A role for a novel calmodulinrelated protein in plant environmental response

1
A role for a novel calmodulin-related protein in
plant environmental response?

• Keith A. Johnson, Ph.D.
• Department of Biology
• Colby College

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Plant Development is Influenced by Abiotic
Stresses
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Tendrils Coiling
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Mimosa the Sensitive Plant
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Nutritional Response to Touch
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Repeated Touch Response in Beans
0 2 5 10 20 30
The effects of the number of rubs of the stem on
the growth of young bean plants
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Arabidopsis Responds to Repeated Mechanical
Stimulation
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TCH Genes
Discovered because they were touch-inducible. Exp
ression touch cold heat darkness
calcium growth regulators and hormones
osmotic shock The predicted proteins
encode TCH1 calmodulin TCH2
calmodulin-related TCH3 calmodulin-related TCH
4 xyloglucan endotransglycosylase TCH5
unknown
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Environmental Stimuli
TCH Gene Expression
TCH Protein Function
Developmental Alterations
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Outline
1. What does TCH2 encode?
2. How is TCH2 regulated?
3. Where is TCH2 expressed?
4. What are possible roles for TCH2 in plant
development?
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Schematic of Calmodulin Structure
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Calcium Ion Concentration Flux Acts as a Signal
for Cellular Activation
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Calcium Activation of Calmodulin
Ca 2 lt0.1 µM
Ca 2 1-10 µM
Calmodulin
Calmodulin-Ca 2
Activated protein kinases Phosphorylate
proteins (altered functions)
Modulate enzymes Adenylate cyclase Phosphodiester
ase NAD kinase Ca 2 -ATPase
Physiological functions
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Computer Model of TCH2 Tertiary Structure
similar to calmodulin
4 potential Ca2binding sites
increase linker flexibility (4 Glycines)
potential disulfide bond
enriched Lys and Arg residues (loss of
acidic residues)
suggests a unique set of target proteins
Khan, Johnson, Braam, and James, 1997
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Flexibility of Linker Domain
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TCH2 encodes a novel calmodulin-related protein
The predicted sequence and structure of TCH2
suggest that it will recognize and bind to a
unique class of target proteins.
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Outline
1. What does TCH2 encode?
2. How is TCH2 regulated?
3. Where is TCH2 expressed?
4. What are possible roles for TCH2 in plant
development?
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TCH2 Accumulation in Response to Different Stimuli
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Delimiting Regulatory Sequences
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TCH2 accumulates rapidly and transiently
following a variety of exogenous stimuli.
The regulatory sequences sufficient for TCH2
accumulation following touch and darkness are
contained within 250 bp of the transcription
start site.
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Outline
1. What does TCH2 encode?
2. How is TCH2 regulated?
3. Where is TCH2 expressed?
4. What are possible roles for TCH2 in plant
development?
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Reporter Gene Activity in Branch Points
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Guttation
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Reporter Gene Activity in Hydathodes
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GUS Activity in TCH2GUS Plants During Floral
Development



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Inducible Staining in Guard Cells
Control
Touch
Darkness
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Reporter Gene Staining in Response to Dehydration
control
30 dehydration
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GUS Activity in PEG Treated Stomata
TCH2GUS 2 PEG8000
Control
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Expression Data Conclusions
Expression of the reporter gene is visualized
in branch points, hydathodes and developing
flowers. Basal expression also is visualized in
developing trichomes, root tips and lateral
roots, and in the root/shoot junction (data not
shown)..
Inducible expression is also seen in guard
cells following stimuli that result in stomatal
closure.

• a commonality is water relations
• general cell expansion
• compression in branch points
• guttation (regulation or ion exchange?)
• turgor maintenance in guard cells

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Outline
1. What does TCH2 encode?
2. How is TCH2 regulated?
3. Where is TCH2 expressed?
4. What are possible roles for TCH2 in plant
development?
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Western Analysis of TCH2 Transgenic Plants
T2over
T2under
3
4
6
10
RLD
7
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Addressing a Role for TCH2 in Plant Water
Relations
Two areas of focus
turgor maintenance in guard cells
role in pollen hydration
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Role of Calcium in Stomata
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Altered Expression of TCH2 Affects the Regulation
of Stomatal Aperture
Width/length ratio (se)
Transgenic plants show slow stomatal closure to
dark (shown here) and abscisic acid (ABA).
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Conclusions
1. TCH2 encodes a novel CaM-related protein.
a. Several novel features of interest, including
potential disulfide bonds.
b. Most likely will have unique target proteins.
2. Regulatory sequences important for TCH2
accumulation following touch and darkness are
contained within 250 bp of the transcriptional
start site.
3. Expression of a TCH2-driven GUS reporter gene
indicates that TCH2 may be involved in turgor
maintenance.
4. Altered expression of TCH2 supports a role
for this calmodulin-related protein in stomatal
aperture regulation and possibly in pollen
hydration.
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Possible TCH Protein Functions
wall modification (xyloglucan
transglycosylation)
wall synthesis (xyloglucan incorporation)
vesicle transport of wall materials
turgor maintenance / recovery
wall modifications
loosening or tightening
(xyloglucan endotransglycosylation)
wall synthesis
turgor maintenance / recover
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Model of TCH2 Function
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Calmodulin-Related Gene Family











































CaBP22
MSNVSF
MSAKRV
MSVAEI
MSMAEI
CMR2
MNNMSL
TCH2
MSKNVSR
CMR5
MFNKNQ
MASANP
MANTNL
CMR3
MKLAKL
PM129
MKLAAS
MEINNE
CMR1
MDSTEL
CMR6
MVRVFL
MVRIFL
MDRGEL
MDQAEL
MKNTQR
MKNNTQ
MATQKE
D-RICH
MAKVVV
MCPSGR
MADATE
MSHKVS
CMR4
MSCDGG
MEDQIR
MASTKP
CaM9
TCH3
CaM8
CaM3
CaM2
CaM6
Calmodulins
CaM7
CaM4
CaM1
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Overview of CMR Work

• Protein Characterization
• Cloning and production
• Characterization
• Ca2-binding
• Disulfide
• CMR targets

• RNA Expression
• Environmental induction
• Tissues
• Reporter gene fusions
• Knockouts constructs
• Altered expression constructs

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Acknowledgments
Collaborators Dr. Janet Braam, Rice
University Dr. Dan Roberts, University of
Tennessee Dr. Michael James, University of Alberta
This work was supported by the Rice NSCORT in
Gravitational Biology, Department of Biology at
Colby College, HHMI, and NSF.