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.

Structural changes in cell wall pectic polymers contribute to freezing tolerance induced by cold acclimation in plants.

Subzero temperatures are often lethal to plants. Many temperate herbaceous plants have a cold acclimation mechanism that allows them to sense a drop in temperature and prepare for freezing stress through accumulation of soluble sugars and cryoprotective proteins. As ice formation primarily occurs in the apoplast (the cell wall space), cell wall functional properties are important for plant freezing tolerance. Although previous studies have shown that the amounts of constituent sugars of the cell wall, in particular those of pectic polysaccharides, are altered by cold acclimation, the significance of this change during cold acclimation has not been clarified. We found that ß-1,4-galactan, which forms neutral side chains of the acidic pectic rhamnogalacturonan-I, accumulates in the cell walls of Arabidopsis and various freezing-tolerant vegetables during cold acclimation. The gals1 gals2 gals3 triple mutant, which has reduced ß-1,4-galactan in the cell wall, exhibited impaired freezing tolerance compared with wild-type Arabidopsis during initial stages of cold acclimation. Expression of genes involved in the galactan biosynthesis pathway, such as galactan synthases and UDP-glucose 4-epimerases, was induced during cold acclimation in Arabidopsis, explaining the galactan accumulation. Cold acclimation resulted in a decrease in extensibility and an increase in rigidity of the cell wall in the wild type, whereas these changes were not observed in the gals1 gals2 gals3 triple mutant. These results indicate that the accumulation of pectic ß-1,4-galactan contributes to acquired freezing tolerance by cold acclimation, likely via changes in cell wall mechanical properties.

The ability to induce heat shock transcription factor-regulated genes in response to lethal heat stress is associated with thermotolerance in tomato cultivars.

Heat stress is a severe challenge for plant production, and the use of thermotolerant cultivars is critical to ensure stable production in high-temperature-prone environments. However, the selection of thermotolerant cultivars is difficult due to the complex nature of heat stress and the time and space needed for evaluation. In this study, we characterized genome-wide differences in gene expression between thermotolerant and thermosensitive tomato cultivars and examined the possibility of selecting gene expression markers to estimate thermotolerance among different tomato cultivars. We selected one thermotolerant and one thermosensitive cultivar based on physiological evaluations and compared heat-responsive gene expression in these cultivars under stepwise heat stress and acute heat shock conditions. Transcriptomic analyses reveled that two heat-inducible gene expression pathways, controlled by the heat shock element (HSE) and the evening element (EE), respectively, presented different responses depending on heat stress conditions. HSE-regulated gene expression was induced under both conditions, while EE-regulated gene expression was only induced under gradual heat stress conditions in both cultivars. Furthermore, HSE-regulated genes showed higher expression in the thermotolerant cultivar than the sensitive cultivar under acute heat shock conditions. Then, candidate expression biomarker genes were selected based on the transcriptome data, and the usefulness of these candidate genes was validated in five cultivars. This study shows that the thermotolerance of tomato is correlated with its ability to maintain the heat shock response (HSR) under acute severe heat shock conditions. Furthermore, it raises the possibility that the robustness of the HSR under severe heat stress can be used as an indicator to evaluate the thermotolerance of crop cultivars.

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.

Transcriptional regulatory network of plant cold-stress responses.

Recent studies have revealed the complex and flexible transcriptional regulatory network involved in cold-stress responses. Focusing on two major signaling pathways that respond to cold stress, we outline current knowledge of the transcriptional regulatory network and the post-translational regulation of transcription factors in the network. Cold-stress signaling pathways are closely associated with other signaling pathways such as those related to the circadian clock, and large amounts of data on their crosstalk and tradeoffs are available. However, it remains unknown how plants sense and transmit cold-stress signals to regulate gene expression. We discuss recent reports on cold-stress sensing and associated signaling pathways that regulate the network. We also emphasize future directions for developing abiotic stress-tolerant crop plants.

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.

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.

DNA demethylase ROS1 prevents inheritable DREB1A/CBF3 repression by transgene-induced promoter methylation in the Arabidopsis ice1-1 mutant.

KEY MESSAGE: 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.

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.

A gene-stacking approach to overcome the trade-off between drought stress tolerance and growth in Arabidopsis.

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.

Heat-induced inhibition of phosphorylation of the stress-protective transcription factor DREB2A promotes thermotolerance of Arabidopsis thaliana.

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.

BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM-knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

Different Cold-Signaling Pathways Function in the Responses to Rapid and Gradual Decreases in Temperature.

In plants, cold temperatures trigger stress responses and long-term responses that result in cold tolerance. In Arabidopsis thaliana, three dehydration-responsive element (DRE) binding protein 1/C-repeat binding factors (DREB1/CBFs) act as master switches in cold-responsive gene expression. Induction of DREB1 genes triggers the cold stress-inducible transcriptional cascade, followed by the induction of numerous genes that function in the cold stress response and cold tolerance. Many regulatory factors involved in DREB1 induction have been identified, but how these factors orchestrate the cold stress-specific expression of DREB1s has not yet been clarified. Here, we revealed that plants recognize cold stress as two different signals, rapid and gradual temperature decreases, and induce expression of the DREB1 genes. CALMODULIN BINDING TRANSCRIPTION ACTIVATOR3 (CAMTA3) and CAMTA5 respond to a rapid decrease in temperature and induce the expression of DREB1s, but these proteins do not respond to a gradual decrease in temperature. Moreover, they function during the day and night, in contrast to some key circadian components, including CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL, which regulate cold-responsive DREB1 expression as transcriptional activators only during the day. Thus, plants efficiently control the acquisition of freezing tolerance using two different signaling pathways in response to a gradual temperature decrease during seasonal changes and a sudden temperature drop during the night.

ABA-unresponsive SnRK2 protein kinases regulate mRNA decay under osmotic stress in plants.

Rapid changes in messenger RNA population are vital for plants to properly exert multiple adaptive responses under continuously changing stress conditions. Transcriptional activation mediated by the 'abscisic acid (ABA)-activated SnRK2 protein kinases-ABA-responsive element (ABRE)-binding proteins/ABRE-binding factors (AREB/ABFs)' signalling module is a crucial step in the expression of stress-inducible genes under osmotic stress conditions in Arabidopsis1-4. In addition to transcriptional control, proper transcript levels of individual genes can be achieved by post-transcriptional regulation, but how this regulation functions under stress conditions and the underlying molecular mechanisms remain elusive. Here, we show that ABA-unresponsive osmotic stress-activated subclass I SnRK2s and their downstream substrate, VARICOSE (VCS), an mRNA decapping activator, regulate mRNA decay under osmotic stress conditions. The expression of many stress-responsive genes was similarly misregulated in a mutant lacking all functional subclass I SnRK2s and in VCS-knockdown plants. Additionally, the mRNA decay of the transcripts of these genes was impaired in these plants under osmotic stress conditions. Furthermore, these plants showed growth retardation under osmotic stresses. Notably, subclass I-type SnRK2s have been identified in seed plants but not in lycophytes or mosses. Therefore, the post-transcriptional regulation mediated by the 'subclass I SnRK2s-VARICOSE' signalling module represents an additional mechanism of gene expression control that facilitates drastic changes in mRNA populations under osmotic stresses and might enhance the adaptability of seed plants to stress conditions.

Temporal and spatial changes in gene expression, metabolite accumulation and phytohormone content in rice seedlings grown under drought stress conditions.

In order to analyze the molecular mechanisms underlying the responses of plants to different levels of drought stress, we developed a soil matric potential (SMP)-based irrigation system that precisely controls soil moisture. Using this system, rice seedlings were grown under three different drought levels, denoted Md1, Md2 and Md3, with SMP values set to -9.8, -31.0 and -309.9 kPa, respectively. Although the Md1 treatment did not alter the visible phenotype, the Md2 treatment caused stomatal closure and shoot growth retardation (SGR). The Md3 treatment markedly induced SGR, without inhibition of photosynthesis. More severe drought (Sds) treatment, under which irrigation was terminated, resulted in the wilting of leaves and inhibition of photosynthesis. Metabolome analysis revealed the accumulation of primary sugars under Md3 and Sds and of most amino acids under Sds. The starch content was increased under Md3 and decreased under Sds. Transcriptome data showed that the expression profiles of associated genes supported the observed changes in photosynthesis and metabolites, suggesting that the time lag from SGR to inhibition of photosynthesis might lead to the accumulation of photosynthates under Md3, which can be used as osmolytes under Sds. To gain further insight into the observed SGR, transcriptome and hormonome analyses were performed in specific tissues. The results showed specific decreases in indole-3-acetic acid (IAA) and cytokinin levels in Md2-, Md3- and Sds-treated shoot bases, though the expression levels of hormone metabolism-related genes were not reflected in IAA and cytokinin contents. These observations suggest that drought stress affects the distribution or degradation of cytokinin and IAA molecules.

Double overexpression of DREB and PIF transcription factors improves drought stress tolerance and cell elongation in transgenic plants.

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.

The Arabidopsis transcriptional regulator DPB3-1 enhances heat stress tolerance without growth retardation in rice.

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 Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression.

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.

Two distinct families of protein kinases are required for plant growth under high external Mg2+ concentrations in Arabidopsis.

Protein phosphorylation events play key roles in maintaining cellular ion homeostasis in higher plants, and the regulatory roles of these events in Na(+) and K(+) transport have been studied extensively. However, the regulatory mechanisms governing Mg(2+) transport and homeostasis in higher plants remain poorly understood, despite the vital roles of Mg(2+) in cellular function. A member of subclass III sucrose nonfermenting-1-related protein kinase2 (SnRK2), SRK2D/SnRK2.2, functions as a key positive regulator of abscisic acid (ABA)-mediated signaling in response to water deficit stresses in Arabidopsis (Arabidopsis thaliana). Here, we used immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry analyses to identify Calcineurin B-like-interacting protein kinase26 (CIPK26) as a novel protein that physically interacts with SRK2D. In addition to CIPK26, three additional CIPKs (CIPK3, CIPK9, and CIPK23) can physically interact with SRK2D in planta. The srk2d/e/i triple mutant lacking all three members of subclass III SnRK2 and the cipk26/3/9/23 quadruple mutant lacking CIPK26, CIPK3, CIPK9, and CIPK23 showed reduced shoot growth under high external Mg(2+) concentrations. Similarly, several ABA biosynthesis-deficient mutants, including aba2-1, were susceptible to high external Mg(2+) concentrations. Taken together, our findings provided genetic evidence that SRK2D/E/I and CIPK26/3/9/23 are required for plant growth under high external Mg(2+) concentrations in Arabidopsis. Furthermore, we showed that ABA, a key molecule in water deficit stress signaling, also serves as a signaling molecule in plant growth under high external Mg(2+) concentrations. These results suggested that SRK2D/E/I- and CIPK26/3/9/23-mediated phosphorylation signaling pathways maintain cellular Mg(2+) homeostasis.