Danforth symposium

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Functional Genomics of Root Growth and Root
Signaling Under Drought
http//rootgenomics.missouri.edu
Henry Nguyen Robert Sharp Georgia Davis Gordon
Springer
Hans Bohnert
Daniel Schachtman
Collaborators Yajun Wu, Utah State Univ. Sixue
Chen, Danforth Center Dong Xu, Univ.
Missouri-Columbia Roberto Tuberosa, Univ.
Bologna, Italy Steve Quarrie, Univ. Belgrade,
Yugoslavia John-Marcel Ribaut, CIMMYT, Mexico
University of Missouri, Columbia
University of Illinois, Urbana-Champaign
Donald Danforth Plant Science Center, St Louis,
Missouri
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A
Dry land Fully irrigated
B
Root system of maize about 8 weeks old Weaver JE
(1926) Root Development of Field Crops
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Well watered
Water stressed
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The Humid Room
Maize seedlings
After germination, transplanted to vermiculite at
various water contents, and grown under
non-transpiring conditions (darkness and
near-saturation humidity) to achieve precise,
constant and reproducible water stress
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Root growth objectives

• Genetic diversity in root growth response to
  water stress
• Kinematic analysis
• Transcript profiles in the root growth zone
• Cell wall protein profiles in the root growth
  zone
• Role of ABA in root growth maintenance

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Root growth objectives

• Genetic diversity in root growth response to
  water stress
• Kinematic analysis
• Gene expression profiles
• Cell wall protein profiles
• Role of ABA in root growth maintenance

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Taking advantage of a kinematic approach
1 cm
WATER STRESSED (-1.6 MPa)
WELL WATERED
Sharp RE et al. (1988) Plant Physiol 87 50-57
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WELL WATERED (WW)
WATER STRESSED (WS)
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DISTANCE FROM ROOT APEX (mm)
Root apex
End of growth zone, WS
End of growth zone, WW
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1
2
3
4
WELL WATERED (WW)
WATER STRESSED (WS)
Region 1, elongation completely maintained in WS
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1
2
3
4
WELL WATERED (WW)
WATER STRESSED (WS)
Region 1, elongation completely maintained in WS
Region 2, maximum elongation in WW, inhibition in
WS
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1
2
3
4
WELL WATERED (WW)
WATER STRESSED (WS)
Region 1, elongation completely maintained in WS
Region 2, maximum elongation in WW, inhibition in
WS
Region 3, deceleration in WW, cessation in WS
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1
2
3
4
WELL WATERED (WW)
WATER STRESSED (WS)
Region 1, elongation completely maintained in WS
Region 2, maximum elongation in WW, inhibition in
WS
Region 3, deceleration in WW, cessation in WS
Region 4, non-elongating in WW and WS
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Root growth objectives

• Genetic diversity in root growth response to
  water stress
• Kinematic analysis
• Gene expression profiles (Bohnert and Nguyen
  labs)
• Cell wall protein profiles
• Role of ABA in root growth maintenance

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Root growth objectives

• Genetic diversity in root growth response to
  water stress
• Kinematic analysis
• Gene expression profiles
• Cell wall protein profiles
• Role of ABA in root growth maintenance

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Cell wall extensibility is increased in the
apical region of water-stressed roots (Spollen
and Sharp, 1991 Wu et al., 1996)

• Increased activities of wall loosening proteins
  XET (Wu et al., 1994), expansins (Wu et al.,
  1996)
• Increased expansin gene expression (Wu et al.,
  2001)
• Proteomic analysis
• how many cell wall proteins are involved?

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Challenges for extraction of cell wall proteins
for proteomic analysis - low abundance
(fraction 1 protein yield 1 of cytosolic
proteins) - avoidance of cytosolic
contamination
Fraction 1
Fraction 3
Fraction 2
Water soluble lightly ionically- bound (vacuum
infiltration with 0.2 M KCl followed by low-speed
centrifugation)
Tightly ionically- bound
Covalently- bound
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2D-gel of fraction 1 cell wall proteins
extracted from the elongation zone of
well-watered roots
No evidence of cytosolic contamination
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A Fraction 1 cell wall proteins
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• Region-specific cell wall protein profiling
• in well-watered and water-stressed roots
• is in progress
• (collaboration with Daniel Schachtman and
• Sixue Chen Danforth Center and Yajun Wu
• Utah State Univ.)

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Root growth objectives

• Genetic diversity in root growth response to
  water stress
• Kinematic analysis
• Gene expression profiles
• Cell wall protein profiles
• Role of ABA (abscisic acid) in root growth
  maintenance

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• ABA is a stress hormone, and is
• required for root growth
• maintenance under water stress
• (Saab et al., 1990 1992 Sharp et al., 1994)

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phytoene phytofluene z-carotene neurosporene l
ycopene d-,g-carotene a-,b-carotene
zeaxanthin antheraxanthin all-trans-violaxanthin
possible oxidative cleavage steps in planta
reactions catalyzed by NCED (9-cis-epoxycaroteno
id dioxygenase)
vp5 (maize) fluridone (FLU)
9-cis-violaxanthin
all-trans-neoxanthin 9-cis-neoxanthin

vp14 (maize)

xanthoxin ABA-aldehyde ABA
Modified from Taylor et al. (2000) J Exp Bot 51
1563-74
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ROOT TIP ABA CONTENT (ng g-1 H2O)
Sharp et al. (1994) J Exp Bot 45 1743-51
21 5
96 29
118 18
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(Sébastien Thomine)
(and ABA)
ABA
VIT C
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Under water stress conditions, ABA stimulates
the antioxidant system to prevent excess reactive
oxygen species (ROS)
In roots of ABA-deficient mutant plants, ROS
levels increase and cause cell membrane damage
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Imaging of ROS using carboxy-H2DCFDA
HighROS
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High ROS
Membrane damage
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Plants carrying a vp14 mutant allele
displayed Burnt tassel at emergence Leaf burn
at the time when ears begin to appear Leaf burn
resembles les mutants of maize which have
necrotic lesions initiated/enhanced by sunlight
Courtesy of Georgia Davis, Univ. Missouri-Columbia
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vp14
The vp14 lesion phenotype shares some features
with several of the maize les mutants
les mutant images courtesy of the Maize Genome
Database
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• Phil Mullineaux and Uli Bechtold, Univ. of Essex,
  UK (personal communication)
• In Arabidopsis, all the excess light
  responsive genes studied (62) were ABA responsive
  and all have ABRE elements

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Excess light responsive gene expression All are
responsive to ABA
Microarray analysis of RNA from excess
light-stressed leaves yielded a pool of 199 genes
altered by 2-fold
Verified expression of 62 by qRT-PCR and also
showed all responsive to ABA
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Excess light
HSP 17.6
1
Alteration in expression (log treatment/ LL)
0.1
Control - low light
0.01
ABA
H2O2
EL
DCMU
DCMU EL
controls
GEM-1 cDNAs
Treatment
Phil Mullineaux and Uli Bechtold
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Summary

• Complexity of mechanisms involved in root growth
  maintenance during water deficits
• Understanding is being enhanced by an integrated
  approach physiology to functional genomics
• Application of kinematics to transcript and
  protein profiling

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Cell wall extensibility is increased in the
apical region of water-stressed roots (Spollen
and Sharp, 1991 Wu et al., 1996)

• Increased activities of wall loosening proteins
  XET (Wu et al., 1994), expansins (Wu et al.,
  1996)
• Increased expansin gene expression (Wu et al.,
  2001)
• Proteomic analysis
• how many cell wall proteins are involved?

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Cosgrove DJ (1989) Planta 177 121-130
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Wu et al. (1996) Plant Physiol 111 765-772
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Cell wall extensibility is increased in the
apical region of water-stressed roots (Spollen
and Sharp, 1991 Wu et al., 1996)

• Increased activities of wall loosening proteins
  XET (Wu et al., 1994), expansins (Wu et al.,
  1996)
• Increased expansin gene expression (Wu et al.,
  2001)
• Proteomic analysis
• how many cell wall proteins are involved?

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5
10
20 mm
A
B
C

• WW, region A
• WS, region A
• ? WW, region B
• ? WS, region B

Wu Y et al. (2001) Plant Physiol 126 1471-9