l-Glutamate activates salicylic acid signaling to promote stomatal closure and PR1 expression in Arabidopsis.

Glutamate (l-Glu), an animal neurotransmitter, plays an essential role in plant signaling and regulates various plant physiological responses. We previously showed that l-Glu regulates stomatal closure in Arabidopsis via the glutamate receptor-like 3.5 gene (GLR3.5). Here, we showed that l-Glu activates salicylic acid (SA) signaling in Arabidopsis. l-Glu not only promoted stomatal closure but also triggered the expression of the PR1 gene via GLR3.5. These l-Glu-dependent actions were strongly suppressed in SA-insensitive npr1-1 and SA-deficient sid2-2 mutants, indicating that SA is involved in l-Glu signaling. A loss-of-function mutant of the gene encoding the SRK2E/OST1 kinase, which plays a pivotal role in abscisic acid signaling, was insensitive to both l-Glu-induced stomatal closure and PR1 expression. The glr3.5 mutants did not alleviate SA-induced stomatal closure, indicating that SA may function downstream of GLR3.5. These results indicate that l-Glu activates SA signaling, and that SRK2E/OST1 may play pivotal roles in such signaling.

Possible roles for phytohormones in controlling the stomatal behavior of Mesembryanthemum crystallinum during the salt-induced transition from C3 to crassulacean acid metabolism.

The halophyte ice plant (Mesembryanthemum crystallinum) converts its mode of photosynthesis from C3 to crassulacean acid metabolism (CAM) during severe water stress. During the transition to CAM, the plant induces CAM-related genes and changes its diurnal stomatal behavior to take up CO2 efficiently at night. However, limited information concerning this signaling exists. Here, we investigated the changes in the diurnal stomatal behavior of M. crystallinum during its shift in photosynthesis using a detached epidermis. M. crystallinum plants grown under C3 conditions opened their stomata during the day and closed them at night. However, CAM-induced plants closed their stomata during the day and opened them at night. Quantitative analysis of endogenous phytohormones revealed that trans-zeatin levels were high in CAM-induced plants. In contrast, the levels of jasmonic acid (JA) and JA-isoleucine were severely reduced in CAM-induced plants, specifically at night. CAM induction did not alter the levels of abscisic acid; however, inhibitors of abscisic acid synthesis suppressed CAM-induced stomatal closure. These results indicate that M. crystallinum regulates the diurnal balance of cytokinin and JA during CAM transition to alter stomatal behavior.

WRKY45 phosphorylation at threonine 266 acts negatively on WRKY45-dependent blast resistance in rice.

WRKY45 is a central regulator of disease resistance mediated by salicylic acid signaling in rice and its activation involves phosphorylation by OsMPK6. OsMPK6 phosphorylates WRKY45 at Thr266, Ser294, and Ser299 in vitro. Phosphorylation of Ser294 and/or Ser299 is required for full activation of WRKY45, but the importance of Thr266 phosphorylation has remained unknown. Here, we report on the characterization of Thr266 phosphorylation of WRKY45 in rice. Transient expression of mutant WRKY45 revealed that Thr266 is phosphorylated in vivo, together with Ser294/299. Replacement of Thr266 by Asn did not affect the enhanced Magnaporthe oryzae resistance afforded by WRKY45 overexpression. By contrast, replacement by Asp negated the enhancement of M. oryzae resistance. These results suggest that Thr266 phosphorylation acts negatively on WRKY45-dependent disease resistance.

Glutamate functions in stomatal closure in Arabidopsis and fava bean.

Guard cells are indispensable for higher plants because they control gas exchange and water balance to maintain photosynthetic activity. The signaling processes that govern their movement are controlled by several factors, such as abscisic acid (ABA), blue light, pathogen-associated molecular patterns (PAMPs), and carbon dioxide. Herein, we demonstrated that the amino acid glutamate (Glu), a well-known mammalian neurotransmitter, functions as a novel signaling molecule in stomatal closure in both Arabidopsis and fava bean (Vicia faba L.). Pharmacological and electrophysiological analyses provided important clues for the participation of Glu-receptors, Ca(2+), and protein phosphorylation during the signaling process. Genetic analyses using Arabidopsis ABA-deficient (aba2-1) and ABA-insensitive (abi1-1 and abi2-1) mutants showed that ABA is not required for Glu signaling. However, loss-of-function of the Arabidopsis gene encoding Slow Anion Channel-Associated 1 (SLAC1) and Calcium-Dependent Protein Kinase 6 (CPK6) impaired the Glu response. Moreover, T-DNA knockout mutations of the Arabidopsis Glu receptor-like gene (GLR), GLR3.5, lost their sensitivity to Glu-dependent stomatal closure. Our results strongly support functional Glu-signaling in stomatal closure and the crucial roles of GLRs in this signaling process.

Abiotic Stresses Antagonize the Rice Defence Pathway through the Tyrosine-Dephosphorylation of OsMPK6.

Plants, as sessile organisms, survive environmental changes by prioritizing their responses to the most life-threatening stress by allocating limited resources. Previous studies showed that pathogen resistance was suppressed under abiotic stresses. Here, we show the mechanism underlying this phenomenon. Phosphorylation of WRKY45, the central transcription factor in salicylic-acid (SA)-signalling-dependent pathogen defence in rice, via the OsMKK10-2-OsMPK6 cascade, was required to fully activate WRKY45. The activation of WRKY45 by benzothiadiazole (BTH) was reduced under low temperature and high salinity, probably through abscisic acid (ABA) signalling. An ABA treatment dephosphorylated/inactivated OsMPK6 via protein tyrosine phosphatases, OsPTP1/2, leading to the impaired activation of WRKY45 and a reduction in Magnaporthe oryzae resistance, even after BTH treatment. BTH induced a strong M. oryzae resistance in OsPTP1/2 knockdown rice, even under cold and high salinity, indicating that OsPTP1/2 is the node of SA-ABA signalling crosstalk and its down-regulation makes rice disease resistant, even under abiotic stresses. These results points to one of the directions to further improve crops by managing the tradeoffs between different stress responses of plants.

8-Mercapto-Cyclic GMP Mediates Hydrogen Sulfide-Induced Stomatal Closure in Arabidopsis.

Plants are exposed to hydrogen sulfide (H2S) both exogenously, as it exists as a pollutant gas in the environment, and endogenously, as it is synthesized in cells. H2S has recently been found to function as a gaseous signaling molecule, but its signaling cascade remains unknown. Here, we examined H2S-mediated guard cell signaling in Arabidopsis. The H2S donor GYY4137 (morpholin-4-ium-4-methoxyphenyl [morpholino] phosphinodithioate) induced stomatal closure, which peaked after 150 min at 1 µM or after 90 min at 10 and 100 µM. After reaching maximal closure, stomatal apertures gradually increased in size in response to further exposure to GYY4137. GYY4137 induced nitric oxide (NO) generation in guard cells, and GYY4137-induced stomatal closure was reduced by an NO scavenger and inhibitors of NO-producing enzymes. Mass spectrometry analyses showed that GYY4137 induces the synthesis of 8-nitro-cGMP and 8-mercapto-cGMP and that this synthesis is mediated by NO. In addition, 8-mercapto-cGMP triggered stomatal closure. Moreover, inhibitor and genetic studies showed that calcium, cADP ribose and slow anion channel 1 act downstream of 8-mercapto-cGMP. This study therefore demonstrates that 8-mercapto-cGMP mediates the H2S signaling cascade in guard cells.

WRKY45-dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction.

Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice-Magnaporthe oryzae interaction. The DP biosynthetic genes were barely upregulated in BTH-treated rice plants, but were induced rapidly after M. oryzae infection in a WRKY45-dependent manner. These results indicate that the DP biosynthetic genes were primed by BTH through WRKY45. Rapid induction of the DP biosynthetic genes was also observed after M. oryzae infection to WRKY45-overexpressing (WRKY45-ox) plants. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants.

MAP kinases phosphorylate rice WRKY45.

WRKY45 transcription factor is a central regulator of disease resistance mediated by the salicylic acid (SA) signaling pathway in rice. SA-activated WRKY45 protein induces the accumulation of its own mRNA. However, the mechanism underlying this regulation is still unknown. Here, we report three lines of evidence showing that a mitogen-activated protein kinase (MAPK) cascade is involved in this regulation. An inhibitor of MAPK kinase (MAPKK) suppressed the increase in WRKY45 transcript level in response to SA. Two MAPKs, OsMPK4 and OsMPK6, phosphorylated WRKY45 protein in vitro. The activity of OsMPK6 was rapidly upregulated by SA treatment in rice cells. These results suggest that WRKY45 is regulated by MAPK-dependent phosphorylation in the SA pathway.

Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis.

Rapid recognition and signal transduction of mechanical wounding through various signaling molecules, including calcium (Ca²+), protein phosphorylation, and reactive oxygen species (ROS), are necessary early events leading to stress resistance in plants. Here we report that an Arabidopsis mitogen-activated protein kinase 8 (MPK8) connects protein phosphorylation, Ca²+, and ROS in the wound-signaling pathway. MPK8 is activated through mechanical wounding, and this activation requires direct binding of calmodulins (CaMs) in a Ca²+-dependent manner. MPK8 is also phosphorylated and activated by a MAPKK MKK3 in the prototypic kinase cascade, and full activation of MPK8 needs both CaMs and MKK3 in planta. The MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Rboh D gene. These findings suggest that two major activation modes in eukaryotes, Ca²+/CaMs and the MAP kinase phosphorylation cascade, converge at MPK8 to monitor or maintain an essential part of ROS homeostasis.

Molecular breeding of tomato lines for mass production of miraculin in a plant factory.

A transgenic tomato line (56B, "Moneymaker") that expresses the miraculin gene driven by the CaMV 35S promoter was crossed with a dwarf tomato ("Micro-Tom") for the molecular breeding of cultivars that are suitable for miraculin production in a closed cultivation system. Plant size, miraculin accumulation, and self-pruning growth were used as selection indicators for F2 plants. Two lines were chosen for further analysis, bred to the F6 or F7 generation and cultivated in a closed cultivation system. In 56B and the two crossed lines, the concentrations of miraculin in the pericarp were 140, 367, and 343 microg/g FW, respectively. We also estimated that 26.2, 73.6, and 45.9 kg FW/m2 of tomatoes and 2.2, 16.6, and 9.8 mg/m2 of miraculin in the pericarp, respectively, could be harvested per year. These two crossed lines will be useful for the mass production of miraculin, especially in a closed cultivation system.

Abscisic acid interacts antagonistically with salicylic acid signaling pathway in rice-Magnaporthe grisea interaction.

Plant hormones play pivotal signaling roles in plant-pathogen interactions. Here, we report characterization of an antagonistic interaction of abscisic acid (ABA) with salicylic acid (SA) signaling pathways in the rice-Magnaporthe grisea interaction. Exogenous application of ABA drastically compromised the rice resistance to both compatible and incompatible M. grisea strains, indicating that ABA negatively regulates both basal and resistance gene-mediated blast resistance. ABA markedly suppressed the transcriptional upregulation of WRKY45 and OsNPR1, the two key components of the SA signaling pathway in rice, induced by SA or benzothiadiazole or by blast infection. Overexpression of OsNPR1 or WRKY45 largely negated the enhancement of blast susceptibility by ABA, suggesting that ABA acts upstream of WRKY45 and OsNPR1 in the rice SA pathway. ABA-responsive genes were induced during blast infection in a pattern reciprocal to those of WRKY45 and OsPR1b in the compatible rice-blast interaction but only marginally in the incompatible one. These results suggest that the balance of SA and ABA signaling is an important determinant for the outcome of the rice-M. grisea interaction. ABA was detected in hyphae and conidia of M. grisea as well as in culture media, implying that blast-fungus-derived ABA could play a role in triggering ABA signaling at host infection sites.

Punctual coordination: switching on and off for flowering during a day.

The photoperiodic flowering of Arabidopsis is shown to be explained in part by the Bünning's external coincidence model in which clock-controlled expression of CO and stabilization of CO protein by light have important roles. The floral activators, GI and CO, together with ZTL, FKF and CDF1 have been shown to be central for the induction of FT expression during evening to promote the photoperiodic flowering of Arabidopsis. Here we discuss a role of diurnal accumulation of a floral repressor SVP protein in the repression of the FT and SOC1 expression during daytime. A punctual coordination of the diurnal regulation of both positive and negative regulators by circadian clock appears to be important for the photoperiodic flowering in Arabidopsis.

Possible role of early flowering 3 (ELF3) in clock-dependent floral regulation by short vegetative phase (SVP) in Arabidopsis thaliana.

Circadian clock proteins play key roles in adaptations of plants to diurnal environmental conditions. The photoperiodic flowering response is one of the mechanisms of adaptation to seasonal changes in the lengths of day and night. Double mutations in two clock genes, late elongated hypocotyl (LHY) and circadian clock associated 1 (CCA1), accelerated flowering under short days (SDs) but delayed flowering under continuous light (LL) in Arabidopsis thaliana. The mechanism underlying the late flowering of lhy;cca1 mutants under LL was investigated here. Late flowering of plants with overexpression of short vegetative phase (SVP) was much more pronounced under SDs and enhanced by constans 2 (co-2) under long days (LDs), suggesting that SVP and CO act independently in the photoperiodic flowering pathway. However, how SVP and flowering locus C (FLC) mediated the effects of LHY/CCA1 and thus influenced flowering time was not completely clear. A mutant line lhy;cca1 in the Landsberg erecta (Ler) background was established, ethyl methanesulfonate (EMS)-mutagenized and used to screen suppressors of late flowering of lhy;cca1 under LL. Mutations in the clock gene early flowering 3 (ELF3) were identified as suppressors. Overexpression and loss-of-function of ELF3 influenced SVP protein accumulation. Therefore, we propose that, as well as the classical GIGANTEA (GI)-CO pathway, LHY/CCA1 regulates a pathway negatively controlling flowering locus T (FT), possibly via ELF3-SVP/FLC.

Circadian clock proteins LHY and CCA1 regulate SVP protein accumulation to control flowering in Arabidopsis.

The floral regulators GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) play key roles in the photoperiodic flowering responses of the long-day plant Arabidopsis thaliana. The GI-CO-FT pathway is highly conserved in plants. Here, we demonstrate that the circadian clock proteins LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) not only repressed the floral transition under short-day and long-day conditions but also accelerated flowering when the plants were grown under continuous light (LL). LHY and CCA1 accelerated flowering in LL by promoting FT expression through a genetic pathway that appears to be independent of the canonical photoperiodic pathway involving GI and CO proteins. A genetic screen revealed that the late-flowering phenotype of the lhy;cca1 double mutant under LL was suppressed through mutations in SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor. Yeast two-hybrid analysis demonstrated an interaction between SVP and FLOWERING LOCUS C, and genetic analysis indicated that these two proteins act as partially redundant repressors of flowering time. SVP protein accumulated in lhy;cca1 plants under LL. We propose a model in which LHY and CCA1 accelerate flowering in part by reducing the abundance of SVP and thereby antagonizing its capacity to repress FT expression under LL.

Characterization of growth-phase-specific responses to cold in Arabidopsis thaliana suspension-cultured cells.

To understand complex responses of plant cells to low temperatures, suspension-cultured cells of Arabidopsis thaliana (line T87) were characterized during cold treatment. Freezing tolerance of cells collected at the lag or log phase was quite different: an increase in freezing tolerance during cold treatment was only detectable with cells at the lag phase. Although there were little differences in the osmolality of cells at the two growth phases, sugar content increased during cold treatment only in lag phase cells. Abscisic acid (ABA) content was greater at the lag phase than at the log phase throughout the cold treatment, and increased only in lag phase cells. Interestingly, ABA treatment resulted in an increase in freezing tolerance only in lag phase cells. Expression of cold-responsive genes such as DREB1A/CBF3, COR15a and RD29A occurred in cells at the two growth phases; however, the extent of the induction of COR15a and RD29A was somewhat greater in cells at the lag phase than at the log phase. Microarray analysis revealed that cold-regulated genes (COR genes) were categorized into three groups: up- or down-regulated consistently during cold treatment, transiently after 1 d and later after 2 d of cold treatment. Among genes that were up-regulated throughout or transiently after 1 d of cold treatment only in lag phase cells, functions in upstream of signal transduction such as kinase and transcription factor were often deduced. These results collectively suggest that these genes seem to be associated with induction of freezing tolerance by cold treatment in cells at the lag phase.

The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis.

The plant hormone jasmonic acid (JA) plays a key role in the environmental stress responses and developmental processes of plants. Although ATMYC2/JASMONATE-INSENSITIVE1 (JIN1) is a major positive regulator of JA-inducible gene expression and essential for JA-dependent developmental processes in Arabidopsis thaliana, molecular mechanisms underlying the control of ATMYC2/JIN1 expression remain largely unknown. Here, we identify a mitogen-activated protein kinase (MAPK) cascade, MAPK KINASE 3 (MKK3)-MAPK 6 (MPK6), which is activated by JA in Arabidopsis. We also show that JA negatively controls ATMYC2/JIN1 expression, based on quantitative RT-PCR and genetic analyses using gain-of-function and loss-of-function mutants of the MKK3-MPK6 cascade. These results indicate that this kinase unit plays a key role in JA-dependent negative regulation of ATMYC2/JIN1 expression. Both positive and negative regulation by JA may be used to fine-tune ATMYC2/JIN1 expression to control JA signaling. Moreover, JA-regulated root growth inhibition is affected by mutations in the MKK3-MPK6 cascade, which indicates important roles in JA signaling. We provide a model explaining how MPK6 can convert three distinct signals - JA, pathogen, and cold/salt stress - into three different sets of responses in Arabidopsis.

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis.

ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to negatively regulate many aspects of abscisic acid (ABA) signaling, including stomatal closure in Arabidopsis. In contrast, SRK2E/OST1/SnRK2.6 encodes an Arabidopsis SnRK2 protein kinase and acts as a positive regulator in the ABA-induced stomatal closure. SRK2E/OST1 is activated by osmotic stress as well as by ABA, but the independence of the two activation processes has not yet been determined. Additionally, interaction between SRK2E/OST1 and PP2C-type phosphatases (ABI1 and ABI2) is not understood. In the present study, we demonstrated that the abi1-1 mutation, but not the abi2-1 mutation, strongly inhibited ABA-dependent SRK2E/OST1 activation. In contrast, osmotic stress activated SRK2E/OST1 even in abi1-1 and aba2-1 plants. The C-terminal regulatory domain of SRK2E/OST1 was required for its activation by both ABA and osmotic stress in Arabidopsis. The C-terminal domain was functionally divided into Domains I and II. Domain II was required only for the ABA-dependent activation of SRK2E/OST1, whereas Domain I was responsible for the ABA-independent activation. Full-length SRK2E/OST1 completely complemented the wilty phenotype of the srk2e mutant, but SRK2E/OST1 lacking Domain II did not. Domain II interacted with the ABI1 protein in a yeast two-hybrid assay. Our results suggested that the direct interaction between SRK2E/OST1 and ABI1 through Domain II plays a critical role in the control of stomatal closure.

SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana.

Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Here, we showed that a SNF1-related protein kinase 2 (SnRK2), SRK2C, is an osmotic-stress-activated protein kinase in Arabidopsis thaliana that can significantly impact drought tolerance of Arabidopsis plants. Knockout mutants of SRK2C exhibited drought hypersensitivity in their roots, suggesting that SRK2C is a positive regulator of drought tolerance in Arabidopsis roots. Additionally, transgenic plants with CaMV35S promoter::SRK2C-GFP displayed higher overall drought tolerance than control plants. Whereas stomatal regulation in 35S::SRK2C-GFP plants was not altered, microarray analysis revealed that their drought tolerance coincided with up-regulation of many stress-responsive genes, for example, RD29A, COR15A, and DREB1A/CBF3. From these results, we concluded that SRK2C is capable of mediating signals initiated during drought stress, resulting in appropriate gene expression. Our present study reveals new insights around signal output from osmotic-stress-activated SnRK2 protein kinase as well as supporting feasibility of manipulating SnRK2 toward improving plant osmotic-stress tolerance.