A Lignin Molecular Brace Controls Precision Processing of Cell Walls Critical for Surface Integrity in Arabidopsis.

The cell wall, a defining feature of plants, provides a rigid structure critical for bonding cells together. To overcome this physical constraint, plants must process cell wall linkages during growth and development. However, little is known about the mechanism guiding cell-cell detachment and cell wall remodeling. Here, we identify two neighboring cell types in Arabidopsis that coordinate their activities to control cell wall processing, thereby ensuring precise abscission to discard organs. One cell type produces a honeycomb structure of lignin, which acts as a mechanical "brace" to localize cell wall breakdown and spatially limit abscising cells. The second cell type undergoes transdifferentiation into epidermal cells, forming protective cuticle, demonstrating de novo specification of epidermal cells, previously thought to be restricted to embryogenesis. Loss of the lignin brace leads to inadequate cuticle formation, resulting in surface barrier defects and susceptible to infection. Together, we show how plants precisely accomplish abscission.

Regulation of stomatal development by stomatal lineage miRNAs.

Stomata in the plant epidermis play a critical role in growth and survival by controlling gas exchange, transpiration, and immunity to pathogens. Plants modulate stomatal cell fate and patterning through key transcriptional factors and signaling pathways. MicroRNAs (miRNAs) are known to contribute to developmental plasticity in multicellular organisms; however, no miRNAs appear to target the known regulators of stomatal development. It remains unclear as to whether miRNAs are involved in stomatal development. Here, we report highly dynamic, developmentally stage-specific miRNA expression profiles from stomatal lineage cells. We demonstrate that stomatal lineage miRNAs positively and negatively regulate stomatal formation and patterning to avoid clustered stomata. Target prediction of stomatal lineage miRNAs implicates potential cellular processes in stomatal development. We show that miR399-mediated PHO2 regulation, involved in phosphate homeostasis, contributes to the control of stomatal development. Our study demonstrates that miRNAs constitute a critical component in the regulatory mechanisms controlling stomatal development.

Methionine synthase 1 provides methionine for activation of the GLR3.5 Ca2+ channel and regulation of germination in Arabidopsis.

Seed germination is a developmental process regulated by numerous internal and external cues. Our previous studies have shown that calcium influx mediated by the Arabidopsis glutamate receptor homolog 3.5 (AtGLR3.5) modulates the expression of the ABSCISIC ACID INSENSITIVE 4 (ABI4) transcription factor during germination and that L-methionine (L-Met) activates AtGLR3.1/3.5 Ca2+ channels in guard cells. However, it is not known whether L-Met participates in regulation of germination and what cellular mechanism is responsible for Met production during germination. Here, we describe Arabidopsis methionine synthase 1 (AtMS1), which acts in the final step of Met biosynthesis, synthesizes the Met required for the activation of AtGLR3.5 Ca2+ channels whose expression is up-regulated during germination, leading to the regulation of seed germination. We show that exogenous L-Met promotes germination in an AtGRL3.5-dependent manner. We also demonstrate that L-Met directly regulates the AtGLR3.5-mediated increase in cytosolic Ca2+ level in seedlings. We provide pharmacological and genetic evidence that Met synthesized via AtMS1 acts upstream of the AtGLR3.5-mediated Ca2+ signal and regulates the expression of ABI4, a major regulator in the abscisic acid response in seeds. Overall, our results link AtMS1, L-Met, the AtGLR3.5 Ca2+ channel, Ca2+ signals, and ABI4, and shed light on the physiological role and molecular mechanism of L-Met in germination.

POWERDRESS and HDA9 interact and promote histone H3 deacetylation at specific genomic sites in Arabidopsis.

Histone acetylation is a major epigenetic control mechanism that is tightly linked to the promotion of gene expression. Histone acetylation levels are balanced through the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Arabidopsis HDAC genes (AtHDACs) compose a large gene family, and distinct phenotypes among AtHDAC mutants reflect the functional specificity of individual AtHDACs However, the mechanisms underlying this functional diversity are largely unknown. Here, we show that POWERDRESS (PWR), a SANT (SWI3/DAD2/N-CoR/TFIII-B) domain protein, interacts with HDA9 and promotes histone H3 deacetylation, possibly by facilitating HDA9 function at target regions. The developmental phenotypes of pwr and hda9 mutants were highly similar. Three lysine residues (K9, K14, and K27) of H3 retained hyperacetylation status in both pwr and hda9 mutants. Genome-wide H3K9 and H3K14 acetylation profiling revealed elevated acetylation at largely overlapping sets of target genes in the two mutants. Highly similar gene-expression profiles in the two mutants correlated with the histone H3 acetylation status in the pwr and hda9 mutants. In addition, PWR and HDA9 modulated flowering time by repressing AGAMOUS-LIKE 19 expression through histone H3 deacetylation in the same genetic pathway. Finally, PWR was shown to physically interact with HDA9, and its SANT2 domain, which is homologous to that of subunits in animal HDAC complexes, showed specific binding affinity to acetylated histone H3. We therefore propose that PWR acts as a subunit in a complex with HDA9 to result in lysine deacetylation of histone H3 at specific genomic targets.

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana.

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.

Molecular and systems approaches towards drought-tolerant canola crops.

1169 I. 1170 II. 1170 III. 1172 IV. 1176 V. 1181 VI. 1182 1183 References 1183 SUMMARY: Modern agriculture is facing multiple challenges including the necessity for a substantial increase in production to meet the needs of a burgeoning human population. Water shortage is a deleterious consequence of both population growth and climate change and is one of the most severe factors limiting global crop productivity. Brassica species, particularly canola varieties, are cultivated worldwide for edible oil, animal feed, and biodiesel, and suffer dramatic yield loss upon drought stress. The recent release of the Brassica napus genome supplies essential genetic information to facilitate identification of drought-related genes and provides new information for agricultural improvement in this species. Here we summarize current knowledge regarding drought responses of canola, including physiological and -omics effects of drought. We further discuss knowledge gained through translational biology based on discoveries in the closely related reference species Arabidopsis thaliana and through genetic strategies such as genome-wide association studies and analysis of natural variation. Knowledge of drought tolerance/resistance responses in canola together with research outcomes arising from new technologies and methodologies will inform novel strategies for improvement of drought tolerance and yield in this and other important crop species.

Arabidopsis glutamate receptor homolog3.5 modulates cytosolic Ca2+ level to counteract effect of abscisic acid in seed germination.

Seed germination is a critical step in a plant's life cycle that allows successful propagation and is therefore strictly controlled by endogenous and environmental signals. However, the molecular mechanisms underlying germination control remain elusive. Here, we report that the Arabidopsis (Arabidopsis thaliana) glutamate receptor homolog3.5 (AtGLR3.5) is predominantly expressed in germinating seeds and increases cytosolic Ca2+ concentration that counteracts the effect of abscisic acid (ABA) to promote germination. Repression of AtGLR3.5 impairs cytosolic Ca2+ concentration elevation, significantly delays germination, and enhances ABA sensitivity in seeds, whereas overexpression of AtGLR3.5 results in earlier germination and reduced seed sensitivity to ABA. Furthermore, we show that Ca2+ suppresses the expression of ABSCISIC ACID INSENSITIVE4 (ABI4), a key transcription factor involved in ABA response in seeds, and that ABI4 plays a fundamental role in modulation of Ca2+-dependent germination. Taken together, our results provide molecular genetic evidence that AtGLR3.5-mediated Ca2+ influx stimulates seed germination by antagonizing the inhibitory effects of ABA through suppression of ABI4. These findings establish, to our knowledge, a new and pivotal role of the plant glutamate receptor homolog and Ca2+ signaling in germination control and uncover the orchestrated modulation of the AtGLR3.5-mediated Ca2+ signal and ABA signaling via ABI4 to fine-tune the crucial developmental process, germination, in Arabidopsis.

R. S. WebTool, a web server for random sampling-based significance evaluation of pairwise distances.

Pairwise comparison of data vectors represents a large part of computational biology, especially with the continuous increase in genome-wide approaches yielding more information from more biological samples simultaneously. Gene clustering for function prediction as well as analyses of signalling pathways and the time-dependent dynamics of a system are common biological approaches that often rely on large dataset comparison. Different metrics can be used to evaluate the similarity between entities to be compared, such as correlation coefficients and distances. While the latter offers a more flexible way of measuring potential biological relationships between datasets, the significance of any given distance is highly dependent on the dataset and cannot be easily determined. Monte Carlo methods are robust approaches for evaluating the significance of distance values by multiple random permutations of the dataset followed by distance calculation. We have developed R. S. WebTool (http://rswebtool.kwaklab.org), a user-friendly online server for random sampling-based evaluation of distance significances that features an array of visualization and analysis tools to help non-bioinformaticist users extract significant relationships from random noise in distance-based dataset analyses.

Acetylated 1,3-diaminopropane antagonizes abscisic acid-mediated stomatal closing in Arabidopsis.

Faced with declining soil-water potential, plants synthesize abscisic acid (ABA), which then triggers stomatal closure to conserve tissue moisture. Closed stomates, however, also create several physiological dilemmas. Among these, the large CO2 influx required for net photosynthesis will be disrupted. Depleting CO2 in the plant will in turn bias stomatal opening by suppressing ABA sensitivity, which then aggravates transpiration further. We have investigated the molecular basis of how C3 plants resolve this H2 O-CO2 conflicting priority created by stomatal closure. Here, we have identified in Arabidopsis thaliana an early drought-induced spermidine spermine-N(1) -acetyltransferase homolog, which can slow ABA-mediated stomatal closure. Evidence from genetic, biochemical and physiological analyses has revealed that this protein does so by acetylating the metabolite 1,3-diaminopropane (DAP), thereby turning on the latter's intrinsic activity. Acetylated DAP triggers plasma membrane electrical and ion transport properties in an opposite way to those by ABA. Thus in adapting to low soil-water availability, acetyl-DAP could refrain stomates from complete closure to sustain CO2 diffusion to photosynthetic tissues.

Crosstalk between the circadian clock and innate immunity in Arabidopsis.

The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.

Suppression of Arabidopsis AtPUB30 resulted in increased tolerance to salt stress during germination.

KEY MESSAGE: The Arabidopsis U-box E3 Ub ligase AtPUB30 participates in the salt stress tolerance as a negative factor in an ABA-independent manner during germination. Based on the in silico expression data, the U-box protein 30 (AtPUB30) from Arabidopsis thaliana was identified as a gene that responds to salt stress. The deduced AtPUB30 protein consists of 448 amino acids with a single U-box motif and five ARM-repeat domains. An in vitro self-ubiquitination assay demonstrated that bacterially expressed AtPUB30 exhibited E3 ubiquitin (Ub) ligase activity and that the U-box domain was essential for the activity. Real-time qRT-PCR and promoter-GUS analyses showed that AtPUB30 was induced by high salinity, but not by drought, cold, or abscisic acid (ABA), in roots but not in shoots. These results suggest that AtPUB30 is an Arabidopsis U-box E3 Ub ligase, the expression of which is selectively enhanced by salt stress in roots. T-DNA-inserted loss-of-function atpub30 mutant plants (atpub30-1 and atpub30-2) were more tolerant to salt stress in the germination stage, as identified by radicle emergence, cotyledon opening, and more vigorous early root growth relative to wild-type plants. Thus, it is likely that AtPUB30 plays a negative role in high salinity tolerance in the germination process. Wild type and mutant plants displayed very similar germination rates when treated with ABA, suggesting that the action of AtPUB30 in the germination stage is ABA independent. The post-germination growth of NaCl-stressed wild type and mutant plants were indistinguishable. Overall, our data suggest that the Arabidopsis U-box E3 Ub ligase AtPUB30 participates in the salt stress tolerance as a negative factor in the germination stage in root tissues.

In situ metabolic analysis of single plant cells by capillary microsampling and electrospray ionization mass spectrometry with ion mobility separation.

Advances in single cell analysis techniques have demonstrated cell-to-cell variability in both homogeneous and heterogeneous cell populations strengthening our understanding of multicellular organisms and individual cell behaviour. However, additional tools are needed for non-targeted metabolic analysis of live single cells in their native environment. Here, we combine capillary microsampling with electrospray ionization (ESI) mass spectrometry (MS) and ion mobility separation (IMS) for the analysis of various single A. thaliana epidermal cell types, including pavement and basal cells, and trichomes. To achieve microsampling of different cell types with distinct morphology, custom-tailored microcapillaries were used to extract the cell contents. To eliminate the isobaric interferences and enhance the ion coverage in single cell analysis, a rapid separation technique, IMS, was introduced that retained ions based on their collision cross sections. For each cell type, the extracted cell material was directly electrosprayed resulting in ∼200 peaks in ESI-MS and ∼400 different ions in ESI-IMS-MS, the latter representing a significantly enhanced coverage. Based on their accurate masses and tandem MS, 23 metabolites and lipids were tentatively identified. Our results indicated that profound metabolic differences existed between the trichome and the other two cell types but differences between pavement and basal cells were hard to discern. The spectra indicated that in all three A. thaliana cell types the phenylpropanoid metabolism pathway had high coverage. In addition, metabolites from the subpathway, sinapic acid ester biosynthesis, were more abundant in single pavement and basal cells, whereas compounds from the kaempferol glycoside biosynthesis pathway were present at significantly higher level in trichomes. Our results demonstrate that capillary microsampling coupled with ESI-IMS-MS captures metabolic differences between A. thaliana epidermal cell types, paving the way for the non-targeted analysis of single plant cells and subcellular compartments.

Direct force measurement of single DNA-peptide interactions using atomic force microscopy.

The selective interactions between DNA and miniature (39 residues) engineered peptide were directly measured at the single-molecule level by using atomic force microscopy. This peptide (p007) contains an α-helical recognition site similar to leucine zipper GCN4 and specifically recognizes the ATGAC sequence in the DNA with nanomolar affinity. The average rupture force was 42.1 pN, which is similar to the unbinding forces of the digoxigenin-antidigoxigenin complex, one of the strongest interactions in biological systems. The single linear fit of the rupture forces versus the logarithm of pulling rates showed a single energy barrier with a transition state located at 0.74 nm from the bound state. The smaller koff compared with that of other similar systems was presumably due to the increased stability of the helical structure by putative folding residues in p007. This strong sequence-specific DNA-peptide interaction has a potential to be utilized to prepare well-defined mechanically stable DNA-protein hybrid nanostructures.

Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH.

CATION EXCHANGERs CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3 in regulating apoplastic pH and describe how they contribute to auxin transport using the guard cell's response as readout of hormone signaling and cross talk. We show that indole-3-acetic acid (IAA) inhibition of abscisic acid (ABA)-induced stomatal closure is impaired in cax1, cax3, and cax1/cax3. These mutants exhibited constitutive hypopolarization of the plasma membrane, and time-course analyses of membrane potential revealed that IAA-induced hyperpolarization of the plasma membrane is also altered in these mutants. Both ethylene and 1-naphthalene acetic acid inhibited ABA-triggered stomatal closure in cax1, cax3, and cax1/cax3, suggesting that auxin signaling cascades were functional and that a defect in IAA transport caused the phenotype of the cax mutants. Consistent with this finding, chemical inhibition of AUX1 in wild-type plants phenocopied the cax mutants. We also found that cax1/cax3 mutants have a higher apoplastic pH than the wild type, further supporting the hypothesis that there is a defect in IAA import in the cax mutants. Accordingly, we were able to fully restore IAA inhibition of ABA-induced stomatal closure in cax1, cax3, and cax1/cax3 when stomatal movement assays were carried out at a lower extracellular pH. Our results suggest a network linking the vacuolar cation exchangers to apoplastic pH maintenance that plays a crucial role in cellular processes.

Roles of four Arabidopsis U-box E3 ubiquitin ligases in negative regulation of abscisic acid-mediated drought stress responses.

AtPUB18 and AtPUB19 are homologous U-box E3 ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). AtPUB19 is a negative regulator of abscisic acid (ABA)-mediated drought responses, whereas the role of AtPUB18 in drought responses is unknown. Here, loss-of-function and overexpression tests identified AtPUB18 as a negative regulator in ABA-mediated stomatal closure and water stress responses. The atpub18-2atpub19-3 double mutant line displayed more sensitivity to ABA and enhanced drought tolerance than each single mutant plant; therefore, AtPUB18 and AtPUB19 are agonistic. Stomatal closure of the atpub18-2atpub19-3 mutant was hypersensitive to hydrogen peroxide (H(2)O(2)) but not to calcium, suggesting that AtPUB18 and AtPUB19 exert negative effects on the ABA signaling pathway downstream of H(2)O(2) and upstream of calcium. AtPUB22 and AtPUB23 are other U-box E3 negative regulators of drought responses. Although atpub22atpub23 was more tolerant to drought stress relative to wild-type plants, its ABA-mediated stomatal movements were highly similar to those of wild-type plants. The atpub18-2atpub19-3atpub22atpub23 quadruple mutant exhibited enhanced tolerance to drought stress as compared with each atpub18-2atpub19-3 and atpub22atpub23 double mutant progeny; however, its stomatal behavior was almost identical to the atpub18-2atpub19-3 double mutant in the presence of ABA, H(2)O(2), and calcium. Overexpression of AtPUB18 and AtPUB19 in atpub22atpub23 effectively hindered ABA-dependent stomatal closure, but overexpression of AtPUB22 and AtPUB23 in atpub18-2atpub19-3 did not inhibit ABA-enhanced stomatal closure, highlighting their ABA-independent roles. Overall, these results suggest that AtPUB18 has a linked function with AtPUB19, but is independent from AtPUB22 and AtPUB23, in negative regulation of ABA-mediated drought stress responses.

MAP kinases, MPK9 and MPK12, regulate chitosan-induced stomatal closure.

Chitosan (CHT)-induced stomatal closure was inhibited by an MAPKK inhibitor, PD98059, and was impaired in mpk9 mpk12 but not in mpk9 or mpk12. CHT induced the production of reactive oxygen species, cytosolic alkalization, and cytosolic Ca(2+) oscillation in mpk9 mpk12. These results suggest that MPK9 and MPK12 are involved in CHT-induced stomatal closure.

MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling.

Reactive oxygen species (ROS) mediate abscisic acid (ABA) signaling in guard cells. To dissect guard cell ABA-ROS signaling genetically, a cell type-specific functional genomics approach was used to identify 2 MAPK genes, MPK9 and MPK12, which are preferentially and highly expressed in guard cells. To provide genetic evidence for their function, Arabidopsis single and double TILLING mutants that carry deleterious point mutations in these genes were isolated. RNAi-based gene-silencing plant lines, in which both genes are silenced simultaneously, were generated also. Mutants carrying a mutation in only 1 of these genes did not show any altered phenotype, indicating functional redundancy in these genes. ABA-induced stomatal closure was strongly impaired in 2 independent RNAi lines in which both MPK9 and MPK12 transcripts were significantly silenced. Consistent with this result, mpk9-1/12-1 double mutants showed an enhanced transpirational water loss and ABA- and H(2)O(2)-insensitive stomatal response. Furthermore, ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA signaling. An MPK12-YFP fusion construct rescued the ABA-insensitive stomatal response phenotype of mpk9-1/12-1, demonstrating that the phenotype was caused by the mutations. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H(2)O(2) treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively.

Stomatal Lineage Control by Developmental Program and Environmental Cues.

Stomata are micropores that allow plants to breathe and play a critical role in photosynthesis and nutrient uptake by regulating gas exchange and transpiration. Stomatal development, therefore, is optimized for survival and growth of the plant despite variable environmental conditions. Signaling cascades and transcriptional networks that determine the birth, proliferation, and differentiation of a stomate have been identified. These networks ensure proper stomatal patterning, density, and polarity. Environmental cues also influence stomatal development. In this review, we highlight recent findings regarding the developmental program governing cell fate and dynamics of stomatal lineage cells at the cell state- or single-cell level. We also overview the control of stomatal development by environmental cues as well as developmental plasticity associated with stomatal function and physiology. Recent advances in our understanding of stomatal development will provide a route to improving photosynthesis and water-stress resilience of crop plants in the climate change we currently face.

De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure.

Cytosolic Ca(2+) ([Ca(2+)](cyt)) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca(2+)](cyt) has been shown to regulate stomatal movements, and a defined window of Ca(2+) oscillation kinetic parameters encodes necessary information for long-term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca(2+) signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over-expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca(2+)-induced stomatal closure. Cytosolic Ca(2+) activation of S-type anion channels, which play a central role in Ca(2+)-reactive stomatal closure, was normal in the AtGLR3.1 over-expressing plants. Interestingly, AtGLR3.1 over-expression did not affect Ca(2+)-induced Ca(2+) oscillation kinetics, but resulted in a failure to maintain long-term 'Ca(2+)-programmed' stomatal closure when Ca(2+) oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short-term Ca(2+)-reactive closure was not affected in AtGLR3.1 over-expressing plants. In wild-type plants, the translational inhibitor cyclohexamide partially inhibited Ca(2+)-programmed stomatal closure induced by experimentally imposed Ca(2+) oscillations without affecting short-term Ca(2+)-reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over-expressing plants. Our results suggest that over-expression of AtGLR3.1 impairs Ca(2+) oscillation-regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long-term Ca(2+)-programmed stomatal closure.

Arabidopsis annexins AnnAt1 and AnnAt4 interact with each other and regulate drought and salt stress responses.

Annexins are Ca2+--and phospholipid-binding proteins that form an evolutionarily conserved multigene family throughout the animal and plant kingdoms. Two annexins, AnnAt1 and AnnAt4, have been identified as components in osmotic stress and abscisic acid signaling in Arabidopsis. Here, we report that AnnAt1 and AnnAt4 regulate plant stress responses in a light-dependent manner. The single-mutant annAt1 and annAt4 plants showed tolerance to drought and salt stress, which was greatly enhanced in double-mutant annAt1annAt4 plants, but AnnAt4-overexpressing transgenic plants (35S:AnnAt4) were more sensitive to stress treatments under long day conditions. Furthermore, expression of stress-related genes was altered in these mutant and transgenic plants. Upon dehydration and salt treatment, AtNCED3, encoding 9-cis-epoxycarotenoid dioxygenase, and P5CS1, encoding Δ-1-pyrroline-5-carboxylate synthase, which are key enzymes in ABA and proline synthesis, respectively, were highly induced in annAt1annAt4 plants and to a lesser extent in annAt1 and annAt4 plants, but not in 35S:AnnAt4 plants. While annAt1 plants were more drought sensitive, annAt4 plants were more tolerant in short days than in long days. In vitro and in vivo binding assays revealed that AnnAt1 and AnnAt4 bind to each other in a Ca2+-dependent manner. Our results suggest that AnnAt1 and AnnAt4 function cooperatively in response to drought and salt stress and their functions are affected by photoperiod.