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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 Dudits Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, .

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slide1

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 Dudits

Institute 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

slide3

Umezawa et al 2006, 17:113-122

Strategiesforthegeneticengineering

ofdroughttolerance.

series of r esponses to drought stress
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

slide8
Fig.1 Aschematicrepresentationofcellularsignaltransduction
  • Pathwaysbetweenstresssignalperceptionandgeneexpressionandthe cis-and trans-elementsinvolvedinstressresponsivegeneexpression.DREB1/CBFandDREB2distinguishtwodifferentsignaltransductionpathwaysinresponsetocoldanddroughtstresses,respectively. DRE:droughtresponsiveelement,ABRE:abscisicacidresponsivebindingelement,MYBRS:MYBrecognitionsite,MYCRS:MYCrecognitionsite,bZIP:basic-domainleucine-zipper
  • Agarwal et al. PlantCellRep(2006)25:1263–1274
slide15

Osmotic stress of wheat plantlets in

hydroponics

0 day

(10

days

old plantlets)

Untreated

PEG

-

treated:

100

mOsm

2.

days

200

mOsm

4.

days

400

mOsm

7.

days

9.

days

11.

days

14.

days

Sampling

rice chip app 16 000 unigene
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
slide18

6

5

Unknown

ACC

synthase

5

4

4

3

3

2

2

1

1

0

0

0 day

4 days

9 days

0 day

4 days

9 days

14

5

ADH

GST

12

4

10

3

8

6

2

4

1

2

0

0

0 day

4 days

9 days

0 day

4 days

9 days

Kobomugi

Kobo

PEG

Plainsmann

Pla

PEG

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

Q-PCR approved

slide22

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

„0 day” (16 day old plantlets)

Normalirrigation

Reduced irrigation (30%)

1. week

2. week

3. week

4. week

Sampling

slide23

KOBOMUGI AND PLAISMANN GENOTYPES DIFFER IN VARIETY

OF PHYSIOLOGICAL PARAMETERS UNDER

WATER STRESS (50% WATER SUPPLY)

Kobomugi

Plainsmann

É. Sárvári et al.

slide24

Kobomugi

Plainsman V

Growth rate of two

genotypes under

water limitation

(30% water supply)

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

(percentage of 10 500 clones)

functional classification of genes upregulated in one genotype only

6%

19%

29%

6%

11%

9%

34%

12%

Stress and defense

Kobomugi

Protein synthesis

Functional classification of genes upregulated in one genotype only

Protein degradation

Gene expression

Signal transd.

Transport

Cytosceleton and cell wall

Cell growth and division

Metabolism

Plainsman

slide28

Cluster analysis

Kobomugi

Plainsman

Kobomugi

Plainsman

  • 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

slide29

Kobomugi

Plainsman

Kobomugi

Plainsman

Kobomugi

Plainsman

Kobomugi

Plainsman

18

conclusions
Conclusions

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.

promoter elements in rice ortholog ue of oda1 gene

Osmotic and Drought Adaptation induced clone

Promoter elements in rice orthologue of ODA1 gene

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

standard system for water limitation
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
slide34
Relative transcript levels in shoots of Kobomugi after two weeks of moderate drought stress (limited water supply)
slide35

Daily change in transcript profile during water limitation in roots of rice

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

Azsuka Bioryza H Sandora Marilla

slide36

Genes induced during the day in rice roots under water limitation

And 7 genes with unknown function

slide41

Genetic engineering of the

glyoxalase pathway in tobacco

leads to enhanced salinity

tolerance

Singla-Pareek et al.

PNAS 100, 14672-14677(2003)

about the aldose reductase superfamily in general
About the aldose reductase superfamily in general:
  • Wide range of substrate specificity
  • Highly conserved structure (NADH or NADPH binding region, catalytic tetrad)
  • Occurrence: from bacteria to Homo sapiens

polyol pathway

detoxification of

reactive aldehydes

slide45

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)
slide46

Regeneration of the ALR transformants and growth of mature plants

Development of first

shoots on AAR medium

Transgenic

plantlets

in soil

Fertile ALR spikes

slide47

IMPROVED PHOTOSYNTHETIC FUNCTION OF WHEAT ALR

TRANSFORMANTS (4310-B) AFTER 15 DAYS OF WATER STRESS

120

120

control

100

4310-b

100

80

80

60

Electrontransport (a. u.)

Electrontransport (a. u.)

60

40

control

40

4310-b

20

20

0

0

0

500

1000

1500

2000

0

500

1000

1500

2000

PAR (mmol m-2 s-1)

PAR (mmol m-2 s-1)

E. Hideg et al.

slide49

HARVEST INDEX

(% DROUGHT STR./UNSTR.)

110

105

THOUSAND KERNEL WEIGHT

(% DROUGHT STR./UNSTR.)

ALR ACTIVITY IN LEAF EXTRACTS

100

(% DROUGHT STR./UNSTR.)

50

125

40

115

TR288

CTR

TR304

105

ALR ACT. (A. U.)

30

(% DROUGHT STR./UNSTR.)

20

95

95

90

85

10

TR288

TR288

CTR

CTR

TR304

TR304

MsALR EXPRESSING TRANSGENIC WHEAT LINES WITH

IMPROVED DROUGHT TOLERANCE

slide50

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