Reciprocal inhibition of expression between RAV1 and BES1 modulates plant growth and development in Arabidopsis.

RAV1 (Related to ABI3/VP1) is a plant-specific B3 and AP2 domain-containing transcription factor that acts as a negative regulator of growth in many plant species. The expression of RAV1 is downregulated by brassinosteroids (BRs); large-scale transcriptome analyses have shown that the expression of RAV1 was previously targeted by BRI1-EMS-SUPPRESOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), which are critical transcription factors for the BR-signaling process. Using RAV1-overexpressing transgenic plants, we showed that RAV1 overexpression reduced the BR signaling capacity, resulting in the downregulation of BR biosynthetic genes and BES1 expression. Furthermore, we demonstrated that BES1, not BZR1, is directly bound to the RAV1 promoter and repressed RAV1 expression, and vice versa; RAV1 is also bound to the BES1 promoter and repressed BES1 expression. This mutual inhibition was specific to RAV1 and BES1 because RAV1 exhibited binding activity to the BZR1 promoter but did not repress BZR1 expression. We observed that constitutively activated BR signaling phenotypes in bes1-D were attenuated by the repression of endogenous BES1 expression in transgenic bes1-D plants overexpressing RAV1. RNA-sequencing analysis of RAV1-overexpressing transgenic plants and bes1-D mutant plants revealed differentially expressed genes by RAV1 and BES1 and genes that were oppositely co-regulated by RAV1 and BES1. RAV1 and BES1 regulated different transcriptomes but co-regulated a specific set of genes responsible for the balance between growth and defense. These results suggested that the mutual inhibitory transcriptional activities of RAV1 and BES1 provide fine regulatory mechanisms for plant growth and development.

BAK1-induced RPK1 phosphorylation is essential for RPK1-mediated cell death in Arabidopsis.

Being sessile, plants must deploy highly exquisite systems to respond to various internal and external signals for modulating growth and development throughout their lifespan. Many studies on Arabidopsis have shown that leucine-rich repeat-containing receptor-like kinases, including BRI1-associated receptor kinase 1 (BAK1) and receptor-like protein kinase 1 (RPK1), are suitable for such pleiotropic demands of plants. Previously, BAK1 and RPK1 were independently proven to be involved in the regulation of premature cell death. BAK1 inhibits spontaneous cell death and promotes defense-induced cell death. Meanwhile, RPK1 mediates reactive oxygen species (ROS) production through complexation with CaM4 and RbohF in an age-dependent manner. In the present study, RPK1-induced cell death and growth retardation were abolished both with respect to the phenotype and ROS production in bak1 mutants. Moreover, BAK1 interacts with RPK1 and mediates its unidirectional phosphorylation in plants. Further, BAK1-mediated RPK1 phosphorylation is indispensable for RPK1-CaM4 interaction, which is vital for ROS production, resulting in cell death. The presence of BAK1 enhanced the expression of cell death- and senescence-related genes, such as ORE1, PR1, SAG12, and SIRK in RPK1-mediated signaling cascades. Overall, in Arabidopsis, in addition to independent cell death regulation by BAK1 and RPK1, multiple-layers control cell death and premature senescence via the coordinated action of BAK1 and RPK1.

Open stomata 1 exhibits dual serine/threonine and tyrosine kinase activity in regulating abscisic acid signaling.

Open Stomata 1 (OST1)/SnRK2.6 is a critical component connecting abscisic acid (ABA) receptor complexes and downstream components, including anion channels and transcription factors. Because OST1 is a serine/threonine kinase, several autophosphorylation sites have been identified, and S175 is known to be critical for its kinase activity. We previously reported that BAK1 interacts with and phosphorylates OST1 to regulate ABA signaling. Here, we mapped additional phosphosites of OST1 generated by autophosphorylation and BAK1-mediated transphosphorylation in Arabidopsis. Many phosphosites serve as both auto- and transphosphorylation sites, especially those clustered in the activation loop region. Phospho-mimetic transgenic plants containing quadruple changes in Y163, S164, S166, and S167 rescued ost1 mutant phenotypes, activating ABA signaling outputs. Moreover, we found that OST1 is an active tyrosine kinase, autophosphorylating the Y182 site. ABA induced tyrosine phosphorylation of Y182 in OST1; this event is catalytically important for OST1 activity in plants. ABA-Insensitive 1 (ABI1) and its homologs ABI2 and HAB1, PP2C serine/threonine phosphatases that are known to dephosphorylate OST1 at S175, function as tyrosine phosphatases acting on the phosphorylated Y182 site. Our results indicate that phosphorylation cycles between OST1 and ABI1, which have dual specificity for tyrosine and serine/threonine, coordinately control ABA signaling in Arabidopsis.

Receptor-like protein kinases RPK1 and BAK1 sequentially form complexes with the cytoplasmic kinase OST1 to regulate ABA-induced stomatal closure.

Regulation of plant water status occurs via abscisic acid (ABA)-induced stomatal closure. Open Stomata 1 (OST1) is a critical ABA signaling component regulating this process in guard cells. We previously reported that BRI1-associated receptor kinase 1 (BAK1) positively regulates ABA-induced stomatal closure by interacting with and phosphorylating OST1. Here, using Arabidopsis, we show that the receptor-like protein kinase 1 (RPK1), previously known to be induced by ABA, is a positive ABA-signaling component in guard cell movement, and interacts with OST1. ABA-inducible expression patterns were observed in RPK1 and OST1, but not in BAK1. We investigated the underlying mechanisms by which the RPK1-OST1 and BAK1-OST1 complexes interact in stomatal guard cells by monitoring the complex formation continuously using fluorescence resonance energy transfer analyses. We found that the BAK1-OST1 complex was formed earlier than the RPK1-OST1 complex in response to ABA. In vitro and semi-in vivo kinase assays revealed that a transphosphorylation event occurred in the RPK1-OST1 complex, which differs from that in the BAK1-OST1 complex, wherein only OST1 phosphorylation occurred via BAK1. ABA-insensitive 1 (ABI1) only dephosphorylated OST1 in the BAK1-OST1 complex, but dephosphorylated both RPK1 and OST1 proteins in the RPK1-OST1 complex. Our results suggest that there are multiple coordinated ABA signaling systems to regulate stomatal movement.

Brassinosteroid reduces ABA accumulation leading to the inhibition of ABA-induced stomatal closure.

Proper regulation of stomatal movement in response to various environmental stresses or developmental status is critical for the adaptation of many plant species to land. In plants, abscisic acid (ABA)-induced stomatal closure is a well-adapted method of regulating water status. In addition to ABA, we previously showed that plant-specific steroidal hormone, brassinosteroid (BR), also induces stomatal closure; however, BR modulates ABA-induced stomatal closure negatively at high concentrations. In this study, we further investigated the cross-talk between ABA and BR in relation to stomatal movement. In contrast to previous reports that ABA-induced stomatal closure was inhibited by brassinolide (BL), the most active BR, we showed that BL-induced stomatal closure was enhanced by ABA, indicating that the sequence of ABA or BL treatments led to different results. We found that this phenomenon occurred because the guard cells still had the capacity to be closed further by ABA, as the degree of stomatal closure by BL was always less than that by ABA. We also found that BL-induced stomatal closure required Open Stomata 1 (OST1) activity and the induced expression of OST1 was indifferent to the sequence of ABA and/or BL treatments. In addition, we examined the underlying mechanism by which inhibition of ABA-induced stomatal closure by BL occurred. We revealed that the downregulation of ABA-biosynthetic genes by BL resulted in a lower accumulation of ABA. These results suggested that the regulation of stomatal movement is finely controlled by the combined effects of plant hormones, ABA and BR.

Overexpression of BAK1 causes salicylic acid accumulation and deregulation of cell death control genes.

Since the BRI1-Associated Receptor Kinase 1 (BAK1) was firstly identified as a co-receptor of BRI1 that mediates brassinosteroids (BR) signaling, the functional roles of BAK1, as a versatile co-receptor for various ligand-binding leucine-rich repeat (LRR)-containing receptor-like kinase (RLKs), are being extended to involvement with plant immunity, cell death, stomatal development and ABA signaling in plants. During more than a decade of research on the BAK1, it has been known that transgenic Arabidopsis plants overexpressing BAK1 tagged with various reporters do not fully represent its natural functions. Therefore, in this study, we characterized the transgenic plants in which native BAK1 is overexpressed driven by its own promoter. We found that those transgenic plants were more sensitive to BR signaling but showed reduced growth patterns accompanied with spontaneous cell death features that are different from those seen in BR-related mutants. We demonstrated that more salicylic acid (SA) and hydrogen peroxide were accumulated and that expressions of the genes that are known to regulate cell death, such as BONs, BIRs, and SOBIR, were increased in the BAK1-overexpressing transgenic plants. These results suggest that pleiotropic phenotypic alterations shown in the BAK1- overexpressing transgenic plants result from the constitutive activation of SA-mediated defense responses.

BRI1-EMS-suppressor 1 gain-of-function mutant shows higher susceptibility to necrotrophic fungal infection.

Brassinosteroids (BRs) are plant-specific steroids that are involved in plant growth and defense responses. However, the exact roles of BR in plant defense are unclear. We used the bes1-D gain-of-function mutant to define the underlying relationship between plant growth and defense through BR signaling and innate immunity. In bes1-D, further downstream component BES1 transcription factor is stabilized, leading to the activation of BR signaling. Previous reports on BES1 target genes showed that approximately 10% are related to biotic stress responses. Therefore, the bes1-D PTI responses were examined. The bes1-D mutant was specifically susceptible to Alternaria brassicicola, a necrotrophic fungus, which successfully produced spore, resulting in considerable cell death. However, it was not affected by a biotrophic pathogen, Pseudomonas syringae pv. tomato (Pst) DC3000. Instead of a ROS burst, a representative initial PTI responses, higher ROS accumulation was sustained in bes1-D than in the wild type plant. PDF1.2 expression was not induced in response to fungal pathogen infection in bes1-D. These results suggest that BES1 is also involved in JA-related defense responses, especially in response to necrotrophic pathogens.

Brassinosteroids modulate ABA-induced stomatal closure in Arabidopsis.

Stomatal movement in response to water availability is an important physiological process in the survival of land plants. The plant hormone abscisic acid (ABA) and brassinosteroids (BRs) regulate stomatal closure. The physiological functions of ABA and BRs, including germination, cell elongation and stomatal movement, are generally known to be antagonistic. Here, we investigated how BRs affect stomatal movement alone and in combination with ABA. We demonstrate that brassinoslide (BL), the most active BR, promotes stomatal closure in an ABA-independent manner. Interestingly, BL also inhibited ABA-induced stomatal closure when a high concentration of BL was added to ABA. Furthermore, we found that the induction of some genes for reactive oxygen species (ROS) generation by ABA (AtrbohD, NIA1 and NIA2) and subsequent ROS production were repressed by BL treatment. The BR signaling mutant bri1-301 failed to inhibit ABA-induced stomatal closure upon BL treatment. However, BRI1-overexpressing transgenic plants were hypersensitive to ABA during stomatal closure, and BL reversed ABA-induced stomatal closure more completely than in wild type plants. Taken together, these results suggest that BRs can positively and negatively modulate ABA-induced stomatal closure. Therefore, interactions between ABA and BR signaling are important for the regulation of stomatal closure.

A subset of Arabidopsis RAV transcription factors modulates drought and salt stress responses independent of ABA.

Arabidopsis RAV1, RAV1L and RAV2/TEM2 are Related to ABI3/VP1 (RAV) transcription factors that contain both plant-specific B3 and AP2 domains. RAV1 was known to be a negative regulator of growth and its transcript level was repressed by brassinolide (BL). In this study, we found that the expressions of RAV1, and its closest homologs RAV1L and RAV2 were also regulated by other plant hormones, and especially repressed significantly by BL and abscisic acid (ABA), which mediate various abiotic stress responses in plants. Therefore, to further investigate the physiological functions of RAV1, RAV1L and RAV2 in abiotic stress responses, we isolated T-DNA insertional knockout mutants of each gene and produced transgenic plants overexpressing the RAVs. Under normal conditions, each single mutant showed slightly promoted growth patterns only at an early stage of development. In comparison, the RAV1-overexpressing plants exhibited strong growth retardation with semi-dwarfed stature. In drought conditions, RAVs-overexpressing transgenic plants exhibited higher transpirational water loss than the wild type. In salt conditions, seed germination of the RAVs-overexpressing transgenic plants was more inhibited than that of the wild type, while ravs mutants showed promoted seed germination. We also found that RAVs expressions were reduced by dryness and salt. RAV1-overexpressing plants showed the same patterns of increased expression as stress-inducible genes such as RD29A, RD29B and the genes encoding ABA biosynthetic enzymes, as did the wild type and rav1 mutant. However, the RAV1-overexpressing transgenic plants were insensitive to ABA, regardless of the higher accumulation of ABA even in normal conditions. Taken together, these results suggest that RAVs are versatile negative regulators for growth and abiotic stresses, drought and salt, and that negative regulatory effects of RAVs on abiotic stresses are likely to be operated independently of ABA.

Overexpression of miR172 suppresses the brassinosteroid signaling defects of bak1 in Arabidopsis.

BRI1-Associated Receptor Kinase 1 (BAK1) is a leucine-rich repeat serine/threonine receptor-like kinase (LRR-RLK) that is involved in multiple developmental pathways, such as brassinosteroid (BR) signaling, plant immunity and cell death control in plants. Because the roundish and compact rosette leaves of bak1 mutant plants are characteristic phenotypes for deficient BR signaling, we screened genetic suppressors of bak1 according to changes in leaf shape to identify new components that may be involved in BAK1-mediated BR signaling using the activation-tagging method. Here, we report bak1-SUP1, which exhibited longer and narrower rosette leaves and an increased BR sensitivity compared with those of bak1. Analyses of the T-DNA insertional site and the gene expression that was affected by the T-DNA insertion revealed that a microRNA, namely, miR172, over-accumulates in bak1-SUP1. Detailed phenotypic analyses of bak1-SUP1 and a single mutant in which the bak1 mutation was segregated out (miR172-D) revealed that the overexpression of miR172 promotes leaf length elongation in adult plants and increases the root and hypocotyl growth during the seedling stage compared with that of wild type plants. Taken together with its increased BR sensitivity, these results suggest that miR172 regulates vegetative growth patterns by modulating BR sensitivity as well as by the previously identified developmental phase transition.

Functional implication of ß-carotene hydroxylases in soybean nodulation.

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative ß-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

Identification of GA20ox2 as a target of ATHB2 and TCP13 during shade response.

The shade avoidance syndrome (SAS) is a collective adaptive response of plants under shade highlighted by characteristic phenotypes such as hypocotyl elongation, which is largely mediated by concerted actions of auxin and GA. We identified ATHB2, a homeodomain-leucine zipper (HD-Zip) domain transcription factor known to be rapidly induced under shade condition, as a positive regulator of GA biosynthesis necessary for the SAS by transactivating the expression of GA20ox2, a key gene in the GA biosynthesis pathway. Based on promoter deletion analysis, EMSA and ChIP assay, ATHB2 appears to regulate the GA20ox2 expression as a direct binding target. We also found that the GA20ox2 expression is under negative control by TCP13, the effect of which can be suppressed by presence of ATHB2. Considering a rapid induction kinetics of ATHB2, this relationship between ATHB2 and TCP13 may allow ATHB2 to play a shade-specific activator for GA20ox by derepressing a pre-existing activity of TCP13.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 Modulates Abscisic Acid Signaling by Inducing PYR1 Monomerization and Association With ABI1 in Arabidopsis.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 (BAK1) is a versatile kinase involved in many different plant developmental responses. Previously, we showed that BAK1 interacts with open stomata 1 (OST1), a cytoplasmic kinase, to promote abscisic acid (ABA)-induced stomatal closure. ABA is a plant hormone that primarily regulates stress responses and is recognized by the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENT OF ABA RECEPTORS (RCAR), which activates ABA signaling. Here, we demonstrated that BAK1 interacts with PYR1 and phosphorylates PYR1 in response to ABA in plants. We identified T137 and S142 of PYR1 as the phosphosites targeted by BAK1. Using phosphomimetic (PYR1DD) and phospho-dead (PYR1AA) PYR1 compared with wild-type PYR1, we showed that transgenic plants overexpressing a phosphomimetic PYR1 exhibited hypersensitivity to the inhibition of ABA-induced root growth and seed germination and increased ABA-induced stomatal closure and ABA-inducible gene expression. As underlying reasons for these phenomena, we further demonstrated that phosphorylated PYR1 existed in a monomeric form, in which ABA binding was increased, and the degree of complex formation with ABI1 was also increased. These results suggest that BAK1 positively modulates ABA signaling through interaction with PYR1, in addition to OST1.

Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR signaling.

BACKGROUND: Since the identification of BRI1 (BRASSINOSTEROID-INSENSITIVE1), a brassinosteroids (BRs) receptor, most of the critical roles of BR in plant development have been assessed using various bri1 mutant alleles. The characterization of individual bri1 mutants has shown that both the extracellular and cytoplasmic domains of BRI1 are important to its proper functioning. Particularly, in the extracellular domain, regions near the 70-amino acid island are known to be critical to BR binding. In comparison, the exact function of the leucine rich-repeats (LRR) region located before the 70-amino acid island domain in the extracellular cellular portion of BRI1 has not yet been described, due to a lack of specific mutant alleles. RESULTS: Among the mutants showing altered growth patterns compared to wild type, we further characterized cp3, which displayed defective growth and reduced BR sensitivity. We sequenced the genomic DNA spanning BRI1 in the cp3 and found that cp3 has a point mutation in the region encoding the 13th LRR of BRI1, resulting in a change from serine to phenylalanine (S399F). We renamed it bri1-120. We also showed that overexpression of the wild type BRI1 protein rescued the phenotype of bri1-120. Using a GFP-tagged bri1-120 construct, we detected the bri1-120 protein in the plasma membrane, and showed that the phenotypic defects in the rosette leaves of bri1-301, a kinase-inactive weak allele of BRI1, can be restored by the overexpression of the bri1-120 proteins in bri1-301. We also produced bri1-301 mutants that were wild type in appearance by performing a genetic cross between bri1-301 and bri1-120 plants. CONCLUSIONS: We identified a new bri1 allele, bri1-120, whose mutation site has not yet been found or characterized. Our results indicated that the extracellular LRR regions before the 70-amino acid island domain of BRI1 are important for the appropriate cellular functioning of BRI1. Also, we confirmed that a successful interallelic complementation occurs between the extracellular domain mutant allele and the cytoplasmic kinase-inactive mutant allele of BRI1 in vivo.

ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of Arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene.

Arabidopsis thaliana homeobox 12 (ATHB12) is rapidly induced by ABA and water stress. A T-DNA insertion mutant of ATHB12 with a reduced level of ATHB12 expression in stems had longer inflorescence stems and reduced sensitivity to ABA during germination. A high level of transcripts of gibberellin 20-oxidase 1 (GA20ox1), a key enzyme in the synthesis of gibberellins, was detected in athb12 stems, while transgenic lines overexpressing ATHB12 (A12OX) had a reduced level of GA20ox1 in stems. Consistent with these data, ABA treatment of wild-type plants resulted in decreased GA20ox1 expression whereas ABA treatment of the athb12 mutant gave rise to slightly decreased GA20ox1 expression. Retarded stem growth in 3-week-old A12OX plants was rescued by exogenous GA(9), but not by GA(12), and less GA(9) was detected in A12OX stems than in wild-type stems. These data imply that ATHB12 decreases GA20ox1 expression in stems. On the other hand, the stems of A12OX plants grew rapidly after the first 3 weeks, so that they were almost as high as wild-type plants at about 5 weeks after germination. We also found changes in the stems of transgenic plants overexpressing ATHB12, such as alterations of expression GA20ox and GA3ox genes, and of GA(4) levels, which appear to result from feedback regulation. Repression of GA20ox1 by ATHB12 was confirmed by transfection of leaf protoplasts. ABA-treated protoplasts also showed increased ATHB12 expression and reduced GA20ox1 expression. These findings all suggest that ATHB12 negatively regulates the expression of a GA 20-oxidase gene in inflorescence stems.

Identification of uricase as a potential target of plant thioredoxin: Implication in the regulation of nodule development.

During symbiotic nodule development in legume roots, early signaling events between host and rhizobia serve critical determinants for the proper onset of nodule morphogenesis, nitrogen fixation, and assimilation. Previously we isolated thioredoxin from soybean nodules as one of differentially expressed genes during nodulation and noted its positive role in nitrogen fixation. To identify the target proteins of thioredoxin in nodules, we used thioredoxin affinity chromatography followed by mass spectrometry. Nodulin-35, a subunit of uricase, was found to be a target of thioredoxin. Their interaction was confirmed by pull-down assay and by bimolecular fluorescent complementation. With an increased uricase activity observed also in the presence of thioredoxin, these results appear to implicate a novel role of thioredoxin in the regulation of enzyme activities involved in nodule development and nitrogen fixation.

Constitutive activation of stress-inducible genes in a brassinosteroid-insensitive 1 (bri1) mutant results in higher tolerance to cold.

Many plant hormones are involved in coordinating the growth responses of plants under stress. However, not many mechanistic studies have explored how plants maintain the balance between growth and stress responses. Brassinosteroids (BRs), plant-specific steroid hormones, affect many aspects of plant growth and development over a plant's lifetime. In this study we determined that exogenous treatment of BR helped the plant overcome the cold condition only when pretreated with less than 1 nM, and the brassinosteroid-insensitive 1 (bri1) mutation, which results in defective BR signaling and subsequent dwarfism, generates an increased tolerance to cold. In contrast, BRI1-overexpressing plants were more sensitive to the same stress than wild-type. We found that the bri1 mutant and BRI1-overexpressing transgenic plants contain higher basal level of expression of CBFs/DREB1s than wild-type. However, representative cold stress-related genes were regulated with the same pattern to cold in wild-type, bri1-9 and BRI1 overexpressing plants. To examine the global gene expression and compare the genes that show differential expression pattern in bri1-9 and BRI1-GFP plants other than CBFs/DREB1s, we analyzed differential mRNA expression using the cDNA microarray analysis in the absence of stress. Endogenous expression of both stress-inducible genes as well as genes encoding transcription factors that drive the expression of stress-inducible genes were maintained at higher levels in bri1-9 than either in wild-type or in BRI1 overexpressing plants. This suggests that the bri1-9 mutant could always be alert to stresses that might be exerted at any times by constitutive activation of subsets of defense.

Physiological roles of ERD10 in abiotic stresses and seed germination of Arabidopsis.

While screening for genes affected by cold, we found that Early Responsive to Dehydration 10 (ERD10)was induced by cold treatment. To further investigate the physiological functions of ERD10, we analyzed the T-DNA insertion mutant of ERD10 as well as the expression of ERD10 in response to various stress conditions. The erd10 mutant showed reduced stress tolerance relative to wild-type plants. Activation of the CBF/DREB1 genes by cold stress did not occur in the erd10 mutant, indicating that an increased level of ERD10 is required to subsequently activate the CBF/DREB1 genes and their downstream target genes during treatment with cold stress in Arabidopsis. In addition, we showed that accumulation of the ERD10 transcript in developing seeds was necessary for completion of seed development. Erd10 mutant seeds were abnormally shaped and showed reduced germination. These results suggest that ERD10 can play roles both in protection of the plants from various stresses, including cold and dehydration, and also in seed development and germination.

Modulations of AtGSTF10 expression induce stress tolerance and BAK1-mediated cell death.

Glutathione-S-transferases are essential proteins involved in cellular detoxification. The expression of GSTs has been studied extensively under various environmental stressors including xenobiotics. Here, we have isolated AtGST10, one of the phi classes of AtGSTs on the basis of its interaction with BAK1 in a yeast two-hybrid screen. BAK1 is an LRR-RLK, acting in both brassinosteroid signaling and plant defense responses. We found that AtGSTF10 binds to BAK1 through its N-terminal domain. AtGSTF10 is expressed ubiquitously in plant tissues, and the endogenous transcript level of AtGSTF10 was not induced by plant growth regulators or abiotic stressors, except drought, unlike other GSTs. Overexpression of AtGSTF10 conferred higher tolerance to salt and disturbed redox status of transgenic plants. The down-regulation of AtGSTF10 produced by RNA interference caused reduced tolerance to abiotic stress and an accelerated senescence of transformants, indicating that AtGSTF10 is involved in stress tolerance and the BAK1-mediated spontaneous cell death signaling pathway in Arabidopsis.

An atypical soybean leucine-rich repeat receptor-like kinase, GmLRK1, may be involved in the regulation of cell elongation.

A leucine-rich repeat receptor-like kinase (LRR-RLK) encoded by one of the genes highly expressed in a specific stage of soybean seed development, referred to as GmLRK1, was identified and characterized. Examination of its kinase domain indicated that GmLRK1 may be a catalytically inactive atypical receptor kinase. An autophosphorylation assay confirmed that GmLRK1 is incapable of autophosphorylation in vitro. However, the phosphorylation of GmRLK1 could be induced after incubation with plant protein extracts, suggesting that some plant proteins may interact with GmLRK1 and phosphorylate the protein in vivo. Analyses of the expression profiles of GmLRK1 and its Arabidopsis ortholog At2g36570 revealed that they may be involved in regulation of more fundamental metabolic and/or developmental pathways, rather than a specialized developmental program such as seed development. Our results further indicate that the GmLRK1 and At2g36570 may play a role in the regulation of certain cellular processes that lead to cell elongation and expansion.