Plant Signal Transduction Bulletin - Vol. XI

Bratislav Stankovic braco_stankovic at ncsu.edu
Wed Apr 8 09:51:43 EST 1998

Plant Signal Transduction Bulletin
Vol. XI
April 1998

Greetings to everyone interested in plant signal transduction.  This volume
contains a summary of the talks that were presented at the Calcium, Signal
Transduction and Gravitational Biology Symposium held at the North Carolina
Biotechnology Center on January 24, 1998 and sponsored by the NCSU NSCORT.
 Thanks to the speakers for finding the time to contribute the summaries of
their talks, and thanks to everyone else who sent information and

Bratislav Stankovic


Calcium, Signal Transduction and Gravitational Biology Symposium


Jen Sheen 
Department of Molecular Biology, Wellman 11, MGH, Boston, MA 02114

	In both monocot and dicot plants, stress hormone abscisic acid (ABA) or
environmental stress conditions, such as drought, cold and salinity can
induce the expression of a number of highly conserved genes in vegetative
tissues. The accumulation of these gene products could potentially protect
plants against stress damage. Many of these genes are also expressed at the
late stage of embryogenesis during seed development and thought to be
important for seed desiccation and dormancy. Extensive studies have
identified cis-elements and trans-factors important for the regulation of
these stress-inducible genes. However, the molecular mechanisms underlying
the perception and transduction of the stress signals to the target genes
in the nucleus remain elusive.

	I have established a physiological single cell system using maize leaf
protoplasts in which responses to multiple stress treatments could be
easily monitored using green-fluorescent protein (GFP) as a vital reporter.
A chimeric gene was generated by fusing the barley HVA1 promoter to a
synthetic GFP sequence (HVA1-SGFP). The barley HVA1 gene has been reported
to be activated by multiple stress signals in vegetative tissues. After the
electroporation of the plasmid DNA carrying HVA1-SGFP into maize leaf
protoplasts, the expression of GFP was found to be enhanced by cold, high
salt, darkness, and ABA. Similar results were obtained when luciferase
(HVA1-LUC) was used as a reporter. These responses are specific to
HVA1-SGFP and HVA1-LUC as the internal control with a fusion of the maize
ubiquitin promoter and the beta-glucuronidase gene (UBI-GUS) was not
affected. In addition, the expression of UBI-SGFP was also insensitive to
stress signals. The system provided the opportunity to elucidate the
mechanisms of stress signal transduction using biochemical and molecular
genetic approaches.

	Previous experiments in many other laboratories have suggested that
calcium might be a second messenger in stress responses in plants. The
effect of calcium and ionophores on HVA1-SGFP expression in maize leaf
cells was tested and found to mimic stress signals. Furthermore, the
constitutively active form of two specific and closely related
calcium-dependent protein kinases (CDPK1 and CDPK1a) activated
stress-inducible transcription bypassing stress signals. The effect of
CDPK1 and CDPK1a is highly specific because six distinct plant PKs
including two other CDPKs fail to mimic stress signaling. The activation is
abolished by a CDPK1 mutation (K40M) in the ATP binding site. The results
suggest that different plant CDPKs may play distinct physiological roles
and CDPK1 and CDPK1a are likely the positive regulators controlling plant
stress responses.

	In addition to the finding of CDPK1 and CDPK1a as positive regulators, the
specific role of protein phosphatase 2C (PP2C), a class of ubiquitous and
evolutionarily conserved serine/threonine PP, as a negative regulator in
stress responses mediated by ABA was also presented. Specifically,
mutational analyses of two Arabidopsis thaliana PP2Cs, encoded by ABI1 and
AtPP2C involved in the plant stress hormone ABA signaling, were carried out
in maize leaf protoplasts. Consistent with the crystal structure of the
human PP2C, the mutation of two conserved motifs in ABI1, predicted to be
involved in metal binding and catalysis, abolished PP2C activity.
Surprisingly, although the DGH177-179KLN mutant lost the ability to be a
negative regulator in ABA signaling, the MED141-143IGH mutant still
inhibited ABA-inducible transcription, perhaps through a dominant
interfering effect. Moreover, two G to D mutations near the DGH motif
eliminated PP2C activity but displayed opposite effects on ABA signaling.
The G174D mutant had no effect but the G180D mutant showed strong
inhibitory effect on ABA-inducible transcription. Based on the results that
a constitutive PP2C blocks but constitutive CDPKs activate ABA responses,
the MED141-143IGH and G180D dominant mutants are unlikely to impede the
wild-type PP2C and cause hyperphosphorylation of substrates. In contrast,
these dominant mutants could trap cellular targets and prevent
phosphorylation by PKs required for ABA signaling. The equivalent mutations
in AtPP2C showed similar effects on ABA responses. This study suggests a
novel mechanism for the action of dominant PP2C mutants that could serve as
valuable tools to understand protein-protein interactions mediating ABA
signal transduction in higher plants.

	In summary, I have used the maize leaf cells to show that specific CDPKs
activate whereas multiple PP2C repress stress and ABA signaling. The study
reveals a novel mechanism for the action of dominant interfering mutants
that does not impede the wild-type PP2C activity in the same cells. These
PP2C mutations could serve as valuable tools to identify other essential
components in the ABA signal transduction pathway in higher plants. The
system provides powerful tools to complement the classical genetic approach
in dissecting the stress and ABA signal transduction pathways in leaf cells
especially when redundant functions are involved.

Sheen, J., Hwang, S, Niwa, Y., Kobayashi, H., Galbraith, D.W. 1995. Green-
             fluorescent protein as a new vital marker in plant cells.
Plant J. 8: 777-784.
Chiu, W.-L. , Niwa, Y, Zeng, W, Hirano, T., Kobayashi, H, Sheen, J. 1996.
             GFP as a vital reporter in plants. Current Biol. 6: 325-330.
Sheen, J. 1996. Specific Ca2+-dependent protein kinase in stress signal
	Science. 274: 1900-1902.
Sheen, J. 1998. Mutational analysis of two protein phosphatases involved in
	signal transduction in higher plants. Proc. Natl. Acad. Sci. USA. 95:

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