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Soybean is an important commodity worldwide. Abiotic conditions can adversely disturb crop growth and final yield. The transcription factor Dehydration-Responsive Element-Binding Proteins 2 (DREB2) act as a regulator of drought-responses. This study aimed to characterize soybean plants genetically modified with GmDREB2A;2 FL and GmDREB2A;2 CA for molecular, physiological, and agronomic responses, at different developmental periods. Results showed that seedlings from GmDREB2A;2 FL event presented lower growth reduction under osmotic treatment during germination. The GmDREB2A;2 FL and GmDREB2A;2 CA events showed improved performance in experiments of water deficit imposed in the vegetative period and higher rates in physiological parameters. In the reproductive period, there was a trend of higher yield compounds in GM GmDREB2A;2 FL event when compared to other genotypes and treatments. It was suggested that GmDREB2A;2 FL event presented superior performance due to the higher expression levels of the cisgene and drought-induced genes.

Significance The mechanisms of eukaryotic circadian clocks rely on transcriptional-translational feedback loops (TTFLs), but components of TTFLs from different phylogenetic lineages are thought to be evolutionarily diverse. Posttranslational modification is also required for clock function, but those within the plant clock are less studied, likely due to genetic redundancy. Here, we identified a small synthetic molecule that lengthened the Arabidopsis circadian period. Using an affinity probe, we found that the molecule inhibited multiple members of the casein kinase I (CK1) family, which is also essential in animal, fungal, and algal clocks. The CK1 family modulated plant-specific clock-associated transcriptional repressors. With other studies, our results established the prominent role of CK1 family to control circadian clocks among vastly divergent phylogenetic lineages., The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

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.

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.

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.

Drought is a major environmental factor limiting cotton (Gossypium hirsutum L.) productivity worldwide and projected climate changes could increase their negative effects in the future. Thus, targeting the molecular mechanisms correlated with drought tolerance without reducing productivity is a challenge for plant breeding. In this way, we evaluated the effects of water deficit progress on AtDREB2A-CA transgenic cotton plant responses, driven by the stress-inducible rd29 promoter. Besides shoot and root morphometric traits, gas exchange and osmotic adjustment analyses were also included. Here, we present how altered root traits shown by transgenic plants impacted on physiological acclimation responses when submitted to severe water stress. The integration of AtDREB2A-CA into the cotton genome increased total root volume, surface area and total root length, without negatively affecting shoot morphometric growth parameters and nor phenotypic evaluated traits. Additionally, when compared to wild-type plants, transgenic plants (17-T0 plants and its progeny) highlighted a gradual pattern of phenotypic plasticity tosome photosynthetic parameters such as photosynthetic rate and stomatal conductance with water deficit progress. Transgene also promoted greater shoot development and root robustness (greater and deeper root mass) allowing roots to grow into deeper soil layers. The same morpho-physiological trend was observed in the subsequent generation (17.6-T2). Our results suggest that the altered root traits shown by transgenic plants are the major contributors to higher tolerance response, allowing the AtDRE2A-CA-cotton plants to maintain elevated stomatal conductance and assimilate rates and, consequently, reducing their metabolic costs involved in the antioxidant responses activation. These results also suggest that these morpho-physiological changes increased the number of reproductive structures retained per plant (26% higher) when compared with its non-transgenic counterpart. This is the first report of cotton plants overexpressing the AtDRE2A-CA transcription factor, demonstrating a morpho-physiological and yield advantages under drought stress, without displaying any yield penalty under irrigated conditions. The mechanisms by which the root traits influenced the acclimation of the transgenic plants to severe water deficit conditions are also discussed. These data present an opportunity to use this strategy in cotton breeding programs in order to improve drought adaptation toward better rooting features.

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.

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.

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.

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.

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.

Brazil is the second largest soybean-producing country, but yields have recently been unstable because of droughts. The objective of this project was to develop drought-tolerant soybean lines based on information from earlier molecular studies involving model plants. We also searched the soybean genome for genes conferring drought tolerance and elucidated the mechanisms regulating the identified genes. Based on our findings, we generated new soybean lines, which were then evaluated under greenhouse and field conditions to identify the most drought-tolerant lines. We analyzed the functions of drought tolerance genes in Arabidopsis thaliana and identified soybean genes exhibiting similar properties. We also comprehensively investigated soybean gene expression levels in stressed plants. Additionally, we determined the best combinations of drought tolerance genes and promoters and introduced these combinations into soybean cells using biolistic and Agrobacterium tumefaciens-based methods. We evaluated the stress tolerance of the resulting transgenic plants in a greenhouse and in the field and observed that some transgenic soybean lines exhibited increased drought tolerance. We developed a new technique for generating genetically modified soybean lines that are more tolerant to environmental stresses such as drought. These lines may be useful for mitigating the effects of climate changes. The developed technique and generated transgenic soybean lines may help stabilize or increase soybean production in Brazil.

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 Mg2+ transport and homeostasis in higher plants remain poorly understood, despite the vital roles of Mg2+ 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 Mg2+ concentrations. Similarly, several ABA biosynthesis-deficient mutants, including aba2-1, were susceptible to high external Mg2+ 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 Mg2+ 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 Mg2+ concentrations. These results suggested that SRK2D/E/I- and CIPK26/3/9/23-mediated phosphorylation signaling pathways maintain cellular Mg2+ homeostasis.

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.

Drought is one of the most stressful environmental factor causing yield and economic losses in many soybean-producing regions. In the last decades, transcription factors (TFs) are being used to develop genetically modified plants more tolerant to abiotic stresses. Dehydration responsive element binding (DREB) and ABA-responsive element-binding (AREB) TFs were introduced in soybean showing improved drought tolerance, under controlled conditions. However, these results may not be representative of the way in which plants behave over the entire season in the real field situation. Thus, the objectives of this study were to analyze agronomical traits and physiological parameters of AtDREB1A (1Ab58), AtDREB2CA (1Bb2193), and AtAREB1 (1Ea2939) GM lines under irrigated (IRR) and non-irrigated (NIRR) conditions in a field experiment, over two crop seasons and quantify transgene and drought-responsive genes expression. Results from season 2013/2014 revealed that line 1Ea2939 showed higher intrinsic water use and leaf area index. Lines 1Ab58 and 1Bb2193 showed a similar behavior to wild-type plants in relation to chlorophyll content. Oil and protein contents were not affected in transgenic lines in NIRR conditions. Lodging, due to plentiful rain, impaired yield from the 1Ea2939 line in IRR conditions. qPCR results confirmed the expression of the inserted TFs and drought-responsive endogenous genes. No differences were identified in the field experiment performed in crop season 2014/2015, probably due to the optimum rainfall volume during the cycle. These field screenings showed promising results for drought tolerance. However, additional studies are needed in further crop seasons and other sites to better characterize how these plants may outperform the WT under field water deficit.

Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty.

Phosphatidylethanolamine is the predominant phospholipid of the mitochondrial inner membrane. In Arabidopsis, pect1-4 mutants exhibit reduced cellular phosphatidylethanolamine levels owing to reduced CTP:phosphorylethanolamine cytidylyltransferase (PECT; EC 2.7.7.14) activity. Consequently, pect1-4 mutants may have decreased mitochondrial phosphatidylethanolamine levels, thereby affecting respiration capacity. Wild-type and pect1-4 plants grew similarly under a short-day condition until 5 weeks, when pect1-4 leaves had slightly less Chl. Total respiration was comparable between wild-type and pect1-4 leaves at 3 weeks and then increased 2-fold in the wild-type but only 1.1-fold in pect1-4 leaves. Compared with the wild type, the Cyt oxidase pathway capacity was reduced by 36% in pect1-4 leaves at 5 weeks and by 43% in pect1-4 mitochondria in 5-week-old rosette leaves. Maximal Cyt c oxidase (COX) activity was 20% lower in pect1-4 mitochondria than in wild-type mitochondria at 5 weeks despite comparable COX II protein levels in mitochondria at that time. Furthermore, COX II protein levels doubled in both wild-type and pect1-4 mitochondria between 3 and 5 weeks. Phosphatidylethanolamine levels were similar between mitochondria from these plants at 3 weeks and then increased by 6.4% in wild-type mitochondria and decreased by 6.5% in pect1-4 mitochondria by 5 weeks. Phosphatidylcholine levels compensated for the decreases in phosphatidylethanolamine levels. COX activity was lower in pect1-4 mitochondria at 5 weeks, most probably due to reduced phosphatidylethanolamine levels and/or an altered phosphatidylethanolamine:phosphatidylcholine ratio. Thus, PECT1 regulates mitochondrial phosphatidylethanolamine levels, which are important for maintaining respiration capacity in Arabidopsis leaves during prolonged growth under short-day conditions.

Soybean (Glycine max) is an important crop around the world. Abiotic stress conditions, such as drought and heat, adversely affect its survival, growth, and production. The DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2 (DREB2) group includes transcription factors that contribute to drought and heat stress tolerance by activating transcription through the cis-element dehydration-responsive element (DRE) in response to these stress stimuli. Two modes of regulation, transcriptional and posttranslational, are important for the activation of gene expression by DREB2A in Arabidopsis (Arabidopsis thaliana). However, the regulatory system of DREB2 in soybean is not clear. We identified a new soybean DREB2 gene, GmDREB2A;2, that was highly induced not only by dehydration and heat but also by low temperature. GmDREB2A;2 exhibited a high transactivation activity via DRE and has a serine/threonine-rich region, which corresponds to a negative regulatory domain of DREB2A that is involved in its posttranslational regulation, including destabilization. Despite the partial similarity between these sequences, the activity and stability of the GmDREB2A;2 protein were enhanced by removal of the serine/threonine-rich region in both Arabidopsis and soybean protoplasts, suggestive of a conserved regulatory mechanism that involves the recognition of serine/threonine-rich sequences with a specific pattern. The heterologous expression of GmDREB2A;2 in Arabidopsis induced DRE-regulated stress-inducible genes and improved stress tolerance. However, there were variations in the growth phenotypes of the transgenic Arabidopsis, the induced genes, and their induction ratios between GmDREB2A;2 and DREB2A. Therefore, the basic function and regulatory machinery of DREB2 have been maintained between Arabidopsis and soybean, although differentiation has also occurred.

Summary To analyze the molecular mechanisms underlying plant responses 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 photosynthesis inhibition. More severe drought (Sds) treatment, under which irrigation was terminated, resulted in leaf wilting and photosynthesis inhibition. 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 photosynthesis inhibition might lead to 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 in specific tissues were performed. The results showed specific decreases in 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. This article is protected by copyright. All rights reserved.

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.

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.

In terrestrial environments, temperature and water conditions are highly variable, and extreme temperatures and water conditions affect the survival, growth and reproduction of plants. To protect cells and sustain growth under such conditions of abiotic stress, plants respond to unfavourable changes in their environments in developmental, physiological and biochemical ways. These responses require expression of stress-responsive genes, which are regulated by a network of transcription factors. The AP2/ERF family is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain. This transcription factor family includes DRE-binding proteins (DREBs), which activate the expression of abiotic stress-responsive genes via specific binding to the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element in their promoters. In this review, we discuss the functions of the AP2/ERF-type transcription factors in plant abiotic stress responses, with special emphasis on the regulations and functions of two major types of DREBs, DREB1/CBF and DREB2. In addition, we summarise the involvement of other AP2/ERF-type transcription factors in abiotic stress responses, which has recently become clear. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.

The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (4(6) = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.

The SPINDLY (SPY) gene was first identified as a negative regulator of plant gibberellic acid (GA) signaling because mutation of this gene phenocopies plants treated with an overdose of bioactive GA and results in insensitivity to a GA inhibitor during seed germination. The SPY gene encodes an O-linked N-acetylglucosamine transferase that can modify the target protein and modulate the protein activity in cells. In this study, we describe the strong salt and drought tolerance phenotypes of Arabidopsis (Arabidopsis thaliana) spy-1 and spy-3 mutants in addition to their GA-related phenotypes. SPY gene expression was found to be drought stress inducible and slightly responsive to salt stress. Transcriptome analysis of spy-3 revealed that many GA-responsive genes were up-regulated, which could explain the GA-overdosed phenotype of spy-3. Some stress-inducible genes were found to be up-regulated in spy-3, such as genes encoding late embryogenesis abundant proteins, Responsive to Dehydration20, and AREB1-like transcription factor, which may confer stress tolerance on spy-3. CKX3, a cytokinin (CK) catabolism gene, was up-regulated in spy-3; this up-regulation indicates that the mutant possesses reduced CK signaling, which is consistent with a positive role for SPY in CK signaling. Moreover, overexpression of SPY in transgenics (SPY overexpressing [SPY-OX]) impaired plant drought stress tolerance, opposite to the phenotype of spy. The expression levels of several genes, such as DREB1E/DDF1 and SNH1/WIN1, were decreased in SPY-OX but increased in spy-3. Taken together, these data indicate that SPY plays a negative role in plant abiotic stress tolerance, probably by integrating environmental stress signals via GA and CK cross talk.

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using beta-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.

Transcription factors of the DRE-Binding1 (DREB1)/C-repeat binding factor family specifically interact with a cis-acting dehydration-responsive element/C-repeat involved in low-temperature stress-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Expression of DREB1s is induced by low temperatures and is regulated by the circadian clock under unstressed conditions. Promoter sequences of DREB1s contain six conserved motifs, boxes I to VI. We analyzed the promoter region of DREB1C using transgenic plants and found that box V with the G-box sequence negatively regulates DREB1C expression under circadian control. The region around box VI contains positive regulatory elements for low-temperature-induced expression of DREB1C. Using yeast one-hybrid screens, we isolated cDNA encoding the transcriptional factor Phytochrome-Interacting Factor7 (PIF7), which specifically binds to the G-box of the DREB1C promoter. The PIF7 gene was expressed in rosette leaves, and the PIF7 protein was localized in the nuclei of the cells. Transactivation experiments using Arabidopsis protoplasts indicated that PIF7 functions as a transcriptional repressor for DREB1C expression and that its activity is regulated by PIF7-interacting factors TIMING OF CAB EXPRESSION1 and Phytochrome B, which are components of the circadian oscillator and the red light photoreceptor, respectively. Moreover, in the pif7 mutant, expression of DREB1B and DREB1C was not repressed under light conditions, indicating that PIF7 functions as a transcriptional repressor for the expression of DREB1B and DREB1C under circadian control. This negative regulation of DREB1 expression may be important for avoiding plant growth retardation by the accumulation of DREB1 proteins under unstressed conditions.

Acetyl-CoA carboxylase (ACCase) in plastids is a key enzyme regulating the rate of de novo fatty acid biosynthesis in plants. Plastidic ACCase is composed of three nuclear-encoded subunits and one plastid-encoded accD subunit. To boost ACCase levels, we examined whether overexpression of accD elevates ACCase production. Using homologous recombination, we replaced the promoter of the accD operon in the tobacco plastid genome with a plastid rRNA-operon (rrn) promoter that directs enhanced expression in photosynthetic and non-photosynthetic organs, and successfully raised the total ACCase levels in plastids. This result suggests that the level of the accD subunit is a determinant of ACCase levels, and that enzyme levels are in part controlled post-transcriptionally at the level of subunit assembly. The resultant transformants grew normally and the fatty acid content was significantly increased in leaves, but not significantly in seeds. However, the transformants displayed extended leaf longevity and a twofold increase of seed yield over the control value, which eventually almost doubled the fatty acid production per plant of the transformants relative to control and wild-type plants. These findings offer a potential method for raising plant productivity and oil production.

Summary Soybean (Glycine max) is a globally important crop, and its growth and yield are severely reduced by abiotic stresses, such as drought, heat, and cold. The cis-acting element DRE (dehydration-responsive element)/CRT plays an important role in activating gene expression in response to these stresses. The Arabidopsis DREB1/CBF genes that encode DRE-binding proteins function as transcriptional activators in the cold stress responsive gene expression. In this study, we identified 14 DREB1-type transcription factors (GmDREB1s) from a soybean genome database. The expression of most GmDREB1 genes in soybean was strongly induced by a variety of abiotic stresses, such as cold, drought, high salt, and heat. The GmDREB1 proteins activated transcription via DREs (dehydration-responsive element) in Arabidopsis and soybean protoplasts. Transcriptome analyses using transgenic Arabidopsis plants overexpressing GmDREB1s indicated that many of the downstream genes are cold-inducible and overlap with those of Arabidopsis DREB1A. We then comprehensively analyzed the downstream genes of GmDREB1B;1, which is closely related to DREB1A, using a transient expression system in soybean protoplasts. The expression of numerous genes induced by various abiotic stresses were increased by overexpressing GmDREB1B;1 in soybean, and DREs were the most conserved element in the promoters of these genes. The downstream genes of GmDREB1B;1 included numerous soybean-specific stress-inducible genes that encode an ABA receptor family protein, GmPYL21, and translation-related genes, such as ribosomal proteins. We confirmed that GmDREB1B;1 directly activates GmPYL21 expression and enhances ABRE-mediated gene expression in an ABA-independent manner. These results suggest that GmDREB1 proteins activate the expression of numerous soybean-specific stress-responsive genes under diverse abiotic stress conditions.

The Arabidopsis thaliana transcription factor DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A) controls the expression of many genes involved in the plant's response to dehydration and heat stress. Despite the significance of post-translational regulation in DREB2A activation, the mechanism underlying this activation remains unclear. Here, with the aid of a newly produced antibody against DREB2A, we characterized the regulation of DREB2A stability in plants exposed to stress stimuli. Endogenous DREB2A accumulated in wild-type Arabidopsis plants subjected to dehydration and heat stress. A degradation assay using Arabidopsis T87 suspension-cultured cells revealed that DREB2A protein degradation was inhibited at high temperatures. The proteasome-dependent degradation of DREB2A required the import of this protein into the nucleus. The E3 ligases DRIP1 and DRIP2 were involved in this process under both normal and stressful conditions; however, other E3 ligases may have also been involved, at least during the late stages of the heat stress response. Although the constitutive expression of DREB2A resulted in an overproduction of DREB2A and enhanced target gene induction during stress in transgenic plants, the accumulation of DREB2A caused by proteasome inhibitors did not induce target gene expression. Thus, the stabilization of DREB2A is important but not sufficient to induce target gene expression; further activation processes are required.

Abiotic stress is a major factor limiting productivity of rice crops in large areas of the world. Because plants cannot avoid abiotic stress by moving, they have acquired various mechanisms for stress tolerance in the course of their evolution. Enhancing or introducing such mechanisms in rice is one effective way to develop stress-tolerant cultivars. Based on physiological studies on stress responses, recent progress in plant molecular biology has enabled discovery of many genes involved in stress tolerance. These genes include regulatory genes, which regulate stress response (e.g., transcription factors and protein kinases), and functional genes, which protect the cell (e.g., enzymes for generating protective metabolites and proteins). Both kinds of genes are used to increase stress tolerance in rice. In addition, several quantitative trait loci (QTLs) associated with higher stress tolerance have been cloned, contributing to the discovery of significantly important genes for stress tolerance.

Abiotic stress is a major factor limiting productivity of rice crops in large areas of the world. Because plants cannot avoid abiotic stress by moving, they have acquired various mechanisms for stress tolerance in the course of their evolution. Enhancing or introducing such mechanisms in rice is one effective way to develop stress-tolerant cultivars. Based on physiological studies on stress responses, recent progress in plant molecular biology has enabled discovery of many genes involved in stress tolerance. These genes include regulatory genes, which regulate stress response (e.g., transcription factors and protein kinases), and functional genes, which protect the cell (e.g., enzymes for generating protective metabolites and proteins). Both kinds of genes are used to increase stress tolerance in rice. In addition, several quantitative trait loci (QTLs) associated with higher stress tolerance have been cloned, contributing to the discovery of significantly important genes for stress tolerance.

Arabidopsis thaliana DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) functions as a transcriptional activator that increases tolerance to osmotic and heat stresses; however, its expression also leads to growth retardation and reduced reproduction. To avoid these adverse effects, the expression of DREB2A is predicted to be tightly regulated. We identified a short promoter region of DREB2A that represses its expression under nonstress conditions. Yeast one-hybrid screening for interacting factors identified GROWTH-REGULATING FACTOR7 (GRF7). GRF7 bound to the DREB2A promoter and repressed its expression. In both artificial miRNA-silenced lines and a T-DNA insertion line of GRF7, DREB2A transcription was increased compared with the wild type under nonstress conditions. A previously undiscovered cis-element, GRF7-targeting cis-element (TGTCAGG), was identified as a target sequence of GRF7 in the short promoter region of DREB2A via electrophoretic mobility shift assays. Microarray analysis of GRF7 knockout plants showed that a large number of the upregulated genes in the mutant plants were also responsive to osmotic stress and/or abscisic acid. These results suggest that GRF7 functions as a repressor of a broad range of osmotic stress–responsive genes to prevent growth inhibition under normal conditions.

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.

In plants, osmotic stress-responsive transcriptional regulation depends mainly on two major classes of cis-acting elements found in the promoter regions of stress-inducible genes: ABA-responsive elements (ABREs) and dehydration-responsive elements (DREs). ABRE has been shown to perceive ABA-mediated osmotic stress signals, whereas DRE is known to be involved in an ABA-independent pathway. Previously, we reported that the transcription factor DRE-BINDING PROTEIN 2A (DREB2A) regulates DRE-mediated transcription of target genes under osmotic stress conditions in Arabidopsis (Arabidopsis thaliana). However, the transcriptional regulation of DREB2A itself remains largely uncharacterized. To elucidate the transcriptional mechanism associated with the DREB2A gene under osmotic stress conditions, we generated a series of truncated and base-substituted variants of the DREB2A promoter and evaluated their transcriptional activities individually. We found that both ABRE and coupling element 3 (CE3)-like sequences located approximately -100 bp from the transcriptional initiation site are necessary for the dehydration-responsive expression of DREB2A. Coupling our transient expression analyses with yeast one-hybrid and chromatin immunoprecipitation (ChIP) assays indicated that the ABRE-BINDING PROTEIN 1 (AREB1), AREB2 and ABRE-BINDING FACTOR 3 (ABF3) bZIP transcription factors can bind to and activate the DREB2A promoter in an ABRE-dependent manner. Exogenous ABA application induced only a modest accumulation of the DREB2A transcript when compared with the osmotic stress treatment. However, the osmotic stress-induced DREB2A expression was found to be markedly impaired in several ABA-deficient and ABA-insensitive mutants. These results suggest that in addition to an ABA-independent pathway, the ABA-dependent pathway plays a positive role in the osmotic stress-responsive expression of DREB2A.

Abiotic stresses such as drought and high salinity adversely affect the growth and productivity of plants, including crops. The development of stress-tolerant crops will be greatly advantageous for modern agriculture in areas that are prone to such stresses. In recent years, several advances have been made towards identifying potential stress related genes which are capable of increasing the tolerance of plants to abiotic stress. NAC proteins are plant-specific transcription factors and more than 100 NAC genes have been identified in Arabidopsis and rice to date. Phylogenetic analyses indicate that the six major groups were already established at least in an ancient moss lineage. NAC transcription factors have a variety of important functions not only in plant development but also in abiotic stress responses. Stress-inducible NAC genes have been shown to be involved in abiotic stress tolerance. Transgenic Arabidopsis and rice plants overexpressing stress-responsive NAC (SNAC) genes have exhibited improved drought tolerance. These studies indicate that SNAC factors have important roles for the control of abiotic stress tolerance and that their overexpression can improve stress tolerance via biotechnological approaches. Although these transcription factors can bind to the same core NAC recognition sequence, recent studies have demonstrated that the effects of NAC factors for growth are different. Moreover, the NAC proteins are capable of functioning as homo- or hetero-dimer forms. Thus, SNAC factors can be useful for improving stress tolerance in transgenic plants, although the mechanism for mediating the stress tolerance of these homologous factors is complex in plants. Recent studies also suggest that crosstalk may exist between stress responses and plant growth. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.

Arabidopsis DREB2A is a key transcription factor of heat- and drought-responsive gene expression, and DREB2A expression is induced by these stresses. We analyzed the DREB2A promoter and found a heat shock element that functions as a cis-acting element in the heat shock (HS)-responsive expression of DREB2A. Among the 21 Arabidopsis heat shock factors, we chose 4 HsfA1-type proteins as candidate transcriptional activators (HsfA1a, HsfA1b, HsfA1d, and HsfA1e) based on transactivation activity and expression patterns. We generated multiple mutants and found that the HS-responsive expression of DREB2A disappeared in hsfa1a/b/d triple and hsfa1a/b/d/e quadruple mutants. Moreover, HS-responsive gene expression, including that of molecular chaperones and transcription factors, was globally and drastically impaired in the hsfa1a/b/d triple mutant, which exhibited greatly reduced tolerance to HS stress. HsfA1 protein accumulation in the nucleus was negatively regulated by their interactions with HSP90, and other factors potentially strongly activate the HsfA1 proteins under HS stress. The hsfa1a/b/d/e quadruple mutant showed severe growth retardation, and many genes were downregulated in this mutant even under non-stress conditions. Our study indicates that HsfA1a, HsfA1b, and HsfA1d function as main positive regulators in HS-responsive gene expression and four HsfA1-type proteins are important in gene expression for normal plant growth.

Yeast Smt3 is a ubiquitin-like protein similar to the mammalian SUMO-1. Cdc3, a septin component, is known to be modified by Smt3. The level of this modification was affected by Smt3-specific protease mutation ulp1-ts or overexpression of ULP1. By two-hybrid screening, we isolated 5 UIP (Ulp1 interacting protein) genes. UIP1 was identical to NUP42 encoding a component of the nuclear pore complex (NPC). Gle1, another NPC-associating component, also interacted with Ulp1 in the two-hybrid system and co-immunoprecipitation experiment. Thus Ulp1 associates with nucleoporins and may interact with septin rings in the telophase.

A myriad of drought stress-inducible genes have been reported, and many of these are activated by abscisic acid (ABA). In the promoter regions of such ABA-regulated genes, conserved cis-elements, designated ABA-responsive elements (ABREs), control gene expression via bZIP-type AREB/ABF transcription factors. Although all three members of the AREB/ABF subfamily, AREB1, AREB2, and ABF3, are upregulated by ABA and water stress, it remains unclear whether these are functional homologs. Here, we report that all three AREB/ABF transcription factors require ABA for full activation, can form hetero- or homodimers to function in nuclei, and can interact with SRK2D/SnRK2.2, an SnRK2 protein kinase that was identified as a regulator of AREB1. Along with the tissue-specific expression patterns of these genes and the subcellular localization of their encoded proteins, these findings clearly indicate that AREB1, AREB2, and ABF3 have largely overlapping functions. To elucidate the role of these AREB/ABF transcription factors, we generated an areb1 areb2 abf3 triple mutant. Large-scale transcriptome analysis, which showed that stress-responsive gene expression is remarkably impaired in the triple mutant, revealed novel AREB/ABF downstream genes in response to water stress, including many LEA class and group-Ab PP2C genes and transcription factors. The areb1 areb2 abf3 triple mutant is more resistant to ABA than are the other single and double mutants with respect to primary root growth, and it displays reduced drought tolerance. Thus, these results indicate that AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent gene expression for ABA signaling under conditions of water stress.

DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2 s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.

A transcription factor DREB2A functions as a key regulator not only in drought stress responses but also in heat stress (HS) responses, and activates expression of many abiotic stress-responsive-genes involved in drought and HS tolerance. HsfA3 is one of the most up-regulated heat-inducible genes in transgenic plants overexpressing DREB2A. In this study, the analyses of HsfA3 expression profile and the transactivation analysis of HsfA3 showed that the expression of HsfA3 was directly regulated by DREB2A under HS. Microarray analysis using transgenic plants overexpressing HsfA3 also showed that overexpression of HsfA3 induces many heat-inducible genes. Furthermore, we showed that thermotolerance of the HsfA3 overexpressors was increased, and that of the hsfA3 T-DNA tagged mutants was decreased. These results indicate that HsfA3 regulates expression of many heat-inducible genes in the transcriptional cascade downstream of the DREB2A stress-regulatory system and functions in acquisition of thermotolerance under the control of the DREB2A cascade.