accumulation of amino acids, in particular asparagine,

which was verified using High Performance Liquid Chromatography

(HPLC; Paper I, Table 2). Additionally, these results showed that while

darkened plants accumulated almost all detected amino acids compared to

light, individually darkened leaves only accumulated minor amounts of

aromatic and branch-chain amino acids. To see whether this difference was

due to a lack of amino acid transport, the metabolomes of petiole-sap

extracts from leaves of both dark-treatments were compared. Compared to

individually darkened leaves, darkened plants showed enhanced levels of

amino acids in their petiole sap (Paper I, Table 3), which suggests that the

accumulation of amino acids in darkened plants was not caused by reduced

transport of those amino acids.

Together with the metabolomes, transcriptomes of the two dark-

treatments also showed considerable differences (Paper I, Figure 3, Table

S1). Individually darkened leaves showed increased transcript abundance

related to nitrogen metabolism, sulphur assimilation, metabolic regulation

and gluconeogenesis, in particular PEPCK. Combined with the metabolic

changes, these findings indicate that individually darkened leaves use the

galactolipid-containing thylakoid membranes as a source for carbohydrate-

production via gluconeogenesis. While these leaves remain carbon-starved,

24

the relatively increased availability of carbohydrates seemed to reduce the

starvation response and enhance their energy metabolism compared to

darkened plants.

Darkened plants, on the other hand, showed increased abundance of

transcripts related to cell wall expansion and biosynthesis of tetrapyrroles for

both phytochromes and chlorophyll. Along with the metabolic changes, the

transcriptional differences suggest that darkened plants did not use thylakoid

membranes as a carbon source, but instead degraded regular membranes and

cell wall components. Interestingly, many of the cell wall degrading

enzymes specific to darkened plants, such as expansins, pectin lyases, pectin

methylesterases and xyloglucan endotransglucosylase/ hydrolases, have been

connected to elongation responses during the shade avoidance syndrome

(Devlin et al., 2003; Sasidharan et al., 2008; Franklin, 2008). Earlier reports

have mentioned that, compared to individually darkened leaves, darkened

plants show reductions in mitochondrial respiration (Keech et al., 2007) and

degradation of chlorophyll- and protein (Weaver & Amasino 2001). Our

observations additionally show that darkened plants are more carbon-starved

and show strong accumulation of amino acids, enhanced cell wall

degradation and reduced degradation of chlorophyll, protein, and thylakoid

membranes.

With regard to what causes these differences between the two dark-

treatments, two main differences stand out: individually darkened leaves

contain more carbon, which likely accounts for their enhanced metabolism,

and darkened plants contain more amino acids (Paper I, Table 1). The

enhanced carbohydrate content in the petiole sap of individually darkened

leaves could originate from the illuminated parts of the plant and could

possibly supply the darkened leaves. However, our observations that

individually darkened leaves are carbon-starved do not support this idea.

Instead, it seems more likely that the enhanced degradation of thylakoid

membranes and phytol, in combination with gluconeogenesis, causes the

enhanced carbohydrate content in individually darkened leaves. A plausible

reason for why darkened plants do not share this enhanced degradation can

be found in the accumulation of amino acids. Reduced degradation of

photosynthetic machinery, at the cost of forfeiting degradation of thylakoid

membranes and phytol as sources for carbohydrates, might limit further

accumulation of amino acids in the darkened plants. Accumulation of amino

acids in both leaves and petiole-sap of darkened plants suggests that these

plants exhibit a reduced uptake of amino acids from the phloem by nutrient

sinks. Most types of leaf senescence occur in the presence of nutrient sinks,

such as bark during autumn senescence, seeds or roots during somatic

senescence or developing leaves during senescence of individual leaves.

Therefore, the reduced sink strength in darkened plants questions whether

these plants undergo leaf senescence.

25

The results presented above indicate that darkened plants and

individually darkened leaves exhibit different responses: Individually

darkened leaves show a strong degradation of chlorophyll, protein and

thylakoid membranes, as well as a continuous relocation of amino acids,

which all indicate that individually darkened leaves exhibit an accelerated

leaf senescence. Darkened plants on the other hand, exhibit a reduced

relocation of amino acids, which accumulate in the leaves and appear to

delay further degradation of protein and thylakoid membranes. As a result,

these plants become increasingly carbon-starved, slowing down their

metabolism to a 'stand-by mode'.

Based on these conclusions, an experimental setup that is similar to that

of individually darkened leaves appears to be the more appropriate system to

study shade-induced leaf senescence.

Shade-induced leaf senescence depends on the light intensity

A common criticism on the use of darkened leaves as an experimental

system is that sudden shifts from light to complete darkness for several days

are uncommon in nature. Leaf shading, on the other hand, occurs

continuously; both between plants and between individual leaves from the

same plant. To assess whether the individually darkened leaf system would

also work for shading, individual leaves of Arabidopsis ecotype Columbia-0

(Col-0) were covered with envelopes constructed of light filters with

different transmission spectra (Figure 7a). Removing these envelopes after 8

days revealed that the filters transmitting blue, red and green light had

reduced the chlorophyll content by 36, 42 and 57%, respectively (figure 7b).

Combination of the red and blue filters, which together transmitted mainly

FR, reduced the chlorophyll content by 55%, similar to green filters.

Combination of the red and green filters, which together transmitted light of

wavelengths longer than FR, reduced the chlorophyll content by 97%,

comparable to individual leaf darkening (Paper II, Figure 1). These results

suggest that although FR is considered to induce leaf senescence (Rousseaux

et al 1996, 1997), it can significantly reduce the rate of leaf senescence

compared to darkness. Furthermore, the loss in chlorophyll appeared to

depend more on the decrease in light intensity than to the decline in the

R/FR ratio (Figure 7a & b).

To study the effects of light intensity and R/FR ratio in more detail, two

new types of envelopes were constructed using water-resistant paper and

Neutral Density Filter (NDF). Paper was used as it reduced the light intensity

and affecting the R/FR relatively little compared to other filter material

(Figure 7a). On the other hand, while NDF reduces the intensity of visible

light, it fully transmits FR wavelengths, thus greatly reducing the R/FR ratio.

By using multiple layers of these materials, we obtained envelopes that

transmitted visible light of equal light intensities (both 14 and 3.0 µmol m-2

26

s-1

), but with different R/FR ratios (Paper II, figure 3a). Using these

envelopes to cover individual leaves for 6 days showed that, under the tested

light intensities, the loss of chlorophyll was not enhanced by enriched FR,

but by the reduction in light intensity (Paper II, figure 3b).

Figure 7: a) Light transmission spectra of different filter materials used to construct

filter envelopes. Additional transmission spectra of paper and neutral density filter

(NDF) are shown in grey and black, respectively. b and c) Chlorophyll contents after

shading Arabidopsis ecotype Col-0 for 8 days using different filter envelopes,

plotted as a function of either the light intensity (b) or the R/FR ratio (c) that was

transmitted by the filters. Values are means, n > 2. Statistical notations (Spearman

correlation): rs, spearman's ρ; *, p < 0.05; ns, not significant.

Different light intensities induce different hallmarks of senescence

To investigate how light intensity regulates leaf senescence, individual

leaves were subjected to a range of reduced light intensities by covering

them for 6 days with shading envelopes constructed from multiple layers of

paper (Paper II, Figure 3a). Analysis of the shaded leaves revealed that the

different senescence-associated parameters, e.g. loss of chlorophyll, loss of

protein and enhanced expression of SAGs appeared in response to different

shade light intensities. As expected, the chlorophyll content decreased with

the light intensity, but only at shading of moderate and stronger levels of

shade (14 µmol m-2

s-1

and lower), reaching its minimum under complete

darkness (Paper II, Figure 2c). Loss of protein was already visible under

mild shading (37 µmol m-2

s-1

), increased with stronger shade and reached its

minimum (30% of the original content) at strong shade (3 µmol m-2

s-1

;

Paper II, figure 4a). Looking at individual protein levels, protein related to

PSII only degraded under stronger shade (3 µmol m-2

s-1

and below), while

other proteins, including Rubisco, PSI, GS1, GS2 and COXII, degraded also

27

under milder shade (Paper II, Figure 4b). Expression of senescence-

enhanced genes, such as SAG2 and PAO, showed increased expression

under stronger shade (Paper II, Figure 6). Senescence-specific SAG12 was

mildly expressed under very strong shade (0.5 µmol m-2

s-1

) and strongly

induced under darkness.

Differential appearance of hallmarks related to leaf senescence is not

specific to our experimental setup. Similar observations have been made

regarding natural leaf senescence, where protein loss also occurred before

loss of chlorophyll and enhanced expression of SAGs (Batt & Woolhouse,

1975; Hensel et al., 1993, Masclaux et al., 2000). Furthermore, the

expression of individual SAGs has been shown to be initiated at different

time-points during leaf development (Weaver et al., 1998). With these

observations in mind, it appears that shade induces leaf senescence at a rate

that depends on the residual light intensity.

PRP as part of the senescence process

Interestingly, loss of protein without the visible loss of chlorophyll and

expression of SAGs has also been suggested to be caused by PRP, the

photosynthetic acclimation response that relocates nitrogen from shaded to

unshaded young leaves (Evans 1993, Hikosaka et al., 2005, Terashima et al.,

2005). In theory, PRP differs from leaf senescence based on two suggestions:

PRP selectively degrades photosynthetic protein (Terashima et al., 2005) and

PRP is not induced by FR (Pons & de Jong van Berkel, 2004). Although the

protein lost under mild shade mostly consisted of photosynthesis-related

protein, other proteins, such as COXII and GS1 decreased as well (Paper II,

Figure 4b). This, as well as the inability of FR to enhance chlorophyll loss

under low light and the lack of other senescence hallmarks suggested that the

loss of protein under mild and moderate shade could be classified as PRP.

Such classification implies that similar losses of protein, such as under

stronger shade and darkness and which can encompass more than 50% of the

cellular protein (Paper II, Figure 4a), are a result of PRP as well.

Degradation of other proteins, especially those related to PSII, occurred only

under stronger shade (Paper II, Figure 4b) and was accompanied by the

expression of SAGs (Paper II, Figure 6) and other senescence-hallmarks

(Paper II, Figure 2c, 4d). However, PSII-related protein only encompasses a

minor part of the cellular protein, which disagrees with the notion that

protein degradation is one of the main hallmarks of leaf senescence. The

question thus becomes whether PRP-associated protein degradation could be

considered to be a part of leaf senescence. Indeed, by definition, the two

processes overlap almost completely, except for PRP being an acclimation

response and thus, in theory, reversible. Furthermore, as mentioned

previously, the two proposed differences between leaf senescence and PRP

are not particularly clear. For one, additional FR does not enhance

28

chlorophyll loss under low light (Paper II, Figure 3a), which implies that leaf

senescence is not affected specifically by FR. For the other, protein

degradation associated to PRP is not restricted to photosynthetic protein

(Paper II, Figure 4b). Moreover, large losses of protein are generally not

reversed under natural conditions (van Doorn & Woltering, 2004) and are

eventually followed by leaf death. Since this sequence of events is generally

considered part of the senescence-process (Leopold, 1961, Smart, 1994, Gan

& Amasino, 1997, van Doorn & Woltering, 2004), one could argue that PRP

is a senescence-related process. In this regard, while PRP comprises the bulk

of the protein degradation, the expression of SAGs appears to act as an

auxiliary mechanism to degrade the final part of the photosynthetic protein

and complete the senescence-process.

Leaf senescence overlaps with PA

In addition to losses of chlorophyll and protein, the chlorophyll a/b ratio

decreased as well (Paper II, figure 2e). Chlorophyll b is specific to light

harvesting antennae and the decrease in chl a/b ratio therefore indicates a

relative increase in these antennae, which is a hallmark of PA (Walters,

2005). Additional hallmarks of PA, such as a relative increase in light

harvesting antennae protein compared to photosystem core protein (Walters

2005), a relative increase in PSII over PSI (Walters, 2005, Wagner et al.,

2008) and increased thylakoid stacking (Melis & Harvey 1981, Wagner et

al., 2008) were visible as well (Paper II, figures 5 and S2). Appearing at all

shade light intensities, these hallmarks were visible even as the relative

abundance of photosynthesis-related proteins decreased (Paper II, figure S2)

and expression of SAGs increased (Paper II, figure 6). Additionally, the

increase in both number and size of plastoglobuli under both strong and very

strong shade (Paper II, figure 5) indicated enhanced degradation of thylakoid

membranes, which has been reported to be associated with leaf senescence

(Haber et al., 1969, Wagstaff et al., 2009). However, plastoglobuli have also

been shown to appear in response to PA (Wagner et al., 2008), which

suggests that this hallmark is not specific to leaf senescence. Rather, the

appearance of plastoglobuli and the selective loss of protein and chlorophyll

under shade suggest that PA overlaps with leaf senescence.

The LCP as a threshold to enhance the expression of SAGs

Degradation of photosynthetic protein, prior to the expression of SAGs,

has been suggested to induce leaf senescence by decreasing photosynthesis

(Hensel et al., 1993). The data presented above show that loss of protein and

photosynthetic activity, without expression of SAGs, also occurs in response

to shade and that SAGs are expressed depending on the intensity of the

shade (Paper II, figures 4b, 6 and 7a). Furthermore, the onsets of both age-

and shade-induced leaf senescence have been suggested to relate to the LCP.

29

While shade reduces the light intensity that is required to establish a positive

carbohydrate balance (Veierskov, 1987, Boonman et al., 2006), age-related

loss of photosynthetic protein reduces the capacity to establish this (Hensel

et al., 1993). Under our growth conditions, uncovered leaves had a LCP

around 8 µmol m-2

s-1

(Paper II, figure 7c), which coincided with the light

intensity below which the expression of SAG2 and PAO was enhanced

(figure 6). However, after 6 days of shade, the LCP had decreased to 2-3

µmol m-2

s-1

(Paper III, Figure 7d-f), which likely results from the 50%

decrease in respiration (figure 7b), in combination with PA. This is an

important result, as it shows that leaves acclimate to extend their range of

light intensities at which they have a positive carbon balance. However, this

'acclimated LCP' was below the light level where SAG2 and PAO showed

increased expression, which suggests that the LCP is not the clear-cut border

below which leaf senescence is initiated. On the other hand, expression of

SAG12 and the subsequent occurrence of leaf death were only observed at

light intensities below the acclimated LCP. These results indicate that

parameters associated to visual leaf senescence appear in response to both

mild and strong shading as part of acclimation responses such as PA and

PRP. However, parameters such as SAGs, which are generally correlated to

degradation, showed up only when the intensity of the shade light was lower

than the LCP at growth light and particularly when the shade light intensity

was lower than the acclimated LCP. Such relation between the initiation of

leaf senescence and the LCP suggests that under shade, leaf senescence is

inversely related to the photosynthetic production of carbohydrates.

Shade-induced chlorophyll loss depends on phytochromes

FR does not enhance chlorophyll loss under low light

As mentioned previously, FR could inhibit leaf senescence compared to

darkness (Figure 7a). At first sight, these results on FR-enriched light

inhibiting chlorophyll loss differed from earlier reports that showed FR-

enriched light to enhance chlorophyll loss of individually shaded sunflower

leaves (Rousseaux et al., 1996, 1997). However, the results on sunflower

leaves were obtained at light intensities above 200 µmol m-2

s-1

. At high

growth light intensities (250 µmol m-2 s-1), additional FR (300 µmol m-2

s-1

)

decreased the chlorophyll content of individual Arabidopsis leaves (ecotpye

Wassilewskija, Ws) by 20% after 12 days (Paper III, figure 2c), similar to

results in sunflower (Rousseaux et al., 1997). In contrast, at lower light

intensities, such as within filter envelopes or dense maize crops, decreased

R/FR ratios did not seem to enhance leaf senescence (Borrás et al., 2003,

figure 7). These results indicate that FR induces chlorophyll loss via

different signaling pathways that depend on the intensity of the visible light.

30

PHYA inhibits chlorophyll loss

In sunflower, FR-induced chlorophyll loss has been shown to be

inhibited by overexpression of PHYA (Rousseaux et al., 1997). As mentioned

in the introduction, PHYA is labile in light, but shows enhanced signaling at

low light intensities and low R/FR ratios (Rausenberger et al., 2011),

conditions under which light-stable phytochromes (PHYB-E) are inactivated

(Franklin & Quail 2010). Based on this, PHYA is a good candidate to explain

the lack of FR-induced chlorophyll loss at low light.

To investigate whether PHYA plays a role during shade-induced leaf

senescence, individual leaves of different phytochrome mutants were shaded

using paper envelopes (Paper III, figure 1). In this chapter, Arabidopsis

ecotype Wassilewskija (Ws) was used as it is a natural phyD-mutant that

showed less sensitivity to pathogens, which could cause unrelated leaf

senescence. The results showed that while the phyD-mutation had no effect

during shade (Paper III, Figure 1a), the phyA-mutant exhibited an enhanced

loss of chlorophyll under low light (Paper III, Figure 1b). Under darkness,

this mutant showed a similar loss of chlorophyll as the wt, consistent with

earlier results from Weaver & Amasino (2001). In contrast to the phyA-

mutant, the phyB-mutant showed a reduced chlorophyll content at higher

light intensities that was slightly increased under low light (Paper III, Figure

1c). However, while the phyA-mutant behaved like their respective wt, a

phyAphyB double-mutant showed reduced chlorophyll contents under all

tested light intensities compared to the phyB-mutant (Paper III, Figure 1d).

Earlier reports have mentioned that phyB-mutant seedlings show enhanced

expression of PHYA (Devlin et al., 2003) and an increased stability of the

protein (Franklin et al., 2007). The enhanced chlorophyll content in phyB-

mutant plant compared to phyAphyB double-mutant plant could be caused by

enhanced levels of PHYA protein. Nevertheless, the similar response of

phyA- and phyAphyB double-mutants under low light suggested that PHYA is

involved in delaying senescence-associated chlorophyll loss under low light.

PHYA inhibits chlorophyll loss via the FR-HIR

As mentioned in the introduction, PHYA-signaling can occur via two

signaling pathways: the VLFR and the FR-HIR, which can be separated by

pulsed FR (pFR) and continuous FR (cFR), respectively (Casal et al., 2000).

To estimate which of these signaling pathways was responsible for the

PHYA-specific inhibition of chlorophyll loss, wt and phyA-mutant plants

were partially covered using a dark box that allowed illumination with FR

LED-arrays (Paper III, Figure S1a). These arrays were used to illuminate the

darkened parts of the plants with either pFR or cFR. While pulsed FR

induced a similar loss of chlorophyll loss as darkness, continuous FR

induced an enhanced loss of chlorophyll in the phyA-mutant leaves (Paper

31

III, figure 2a). However, continuous FR did not reduce the loss of

chlorophyll to a level that was significantly different from darkness, which

disputes an inhibitory action of FR. In response to both shade and darkness,

Ws showed a reduced loss of chlorophyll compared to the ecotypes Col-0

(Paper II, figure 2c) and Landsberga erecta (Paper III, figure 3d), which

suggested ecotype-specific differences may have masked the inhibitory

response of FR in Ws. Repetition of this experiment in Ler revealed that FR

can effectively reduce dark-induced chlorophyll loss depending on the

presence and activation of PHYA via the FR-HIR (Paper II, figure 2b).

To see how the stability and functionality of PHYA affects the FR-HIR

during the shading treatments, a logical next step would have been to study

the PHYA protein contents under these conditions. However, the PHYA

protein content decreases rapidly to undetectable levels as plants are

transferred to light (Franklin et al., 2007) and could not be detected in wt

protein extracts (data not shown). To bypass the low amount of PHYA

protein, while still being able to study PHYA protein-related effects, a

number of phyA missense-mutation lines were used. Each of these lines

contained a point-mutation in the PHYA-gene causing a single amino acid

change in the PHYA protein that led to specific alterations with regard to

PHYA-stability and -signaling. For example, the PHYA in phyA-401 has

been reported to have an increased stability, leading to a stronger HIR

(Dieterle et al., 2005), whereas the PHYA in phyA-402 has a reduced

persistence of both its Pfr and HIR, particularly in darkness (Müller et al.,

2009). The HIR-related changes described in these lines were also visible

when their leaves were individually shaded; compared to their respective

wildtypes, chlorophyll loss in phyA-401 was reduced in response to both

shade and darkness, whereas in phyA-402 it was enhanced in response to

darkness (Paper III, Figure 3a,b). Additionally, in a third missense line

(phyA-302), the mutated PHYA fails to form nuclear spots in the nucleus and

as a result has no detectable HIR, while retaining its VLFR (Yanovsky et al.,

2002). Shade-induced chlorophyll loss in this line was similar to that of the

null mutant phyA-201 (Paper III, figure 3b), suggesting that the mechanisms

disrupted in phyA-302 were essential for PHYA-mediated inhibition of

chlorophyll loss during shading. These results further support the role of the

PHYA-mediated HIR in reducing the loss of chlorophyll loss in response to

shade and darkness.

Altogether, the results described above indicate that the differences in

chlorophyll retention between wt and phyA-mutants in response to shade are

mediated via a PHYA-mediated FR-HIR.

32

PHYA does not directly inhibit the expression of SAGs

Loss of chlorophyll after shading or illuminating mature leaves with FR

has often been attributed as leaf senescence as it usually ends in leaf death

(Rousseaux et al., 1996, 1997). However, additional FR has also been

described to decrease the accumulation of chlorophyll and protein, as well as

the photosynthetic activity in developing primary leaves of soybean, as part

of a light acclimation process (Barreiro et al., 1992). To assess whether the

PHYA-mediated retention of chlorophyll was accompanied by an actual

change in the senescence process, individually shaded leaves of wt and

phyA-mutant plants were compared for changes in both protein and

expression of senescence-associated genes. Interestingly, the phyA-mutant

showed no significant changes in either total protein or the expression of

SAG2, SAG12 and PAO (Paper III, Figure 4a-d). While under very strong

shade (0.5 µmol m-2

s-1

) and darkness these senescence-associated

parameters seemed to increase slightly in the phyA-mutants, they did not

increase significantly (Paper III, Figure 4a-d). Since these increases occurred

below the acclimated LCP of Col-0 wt (Paper II, Figure 7) and reductions in

chlorophyll could affect photosynthesis, the LCP of Ws wt and the phyA-

mutant was determined. Under strong shade, photosynthesis in phyA-mutants

had decreased, whereas the acclimated LCP had significantly increased

(Paper III, Figure S2). Such decrease in photosynthesis and increase in LCP

implies that the phyA-mutants had a reduced production of carbohydrates

and therefor underwent increased starvation under very strong shade. Since

carbohydrate starvation has earlier been connected to leaf senescence (Paper

I), it could explain the enhanced expression of senescence-associated genes

in the phyA-mutant under very strong shade and be an indirect consequence

of the lack of PHYA. However, since the added loss of chlorophyll in the

phyA-mutants also occurred under milder shading conditions, it appears that

PHYA does not directly inhibit the expression of SAGs.

Could PHYA inhibit leaf senescence via chlorophyll biosynthesis?

Besides inhibiting shade-induced leaf yellowing, phytochromes are

mainly known to regulate the expression of various components of the

photosynthetic machinery during photomorphogenesis (Moon et al., 2008;

Shin et al 2009; Stephenson et al., 2009). As a result, plants that overexpress

PHYA or PHYB show enhanced abundance of photosynthetic proteins and

chlorophyll (Sharkey et al., 1991; Thiele et al., 1999). Two phytochrome-

regulated genes related to the biosynthesis of chlorophyll are HEMA1 and

PORB. In response to shading of individual leaves, both of these genes were

strongly down-regulated (Paper III, Figure 4ef) and PORB was significantly

reduced in the phyA-mutant compared to the wt (Paper III, figure 4f). To see

whether the reduction in PORB-expression related to the additional

chlorophyll loss in the phyA-mutants compared to the wt, the chlorophyll

33

content was plotted against the expression of PORB, which revealed two

overlapping lines (Paper III, figure 5c). This overlap indicated that the

difference in chlorophyll loss between wt and the phyA-mutant could be

explained by the reduced expression of PORB in the phyA-mutant. Similar

correlations, using the expression of HEMA1 and PAO instead of PORB,

revealed seemingly overlapping correlation patterns for HEMA1, while they

diverged for PAO (Paper III, figure 5c and a, respectively). These data show

that the additional chlorophyll loss in the phyA-mutant is not met by an

increase in the expression of PAO. Together, these data suggest that the

difference in chlorophyll loss between wt and the phyA-mutant during

shading may be caused by the decreased expression of chlorophyll

biosynthesis-related genes such as HEMA1 and PORB, independently of the

expression of chlorophyll degradation-related genes such as PAO. However,

limited expression data by themselves provide insufficient proof to claim

this.

To provide further confirmation that chlorophyll biosynthesis contributes

during shading, chlorophyll intermediates were preliminary analyzed. Since

the chlorophyll biosynthesis in mature leaves was expected to be relatively

low, mature leaves that had been shaded for 6 days were fed with

aminolevunilic acid (ALA), a precursor of chlorophyll biosynthesis. Hereto,

the shaded leaves were detached under green safe-light just before the dark-

period and placed overnight with their petioles in an ALA solution. In the

ALA-fed leaves, chlorophyll intermediates were detectable for both

illuminated and moderately shaded leaves, but not for strongly shaded

leaves. This indicates that the chlorophyll biosynthesis machinery is still

functional in mature leaves. However, further analysis is required to check

whether the ALA-processing capacity differs between wt and phyA-mutant

leaves after shading.

In addition, a comparison of the re-greening capacity between darkened

wt and mutants in a type 5 serine/threonine protein phosphatase (papp5)

seems to support that phytochromes influence chlorophyll biosynthesis in

mature leaves. PAPP5 is a protein that stabilizes the active form of

phytochrome and stimulates its signal transduction (Ryu et al., 2005).

Darkening papp5-mutants for 6 days showed a detectable loss in

chlorophyll, but yielded no visible differences compared to wt. However,

when left to re-green for 2 weeks, the younger wt leaves re-greened slowly,

while those of the papp5-mutant re-greened at a visibly lower rate (data not

shown).

Further experimentation is required to confirm the preliminary

observations that phytochrome A, and potentially other phytochromes, delay

visible senescence by stimulating the biosynthesis of chlorophyll.

Nevertheless, the presented data support the extensive literature on how

34

phytochrome action promotes biosynthesis of the photosynthetic machinery.

The reduced photosynthetic capacity and 'acclimated LCP' in phyA-mutants,

which is accompanied by enhanced leaf senescence under very strong shade,

further suggests that the effect of PHYA on leaf senescence operates via

photosynthesis.

Shedding light on leaf senescence

The overall aim of this thesis is to increase the understanding of how

photosynthesis, photomorphogenesis and transpiration work together to

decide when leaf senescence should occur. Hereto these processes were

combined into a single scheme, which was modified based on the earlier

presented data and information from other experimental systems to predict

the interaction between the different processes (Figure 8).

In this scheme, leaf senescence (Figure 8, in red) is represented by the

degradation of protein, chlorophyll, cell wall and lipids that provide amino

acids and carbohydrates for relocation to sink tissues via the phloem. Since

most of the degraded protein and chlorophyll is part of the photosynthetic

machinery, leaf senescence has a direct inhibitory effect on photosynthesis.

In turn, photosynthesis (Figure 8, in green) has been described to

regulate leaf senescence. In this regulation, photosynthetic light reactions

seem to play a non-essential part, as their inhibition by DCMU and DBMIB

in attached leaves does not induce leaf senescence (Okada & Katoh, 1998).

Carbon fixation on the other hand, plays an important role for two reasons:

first, CO2 is essential for light to inhibit senescence in detached leaves

floating on water (Satler & Thimann, 1983). Second, most senescence

markers show significant up-regulation around the acclimated LCP, when

CO2-fixation yields no net carbohydrates (Veierskov, 1989, Paper II). These

observations suggest that photosynthesis may inhibit leaf senescence through

the production of carbohydrates (sugars).

Photomorphogenesis (Figure 8, in blue) has been connected to leaf

senescence based on the role of phytochromes in inhibiting the loss of

photosynthetic machinery (Biswal & Biswal 1984; Rousseaux et al., 1997).

However, phytochromes and cryptochromes have not been shown to reduce

the expression of SAGs. Instead, they are well-known for regulating

photosynthesis-related genes (Kaufman et al., 1984; McCormac & Terry,

2002; Shin et al., 2009) and promoting assimilation of nitrate into amino

acids (Rousseaux et al., 1997, Lillo et al., 2008) in seedlings. The results

regarding FR and PHYA (Paper III; previous section in this thesis) support

this notion. Together, this knowledge suggests that photomorphogenesis

promotes the biosynthesis of photosynthetic machinery and amino acids in

mature leaves as well (Figure 8, blue dotted arrows). Promotion of such

35

biosynthesis would therefore contribute to a stable photosynthetic system

and carbohydrate production.

Figure 8: Framework presenting how light affects leaf senescence. Transpiration

and phytochromes influence the biosynthesis of the photosynthetic machinery and

thereby exert control over the visual aspects of leaf senescence. Photosynthesis,

nitrate and cytokinins on the other hand influence the levels of both carbohydrates

and amino acids in the leaf by regulating both their production and their unloading

from the phloem. With regard to the degradative aspects of leaf senescence, in

particular the levels of carbohydrates are important, as their over-abundance inhibits

the biosynthesis of the photosynthetic machinery via HXK1, while their limitation

relieves the inhibition of SnRK1-regulated catabolic processes to restore

carbohydrate levels, with amino acids as a by-product that is relocated via the

phloem to sinks elsewhere in the plant. Dashed arrows indicate pathways that have

not been published in mature and senescing leaves. Abbreviations: Chl, chlorophyll;

HXK1, hexokinase 1; SAGs, senescence associated genes; SnRK1, Starvation-

induced sucrose Non-fermenting-1-Related protein Kinase-1.

Transpiration (Figure 8, in yellow) mainly regulates the xylem-mediated

flow of nitrate and cytokinins into the leaf, both of which have been shown

to inhibit leaf senescence (Mothes, 1960; Zavaleta-Mancera et al., 1999,

Boonman et al., 2007). While it remains unclear how nitrate inhibits leaf

senescence, nitrate is known to serve as a substrate for the production of

amino acids and to stimulate the production of cytokinins (Sakakibara et al.,

2006; Figure 8, in black). Cytokinin action is very diverse and influences

36

photosynthesis, photomorphogenesis and transpiration. Photosynthesis and

transpiration are positively affected as cytokinins promote stomatal opening

and gas exchange (Dodd, 2003; Figure 8, black dotted arrows). Furthermore,

cytokinins promote both the abundance of various photosynthetic proteins

(Paul & Foyer, 2001) and the assimilation of nitrate into amino acids

(Sakakibara et al., 2006). These effects overlap with photomorphogenesis,

which in seedlings is enhanced by the stabilizing effect of cytokinins on

HY5 (Sweere et al., 2001; vandenBussche et al., 2007; Figure 8, black

dotted arrows). With regard to leaf senescence, cytokinins have an inhibitory

effect, which has been connected to cytokinins promoting the unloading of

carbohydrates and amino acids from the phloem (Mothes 1960; Balibrea-

Lara et al., 2004; Figure 8, black arrows). Thus, cytokinins appear to inhibit

leaf senescence in two ways; by stimulating photosynthesis,

photosmorphogenesis and transpiration and by promoting the unloading of

sugars to the apoplast, even when the photosynthesis within that leaf is

inhibited.

As outlined above, all three mechanisms by which light has been

described to inhibit shade-induced leaf senescence converge on the

availability of carbohydrates and nitrate (Figure 8, center, black). Reductions

in light, such as under shade, result in reduced carbohydrate levels that may

lead to starvation, as occurs in both darkened detached leaves and

individually darkened leaves, which enhances leaf senescence (Noh &

Amasino, 1999, van Doorn, 2008, Paper I). Reduced carbohydrate levels

relieve the inhibition on SnRK1-signaling (Baena-Gonzales et al., 2007;

Figure 8; red, SnRK1) and thereby stimulate degradation pathways to restore

the carbohydrate content to a functional level (Baena-Gonzales & Sheen,

2008). However, the different degradation strategies employed in darkened

plants and individually darkened leaves suggest that the SnRK1-dependent

regulation is modified by the metabolic state of the plant, possibly related to

the abundance of amino acids (Paper I).

On the other hand, when carbohydrate availability is relatively high

compared to that of nitrogen, such as during girdling, sugar-feeding or strong

illumination, leaf senescence is enhanced (Parrot et al., 2005; Pourteau et al.,

2004; Ono et al., 2001). High contents of the carbohydrate glucose have

been shown to repress photosynthetic genes such as RbcS and Lhcb1 via the

sugar sensor HeXoKinase 1 (HXK1; Moore et al., 2003; Figure 8, red,

HXK1). This is further illustrated by the overexpression of HXK1, which

enhances both sugar-sensitivity and leaf senescence in tomato (Dai et al.,

1999; Swartzberg et al., 2006). Additionally, leaf senescence that is

enhanced by overexpression of HXK1 is not inhibited by senescence-directed

production of cytokinin (Swartzberg et al., 2011). These observations

suggest that HXK1 contributes to the senescence phenotype separately from

37

the starvation-induced leaf senescence.

Besides repressing photosynthetic genes, high carbohydrate availability

has furthermore been indicated to enhance assimilation of nitrate and

production of amino acids (Stitt & Krapp 1999, Wingler et al., 2006).

Correspondingly, addition of nitrate can reduce enhanced sugar contents

(Ono & Watanabe 1997). Thus, carbohydrate levels are balanced by nitrate

in two ways: nitrate can reduce carbohydrate levels, as well as its own, by

stimulating amino acid production and enhance carbohydrate levels by

stimulating the synthesis of cytokinins. These abilities of nitrate further

contribute to the central role carbohydrates appear to play in the induction of

senescence-associated symptoms.

Summary

The scheme presented above illustrates that the decision of the plant to

initiate leaf senescence depends on a variety of factors that are

interdependent and mostly regulated by light. Photomorphogenesis has been

shown to be essential to maintain green leaves, possibly via biosynthesis of

photosynthetic machinery. However, photosynthesis and transpiration are

important as well, since they regulate the actual availability of

carbohydrates. In this regard it is also extremely important to consider the

experimental systems behind the results. While detached leaves floating on

top of a solution may close their phloem near the wound site, their apoplast

remains in direct contact with the solution. This could act as a non-selective

sink and prevent accumulation of nutrients in the leaf. Darkened plants, as a

result of their starvation, accumulate nitrogen-rich amino acids, which

appears to discourage further degradation of protein and inhibits visible leaf

senescence. Individually darkened leaves on the other hand, do contain sinks

in the form of developing leaves on the illuminated part of the plant. These

sinks prevent amino acids to accumulate and thereby promote the

degradation of the photosynthetic machinery, including thylakoid

membranes as a carbon source.

Darkening of individual leaves results in the simultaneous increase of

different characteristics of leaf senescence. However, shading individual

leaves revealed that these hallmarks can be initiated depending on the light

intensity. Furthermore, many hallmarks of senescence overlap with those of

photosynthetic acclimation responses. One of these, PRP, can actually be

considered part of the senescence-process, despite the absence of enhanced

expression of SAGs. The switch between PRP and enhanced SAG-

expression appears to be related to the LCP. The LCP itself can acclimate to

shade and, once acclimated, can act as a threshold below which SAG12 is

expressed and leaf death occurs. By being a threshold for the net-production

of carbohydrates, the LCP indicates that reduced carbohydrate levels control

the enhanced senescence-associated degradation.

38

Besides separating senescence-associated hallmarks, the experimental

setup of shading individual leaves is the first system in which phytochrome

mutants have been shown to have an altered visual leaf senescence. While

mutations in light-stable phytochromes result in light-green or yellow

phenotypes under growth light, these differences disappear with increasing

shade treatments. Mutations in light labile PHYA, on the other hand,

enhance leaf yellowing under shade, indicating that the action of PHYA

inhibits visible leaf senescence. Interestingly, it does so without affecting the

expression of SAGs and several observations suggest that PHYA instead

stimulates chlorophyll biosynthesis, as it has been shown to do in seedlings.

Together, these findings suggest that shade-induced leaf senescence is

not merely a straightforward process of degradation, but an intricate network

of different processes, that work together to maintain an optimal distribution

of nutrients within the plant.

39

CONCLUSIONS AND FUTURE PERSPECTIVES

The work presented in this thesis contributes to the understanding of

how changes in light affect leaf senescence and associated phenotypes.

During the last decades, the process of shade-induced leaf senescence has

been studied using a variety of experimental setups, yielding many and

sometimes conflicting results. Because of this, research within the field was

examined critically, especially with regard to the experimental conditions

and the methods used to assess leaf senescence. Based on this examination,

an experimental setup was designed that allowed attached Arabidopsis leaves

to be shaded in a controlled manner that mimics canopy dynamics.

As a part this examination, the comparison between darkened plants and

individually leaves suggested that both dark-treatments are accompanied by

sucrose-starvation, possibly via SnRK1-related signaling. This is an

interesting notion and should be further studied using SnRK1-mutants and

over-expressing lines. However, such lines can be expected to exhibit

significant alterations in both phenotype and metabolism, which may affect

leaf senescence indirectly from the SnRK1-alteration. In this regard,

studying the effects of SnRK1 in an inducible manner would provide more

direct information concerning a role of SnRK1 in leaf senescence. Using a

similar method, the role of HXK1 during leaf senescence in response to high

sugar levels can be elucidated as well.

While both darkened plants and individually darkened leaves exhibited

sugar starvation, their metabolisms differed significantly. Most obvious was

the accumulation of amino acids in darkened plants, which was suggested to

discourage senescence-associated degradation of the photosynthetic

machinery. To confirm this suggestion, the leaf amino acid content should be

increased in senescing leaves by other methods, such as feeding. However,

transport of amino acids away from the senescing leaf and the capacity of

plants to inter-convert amino acids should be considered. Steam-girdling

petioles of senescing leaves, while feeding either plant or leaf using a

cocktail of certain amino acids, could bypass these issues.

Traditionally, leaf senescence is viewed as a process that enhances

degradation of chlorophyll and protein. However, our results have shown

that large losses of protein could occur despite the absence of other typical

hallmarks of leaf senescence. This is interesting with regard to two things.

First, since the protein loss occurred in absence of other senescence

hallmarks, transcriptomics analysis would be required to estimate whether

the loss of certain proteins under mild shading is accompanied by similar

reductions in gene-expression. Second, the mechanism by which the protein

40

loss occurs have not been established. It would be interesting to see whether

protein degradation under mild shade is accompanied by RCBs or SAVs or

whether these bodies only appear at stronger shade, together with other

hallmarks of senescence.

Studying mutants using the shade-induced system has been successfully

demonstrated using phyA-mutants. Furthermore, this was the first

demonstration of an enhanced senescence-like response in a phytochrome

mutant. However, this enhance in response was not accompanied by

enhanced expression of SAGs, but suggested to be caused by a decreased

chlorophyll biosynthesis. While research on photomorphogenesis in

seedlings has shown that light-receptor signaling influences the biosynthesis

of the photosynthetic machinery, this remains to be confirmed for mature

leaves. To this end, the recently reported phytochrome quintuple mutants,

which turn yellow when shifted from white to red light, would provide very

interesting material. Although these mutations lead to considerable changes

in phenotype, the redundancy between phytochromes and cryptochromes

does allow a functional photosynthetic system to develop. A controlled shift

from white light to red light, both at equal photosynthetically active

irradiance, should keep photosynthesis and transpiration constant, while

disabling photomorphogenesis. While this treatment is already known to

cause leaf yellowing in the phytochrome quintuple mutants, the underlying

cause of this yellowing remains speculative. The questions that now beg to

be answered are whether photosynthesis is still functional under these light

conditions and what changes in gene-expression accompany the leaf

yellowing.

In conclusion, this work illustrates that shade-induced leaf senescence is

not merely a program, but that it is a collection of processes that are closely

tied to the environmental conditions of both plant and leaf. Combining the

progress on different signaling pathways with that of leaf senescence, while

keeping in mind the specifics of the experimental conditions, a hypothetical

model was created (Figure 8). Besides providing interesting new angles with

regard to how leaf senescence appears to be regulated, it also emphasizes the

conflicts that may arise due to overlaps between the light-sensing processes

that influence leaf senescence. By doing so, this work sheds new light on the

processes underlying shade-and dark-induced leaf senescence.

41

ACKNOWLEDGEMENTS

Finally we've made it to the end, although this particular section is probably

the first thing many of you will read. It has been a very interesting journey

since I came to Sweden five and a half years ago and there are a lot of people

I am very grateful to have met. Therefore I would like to say up front:

Thank you for the great times I have had with you all!

However, there are some people who I'd like to thank in particular:

My supervisor, Per Gardeström. Thank you for providing me the possibility

to do my PhD in Sweden, for encouraging me to pursue my own project and

for letting me learn things the hard way. I greatly admire your uncanny

patience, your acceptance and also your seemingly effortless skiing

technique. Thank you very much for this experience!

Olivier Keech. Man, thank you for helping me get started at the beginning

of my thesis: for the nice discussions while potting plants and instructing me

how to work with Arabidopsis and dark-induced leaf senescence. But most

of all, thank you for being there during the final year, scrutinizing my

writing and helping my stubborn head recognize how to improve it. Without

your feedback this thesis would have been a lot harder to digest.

Wouter van Doorn. Your critical review on how sugars affect leaf

senescence opened my eyes and made me start looking for answers beyond

the field of leaf senescence. I sincerely hope that you keep writing such

reviews, as they are ever inspiring.

Gunilla. For the nice talks about the garden lot, for the help with finding

chemicals and equipment and for pushing people to clean up their mess.

Siv, Karin, Monica, Inger, Eva-Maria and the cleaning ladies. You make

UPSC a breathable place and cannot get enough credit. Special thanks also

to Britt-Marie, for all her good advice on plants, soil and bugs.

Jan. For being an awesome office-member and the nice discussions on

movies, music and the intricate workings of Sweden. Also, many thanks for

the excellent help and suggestions on a variety of software that contributed

to the visual aspects of this thesis.

Eva. For all the support and always being there to talk about teaching,

learning, life, chlorophyll, Sweden and the garden lot. It is always nice to

talk to you.

42

Krisztina, Agnieszka, Agnieszka, Boszena and Sabine. For being nice

colleagues in both Lab and Office.

Lars, Matthieu B. and Raik. For all our discussions on photosynthetic

acclimation and leaf senescence, they really helped.

Catherine B. For reeling me into the PhD social life when I had just arrived

and for explaining me what IKSU represents.

Hanna and Anders. For being excellent friends, for allowing me to dig

holes in the garden lot and for the skiing, both in Umeå and in Gällivare.

Special thanks as well to Per-Erik and Lisbeth, for making it possible for

me to experience Dundret Runt.

Peter and Micke. For being good friends and colleagues and for all the nice

times at conferences and parties. Melodiefestivalen will never look like it did

before.

Kristina and Barbara. My personal stylists and etiquette trainers. You

proved that there is hope for everyone.

Catherine and Mark. For the help with Bert and Ernie, for being wonderful

friends and for the excellent dinners and discussions.

Henrik, Ye, Sacha, Franziska, Vicky and Daniel. For all our nice game-

evenings; you are always welcome!

Moniek and Arjan. Who needs good friends when you've got faraway

neighbors? Thank you so very much for visiting so often in Sweden and for

ever providing an excellent stay in Wageningen. Special thanks as well to the

rest of clan 4C; Linda, Thomas and Gerben for always being open to

spontaneously drop by.

My family, for all your support, the many visits in Sweden, the packages

with coffee and pepernoten, and of course for Sinterklaas! It is great to be

around you!

Finally, Márcia. Meu Amor, obrigado por ser quem você é e mais que

obrigado por me proporcionando o equilíbrio que me da força para

continuar. Amo-te tambem.

43

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Fysiologisk Botanik, UPSCUmeå Universitet, 901 87 Umeåwww.umu.se / www.upsc.seISBN: 978-91-7459-437-9