1-Butanol treatment enhances drought stress tolerance in Arabidopsis thaliana.

Abiotic stress is a major factor affecting crop productivity. Chemical priming is a promising strategy to enhance tolerance to abiotic stress. In this study, we evaluated the use of 1-butanol as an effectual strategy to enhance drought stress tolerance in Arabidopsis thaliana. We first demonstrated that, among isopropanol, methanol, 1-butanol, and 2-butanol, pretreatment with 1-butanol was the most effective for enhancing drought tolerance. We tested the plants with a range of 1-butanol concentrations (0, 10, 20, 30, 40, and 50 mM) and further determined that 20 mM was the optimal concentration of 1-butanol that enhanced drought tolerance without compromising plant growth. Physiological tests showed that the enhancement of drought tolerance by 1-butanol pretreatment was associated with its stimulation of stomatal closure and improvement of leaf water retention. RNA-sequencing analysis revealed the differentially expressed genes (DEGs) between water- and 1-butanol-pretreated plants. The DEGs included genes involved in oxidative stress response processes. The DEGs identified here partially overlapped with those of ethanol-treated plants. Taken together, the results show that 1-butanol is a novel chemical priming agent that effectively enhances drought stress tolerance in Arabidopsis plants, and provide insights into the molecular mechanisms of alcohol-mediated abiotic stress tolerance.

Ethanol induces heat tolerance in plants by stimulating unfolded protein response.

KEY MESSAGE: Ethanol priming induces heat stress tolerance by the stimulation of unfolded protein response. Global warming increases the risk of heat stress-related yield losses in agricultural crops. Chemical priming, using safe agents, that can flexibly activate adaptive regulatory responses to adverse conditions, is a complementary approach to genetic improvement for stress adaptation. In the present study, we demonstrated that pretreatment of Arabidopsis with a low concentration of ethanol enhances heat tolerance without suppressing plant growth. We also demonstrated that ethanol pretreatment improved leaf growth in lettuce (Lactuca sativa L.) plants grown in the field conditions under high temperatures. Transcriptome analysis revealed a set of genes that were up-regulated in ethanol-pretreated plants, relative to water-pretreated controls. Binding Protein 3 (BIP3), an endoplasmic reticulum (ER)-stress marker chaperone gene, was among the identified up-regulated genes. The expression levels of BIP3 were confirmed by RT-qPCR. Root-uptake of ethanol was metabolized to organic acids, nucleic acids, amines and other molecules, followed by an increase in putrescine content, which substantially promoted unfolded protein response (UPR) signaling and high-temperature acclimation. We also showed that inhibition of polyamine production and UPR signaling negated the heat stress tolerance induced by ethanol pretreatment. These findings collectively indicate that ethanol priming activates UPR signaling via putrescine accumulation, leading to enhanced heat stress tolerance. The information gained from this study will be useful for establishing ethanol-mediated chemical priming strategies that can be used to help maintain crop production under heat stress conditions.

Ethanol treatment enhances drought stress avoidance in cassava (Manihot esculenta Crantz).

External application of ethanol enhances tolerance to high salinity, drought, and heat stress in various plant species. However, the effects of ethanol application on increased drought tolerance in woody plants, such as the tropical crop "cassava," remain unknown. In the present study, we analyzed the morphological, physiological, and molecular responses of cassava plants subjected to ethanol pretreatment and subsequent drought stress treatment. Ethanol pretreatment induced a slight accumulation of abscisic acid (ABA) and stomatal closure, resulting in a reduced transpiration rate, higher water content in the leaves during drought stress treatment and the starch accumulation in leaves. Transcriptomic analysis revealed that ethanol pretreatment upregulated the expression of ABA signaling-related genes, such as PP2Cs and AITRs, and stress response and protein-folding-related genes, such as heat shock proteins (HSPs). In addition, the upregulation of drought-inducible genes during drought treatment was delayed in ethanol-pretreated plants compared with that in water-pretreated control plants. These results suggest that ethanol pretreatment induces stomatal closure through activation of the ABA signaling pathway, protein folding-related response by activating the HSP/chaperone network and the changes in sugar and starch metabolism, resulting in increased drought avoidance in plants.

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

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

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.

Expression of the CCCH-tandem zinc finger protein gene OsTZF5 under a stress-inducible promoter mitigates the effect of drought stress on rice grain yield under field conditions.

Increasing drought resistance without sacrificing grain yield remains an ongoing challenge in crop improvement. In this study, we report that Oryza sativa CCCH-tandem zinc finger protein 5 (OsTZF5) can confer drought resistance and increase grain yield in transgenic rice plants. Expression of OsTZF5 was induced by abscisic acid, dehydration and cold stress. Upon stress, OsTZF5-GFP localized to the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF5 under the constitutive maize ubiquitin promoter exhibited improved survival under drought but also growth retardation. By introducing OsTZF5 behind the stress-responsive OsNAC6 promoter in two commercial upland cultivars, Curinga and NERICA4, we obtained transgenic plants that showed no growth retardation. Moreover, these plants exhibited significantly increased grain yield compared to non-transgenic cultivars in different confined field drought environments. Physiological analysis indicated that OsTZF5 promoted both drought tolerance and drought avoidance. Collectively, our results provide strong evidence that OsTZF5 is a useful biotechnological tool to minimize yield losses in rice grown under drought 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.

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.

Rice Phytochrome-Interacting Factor-Like1 (OsPIL1) is involved in the promotion of chlorophyll biosynthesis through feed-forward regulatory loops.

In phototrophic plants, the highly conserved and tightly regulated process of chlorophyll (Chl) biosynthesis comprises multi-step reactions involving more than 15 enzymes. Since the efficiency of Chl biosynthesis strongly affects plant productivity, understanding the underlying regulatory mechanisms in crop plants can be useful for strategies to increase grain and biomass yields. Here, we show that rice (Oryza sativa) Phytochrome-Interacting Factor-Like1 (OsPIL1), a basic helix-loop-helix transcription factor, promotes Chl biosynthesis. The T-DNA insertion knockdown ospil1 mutant showed a pale-green phenotype when grown in a natural paddy field. Transcriptome analysis revealed that several genes responsible for Chl biosynthesis and photosynthesis were significantly down-regulated in ospil1 leaves. Using promoter binding and transactivation assays, we found that OsPIL1 binds to the promoters of two Chl biosynthetic genes, OsPORB and OsCAO1, and promotes their transcription. In addition, OsPIL1 directly up-regulates the expression of two transcription factor genes, GOLDEN2-LIKE1 (OsGLK1) and OsGLK2. OsGLK1 and OsGLK2 both bind to the promoters of OsPORB and OsCAO1, as well as some of genes encoding the light-harvesting complex of photosystems, probably promoting their transcription. Thus, OsPIL1 is involved in the promotion of Chl biosynthesis by up-regulating the transcription of OsPORB and OsCAO1 via trifurcate feed-forward regulatory loops involving two OsGLKs.

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.

Four Arabidopsis†AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress.

Under osmotic stress conditions such as drought and high salinity, the plant hormone abscisic acid (ABA) plays important roles in stress-responsive gene expression mainly through three bZIP transcription factors, AREB1/ABF2, AREB2/ABF4 and ABF3, which are activated by SNF1-related kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). However, since the three AREB/ABFs are crucial, but not exclusive, for the SnRK2-mediated gene expression, transcriptional pathways governed by SRK2D/E/I are not fully understood. Here, we show that a bZIP transcription factor, ABF1, is a functional homolog of AREB1, AREB2 and ABF3 in ABA-dependent gene expression in Arabidopsis. Despite lower expression levels of ABF1 than those of the three AREB/ABFs, the areb1 areb2 abf3 abf1 mutant plants displayed increased sensitivity to drought and decreased sensitivity to ABA in primary root growth compared with the areb1 areb2 abf3 mutant. Genome-wide transcriptome analyses revealed that expression of downstream genes of SRK2D/E/I, which include many genes functioning in osmotic stress responses and tolerance such as transcription factors and LEA proteins, was mostly impaired in the quadruple mutant. Thus, these results indicate that the four AREB/ABFs are the predominant transcription factors downstream of SRK2D/E/I in ABA signalling in response to osmotic stress during vegetative growth.

Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress.

The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions.

Comparative functional analysis of six drought-responsive promoters in transgenic rice.

Rice production is greatly affected by environmental stresses such as drought and high salinity. Transgenic rice plants tolerant to such stresses are expected to be produced. Stress-responsive promoters with low expression under normal growth conditions are needed to minimize the adverse effects of stress-tolerance genes on rice growth. We performed expression analyses of drought-responsive genes in rice plants using a microarray, and selected LIP9, OsNAC6, OsLEA14a, OsRAB16D, OsLEA3-1, and Oshox24 for promoter analysis. Transient assays using the promoters indicated that AREB/ABF (abscisic acid (ABA)-responsive element-binding protein/ABA-binding factor) transcription factors enhanced expressions of these genes. We generated transgenic rice plants containing each promoter and the ß-glucuronidase (GUS) reporter gene. GUS assays revealed that the LIP9 and OsNAC6 promoters were induced by drought, high salinity, and ABA treatment, and both promoters showed strong activity under normal growth conditions in the root. The other promoters were strongly induced by stresses and ABA, but showed low activity under normal growth conditions. In seeds, GUS staining showed that Oshox24 expression was low and expressions of the other genes were high. Transgenic rice plants overexpressing OsNAC6 under the control of the Oshox24 promoter showed increased tolerance to drought and high salinity, and no growth defects. These data suggest that the Oshox24 promoter is useful to overexpress stress-tolerance genes without adversely affecting growth.

OsTZF1, a CCCH-tandem zinc finger protein, confers delayed senescence and stress tolerance in rice by regulating stress-related genes.

OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of ß-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with ß-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3' untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes.

GmDREB2A;2, a canonical DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2-type transcription factor in soybean, is posttranslationally regulated and mediates dehydration-responsive element-dependent gene expression.

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.

Application of ethanol alleviates heat damage to leaf growth and yield in tomato.

Chemical priming has emerged as a promising area in agricultural research. Our previous studies have demonstrated that pretreatment with a low concentration of ethanol enhances abiotic stress tolerance in Arabidopsis and cassava. Here, we show that ethanol treatment induces heat stress tolerance in tomato (Solanum lycopersicon L.) plants. Seedlings of the tomato cultivar 'Micro-Tom' were pretreated with ethanol solution and then subjected to heat stress. The survival rates of the ethanol-pretreated plants were significantly higher than those of the water-treated control plants. Similarly, the fruit numbers of the ethanol-pretreated plants were greater than those of the water-treated ones. Transcriptome analysis identified sets of genes that were differentially expressed in shoots and roots of seedlings and in mature green fruits of ethanol-pretreated plants compared with those in water-treated plants. Gene ontology analysis using these genes showed that stress-related gene ontology terms were found in the set of ethanol-induced genes. Metabolome analysis revealed that the contents of a wide range of metabolites differed between water- and ethanol-treated samples. They included sugars such as trehalose, sucrose, glucose, and fructose. From our results, we speculate that ethanol-induced heat stress tolerance in tomato is mainly the result of increased expression of stress-related genes encoding late embryogenesis abundant (LEA) proteins, reactive oxygen species (ROS) elimination enzymes, and activated gluconeogenesis. Our results will be useful for establishing ethanol-based chemical priming technology to reduce heat stress damage in crops, especially in Solanaceae.

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.

Overproduction of the membrane-bound receptor-like protein kinase 1, RPK1, enhances abiotic stress tolerance in Arabidopsis.

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H(2)O(2)-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.

An ABRE promoter sequence is involved in osmotic stress-responsive expression of the DREB2A gene, which encodes a transcription factor regulating drought-inducible genes in Arabidopsis.

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.