CPK6 and CPK3 CDPKs, ABA and stomatal closure.

 

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure.

by Mori I. C.Murata Y.Yang Y.Munemasa S.Wang Y. F.Andreoli S.Tiriac H.Alonso J. M.Harper J. F.Ecker J. R., Kwak J. M., Schroeder J. I. (2006)

in PLoS Biol4: e327 –doi:10.1371/journal.pbio.0040327 – pmid:17032064 –

CrossRefMedline – CrossRefPubMedCAS | – 

https://www.ncbi.nlm.nih.gov/pubmed/17032064

pbio-0040327-g001
Guard Cell Expression of CPK3 and CPK6 CDPKs (A) Expression of CPK3 and CPK6 in guard cell (GC) and mesophyll cell (MC) protoplasts was examined by RT-PCR. Control amplifications of the guard cell-expressed KAT1 gene and the mesophyll-expressed CBP marker genes [54] (Leonhardt et al., 2004) were used to test the purity of cell preparations (see Results). ACTIN2 was used for an internal loading control. To amplify each CDPK-specific band, RT-PCR was performed with primer sets as indicated by arrowheads in (B) for 36 cycles. Plants were sprayed with water (−ABA) or 100 μM ABA (+ABA) 4 h before isolation of protoplasts and RNA extraction. (B) Cartoon showing the T-DNA insertion positions in cpk3 and cpk6 T-DNA insertion alleles. PCR was performed with a left boarder primer of the T-DNA and a gene-specific primer, and the PCR products were sequenced to determine the T-DNA insertion positions. Arrowheads indicate primer locations for RT-PCR in (A) and (C). ATG and TGA indicate start and stop codons. White boxes indicate exons. (C) RT-PCR confirmed that cpk3–1 and cpk6–1 alleles were disruption mutants. PCRs (32 cycles) were performed with primer sets as indicated in (B) (black arrowheads) in the left three panels. Transcripts of wild-type (WT) and cpk6–2 were examined with two sets of primers [white and black arrowheads in (B)] showing that cpk6–2 lacks exon 1 and that the cpk6–2 has 8% or less the mRNA level of wild-type based on densitometry analyses (n = 2). RNA was extracted from leaves of WT, homozygous cpk3–1, cpk6–1, and cpk6–2 single mutants, and the cpk3-1cpk6–1 double mutant. – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592316/bin/pbio.0040327.g001.jpg
Abstract

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear.

Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs.

Two Arabidopsis guard cell-expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells.

Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network.

Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing.

Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.

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Published by

Willem Van Cotthem

Honorary Professor of Botany, University of Ghent (Belgium). Scientific Consultant for Desertification and Sustainable Development.

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