Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca2+ in guard cell ion channel regulation. However, genetic mutants in Ca2+ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca2+-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 Ca2+ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca2+-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca2+-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 Ca2+ oscillation experiments revealed that Ca2+-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca2+-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca2+-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.

Numerous studies have shown that transcription factors are important in regulating plant responses to environmental stress. However, specific functions for most of the genes encoding transcription factors are unclear. In this study, we used mRNA profiles generated from microarray experiments to deduce the functions of genes encoding known and putative Arabidopsis transcription factors. The mRNA levels of 402 distinct transcription factor genes were examined at different developmental stages and under various stress conditions. Transcription factors potentially controlling downstream gene expression in stress signal transduction pathways were identified by observed activation and repression of the genes after certain stress treatments. The mRNA levels of a number of previously characterized transcription factor genes were changed significantly in connection with other regulatory pathways, suggesting their multifunctional nature. The expression of 74 transcription factor genes responsive to bacterial pathogen infection was reduced or abolished in mutants that have defects in salicylic acid, jasmonic acid, or ethylene signaling. This observation indicates that the regulation of these genes is mediated at least partly by these plant hormones and suggests that the transcription factor genes are involved in the regulation of additional downstream responses mediated by these hormones. Among the 43 transcription factor genes that are induced during senescence, 28 of them also are induced by stress treatment, suggesting extensive overlap responses to these stresses. Statistical analysis of the promoter regions of the genes responsive to cold stress indicated unambiguous enrichment of known conserved transcription factor binding sites for the responses. A highly conserved novel promoter motif was identified in genes responding to a broad set of pathogen infection treatments. This observation strongly suggests that the corresponding transcription factors play general and crucial roles in the coordinated regulation of these specific regulons. Although further validation is needed, these correlative results provide a vast amount of information that can guide hypothesis-driven research to elucidate the molecular mechanisms involved in transcriptional regulation and signaling networks in plants.

Over one-quarter of all plant genes encode proteins of unknown function that can be further classified as proteins with obscure features (POFs), which lack currently defined motifs or domains, or proteins with defined features, which contain at least one previously defined domain or motif. Although empirical data in the form of transcriptome and proteome profiling suggest that many of these proteins play important roles in plants, their functional characterization remains one of the main challenges in modern biology. To begin the functional annotation of proteins with unknown function, which are involved in the oxidative stress response of Arabidopsis (Arabidopsis thaliana), we generated transgenic Arabidopsis plants that constitutively expressed 23 different POFs (four of which were specific to Arabidopsis) and 18 different proteins with defined features. All were previously found to be expressed in response to oxidative stress in Arabidopsis. Transgenic plants were tested for their tolerance to oxidative stress imposed by paraquat or t-butyl hydroperoxide, or were subjected to osmotic, salinity, cold, and heat stresses. More than 70% of all expressed proteins conferred tolerance to oxidative stress. In contrast, .90% of the expressed proteins did not confer enhanced tolerance to the other abiotic stresses tested, and approximately 50% rendered plants more susceptible to osmotic or salinity stress. Two Arabidopsis-specific POFs, and an Arabidopsis and Brassica-specific protein of unknown function, conferred enhanced tolerance to oxidative stress. Our findings suggest that tolerance to oxidative stress involves mechanisms and pathways that are unknown at present, including some that are specific to Arabidopsis or the Brassicaceae.

Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of >30% of the Arabidopsis proteome poorly characterized. Although empirical data in the form of mRNA and proteome profiling experiments suggest that many of these proteins play an important role in different biological processes, their functional characterization remains one of the major challenges in modern biology. To expand the annotation of genes with unknown function involved in the response of Arabidopsis to different environmental stress conditions, we selected 1007 such genes and tested the response of their corresponding homozygous T-DNA insertional mutants to salinity, oxidative, osmotic, heat, cold and hypoxia stresses. Depending on the specific abiotic stresses tested, 12-31% of mutants had an altered stress-response phenotype. Interestingly, 832 out of 1007 mutants showed tolerance or sensitivity to more than one abiotic stress treatment, suggesting that genes of unknown function could play an important role in abiotic stress-response signaling, or general acclimation mechanisms. Further analysis of multiple stress-response phenotypes within different populations of mutants revealed interesting links between acclimation to heat, cold and oxidative stresses, as well as between sensitivity to ABA, osmotic, salinity, oxidative and hypoxia stresses. Our findings provide a significant contribution to the biological characterization of genes with unknown function in Arabidopsis and demonstrate that many of these genes play a key role in the response of plants to abiotic stresses.