Transgenic strategies for improvement of drought tolerance of cereals to reduce the consequences of water limitations caused by climate change

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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,

Buchanan, Gruissem, Jones Biochemistry
Molecular Biology of Plants chapter 22, and
results of the Cell cycle and Stress Adaptation
Group
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N. Sreenivasulu et al. / Gene 388 (2007) 113
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Strategies for the genetic engineering of
drought tolerance.
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Buchanan, Gruissem, Jones Biochemistry
Molecular Biology of Plants Fig 22.3
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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

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• Fig.1 A schematic representation of cellular
  signal transduction
• Pathways 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-zipper
• Agarwal et al. Plant Cell Rep (2006)2512631274

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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
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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
• gt2x induction more than 1100 spots
• gt5x induction 345 spots
• gt2x repression more than 400 spots
• gt5x repression 77 spots

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Relative transcript level of four selected genes
exhibiting induction during osmotic stress
Q-PCR approved
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EXPERIMENTAL SYSTEM FOR EXPOSURE OF WHEAT
PLANTLETS TO LONG TERM DROUGHT STRESS IN EXPANDED
PERLITE
0 day (16 day old plantlets)
Normal irrigation
Reduced irrigation (30)
1. week
2. week
3. week
4. week
Sampling
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KOBOMUGI AND PLAISMANN GENOTYPES DIFFER IN
VARIETY OF PHYSIOLOGICAL PARAMETERS UNDER WATER
STRESS (50 WATER SUPPLY)
Kobomugi
Plainsmann
É. Sárvári et al.
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Growth rate of two genotypes under water
limitation (30 water supply)
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P5CS mRNA in shoots
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Transcript level changes in wheat roots during
drought adaptation measured on barley macroarray
(percentage of 10 500 clones)
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Functional classification of genes upregulated in
one genotype only
Stress and defense
Kobomugi
Protein synthesis
Protein degradation
Gene expression
Signal transd.
Transport
Cytosceleton and cell wall
Cell growth and division
Metabolism
Plainsman
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Cluster analysis
Kobomugi
Kobomugi
Plainsman
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

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Kobomugi
Kobomugi
Plainsman
Plainsman
Kobomugi
Kobomugi
Plainsman
Plainsman
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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.

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Promoter elements in rice orthologue of ODA1 gene
Osmotic and Drought Adaptation induced clone
Protein function is not known, similar to LEA
family and WSI18
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Relative transcript levels in roots of Kobomugi
during acute drought stress (desiccation)
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Standard system for water limitation

• Soil ? sandperlite21
• Control plants ? 70-80 soil water saturation
• Moderately stressed plants ? 30-40 soil water
  saturation
• Watering ? daily, weight measurment

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Relative transcript levels in shoots of Kobomugi
after two weeks of moderate drought stress
(limited water supply)
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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
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Genes induced during the day in rice roots under
water limitation
And 7 genes with unknown function
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Genes repressed during the day in rice roots
under water limitation
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Genetic engineering of the glyoxalase pathway in
tobacco leads to enhanced salinity
tolerance Singla-Pareek et al. PNAS 100,
14672-14677(2003)
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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

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• 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 (Hegedus et al.
  2004)
• increased tolerance to the effects of high
  temperature and high light intensity (Horváth
  and Hideg, unpublished)

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Regeneration of the ALR transformants and growth
of mature plants
Development of first shoots on AAR medium
Transgenic plantlets in soil
Fertile ALR spikes
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IMPROVED PHOTOSYNTHETIC FUNCTION OF WHEAT ALR
TRANSFORMANTS (4310-B) AFTER 15 DAYS OF WATER
STRESS
120
120
control
100
4310-b
100
80
80
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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.
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TRANSGENIC WHEAT PLANTS IN THE GREENHOUSE
J. Pauk et al.
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MsALR EXPRESSING TRANSGENIC WHEAT LINES WITH
IMPROVED DROUGHT TOLERANCE
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