Constitutively active B2 Raf-like kinases are required for drought-responsive gene expression upstream of ABA-activated SnRK2 kinases.

Osmotic stresses, such as drought and high salinity, adversely affect plant growth and productivity. The phytohormone abscisic acid (ABA) accumulates in response to osmotic stress and enhances stress tolerance in plants by triggering multiple physiological responses through ABA signaling. Subclass III SNF1-related protein kinases 2 (SnRK2s) are key regulators of ABA signaling. Although SnRK2s have long been considered to be self-activated by autophosphorylation after release from PP2C-mediated inhibition, they were recently revealed to be activated by two independent subfamilies of group B Raf-like kinases, B2-RAFs and B3-RAFs, under osmotic stress conditions. However, the relationship between SnRK2 phosphorylation by these RAFs and SnRK2 autophosphorylation and the individual physiological roles of each RAF subfamily remain unknown. In this study, we indicated that B2-RAFs are constantly active and activate SnRK2s when released from PP2C-mediated inhibition by ABA-binding ABA receptors, whereas B3-RAFs are activated only under stress conditions in an ABA-independent manner and enhance SnRK2 activity. Autophosphorylation of subclass III SnRK2s is not sufficient for ABA responses, and B2-RAFs are needed to activate SnRK2s in an ABA-dependent manner. Using plants grown in soil, we found that B2-RAFs regulate subclass III SnRK2s at the early stage of drought stress, whereas B3-RAFs regulate SnRK2s at the later stage. Thus, B2-RAFs are essential kinases for the activation of subclass III SnRK2s in response to ABA under mild osmotic stress conditions, and B3-RAFs function as enhancers of SnRK2 activity under severe stress conditions.

Clock-regulated coactivators selectively control gene expression in response to different temperature stress conditions in Arabidopsis.

Plants respond to severe temperature changes by inducing the expression of numerous genes whose products enhance stress tolerance and responses. Dehydration-responsive element (DRE)-binding protein 1/C-repeat binding factor (DREB1/CBF) transcription factors act as master switches in cold-inducible gene expression. Since DREB1 genes are rapidly and strongly induced by cold stress, the elucidation of the molecular mechanisms of DREB1 expression is vital for the recognition of the initial responses to cold stress in plants. A previous study indicated that the circadian clock-related MYB-like transcription factors REVEILLE4/LHY-CCA1-Like1 (RVE4/LCL1) and RVE8/LCL5 directly activate DREB1 expression under cold stress conditions. These RVEs function in the regulation of circadian clock-related gene expression under normal temperature conditions. They also activate the expression of HSF-independent heat-inducible genes under high-temperature conditions. Thus, there are thought to be specific regulatory mechanisms whereby the target genes of these transcription factors are switched when temperature changes are sensed. We revealed that NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) proteins act as coactivators of RVEs in cold and heat stress responses in addition to regulating circadian-regulated genes at normal temperatures. We found that among the four Arabidopsis LNKs, LNK1 and LNK2 function under normal and high-temperature conditions, and LNK3 and LNK4 function under cold conditions. Thus, these LNK proteins play important roles in inducing specific genes under different temperature conditions. Furthermore, LNK3 and LNK4 are specifically phosphorylated under cold conditions, suggesting that phosphorylation is involved in their activation.

Complex plant responses to drought and heat stress under climate change.

Global climate change is predicted to result in increased yield losses of agricultural crops caused by environmental conditions. In particular, heat and drought stress are major factors that negatively affect plant development and reproduction, and previous studies have revealed how these stresses induce plant responses at physiological and molecular levels. Here, we provide a comprehensive overview of current knowledge concerning how drought, heat, and combinations of these stress conditions affect the status of plants, including crops, by affecting factors such as stomatal conductance, photosynthetic activity, cellular oxidative conditions, metabolomic profiles, and molecular signaling mechanisms. We further discuss stress-responsive regulatory factors such as transcription factors and signaling factors, which play critical roles in adaptation to both drought and heat stress conditions and potentially function as 'hubs' in drought and/or heat stress responses. Additionally, we present recent findings based on forward genetic approaches that reveal natural variations in agricultural crops that play critical roles in agricultural traits under drought and/or heat conditions. Finally, we provide an overview of the application of decades of study results to actual agricultural fields as a strategy to increase drought and/or heat stress tolerance. This review summarizes our current understanding of plant responses to drought, heat, and combinations of these stress conditions.

Regulatory networks in plant responses to drought and cold stress.

Drought and cold represent distinct types of abiotic stress, each initiating unique primary signaling pathways in response to dehydration and temperature changes, respectively. However, a convergence at the gene regulatory level is observed where a common set of stress-responsive genes is activated to mitigate the impacts of both stresses. In this review, we explore these intricate regulatory networks, illustrating how plants coordinate distinct stress signals into a collective transcriptional strategy. We delve into the molecular mechanisms of stress perception, stress signaling, and the activation of gene regulatory pathways, with a focus on insights gained from model species. By elucidating both the shared and distinct aspects of plant responses to drought and cold, we provide insight into the adaptive strategies of plants, paving the way for the engineering of stress-resilient crop varieties that can withstand a changing climate.

Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity.

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 (bz1728) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro, for rescued root growth. We focused here on nobiro6, which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6/taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR.

BIL9 Promotes Both Plant Growth via BR Signaling and Drought Stress Resistance by Binding with the Transcription Factor HDG11.

Drought stress is a major threat leading to global plant and crop losses in the context of the climate change crisis. Brassinosteroids (BRs) are plant steroid hormones, and the BR signaling mechanism in plant development has been well elucidated. Nevertheless, the specific mechanisms of BR signaling in drought stress are still unclear. Here, we identify a novel Arabidopsis gene, BRZ INSENSITIVE LONG HYPOCOTYL 9 (BIL9), which promotes plant growth via BR signaling. Overexpression of BIL9 enhances drought and mannitol stress resistance and increases the expression of drought-responsive genes. BIL9 protein is induced by dehydration and interacts with the HD-Zip IV transcription factor HOMEODOMAIN GLABROUS 11 (HDG11), which is known to promote plant resistance to drought stress, in vitro and in vivo. BIL9 enhanced the transcriptional activity of HDG11 for drought-stress-resistant genes. BIL9 is a novel BR signaling factor that enhances both plant growth and plant drought resistance.

A small peptide modulates stomatal control via abscisic acid in long-distance signalling.

Mammalian peptide hormones propagate extracellular stimuli from sensing tissues to appropriate targets to achieve optimal growth maintenance 1 . In land plants, root-to-shoot signalling is important to prevent water loss by transpiration and to adapt to water-deficient conditions 2, 3 . The phytohormone abscisic acid has a role in the regulation of stomatal movement to prevent water loss 4 . However, no mobile signalling molecules have yet been identified that can trigger abscisic acid accumulation in leaves. Here we show that the CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED 25 (CLE25) peptide transmits water-deficiency signals through vascular tissues in Arabidopsis, and affects abscisic acid biosynthesis and stomatal control of transpiration in association with BARELY ANY MERISTEM (BAM) receptors in leaves. The CLE25 gene is expressed in vascular tissues and enhanced in roots in response to dehydration stress. The root-derived CLE25 peptide moves from the roots to the leaves, where it induces stomatal closure by modulating abscisic acid accumulation and thereby enhances resistance to dehydration stress. BAM receptors are required for the CLE25 peptide-induced dehydration stress response in leaves, and the CLE25-BAM module therefore probably functions as one of the signalling molecules for long-distance signalling in the dehydration response.

Posttranslational regulation of multiple clock-related transcription factors triggers cold-inducible gene expression in Arabidopsis.

Cold stress is an adverse environmental condition that affects plant growth, development, and crop productivity. Under cold stress conditions, the expression of numerous genes that function in the stress response and tolerance is induced in various plant species, and the dehydration-responsive element (DRE) binding protein 1/C-repeat binding factor (DREB1/CBF) transcription factors function as master switches for cold-inducible gene expression. Cold stress strongly induces these DREB1 genes. Therefore, it is important to elucidate the mechanisms of DREB1 expression in response to cold stress to clarify the perception and response of cold stress in plants. Previous studies indicated that the central oscillator components of the circadian clock, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), are involved in cold-inducible DREB1 expression, but the underlying mechanisms are not clear. We revealed that the clock-related MYB proteins REVEILLE4/LHY-CCA1-Like1 (RVE4/LCL1) and RVE8/LCL5 are quickly and reversibly transferred from the cytoplasm to the nucleus under cold stress conditions and function as direct transcriptional activators of DREB1 expression. We found that CCA1 and LHY suppressed the expression of DREB1s under unstressed conditions and were rapidly degraded specifically in response to cold stress, which suggests that they act as transcriptional repressors and indirectly regulate the cold-inducible expression of DREB1s We concluded that posttranslational regulation of multiple clock-related transcription factors triggers cold-inducible gene expression. Our findings clarify the complex relationship between the plant circadian clock and the regulatory mechanisms of cold-inducible gene expression.

Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2.

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.

Inter-tissue and inter-organ signaling in drought stress response and phenotyping of drought tolerance.

Plant response to drought stress includes systems for intracellular regulation of gene expression and signaling, as well as inter-tissue and inter-organ signaling, which helps entire plants acquire stress resistance. Plants sense water-deficit conditions both via the stomata of leaves and roots, and transfer water-deficit signals from roots to shoots via inter-organ signaling. Abscisic acid is an important phytohormone involved in the drought stress response and adaptation, and is synthesized mainly in vascular tissues and guard cells of leaves. In leaves, stress-induced abscisic acid is distributed to various tissues by transporters, which activates stomatal closure and expression of stress-related genes to acquire drought stress resistance. Moreover, the stepwise stress response at the whole-plant level is important for proper understanding of the physiological response to drought conditions. Drought stress is sensed by multiple types of sensors as molecular patterns of abiotic stress signals, which are transmitted via separate parallel signaling networks to induce downstream responses, including stomatal closure and synthesis of stress-related proteins and metabolites. Peptide molecules play important roles in the inter-organ signaling of dehydration from roots to shoots, as well as signaling of osmotic changes and reactive oxygen species/Ca2+ . In this review, we have summarized recent advances in research on complex plant drought stress responses, focusing on inter-tissue signaling in leaves and inter-organ signaling from roots to shoots. We have discussed the mechanisms via which drought stress adaptations and resistance are acquired at the whole-plant level, and have proposed the importance of quantitative phenotyping for measuring plant growth under drought conditions.

A de novo gene originating from the mitochondria controls floral transition in Arabidopsis thaliana.

De novo genes created in the plant mitochondrial genome have frequently been transferred into the nuclear genome via intergenomic gene transfer events. Therefore, plant mitochondria might be a source of de novo genes in the nuclear genome. However, the functions of de novo genes originating from mitochondria and the evolutionary fate remain unclear. Here, we revealed that an Arabidopsis thaliana specific small coding gene derived from the mitochondrial genome regulates floral transition. We previously identified 49 candidate de novo genes that induce abnormal morphological changes on overexpression. We focused on a candidate gene derived from the mitochondrial genome (sORF2146) that encodes 66 amino acids. Comparative genomic analyses indicated that the mitochondrial sORF2146 emerged in the Brassica lineage as a de novo gene. The nuclear sORF2146 emerged following an intergenomic gene transfer event in the A. thaliana after the divergence between Arabidopsis and Capsella. Although the nuclear and mitochondrial sORF2146 sequences are the same in A. thaliana, only the nuclear sORF2146 is transcribed. The nuclear sORF2146 product is localized in mitochondria, which may be associated with the pseudogenization of the mitochondrial sORF2146. To functionally characterize the nuclear sORF2146, we performed a transcriptomic analysis of transgenic plants overexpressing the nuclear sORF2146. Flowering transition-related genes were highly regulated in the transgenic plants. Subsequent phenotypic analyses demonstrated that the overexpression and knockdown of sORF2146 in transgenic plants resulted in delayed and early flowering, respectively. These findings suggest that a lineage-specific de novo gene derived from mitochondria has an important regulatory effect on floral transition.

DREB1A/CBF3 Is Repressed by Transgene-Induced DNA Methylation in the Arabidopsis ice1 -1 Mutant.

DREB1/CBFs are key transcription factors involved in plant cold stress adaptation. The expression of DREB1/CBFs triggers a cold-responsive transcriptional cascade, after which many stress tolerance genes are expressed. Thus, elucidating the mechanisms of cold stress-inducible DREB1/CBF expression is important to understand the molecular mechanisms of plant cold stress responses and tolerance. We analyzed the roles of a transcription factor, INDUCER OF CBF EXPRESSION1 (ICE1), that is well known as an important transcriptional activator in the cold-inducible expression of DREB1A/CBF3 in Arabidopsis (Arabidopsis thaliana). ice1-1 is a widely accepted mutant allele known to abolish cold-inducible DREB1A expression, and this evidence has strongly supported ICE1-DREB1A regulation for many years. However, in ice1-1 outcross descendants, we unexpectedly discovered that ice1-1 DREB1A repression was genetically independent of the ice1-1 allele ICE1(R236H). Moreover, neither ICE1 overexpression nor double loss-of-function mutation of ICE1 and its homolog SCRM2 altered DREB1A expression. Instead, a transgene locus harboring a reporter gene in the ice1-1 genome was responsible for altering DREB1A expression. The DREB1A promoter was hypermethylated due to the transgene. We showed that DREB1A repression in ice1-1 results from transgene-induced silencing and not genetic regulation by ICE1. The ICE1(R236H) mutation has also been reported as scrm-D, which confers constitutive stomatal differentiation. The scrm-D phenotype and the expression of a stomatal differentiation marker gene were confirmed to be linked to the ICE1(R236H) mutation. We propose that the current ICE1-DREB1 regulatory model should be revalidated without the previous assumptions.

Long-distance stress and developmental signals associated with abscisic acid signaling in environmental responses.

Flowering plants consist of highly differentiated organs, including roots, leaves, shoots and flowers, which have specific roles: root system for water and nutrient uptake, leaves for photosynthesis and gas exchange and reproductive organs for seed production. The communication between organs through the vascular system, by which water, nutrient and signaling molecules are transported, is essential for coordinated growth and development of the whole plant, particularly under adverse conditions. Here, we highlight recent progress in understanding how signaling pathways of plant hormones are associated with long-distance stress and developmental signals, with particular focus on environmental stress responses. In addition to the root-to-shoot peptide signal that induces abscisic acid accumulation in leaves under drought stress conditions, we summarize the diverse stress-responsive peptide signals reported to date to play a role in environmental responses.

CIN-like TCP13 is essential for plant growth regulation under dehydration stress.

KEY MESSAGE: A dehydration-inducible Arabidopsis CIN-like TCP gene, TCP13, acts as a key regulator of plant growth in leaves and roots under dehydration stress conditions. Plants modulate their shape and growth in response to environmental stress. However, regulatory mechanisms underlying the changes in shape and growth under environmental stress remain elusive. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family of transcription factors (TFs) are key regulators for limiting the growth of leaves through negative effect of auxin response. Here, we report that stress-inducible CIN-like TCP13 plays a key role in inducing morphological changes in leaves and growth regulation in leaves and roots that confer dehydration stress tolerance in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing TCP13 (35Spro::TCP13OX) exhibited leaf rolling, and reduced leaf growth under osmotic stress. The 35Spro::TCP13OX transgenic leaves showed decreased water loss from leaves, and enhanced dehydration tolerance compared with their control counterparts. Plants overexpressing a chimeric repressor domain SRDX-fused TCP13 (TCP13pro::TCP13SRDX) showed severely serrated leaves and enhanced root growth. Transcriptome analysis of TCP13pro::TCP13SRDX transgenic plants revealed that TCP13 affects the expression of dehydration- and abscisic acid (ABA)-regulated genes. TCP13 is also required for the expression of dehydration-inducible auxin-regulated genes, INDOLE-3-ACETIC ACID5 (IAA5) and LATERAL ORGAN BOUNDARIES (LOB) DOMAIN 1 (LBD1). Furthermore, tcp13 knockout mutant plants showed ABA-insensitive root growth and reduced dehydration-inducible gene expression. Our findings provide new insight into the molecular mechanism of CIN-like TCP that is involved in both auxin and ABA response under dehydration stress.

Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants.

Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.

Characterization of photosystem II assembly complexes containing ONE-HELIX PROTEIN1 in Arabidopsis thaliana.

The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and D2 subunits of PSII together with a few auxiliary proteins including at least ONE-HELIX PROTEIN1 (OHP1), OHP2, and HIGH-CHLOROPHYLL FLUORESCENCE 244 (HCF244) proteins. Herein, we report the basic characterization of the assembling intermediates, which we purified from Arabidopsis transgenic plants overexpressing a tagged OHP1 protein and named the OHP1 complexes. We analyzed two major forms of OHP1 complexes by mass spectrometry, which revealed that the complexes consist of OHP1, OHP2, and HCF244 in addition to the PSII subunits D1, D2, and cytochrome b559. Analysis of chlorophyll fluorescence showed that a major form of the complex binds chlorophyll a and carotenoids and performs quenching with a time constant of 420 ps. To identify the localization of the auxiliary proteins, we solubilized thylakoid membranes using a digitonin derivative, glycodiosgenin, and separated them into three fractions by ultracentrifugation, and detected these proteins in the loose pellet containing the stroma lamellae and the grana margins together with two chlorophyll biosynthesis enzymes. The results indicated that chlorophyll biosynthesis and assembly may take place in the same compartments of thylakoid membranes. Inducible suppression of the OHP2 mRNA substantially decreased the OHP2 protein in mature Arabidopsis leaves without a significant reduction in the maximum quantum yield of PSII under low-light conditions, but it compromised the yields under high-light conditions. This implies that the auxiliary protein is required for acclimation to high-light conditions.

Functional genomics in plant abiotic stress responses and tolerance: From gene discovery to complex regulatory networks and their application in breeding.

Land plants have developed sophisticated systems to cope with severe stressful environmental conditions during evolution. Plants have complex molecular systems to respond and adapt to abiotic stress, including drought, cold, and heat stress. Since 1989, we have been working to understand the complex molecular mechanisms of plant responses to severe environmental stress conditions based on functional genomics approaches with Arabidopsis thaliana as a model plant. We focused on the function of drought-inducible genes and the regulation of their stress-inducible transcription, perception and cellular signal transduction of stress signals to describe plant stress responses and adaptation at the molecular and cellular levels. We have identified key genes and factors in the regulation of complex responses and tolerance of plants in response to dehydration and temperature stresses. In this review article, we describe our 30-year experience in research and development based on functional genomics to understand sophisticated systems in plant response and adaptation to environmental stress conditions.

CIPK23 regulates blue light-dependent stomatal opening in Arabidopsis thaliana.

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K+ (K+ in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+ -ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+ -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

Metabolite/phytohormone-gene regulatory networks in soybean organs under dehydration conditions revealed by integration analysis.

Metabolites, phytohormones, and genes involved in dehydration responses/tolerance have been predicted in several plants. However, metabolite/phytohormone-gene regulatory networks in soybean organs under dehydration conditions remain unclear. Here, we analyzed the organ specificity of metabolites, phytohormones, and gene transcripts and revealed the characteristics of their regulatory networks in dehydration-treated soybeans. Our metabolite/phytohormone analysis revealed the accumulation of raffinose, trehalose, and cis-zeatin (cZ) specifically in dehydration-treated roots. In dehydration-treated soybeans, raffinose, and trehalose might have additional roles not directly involved in protecting the photosynthetic apparatus; cZ might contribute to root elongation for water uptake from the moisture region in soil. Our integration analysis of metabolites-genes indicated that galactinol, raffinose, and trehalose levels were correlated with transcript levels for key enzymes (galactinol synthase, raffinose synthase, trehalose 6-phosphate synthase, trehalose 6-phosphate phosphatase) at the level of individual plants but not at the organ level under dehydration. Genes encoding these key enzymes were expressed in mainly the aerial parts of dehydration-treated soybeans. These results suggested that raffinose and trehalose are transported from aerial plant parts to the roots in dehydration-treated soybeans. Our integration analysis of phytohormones-genes indicated that cZ and abscisic acid (ABA) levels were correlated with transcript levels for key enzymes (cytokinin nucleoside 5'-monophosphate phosphoribohydrolase, cytokinin oxidases/dehydrogenases, 9-cis-epoxycarotenoid dioxygenase) at the level of individual plants but not at the organ level under dehydration conditions. Therefore, processes such as ABA and cZ transport, among others, are important for the organ specificity of ABA and cZ production under dehydration conditions.

Cytosolic HSC70s repress heat stress tolerance and enhance seed germination under salt stress conditions.

Heat shock factor A1 (HsfA1) family proteins are the master regulators of the heat stress-responsive transcriptional cascade in Arabidopsis. Although 70 kDa heat shock proteins (HSP70s) are known to participate in repressing HsfA1 activity, the mechanisms by which they regulate HsfA1 activity have not been clarified. Here, we report the physiological functions of three cytosolic HSP70s, HSC70-1, HSC70-2 and HSC70-3, under normal and stress conditions. Expression of the HSC70 genes was observed in whole seedlings, and the HSC70 proteins were observed in the cytoplasm and nucleus under normal and stress conditions, as were the HsfA1s. hsc70-1/2 double and hsc70-1/2/3 triple mutants showed higher thermotolerance than the wild-type (WT) plants. Transcriptomic analysis revealed the upregulation of heat stress-responsive HsfA1-downstream genes in hsc70-1/2/3 mutants under normal growth conditions, demonstrating that these HSC70s redundantly function as repressors of HsfA1 activity. Furthermore, hsc70-1/2/3 plants showed a more severe growth delay during the germination stage than the WT plants under high-salt stress conditions, and many seed-specific cluster 2 genes that exhibited suppressed expression during germination were expressed in hsc70-1/2/3 plants, suggesting that these HSC70s also function in the developmental transition from seed to seedling under high-salt conditions by suppressing the expression of cluster 2 genes.