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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.

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.

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 b

SNF1-related protein kinases 2 (SnRK2s) are key regulators governing the plant adaptive responses to osmotic stresses, such as drought and high salinity. Subclass III SnRK2s function as central regulators of abscisic acid (ABA) signalling and orchestrate ABA-regulated adaptive responses to osmotic stresses. Seed plants have acquired other types of osmotic stress-activated but ABA-unresponsive subclass I SnRK2s that regulate mRNA decay and promote plant growth under osmotic stresses. In contrast to subclass III SnRK2s, the regulatory mechanisms underlying the rapid activation of subclass I SnRK2s in response to osmotic stress remain elusive. Here, we report that three B4 Raf-like MAP kinase kinase kinases (MAPKKKs) phosphorylate and activate subclass I SnRK2s under osmotic stress. Transcriptome analyses reveal that genes downstream of these MAPKKKs largely overlap with subclass I SnRK2-regulated genes under osmotic stress, which indicates that these MAPKKKs are upstream factors of subclass I SnRK2 and are directly activated by osmotic stress., SnRK2 protein kinases play key roles in signaling during plant responses to abiotic stress. Here Soma et al. report three Arabidopsis Raf-like MAP kinase kinase kinases phosphorylate and activate a subclass of SnRK2s that rapidly respond to osmotic stress independently of ABA signaling.

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.

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. Abstract 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.

Significance SNF1-related protein kinase 2s (SnRK2s) are central regulators in abscisic acid (ABA) signaling and stress responses in plants. However, SnRK2 substrates are largely unknown. Here, we identified Arabidopsis group C Raf-like kinases Raf36 and Raf22 as SnRK2 substrates. Reverse genetic analyses showed that Raf36 and Raf22 negatively regulate ABA signaling. Under optimal growth conditions, Raf36 accumulates in the cell and phosphorylates substrates to suppress ABA responses. Conversely, under abiotic stress conditions, ABA-activated SnRK2s phosphorylate Raf36 and promote Raf36 degradation, leading to stronger ABA responses. Taken together, this study provides insight into molecular and physiological functions of group C Raf kinases in ABA signaling., 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.

Functional diversification of transcription factors allows the precise regulation of transcriptomic changes under different environmental conditions. The NUCLEAR FACTOR Y (NF-Y) transcription factor comprises three subunits, NF-YA, NF-YB, and NF-YC, and is broadly diversified in plant species, whereas Humans (Homo sapiens) have one protein for each subunit. However, there remains much to be learned about the diversified functions of each subunit in plants. Here, we found that NF-YB2 and NF-YB3, which have the greatest sequence similarity to each other among NF-YB family proteins in Arabidopsis (Arabidopsis thaliana), are functionally diversified and specifically activate dehydration-inducible and heat-inducible genes, according to environmental conditions. Overexpression of NF-YB2 and NF-YB3 specifically enhanced drought and heat stress tolerance, respectively, and each single knockout mutant showed adverse stress-sensitive phenotypes. Transcriptomic analyses confirmed that overexpression of NF-YB2 and NF-YB3 largely affected the transcriptomic changes under dehydration and heat stress conditions, respectively. The DNA-binding profiles of each protein in planta also suggested that dehydration and heat stress increased the DNA-binding activity of NF-YB2 and NF-YB3 to dehydration-inducible and heat stress-inducible target genes, respectively. Moreover, phylogenetic analysis suggested that the NF-YB proteins of angiosperm plants belong to divergent NF-YB2 and NF-YB3 subgroups. These results demonstrate the functional diversification of NF-Y through evolutionary processes and how plants adapt to various abiotic stresses under fluctuating environments.

Plants in Mongolian grasslands are exposed to short, dry summers and long, cold winters. These plants should be prepared for fast germination and growth activity in response to the limited summer rainfall. The wild plant species adapted to the Mongolian grassland environment may allow us to explore useful genes, as a source of unique genetic codes for crop improvement. Here, we identified the Chloris virgata Dornogovi accession as the fastest germinating plant in major Mongolian grassland plants. It germinated just 5 h after treatment for germination initiation and showed rapid growth, especially in its early and young development stages. This indicates its high growth potential compared to grass crops such as rice and wheat. By assessing growth recovery after animal bite treatment (mimicked by cutting the leaves with scissors), we found that C. virgata could rapidly regenerate leaves after being damaged, suggesting high regeneration potential against grazing. To analyze the regulatory mechanism involved in the high growth potential of C. virgata, we performed RNA-seq-based transcriptome analysis and illustrated a comprehensive gene expression map of the species. Through de novo transcriptome assembly with the RNA-seq reads from whole organ samples of C. virgata at the germination stage (2 days after germination, DAG), early young development stage (8 DAG), young development stage (17 DAG), and adult development stage (28 DAG), we identified 21,589 unified transcripts (contigs) and found that 19,346 and 18,156 protein-coding transcripts were homologous to those in rice and Arabidopsis, respectively. The best-aligned sequences were annotated with gene ontology groups. When comparing the transcriptomes across developmental stages, we found an over-representation of genes involved in growth regulation in the early development stage in C. virgata. Plant development is tightly regulated by phytohormones such as brassinosteroids, gibberellic acid, abscisic acid, and strigolactones. Moreover, our transcriptome map demonstrated the expression profiles of orthologs involved in the biosynthesis of these phytohormones and their signaling networks. We discuss the possibility that C. virgata phytohormone signaling and biosynthesis genes regulate early germination and growth advantages. Comprehensive transcriptome information will provide a useful resource for gene discovery and facilitate a deeper understanding of the diversity of the regulatory systems that have evolved in C. virgata while adapting to severe environmental conditions.

PI-PLCs synthesise the calcium releasing second messenger IP3. We investigated the expression patterns of the Arabidopsis PI-PLC gene family and measured in vitro activity of encoded enzymes. Gene specific RT-PCR and promoter-GUS fusions were used to analyse AtPLC gene expression patterns. The five available AtPLC cDNAs were expressed as fusion proteins in Escherichia coli. All members of the AtPLC gene family were expressed in multiple organs of the plant. AtPLC1, and AtPLC5 expression was localized to the vascular cells of roots and leaves with AtPLC5::GUS also detected in the guard cells. AtPLC4::GUS was detected in pollen and cells of the stigma surface. In seedlings, AtPLC2 and AtPLC3 were constitutively expressed, while AtPLCs 1, 4 and 5 were induced by abiotic stresses. AtPLC1-5 were all shown to have phospholipase C activity in the presence of calcium ions. AtPLCs showed limited tissue specific expression and expression of at least three genes was increased by abiotic stress. The differing calcium sensitivities of recombinant AtPLC protein activities may provide a mechanism for generating calcium signatures. (Less)

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.

Malate efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1) and mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum resistance in Arabidopsis thaliana. Several studies showed that AtALMT1 expression in roots is rapidly observed in response to aluminum; this early induction is an important mechanism to immediately protect roots from aluminum toxicity. Identifying the molecular mechanisms that underlie rapid aluminum resistance responses should lead to a better understanding of plant aluminum sensing and signal transduction mechanisms. In this study, we observed that GFP-tagged STOP1 proteins accumulated in the nucleus soon after aluminum treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1 induction were detected in the presence of a protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2). However STOP1 did not regulate Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for early induction. Finally, we report that GDH1 and 2 which are targets of STOP1, are novel aluminum-resistance genes in Arabidopsis.

Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase–substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.

The drought stress responses of vascular plants are complex regulatory mechanisms because they include various physiological responses from signal perception under water deficit conditions to the acquisition of drought stress resistance at the whole-plant level. It is thought that plants first recognize water deficit conditions in roots and that several molecular signals then move from roots to shoots. Finally, a phytohormone, abscisic acid (ABA) is synthesized mainly in leaves. However, the detailed molecular mechanisms of stress sensors and the regulators that initiate ABA biosynthesis in response to drought stress conditions are still unclear. Another important issue is how plants adjust ABA propagation, stress-mediated gene expression and metabolite composition to acquire drought stress resistance in different tissues throughout the whole plant. In this review, we summarize recent advances in research on drought stress responses, focusing on long-distance signaling from roots to shoots, ABA synthesis and transport, and metabolic regulation in both cellular and whole-plant levels of Arabidopsis and crops. We also discuss coordinated mechanisms for acquiring drought stress adaptations and resistance via tissue-to-tissue communication and long-distance signaling.

Abscisic acid (ABA), a stress hormone produced by plants to cope with various environmental stresses, has potential as a mobile molecule. Recently, several types of ABA transporters have been described. We previously found a membrane transporter, AtABCG25, that is involved in intercellular ABA transport in Arabidopsis thaliana. However, it is not yet known whether there are any homologs of AtABCG25 in different plant species. Here, we identified a homolog of AtABCG25 in Brachypodium distachyon, named BdABCG25, and characterized its function. We examined the ABA transport activity of BdABCG25 and the physiological properties of BdABCG25 expression in Arabidopsis. The results suggest that BdABCG25 is a putative functional homolog of AtABCG25. Regulating intercellular ABA transport may be a novel strategy for breeding stress-tolerant monocot crops.

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.

InArabidopsis thaliana, a mitogen-activated protein kinase pathway, MEKK1–MKK1/MKK2–MPK4, is important for basal resistance, and disruption of this pathway results in dwarf, autoimmune phenotypes. To elucidate the complex mechanisms activated by the disruption of this pathway, we have previously developed a mutant screening system based on a dwarf autoimmune line that overexpressed the N-terminal regulatory domain of MEKK1. Here, we report that the second group of mutants,smn2, had defects in theSMN2gene, encoding a DEAD-box RNA helicase.SMN2is identical toHEN2, whose function is vital for the nuclear RNA exosome because it provides non-ribosomal RNA specificity for RNA turnover, RNA quality control, and RNA processing. AberrantSMN1/RPS6transcripts were detected insmn2andhen2mutants. Disease resistance againstPseudomonas syringaepv.tomatoDC3000 (hopA1), which is conferred bySMN1/RPS6, was decreased insmn2mutants, suggesting a functional connection betweenSMN1/RPS6andSMN2/HEN2. We produced double mutantsmekk1smn2andmpk4smn2to determine whether thesmn2mutations suppress the dwarf, autoimmune phenotypes of themekk1andmpk4mutants, as thesmn1mutations do. As expected, themekk1andmpk4phenotypes were suppressed by thesmn2mutations. These results suggested thatSMN2is involved in proper function ofSMN1/RPS6. The GO enrichment analysis using RNA-seq data showed that defense genes were downregulated insmn2, suggesting positive contribution ofSMN2to genome-wide expression of defense-related genes. In conclusion, this study provides novel insight into plant immunity viaSMN2/HEN2, an essential component of the nuclear RNA exosome.

In Arabidopsis thaliana, a mitogen-activated protein kinase pathway, MEKK1–MKK1/MKK2–MPK4, is important for basal resistance and disruption of this pathway results in dwarf, autoimmune phenotypes. To elucidate the complex mechanisms activated by the disruption of this pathway, we have previously developed a mutant screening system based on a dwarf autoimmune line that overexpressed the N-terminal regulatory domain of MEKK1. Here, we report that the second group of mutants, smn2, had defects in the SMN2 gene, encoding a DEAD-box RNA helicase. SMN2 is identical to HEN2, whose function is vital for the nuclear RNA exosome because it provides non-ribosomal RNA specificity for RNA turnover, RNA quality control and RNA processing. Aberrant SMN1/RPS6 transcripts were detected in smn2 and hen2 mutants. Disease resistance against Pseudomonas syringae pv. tomato DC3000 (hopA1), which is conferred by SMN1/RPS6, was decreased in smn2 mutants, suggesting a functional connection between SMN1/RPS6 and SMN2/HEN2. We produced double mutants mekk1smn2 and mpk4smn2 to determine whether the smn2 mutations suppress the dwarf, autoimmune phenotypes of the mekk1 and mpk4 mutants, as the smn1 mutations do. As expected, the mekk1 and mpk4 phenotypes were suppressed by the smn2 mutations. These results suggested that SMN2 is involved in the proper function of SMN1/RPS6. The Gene Ontology enrichment analysis using RNA-seq data showed that defense genes were downregulated in smn2, suggesting a positive contribution of SMN2 to the genome-wide expression of defense genes. In conclusion, this study provides novel insight into plant immunity via SMN2/HEN2, an essential component of the nuclear RNA exosome.

In the ros1-defective mutant, DREB1A repression by the transgene-induced promoter methylation of ice1-1 became inheritable across generations even in the absence of the causative transgene NICE1. Transgene silencing (TGS) is a widely observed event during plant bioengineering, which is presented as a gradual decrease in ectopic gene expression across generations and occasionally coupled with endogenous gene silencing based on DNA sequence similarity. TGS is known to be established by guided DNA methylation machinery. However, the machinery underlying gene recovery from TGS has not been fully elucidated. We previously reported that in ice1-1 outcross descendants, the expressional repression and recovery of DREB1A/CBF3 were instantly achieved by a newly discovered NICE1 transgene, instead of the formerly proposed ice1-1 mutation in the ICE1 gene. The plants harboring NICE1 produced small RNAs targeting and causing the DREB1A promoter to be hypermethylated and silenced. To analyze the role of the plant-specific active DNA demethylase REPRESSOR OF SILENCING 1 (ROS1) in instant DREB1A recovery, we propagated the NICE1-segregating population upon ros1 dysfunction and evaluated the gene expression and DNA methylation levels of DREB1A through generations. Our results showed that the epigenetic DREB1A repression was substantially sustained in subsequent generations even without NICE1 and stably inherited across generations. Consistent with the gene expression results, only incomplete DNA methylation removal was detected in the same generations. These results indicate that a novel inheritable epiallele emerged by the ros1 dysfunction. Overall, our study reveals the important role of ROS1 in the inheritability of TGS-associated gene repression.

Increase in atmospheric carbon dioxide (CO

Significance Statement This study indicates that CIPK23 is a phototropin‐interacting protein kinase that promotes blue light‐ and phototropin‐dependent stomatal opening in Arabidopsis thaliana. We propose that CIPK23 does not mediate plasma membrane H+‐ATPase activation but mediates inward‐rectifying K+ channel activation in stomatal opening., SUMMARY 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.

ABSTRUCTA phytohormone abscisic acid (ABA) has a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) mediates ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as an interacting protein with SnRK2. A series of reverse genetic and biochemical analyses revealed that Raf36 negatively regulates ABA responses and is directly phosphorylated by SnRK2s. In addition, we found that Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. Comparative phosphoproteomic analysis using Arabidopsis wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results reveal a novel subsection of ABA-responsive phosphosignaling pathways branching from SnRK2.

Stomatal pores, which are surrounded by pairs of guard cells in the plant epidermis, regulate gas exchange between plants and the atmosphere, thereby controlling photosynthesis and transpiration. Blue light works as a signal to guard cells, to induce intracellular signaling and open stomata. Blue light receptor phototropins (phots) are activated by blue light; phot-mediated signals promote plasma membrane (PM) H+-ATPase activity via C-terminal Thr phosphorylation, serving as the driving force for stomatal opening in guard cells. However, the details of this signaling process are not fully understood. In this study, through an in vitro screening of phot-interacting protein kinases, we obtained the CBC1 and CBC2 that had been reported as signal transducers in stomatal opening. Promoter activities of CBC1 and CBC2 indicated that both genes were expressed in guard cells. Single and double knockout mutants of CBC1 and CBC2 showed no lesions in the context of phot-mediated phototropism, chloroplast movement, or leaf flattening. In contrast, the cbc1cbc2 double mutant showed larger stomatal opening under both dark and blue light conditions. Interestingly, the level of phosphorylation of C-terminal Thr of PM H+-ATPase was higher in double mutant guard cells. The larger stomatal openings of the double mutant were effectively suppressed by the phytohormone abscisic acid (ABA). CBC1 and CBC2 interacted with BLUS1 and PM H+-ATPase in vitro. From these results, we conclude that CBC1 and CBC2 act as negative regulators of stomatal opening, probably via inhibition of PM H+-ATPase activity.

High-throughput and accurate measurements of plant traits facilitate identification of gene function. Along with recent advances in quantitative genomics, there is a growing need for precise quantification of multiple traits in plants. However, it is difficult continuously to quantify plant adaptive responses to environmental stress responses such as drought because multiple environmental factors are intricately involved in the phenotype. To solve this problem, we developed an automatic phenotyping system for evaluating the growth responses of individual Arabidopsis plants to a wide range of environmental conditions. The RIKEN Integrated Plant Phenotyping System (RIPPS) controls soil moisture for single plants by automatically weighing and watering 120 continuously rotating pots under controlled light, humidity and temperature growth conditions. RIPPS also records individual rosette size and expansion rate by photographing plants every 2 h. We used RIPPS to establish phenotype evaluation methods for Arabidopsis growth response and water use efficiency under various water conditions, and analyzed the involvement of ABA metabolism in determining water use efficiency. We also used RIPPS to analyze salinity tolerance in Arabidopsis plants.

Under heat stress, polyunsaturated acyl groups, such as α-linolenate (18:3) and hexadecatrienoate (16:3), are removed from chloroplastic glycerolipids in various plant species. Here, we showed that a lipase designated HEAT INDUCIBLE LIPASE1 (HIL1) induces the catabolism of monogalactosyldiacylglycerol (MGDG) under heat stress in Arabidopsis thaliana leaves. Using thermotolerance tests, a T-DNA insertion mutant with disrupted HIL1 was shown to have a heat stress-sensitive phenotype. Lipidomic analysis indicated that the decrease of 34:6-MGDG under heat stress was partially impaired in the hil1 mutant. Concomitantly, the heat-induced increment of 54:9-triacylglycerol in the hil1 mutant was 18% lower than that in the wild-type plants. Recombinant HIL1 protein digested MGDG to produce 18:3-free fatty acid (18:3-FFA), but not 18:0- and 16:0-FFAs. A transient assay using fluorescent fusion proteins confirmed chloroplastic localization of HIL1. Transcriptome coexpression network analysis using public databases demonstrated that the HIL1 homolog expression levels in various terrestrial plants are tightly associated with chloroplastic heat stress responses. Thus, HIL1 encodes a chloroplastic MGDG lipase that releases 18:3-FFA in the first committed step of 34:6 (18:3/16:3)-containing galactolipid turnover, suggesting that HIL1 has an important role in the lipid remodeling process induced by heat stress in plants.

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 conditions2, 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.

The unfolded protein response (UPR) is a eukaryotic transcriptional regulatory network that is activated upon the accumulation of malformed proteins in the endoplasmic reticulum (ER). In Arabidopsis (Arabidopsis thaliana), three bZIP transcription factors modulate the UPR: bZIP17, bZIP28, and bZIP60. Although bZIP28 and bZIP60 have been relatively well studied, the physiological and transcriptional roles of bZIP17 remain largely unknown. Here, we generated a double knockout mutant of bZIP17 and bZIP28 to elucidate the function of bZIP17. The mutant plant exhibited multiple developmental defects, including markedly reduced root elongation and constantly overinduced bZIP60 activity, indicating the essential roles of bZIP17 and bZIP28 in plant development and UPR modulation. Extended analysis of the transcriptomes of three double knockout mutants of bZIP17, bZIP28, and bZIP60 revealed that bZIP28 and bZIP60 are the major activators of the canonical induced UPR. By contrast, bZIP17 functions with bZIP28 to mediate the noninducible expression of multiple genes involved in cell growth, particularly to sustain their expression under stress conditions. Our study reveals pivotal roles of bZIP17 in the plant UPR and vegetative development, with functional redundancy to bZIP28.

Stomatal regulation is important for water transpiration from plants. Stomatal opening and closing are controlled by many transporter proteins in guard cells. AtABCG22 is a member of the ATP-binding cassette (ABC) transporters and is a stomatal regulator; however, the function of AtABCG22 has not yet been determined fully, although a mutant phenotype included a significant effect on stomatal status. Here, we further investigated the function of the AtABCG22 gene and its functional relationships with other subfamily genes. Among close family members, we found a functional relationship of stomatal phenotypes with AtABCG21, which is also expressed specifically in guard cells. Based on an analysis of double mutants, adding the atabcg21 mutation to atabcg22 mutant partially suppressed the open-stomata phenotype of atabcg22. Multiple-mutant analyses indicated that this suppression was independent of abscisic acid signaling in guard cells. We also found that atabcg22 mutant showed a unique time course-dependent phenotype, being defective in maintenance of stomatal status after initial stomatal opening elicited by light signaling. The function of AtABCG22 and its relationship with AtABCG21 in stomatal regulation are considered.

Gibberellin (GA) is a major hormone for plant growth and development. GA response is derived from the degradation of DELLA repressor proteins after GA-dependent complex formation of the GID1 GA receptor with DELLA. Genistein is a known tyrosine (Tyr) kinase inhibitor and inhibits DELLA degradation. However, the biological role of Tyr phosphorylation on the GA response remains unclear. Here, we demonstrate that GARU (GA receptor RING E3 ubiquitin ligase) mediates ubiquitin-dependent degradation of GID1, and that the TAGK2 plant Tyr-kinase is a target of genistein and inhibits GARU–GID1A interactions by phosphorylation of GARU at Tyr321. Genistein induces degradation of GID1 and accumulation of DELLA. Conversely, Arabidopsis garu mutant and TAGK2-overexpressing plants accelerate GID1 stabilization and DELLA degradation. Under salt stress, GARU suppresses seed germination. We propose that GA response is negatively regulated by GARU-dependent GID1 ubiquitination and positively by Tyr phosphorylation of GARU by TAGK2, and genistein inhibits GA signaling by TAGK2 inhibition., Plants respond to gibberellins via GID1-dependent degradation of DELLA proteins. Here, Nemoto et al. show that the gibberellin response is positively regulated by tyrosine phosphorylation of GARU, an E3 ubiquitin ligase that mediates degradation of GID1.

Brassinosteroids (BRs), plant steroid hormones, play important roles in plant cell elongation and differentiation. To investigate the mechanisms of BR signaling, we previously used the BR biosynthesis inhibitor Brz as a chemical biology tool and identified the Brz-insensitive-long hypocotyl4 mutant (bil4). Although the BIL4 gene encodes a seven-transmembrane-domain protein that is evolutionarily conserved in plants and animals, the molecular function of BIL4 in BR signaling has not been elucidated. Here, we demonstrate that BIL4 is expressed in early elongating cells and regulates cell elongation in Arabidopsis. BIL4 also activates BR signaling and interacts with the BR receptor brassinosteroid insensitive 1 (BRI1) in endosomes. BIL4 deficiency increases the localization of BRI1 in the vacuoles. Our results demonstrate that BIL4 regulates cell elongation and BR signaling via the regulation of BRI1 localization.

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non‐transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.

Summary Plant responses to dehydration stress are mediated by highly complex molecular systems involving hormone signaling and metabolism, particularly the major stress hormone abscisic acid (ABA) and ABA-dependent gene expression. To understand the roles of plant hormones and their interactions during dehydration, we analyzed the plant hormone profiles with respect to dehydration responses in Arabidopsis thaliana wild-type (WT) plants and ABA biosynthesis mutants (nced3-2). We developed a procedure for moderate dehydration stress, and then investigated temporal changes in the profiles of ABA, jasmonic acid isoleucine (JA-Ile), salicylic acid (SA), cytokinin (trans-zeatin, tZ), auxin (indole-acetic acid, IAA), and gibberellin (GA4), along with temporal changes in the expression of key genes involved in hormone biosynthesis. ABA levels increased in a bi-phasic pattern (at the early and late phases) in response to moderate dehydration stress. JA-Ile levels increased slightly in WT plants and strongly increased in nced3-2 mutant plants at 72 h after the onset of dehydration. The expression profiles of dehydration-inducible genes displayed temporal responses in an ABA-dependent manner. The early phase of ABA accumulation correlated with the expression of touch-inducible genes and was independent of factors involved in the major ABA regulatory pathway, including the ABA-responsive element-binding (AREB/ABF) transcription factor. JA-Ile, SA, and tZ were negatively regulated during the late dehydration response phase. Transcriptome analysis revealed important roles for hormone-related genes in metabolism and signaling during dehydration-induced plant responses.

Recent works have shed light on the long-distance interorgan signaling by which hormone-like peptides precisely regulate physiological effects in a manner similar to phytohormones. Many such peptides have already been identified in the primary model plant, Arabidopsis thaliana. In addition, Arabidopsis genome reanalysis revealed over 7000 novel candidate small coding genes, some of which are likely to be associated with hormone-like peptides. Hormone-like peptides have also been reported to play critical roles in interorgan communications during morphogenesis and stress responses. In this review, we focus on the functional roles of hormone-like peptides and small coding genes in cell-to-cell and/or long-distance communications during plant stress signaling and development and discuss the evolutionary conservation of these peptides among plants.

The SNF1-related protein kinase 2 (SnRK2) family includes key regulators of osmostress and abscisic acid (ABA) responses in angiosperms and can be classified into three subclasses. Subclass III SnRK2s act in the ABA response while ABA-nonresponsive subclass I SnRK2s are regulated through osmostress. Here we report that an ancient subclass III SnRK2-based signalling module including ABA and an upstream Raf-like kinase (ARK) exclusively protects the moss Physcomitrella patens from drought. Subclass III SnRK2s from both Arabidopsis and from the semiterrestrial alga Klebsormidium nitens, which contains all the components of ABA signalling except ABA receptors, complement Physcomitrella snrk2− mutants, whereas Arabidopsis subclass I SnRK2 cannot. We propose that the earliest land plants developed the ABA/ARK/subclass III SnRK2 signalling module by recruiting ABA to regulate a pre-existing dehydration response and that subsequently a novel subclass I SnRK2 system evolved in vascular plants conferring osmostress protection independently from the ancient system.

Drought can have a devastating impact on agricultural productivity. Plants actively respond to survival under drought conditions via various avenues, ranging from physiological processes to cellular and molecular responses. Here, we describe achievements in the study of the regulation of gene expression in response to drought stress in Arabidopsis and rice. The abscisic acid (ABA) content in vegetative tissues increases in response to drought stress, which enhances drought tolerance. Transcriptome analysis has revealed that the expression levels of numerous genes are up- or downregulated in response to drought stress. These genes are functionally classified into two groups: one group includes genes encoding proteins that are directly involved in drought tolerance, and the other group includes genes encoding proteins that govern stress signaling. Progress has been made in the elucidation of the complex cascades in response to drought stress. The global transcriptional network consists of ABA-dependent and ABA-independent pathways. We summarize these two regulatory pathways and discuss their roles in transcriptional regulatory networks. We also review recent findings on long-distance signaling under drought stress conditions. In addition, genetic and biochemical analyses have shown that mobile peptides mediate long-distance and/or cell-to-cell communication to complement the regulatory functions of ABA.

Interactions between heat shock (HS) factors (HSFs) and heat shock response elements (HSEs) are important during the heat shock response (HSR) of flora and fauna. Moreover, plant HSFs that are involved in heat stress are also involved in abiotic stresses such as dehydration and cold as well as development, cell differentiation and proliferation. Because the specific combination of HSFs and HSEs involved in plants under heat stress remains unclear, the mechanism of their interaction has not yet been utilized in molecular breeding of plants for climate change. For the study reported herein, we compared the sequences of HS-inducible genes and their promoters in Arabidopsis, soybean, rice and maize and then designed an optimal HS-inducible promoter. Our analyses suggest that, for the four species, the abscisic acid-independent, HSE/HSF-dependent transcriptional pathway plays a major role in HS-inducible gene expression. We found that an 18-bp sequence that includes the HSE has an important role in the HSR, and that those sequences could be classified as representative of monocotyledons or dicotyledons. With the HS-inducible promoter designed based on our bioinformatic predictions, we were able to develop an optimal HS-specific inducible promoter for seedlings or single cells in roots. These findings demonstrate the utility of our HS-specific inducible promoter, which we expect will contribute to molecular breeding efforts and cell-targeted gene expression in specific plant tissues.

Summary Although a variety of transgenic plants that are tolerant to drought stress have been generated, many of these plants show growth retardation. To improve drought tolerance and plant growth, we applied a gene‐stacking approach using two transcription factor genes: DEHYDRATION‐RESPONSIVE ELEMENT‐BINDING 1A (DREB1A) and rice PHYTOCHROME‐INTERACTING FACTOR‐LIKE 1 (OsPIL1). The overexpression of DREB1A has been reported to improve drought stress tolerance in various crops, although it also causes a severe dwarf phenotype. OsPIL1 is a rice homologue of Arabidopsis PHYTOCHROME‐INTERACTING FACTOR 4 (PIF4), and it enhances cell elongation by activating cell wall‐related gene expression. We found that the OsPIL1 protein was more stable than PIF4 under light conditions in Arabidopsis protoplasts. Transactivation analyses revealed that DREB1A and OsPIL1 did not negatively affect each other's transcriptional activities. The transgenic plants overexpressing both OsPIL1 and DREB1A showed the improved drought stress tolerance similar to that of DREB1A overexpressors. Furthermore, double overexpressors showed the enhanced hypocotyl elongation and floral induction compared with the DREB1A overexpressors. Metabolome analyses indicated that compatible solutes, such as sugars and amino acids, accumulated in the double overexpressors, which was similar to the observations of the DREB1A overexpressors. Transcriptome analyses showed an increased expression of abiotic stress‐inducible DREB1A downstream genes and cell elongation‐related OsPIL1 downstream genes in the double overexpressors, which suggests that these two transcription factors function independently in the transgenic plants despite the trade‐offs required to balance plant growth and stress tolerance. Our study provides a basis for plant genetic engineering designed to overcome growth retardation in drought‐tolerant transgenic plants.

In addition to improving drought tolerance, improvement of water use efficiency is a major challenge in plant physiology. Due to their trade-off relationships, it is generally considered that achieving stress tolerance is incompatible with maintaining stable growth. Abscisic acid (ABA) is a key phytohormone that regulates the balance between intrinsic growth and environmental responses. Previously, we identified AtABCG25 as a cell-membrane ABA transporter that export ABA from the inside to the outside of cells. AtABCG25-overexpressing plants showed a lower transpiration phenotype without any growth retardation. Here, we dissected this useful trait using precise phenotyping approaches. AtABCG25 overexpression stimulated a local ABA response in guard cells. Furthermore, AtABCG25 overexpression enhanced drought tolerance, probably resulting from maintenance of water contents over the common threshold for survival after drought stress treatment. Finally, we observed enhanced water use efficiency by overexpression of AtABCG25, in addition to drought tolerance. These results were consistent with the function of AtABCG25 as an ABA efflux transporter. This unique trait may be generally useful for improving the water use efficiency and drought tolerance of plants.

Summary The enhancement of heat stress tolerance in crops is an important challenge for food security to facilitate adaptation to global warming. In Arabidopsis thaliana, the transcriptional regulator DNA polymerase II subunit B3‐1 (DPB3‐1)/nuclear factor Y subunit C10 (NF‐YC10) has been reported as a positive regulator of Dehydration‐responsive element binding protein 2A (DREB2A), and the overexpression of DPB3‐1 enhances heat stress tolerance without growth retardation. Here, we show that DPB3‐1 interacts with DREB2A homologues in rice and soya bean. Transactivation analyses with Arabidopsis and rice mesophyll protoplasts indicate that DPB3‐1 and its rice homologue OsDPB3‐2 function as positive regulators of DREB2A homologues. Overexpression of DPB3‐1 did not affect plant growth or yield in rice under nonstress conditions. Moreover, DPB3‐1‐overexpressing rice showed enhanced heat stress tolerance. Microarray analysis revealed that many heat stress‐inducible genes were up‐regulated in DPB3‐1‐overexpressing rice under heat stress conditions. However, the overexpression of DPB3‐1 using a constitutive promoter had almost no effect on the expression of these genes under nonstress conditions. This may be because DPB3‐1 is a coactivator and thus lacks inherent transcriptional activity. We conclude that DPB3‐1, a coactivator that functions specifically under abiotic stress conditions, could be utilized to increase heat stress tolerance in crops without negative effects on vegetative and reproductive growth.

The molecular breeding of drought stress-tolerant crops is imperative for stable food and biomass production. However, a trade-off exists between plant growth and drought stress tolerance. Many drought stress-tolerant plants overexpressing stress-inducible genes, such as DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1A (DREB1A), show severe growth retardation. Here, we demonstrate that the growth of DREB1A-overexpressing Arabidopsis plants could be improved by co-expressing growth-enhancing genes whose expression is repressed under drought stress conditions. We used Arabidopsis GA REQUIRING 5 (GA5), which encodes a rate-limiting gibberellin biosynthetic enzyme, and PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which encodes a transcription factor regulating cell growth in response to light and temperature, for growth improvement. We observed an enhanced biomass and floral induction in the GA5 DREB1A and PIF4 DREB1A double overexpressors compared with those in the DREB1A overexpressors. Although the GA5 DREB1A double overexpressors continued to show high levels of drought stress tolerance, the PIF4 DREB1A double overexpressors showed lower levels of stress tolerance than the DREB1A overexpressors due to repressed expression of DREB1A. A multiomics analysis of the GA5 DREB1A double overexpressors showed that the co-expression of GA5 and DREB1A additively affected primary metabolism, gene expression and plant hormone profiles in the plants. These multidirectional analyses indicate that the inherent trade-off between growth and drought stress tolerance in plants can be overcome by appropriate gene-stacking approaches. Our study provides a basis for using genetic modification to improve the growth of drought stress-tolerant plants for the stable production of food and biomass.

Significance Hormone-like peptides derived from small coding genes (Arabidopsis , we showed that four genes conferred increased salinity stress tolerance when overexpressed in transgenic plants. One of the four genes ( AtPROPEP3 ) was found to induce salinity stress tolerance by treatment with a 13-peptide (KPTPSSGKGGKHN) fragment, providing unique functional evidence for enhanced salinity stress tolerance in plants in response to a peptide treatment. Although the 13-peptide fragment shares homology with known peptides associated with immune response, the other peptides may encode unique hormone-like peptides associated with salinity stress tolerance.

Plants have evolved complex systems to rapidly respond to severe stress conditions, such as heat, cold, and dehydration. Dehydration-responsive element-binding protein 2A (DREB2A) is a key transcriptional activator that induces many heat- and drought-responsive genes, increases tolerance to both heat and drought stress, and suppresses plant growth in Arabidopsis thaliana. DREB2A expression is induced by stress, but stabilization of the DREB2A protein in response to stress is essential for activating the expression of downstream stress-inducible genes. Under nonstress growth conditions, an integral negative regulatory domain (NRD) destabilizes DREB2A, but the mechanism by which DREB2A is stabilized in response to stress remains unclear. Here, based on bioinformatics, mutational, MS, and biochemical analyses, we report that Ser/Thr residues in the NRD are phosphorylated under nonstress growth conditions and that their phosphorylation decreases in response to heat. Furthermore, we found that this phosphorylation is likely mediated by casein kinase 1 and is essential for the NRD-dependent, proteasomal degradation of DREB2A under nonstress conditions. These observations suggest that inhibition of NRD phosphorylation stabilizes and activates DREB2A in response to heat stress to enhance plant thermotolerance. Our study reveals the molecular basis for the coordination of stress tolerance and plant growth through stress-dependent transcriptional regulation, which may allow the plants to rapidly respond to fluctuating environmental conditions.

The cellular functions of two Arabidopsis (Arabidopsis thaliana) one-helix proteins, OHP1 and OHP2 (also named LIGHT-HARVESTING-LIKE2 [LIL2] and LIL6, respectively, because they have sequence similarity to light-harvesting chlorophyll a/b-binding proteins), remain unclear. Tagged null mutants of OHP1 and OHP2 (ohp1 and ohp2) showed stunted growth with pale-green leaves on agar plates, and these mutants were unable to grow on soil. Leaf chlorophyll fluorescence and the composition of thylakoid membrane proteins revealed that ohp1 deletion substantially affected photosystem II (PSII) core protein function and led to reduced levels of photosystem I core proteins; however, it did not affect LHC accumulation. Transgenic ohp1 plants rescued with OHP1-HA or OHP1-Myc proteins developed a normal phenotype. Using these tagged OHP1 proteins in transgenic plants, we localized OHP1 to thylakoid membranes, where it formed protein complexes with both OHP2 and High Chlorophyll Fluorescence244 (HCF244). We also found PSII core proteins D1/D2, HCF136, and HCF173 and a few other plant-specific proteins associated with the OHP1/OHP2-HCF244 complex, suggesting that these complexes are early intermediates in PSII assembly. OHP1 interacted directly with HCF244 in the complex. Therefore, OHP1 and HCF244 play important roles in the stable accumulation of PSII.

Group A1 heat shock transcription factors (HsfA1s) are the master regulators of the heat stress response (HSR) in plants. Upon heat shock, HsfA1s trigger a transcriptional cascade that is composed of many transcription factors. Despite the importance of HsfA1s and their downstream transcriptional cascade in the acquisition of thermotolerance in plants, the molecular basis of their activation remains poorly understood. Here, domain analysis of HsfA1d, one of several HsfA1s in Arabidopsis thaliana, demonstrated that the central region of HsfA1d is a key regulatory domain that represses HsfA1d transactivation activity through interaction with HEAT SHOCK PROTEIN70 (HSP70) and HSP90. We designated this region as the temperature-dependent repression (TDR) domain. We found that HSP70 dissociates from HsfA1d in response to heat shock and that the dissociation is likely regulated by an as yet unknown activation mechanism, such as HsfA1d phosphorylation. Overexpression of constitutively active HsfA1d that lacked the TDR domain induced expression of heat shock proteins in the absence of heat stress, thereby conferring potent thermotolerance on the overexpressors. However, transcriptome analysis of the overexpressors demonstrated that the constitutively active HsfA1d could not trigger the complete transcriptional cascade under normal conditions, thereby indicating that other factors are necessary to fully induce the HSR. These complex regulatory mechanisms related to the transcriptional cascade may enable plants to respond resiliently to various heat stress conditions.

Leaf senescence is the terminal phenotype of plant leaf development, and ethylene is a major plant hormone inducing leaf senescence. Recent studies have shown that abscisic acid (ABA) also induces leaf senescence. However, the detailed mechanisms of ABA-induced leaf senescence remain unclear. We focused on the A subfamily of stress-responsive NAC (SNAC-A) transcription factors, the expression of which is induced by abiotic stresses, particularly ABA. Gene expression analysis revealed that seven SNAC-A genes including ANAC055, ANAC019, ANAC072/RD26, ANAC002/ATAF1, ANAC081/ATAF2, ANAC102 and ANAC032 were induced by long-term treatment with ABA and/or during age-dependent senescence. The SNAC-A septuple mutant clearly showed retardation of ABA-inducible leaf senescence. Microarray analysis indicated that SNAC-As induce ABA- and senescence-inducible genes. In addition, comparison of the expression profiles of the downstream genes of SNAC-As and ABA-responsive element (ABRE)-binding protein (AREB)/ABRE-binding factor (ABF) (AREB/ABFs) indicates that SNAC-As induce a different set of ABA-inducible genes from those mediated by AREB/ABFs. These results suggest that SNAC-As play crucial roles in ABA-induced leaf senescence signaling. We also discuss the function of SNAC-As in the transcriptional change of leaf senescence as well as in ABA response under abiotic stress conditions.

Protein phosphorylation on Tyr residues is a key post-translational modification in mammals. In plants, recent studies have identified Tyr-specific protein phosphatase and Tyr-phosphorylated proteins in Arabidopsis by phosphoproteomic screenings, implying that plants have a Tyr phosphorylation signal pathway. However, little is known about the protein kinases (PKs) involved in Tyr phosphorylation in plants. Here, we demonstrate that Arabidopsis calcium-dependent protein kinase (CDPK/CPK)-related PKs (CRKs) have high Tyr-autophosphorylation activity and that they can phosphorylate Tyr residue(s) on substrate proteins in Arabidopsis. To identify PKs for Tyr phosphorylation, we examined the autophosphorylation activity of 759 PKs using an Arabidopsis protein array based on a wheat cell-free system. In total, we identified 38 PKs with Tyr-autophosphorylation activity. The CRK family was a major protein family identified. A cell-free substrate screening revealed that these CRKs phosphorylate β-tubulin (TBB) 2, TBB7, and certain transcription factors (TFs) such as ethylene response factor 13 (ERF13). All five CRKs tested showed Tyr-auto/trans-phosphorylation activity and especially two CRKs, CRK2 and CRK3, showed a high ERF13 Tyr-phosphorylation activity. A cell-based transient expression assay revealed that Tyr(16/)Tyr(207) sites in ERF13 were phosphorylated by CRK3 and that Tyr phosphorylation of endogenous TBBs occurs in CRK2 overexpressing cells. Furthermore, crk2 and crk3 mutants showed a decrease in the Tyr phosphorylation level of TBBs. These results suggest that CRKs have Tyr kinase activity, and these might be one of the major PKs responsible for protein Tyr phosphorylation in Arabidopsis plants.