Transgenic strategies for improvement of drought

tolerance of cereals to reduce the consequences of water

limitations caused by climate change

János Györgyey, Gábor V. Horváth, Dénes DuditsInstitute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences,

Buchanan, Gruissem, Jones: Biochemistry & Molecular Biology of Plants; chapter 22,and results of the Cell cycle and Stress Adaptation Group

N. Sreenivasulu et al. / Gene 388 (2007) 1–13

Umezawa et al 2006, 17:113-122

Strategies for the genetic engineering of drought tolerance.

Buchanan, Gruissem, Jones: Biochemistry & Molecular Biology of Plants; Fig 22.3

Series of responses to drought stress

ABA peak

Stoma closure

Reduced photosynthetic activity

Block of cell division, elongation

Activation of protective („stress”)

genes (DRE, ABRE elements) e.g. ALR

Accumulation of osmolytes

Long term adaptation

Fig.1 A schematic representation of cellular signal transductionPathways between stress signal perception and gene expression and the cis- and trans-elements involved in stress responsive gene expression. DREB1/CBF and DREB2 distinguish two different signal transduction pathways in response to cold and drought stresses, respectively. DRE: drought responsive element, ABRE: abscisic acid responsive binding element, MYBRS: MYB recognition site, MYCRS: MYC recognition site, bZIP: basic-domain leucine-zipperAgarwal et al. Plant Cell Rep (2006)25:1263–1274

Osmotic stress of wheat plantlets in hydroponics

Untreated PEG-treated:

0 day (10 daysold plantlets)

100 mOsm

200 mOsm

400 mOsm

2. days

4. days

7. days

9. days

11. days

14. days

Sampling

Rice chip – app. 16 000 unigene

- hybridised with PEG-treated / untreated Kobomugi root samples (day 9)- color flip repeat- app. 5300 spots gave measurable data in both case

>2x induction: more than 1100 spots >5x induction: 345 spots

>2x repression: more than 400 spots>5x repression: 77 spots

Relative transcript level of four selected genes exhibiting induction during osmotic stress

ADH

0

2

4

6

8

10

12

14

0 day 4 days 9 days

Kobomugi KoboPEG Plainsmann PlaPEG

ACC synthase

0

1

2

3

4

5

6

0 day 4 days 9 days

GST

0

1

2

3

4

5

0 day 4 days 9 days

Unknown

0

1

2

3

4

5

0 day 4 days 9 days

Q-PCR approved

EXPERIMENTAL SYSTEM FOR EXPOSURE OF WHEAT PLANTLETS TO LONG TERM DROUGHT STRESS IN EXPANDED PERLITE

„0 day” (16 day old plantlets)

1. week

Sampling

Normal irrigatio

n

2. week

3. week

4. week

Reduced irrigation (30%)

68.97

grain yield(% of control)

86.74

13.2stomata conductance

(mmol H2O m-2 s-1)16.2

59.814CO2 fixation

(cpmx10-5)87.4

37.4fructose accumulation

(nmol g-1)206.5

127.9

lipid peroxidation(MDA pmol cm-2

105.6

160.0

CuZn SOD(% of control)

98.0

11Increase in root weight

(g/plant)13

É. Sárvári É. Sárvári et al.et al.

Kobomugi Plainsmann

Growth rate of two genotypes under water limitation

(30% water supply)

Changes in weight of tissues of Kobomugi genotype

0

0.5

1

1.5

2

2.5

Fw[g

]

Shoot Root

Kobomugi

Changes in weights of tissues of Plainsman genotype

0

0.5

1

1.5

2

Fw

[g]

Shoot Root

Plainsman V

P5CS mRNA in shoots

Figure 5.genotypes. Four to fourteen-fold increase could be observed in treated samples (dark green) in all four cases compared to their controls (light green). Maximum level was reached on the third week in all four genotypes.

Relative transcript levels of P5CS mRNAs in shoots of the four

Relative transcript levels of P5CS in shoots of CapelleDesprez genotype

0

1

2

3

4

5

0 1 2 3 4 weeks

rel.

tran

scrip

tlev

el

Relative transcript levels of P5CS in shoots of Öthalomgenotype

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 weeks

rel.

tran

scrip

tlev

el

Relative transcript levels of P5CS in shoot of Kobomugigenotype

0

2

4

6

8

10

12

14

16

0 1 2 3 4 weeks

rel.

tran

scri

ptle

vel

Relative transcript levels of P5CS in shoot of Plainsmangenotype

0

1

2

3

4

56

7

8

9

10

0 1 2 3 4 weeks

rel.

tran

scri

ptle

vel

Transcript level changes in wheat roots during drought adaptation measured on barley macroarray

Plainsmann(adapting, having good yield)

Kobomugi(fast responding „survivor”)

During 4 weeks

not changed

73 78

Change in 1-2 weeks

updown

6.12.7

1.31.6

Change in 3-4 weeks

updown

0.50.5

0.80.4

(percentage of 10 500 clones)

9% 11%

12%34%

19% 6%

6%29%

Functional classification of genes upregulated in one genotype only

Gene expression

Signal transd.

Transport

Cytosceleton and cell wall

Cell growth and division

Metabolism

Kobomugi

Plainsman

Stress and defense

Protein synthesis

Protein degradation

•putative cyclin-dependent kinase B1-1 •expansin EXPB2 •calmodulin-binding heat-shock protein •xyloglucan endotransglycosylase

•Xet3 protein •caffeic acid O-methyltransferase •putative cellulose synthase catalytic subunit •betaine aldehyde dehydrogenase 16

Cluster analysis

KobomugiKobomugi PlainsmanPlainsman

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 1w 2w 3w 4w 0 1w 2w 3w 4w

Fold

-incr

ease

TPA: cysteineproteaseperoxiredoxin

MnSOD

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 1w 2w 3w 4w 0 1w 2w 3w 4w

Fold

-incr

ease

dehydrin4

germin-like protein

lipid transfer protein-like

0

0.5

1

1.5

2

2.5

3

3.5

0 1w 2w 3w 4w 0 1w 2w 3w 4w

Fold

-incr

ease

beta-expansin 2

putative endo-beta-1,3(4)-glucanase

putative cellulose synthase catalyticsubunitxyloglucan endo-1,4-beta-D-glucanase

putative cellulose synthase

0

0.5

1

1.5

2

2.5

3

0 1w 2w 3w 4w 0 1w 2w 3w 4w

Fo

ld-i

ncr

ease

calmodulin-binding heat-shock protein

putative calcium-dependent protein kinase

calcium-dependent protein kinase

putative calmodulin-binding protein

calmodulin2

putative CDPK substrate protein

Kobomugi

Kobomugi

Kobomugi

Kobomugi

Plainsman

Plainsman

Plainsman

Plainsman18

Divergent drought adaptation strategies of the two genotypes are refelected in their transcript profiles.

Long term adaptation is dependent on moderate changes in the expression of large set of genes in a coordinated manner.

Transient gene activation is characteristic to Kobomugi, while genes of the more adaptive Plainsmann genoptype exhibit prolonged upregulation.

Based on the yield performance and photosynthetic activity, Kobomugi represents escaper strategy while Plainsmann cultivar is capable to maintain physiological functions in harmony with gene expression reprogramming.

Conclusions

Promoter elements in rice orthologue of ODA1 gene

No. of motif

Name of motif

Function

13 ABRE ABA-responsive binding site4 C-repeat

DREDrought response element

1 CE1 ABRE motif linked cis-element3 CGTCA Jasmonate response cis element3 GCN4 Endosperm specific expression14 HSE Heat shock response, HSF binding2 LTR Low temperature response2 TCA elem Salicilic acid response3 TGACG Jasmonate response cis element6 WUN Wound response1 As1 Root specific expression

Osmotic and Drought Adaptation induced clone

Protein function is not known, similar to LEA family and WSI18

Osmotic stress:

Kobomugi Plainsman Control PEG-treated Control PEG-treated

4 days 9 days 4 days 9 days 4 days 9 days 4 days 9 days 2,78 2,56 2,57 3,72 1,00 0,41 4,50 4,20

Drought stress:

Kobomugi Plainsman

1 week

2 weeks

3 weeks

4 weeks

4 w. control

1 week

2 weeks

3 weeks

4 weeks

4 w. control

1,62 1,60 16,52 10,05 2,48 0,58 0,63 1,53 4,10 0,72

Relative transcript levels in roots of Kobomugi during acute drought stress (desiccation)

Kobomugi_root_desicc.

020

406080

100

120140160

180200

ODA1 P5CS UP79

0h

1h

3h

9h

Standard system for water limitation

Soil → sand:perlite=2:1

Control plants → 70-80%

soil water saturation

Moderately stressed

plants → 30-40% soil

water saturation

Watering → daily, weight

measurment

Relative transcript levels in shoots of Kobomugi after two weeks of moderate drought stress (limited water supply)

Kobomugi

0

5

10

15

20

25

30

35

ODA1 P5CS UP79

40%

80%

Azsuka Bioryza H Sandora Marilla

- cv. Sandora- control (100%), stressed (20%) water capacity- 3-3 samples (at 8, 14, 18) - 22 k rice oligo-chip

Daily change in transcript profile during water limitation in roots of

rice

Info 1 Info 2

F-box domain, putative 11682.m04419

oxidoreductase, short chain dehydrogenase/reductase family 11670.m04026

AT5g64600/MUB3_12 11686.m00842

jmjC domain, putative 11668.m04545

putative chelatase subunit 11669.m03601

Protein kinase domain, putative 11686.m00607

Similar to C-x8-C-x5-C-x3-H type Zinc finger protein, putative 11669.m01922

And 7 genes with unknown function

Genes induced during the day in rice roots under water limitation

Info 1 Info 2

Hpt domain, putative 11674.m04480

Af10-protein 11667.m05271

putative reductase 11669.m05442

O-methyltransferase, putative 11670.m00047

UDP-glucoronosyl and UDP-glucosyl transferase 11673.m03060

Similar to GMFP5 11676.m02628

profilin a 11676.m01466

hypothetical protein, (thylakoid membrane phosphoprotein 14 kda, chloroplast precursor, putative, expressed ) 11667.m05484

expressed protein, (putrescine-binding periplasmic protein, putative, expressed ) 11670.m05010

putative transport protein particle component 11669.m02901

pectate lyase precursor (ec 4.2.2.2) 11668.m01124

putative oxidase 11669.m03600

receptor-like protein kinase, putative 11674.m01711

Genes repressed during the day in rice roots under water limitation

Genetic engineering of theglyoxalase pathway in tobaccoleads to enhanced salinity tolerance

Singla-Pareek et al.

PNAS 100, 14672-14677(2003)

Wide range of substrate specificityHighly conserved structure (NADH or NADPH binding region, catalytic tetrad)Occurrence: from bacteria to Homo sapiens

polyol pathway

detoxification of reactive aldehydes

About the aldose reductase superfamily in general:

Effects of MsALR overproduction on tobacco plants:

•protection against lipid peroxidation under chemical and drought stresses (Oberschall et al. 2000)

•protection during drought and UV-B stresses (Hideg et al. 2003)

•transgenic plants showed higher tolerance to low temperature and cadmium stress (Hegedűs et al. 2004)

•increased tolerance to the effects of high temperature and high light intensity (Horváth and Hideg, unpublished)

Development of first shoots on AAR medium

Transgenic

plantletsin soil

Regeneration of the ALR transformants and growth of mature plants

Fertile ALR spikes

PAR (mol m-2 s-1)

0 500 1000 1500 2000

0

20

40

60

80

100

120

control4310-b

0 500 1000 1500 2000

0

20

40

60

80

100

120control4310-b

PAR (mol m-2 s-1)

Ele

ctro

ntra

nspo

rt (

a. u

.)

Ele

ctro

ntra

nspo

rt (

a. u

.)

E. Hideg E. Hideg et al.et al.

J. PaukJ. Pauk et al.et al.

MsALR EXPRESSING TRANSGENIC WHEAT LINES WITH

IMPROVED DROUGHT TOLERANCE

CTR

AL

R A

CT

. (A

. U

.)

10

50

ALR ACTIVITY IN LEAF EXTRACTS

20

30

40

TR304 TR288

CTR

(% D

RO

UG

HT

ST

R./

UN

ST

R.)

90

110

HARVEST INDEX(% DROUGHT STR./UNSTR.)

95

100

105

TR304 TR288

CTR

85

125

95

105

115

TR304 TR288

THOUSAND KERNEL WEIGHT(% DROUGHT STR./UNSTR.)

(% D

RO

UG

HT

ST

R./

UN

ST

R.)

Thank you for your attention

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