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.

Overexpression of AtNCED3 gene improved drought tolerance in soybean in greenhouse and field conditions.

Water deficit is an important climatic problem that can impair agriculture yield and economy. Genetically modified soybean plants containing the AtNCED3 gene were obtained aiming drought-tolerance improvement. The NCED3 gene encodes a 9-cis-epoxycarotenoid dioxygenase (NCED, EC 1.13.11.51), an important enzyme in abscisic acid biosynthesis. ABA activates the expression of drought-responsive genes, in water-deficit conditions, targeting defense mechanisms and enabling plants to survive under low water availability. Results from greenhouse experiments showed that the transgene AtNCED3 and the endogenous genes GmAREB1, GmPP2C, GmSnRK2 and GmAAO3 presented higher expression under water deficit (WD) in the event 2Ha11 than in WT-plants. No significant correlation was observed between the plant materials and WD conditions for growth parameters; however, gas exchange measurements decreased in the GM event, which also showed 80% higher intrinsic water use when compared to WT plants. In crop season 2015/16, event 2Ha11 showed higher total number of pods, higher number of pods with seeds and yield than WT plants. ABA concentration was also higher in GM plants under WD. These results obtained in field screenings suggest that AtNCED3 soybean plants might outperform under drought, reducing economic and yield losses, thus being a good candidate line to be incorporated in the soybean-breeding program to develop drought-tolerant cultivars.

Endophytic bacterial microbiome associated with leaves of genetically modified (AtAREB1) and conventional (BR 16) soybean plants.

Plant leaves (phyllosphere) have a great potential for colonization and microbial growth, consisting of a dynamic environment in which several factors can interfere with the microbial population structure. The use of genetically modified (GM) plants has introduced several traits in agriculture, such as the improvement of plant drought tolerance, as observed in the AtAREB1 transcription factor overexpression in soybean (Glycine max L. Merrill). The present study aimed at investigating the taxonomic and functional profile of the leaf microbial community of bacteria found in GM (drought-tolerant event 1Ea2939) and conventional (BR 16) soybean plants. Bacterial DNA was extracted from leaf samples collected from each genotype and used for microbial diversity and richness analysis through the MiSeq Illumina platform. Functional prediction was performed using the PICRUSt tool and the STAMP v 2.1.3 software. The obtainment of the GM event 1Ea2939 showed minimum effects on the microbial community and in the potential for chemical-genetic communication, i.e. in the potential for symbiotic and/or mutualistic interaction between plants and their natural microbiota.

Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field.

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

SNAC-As, stress-responsive NAC transcription factors, mediate ABA-inducible leaf senescence.

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

Arabidopsis growth-regulating factor7 functions as a transcriptional repressor of abscisic acid- and osmotic stress-responsive genes, including DREB2A.

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.

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.

Phenotyping soybean plants transformed with rd29A:AtDREB1A for drought tolerance in the greenhouse and field.

The development of drought tolerant plants is a high priority because the area suffering from drought is expected to increase in the future due to global warming. One strategy for the development of drought tolerance is to genetically engineer plants with transcription factors (TFs) that regulate the expression of several genes related to abiotic stress defense responses. This work assessed the performance of soybean plants overexpressing the TF DREB1A under drought conditions in the field and in the greenhouse. Drought was simulated in the greenhouse by progressively drying the soil of pot cultures of the P58 and P1142 lines. In the field, the performance of the P58 line and of 09D-0077, a cross between the cultivars BR16 and P58, was evaluated under four different water regimes: irrigation, natural drought (no irrigation) and water stress created using rain-out shelters in the vegetative or reproductive stages. Although the dehydration-responsive element-binding protein (DREB) plants did not outperform the cultivar BR16 in terms of yield, some yield components were increased when drought was introduced during the vegetative stage, such as the number of seeds, the number of pods with seeds and the total number of pods. The greenhouse data suggest that the higher survival rates of DREB plants are because of lower water use due to lower transpiration rates under well watered conditions. Further studies are needed to better characterize the soil and atmospheric conditions under which these plants may outperform the non-transformed parental plants.

A Case Study from the Overexpression of OsTZF5, Encoding a CCCH Tandem Zinc Finger Protein, in Rice Plants Across Nineteen Yield Trials.

BACKGROUND: Development of transgenic rice overexpressing transcription factors involved in drought response has been previously reported to confer drought tolerance and therefore represents a means of crop improvement. We transformed lowland rice IR64 with OsTZF5, encoding a CCCH-tandem zinc finger protein, under the control of the rice LIP9 stress-inducible promoter and compared the drought response of transgenic lines and nulls to IR64 in successive screenhouse paddy and field trials up to the T6 generation. RESULTS: Compared to the well-watered conditions, the level of drought stress across experiments varied from a minimum of - 25 to - 75 kPa at a soil depth of 30 cm which reduced biomass by 30-55% and grain yield by 1-92%, presenting a range of drought severities. OsTZF5 transgenic lines showed high yield advantage under drought over IR64 in early generations, which was related to shorter time to flowering, lower shoot biomass and higher harvest index. However, the increases in values for yield and related traits in the transgenics became smaller over successive generations despite continued detection of drought-induced transgene expression as conferred by the LIP9 promoter. The decreased advantage of the transgenics over generations tended to coincide with increased levels of homozygosity. Background cleaning of the transgenic lines as well as introgression of the transgene into an IR64 line containing major-effect drought yield QTLs, which were evaluated starting at the BC3F1 and BC2F3 generation, respectively, did not result in consistently increased yield under drought as compared to the respective checks. CONCLUSIONS: Although we cannot conclusively explain the genetic factors behind the loss of yield advantage of the transgenics under drought across generations, our results help in distinguishing among potential drought tolerance mechanisms related to effectiveness of the transgenics, since early flowering and harvest index most closely reflected the levels of yield advantage in the transgenics across generations while reduced biomass did not.

NAC transcription factors in plant abiotic stress responses.

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.

ABA signaling in stress-response and seed development.

KEY MESSAGE : We review the recent progress on ABA signaling, especially ABA signaling for ABA-dependent gene expression, including the AREB/ABF regulon, SnRK2 protein kinase, 2C-type protein phosphatases and ABA receptors. Drought negatively impacts plant growth and the productivity of crops. Drought causes osmotic stress to organisms, and the osmotic stress causes dehydration in plant cells. Abscisic acid (ABA) is produced under osmotic stress conditions, and it plays an important role in the stress response and tolerance of plants. ABA regulates many genes under osmotic stress conditions. It also regulates gene expression during seed development and germination. The ABA-responsive element (ABRE) is the major cis-element for ABA-responsive gene expression. ABRE-binding protein (AREB)/ABRE-binding factor (ABF) transcription factors (TFs) regulate ABRE-dependent gene expression. Other TFs are also involved in ABA-responsive gene expression. SNF1-related protein kinases 2 are the key regulators of ABA signaling including the AREB/ABF regulon. Recently, ABA receptors and group A 2C-type protein phosphatases were shown to govern the ABA signaling pathway. Moreover, recent studies have suggested that there are interactions between the major ABA signaling pathway and other signaling factors in stress-response and seed development. The control of the expression of ABA signaling factors may improve tolerance to environmental stresses.

Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration.

The loss of soybean yield to Brazilian producers because of a water deficit in the 2011-2012 season was 12.9%. To reduce such losses, molecular biology techniques, including plant transformation, can be used to insert genes of interest into conventional soybean cultivars to produce lines that are more tolerant to drought. The abscisic acid (ABA)-independent Dehydration Responsive Element Binding (DREB) gene family has been used to obtain plants with increased tolerance to abiotic stresses. In the present study, the rd29A:AtDREB2A CA gene from Arabidopsis thaliana was inserted into soybean using biolistics. Seventy-eight genetically modified (GM) soybean lines containing 2-17 copies of the AtDREB2A CA gene were produced. Two GM soybean lines (P1397 and P2193) were analyzed to assess the differential expression of the AtDREB2A CA transgene in leaves and roots submitted to various dehydration treatments. Both GM lines exhibited high expression of the transgene, with the roots of P2193 showing the highest expression levels during water deficit. Physiological parameters examined during water deficit confirmed the induction of stress. This analysis of AtDREB2A CA expression in GM soybean indicated that line P2193 had the greatest stability and highest expression in roots during water deficit-induced stress.

A Case of End-stage Renal Disease in Which Diazoxide was Effective in Treating Hypoglycemia Caused by Late Dumping Syndrome after Gastrectomy.

We herein report a case in which diazoxide was effective in treating reactive hypoglycemia caused by late dumping syndrome in a patient with ESRD. A 50-year-old man with end-stage renal disease (ESRD) and a history of gastrectomy underwent hemodialysis. Although he was administered voglibose to treat recurrent reactive hypoglycemia caused by late dumping syndrome, he had difficulty continuing treatment because of gastrointestinal side effects. When he began diazoxide treatment, the reactive hypoglycemia improved. The dose was gradually increased with no apparent side effects, and the hypoglycemic attacks disappeared one year after the start of treatment.

OsTZF1, a CCCH-tandem zinc finger protein gene, driven under own promoter produces no pleiotropic effects and confers salt and drought tolerance in rice.

Different abiotic stresses induce OsTZF1, a tandem CCCH-type zinc finger domain gene, in rice. Here, we report that transgenic rice plants overexpressing OsTZF1 under own promoter (POsTZF1:OsTZF1-OX [for overexpression]) transferred to soil showed normal growth similar to vector control plants. The POsTZF1:OsTZF1-OX produced normal leaves without any lesion mimic phenotype and exhibited normal seed setting. The POsTZF1:OsTZF1-OX plants showed significantly increased tolerance to salt and drought stresses and enhanced post stress recovery. Microarray analysis revealed a total of 846 genes up-regulated and 360 genes down-regulated in POsTZF1:OsTZF1-OX salt-treated plants. Microarray analysis of POsTZF1:OsTZF1-OX plants showed the regulation of many abiotic stress tolerance genes. These results suggest that OsTZF1-OX under own promoter show abiotic stress tolerance and produces no pleiotropic effect on phenotype of transgenic rice plant.

Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides.

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.

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.

Arabidopsis HsfA1 transcription factors function as the main positive regulators in heat shock-responsive gene expression.

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.

Genetic and Molecular Mechanisms Conferring Heat Stress Tolerance in Tomato Plants.

Climate change is a major threat to global food security. Changes in climate can directly impact food systems by reducing the production and genetic diversity of crops and their wild relatives, thereby restricting future options for breeding improved varieties and reducing the ability to adapt crops to future challenges. The global surface temperature is predicted to rise by an average of 0.3°C during the next decade, and the Paris Agreement (Paris Climate Accords) aims to limit global warming to below an average of 2°C, preferably to 1.5°C compared to pre-industrial levels. Even if the goal of the Paris Agreement can be met, the predicted rise in temperatures will increase the likelihood of extreme weather events, including heatwaves, making heat stress (HS) a major global abiotic stress factor for many crops. HS can have adverse effects on plant morphology, physiology, and biochemistry during all stages of vegetative and reproductive development. In fruiting vegetables, even moderate HS reduces fruit set and yields, and high temperatures may result in poor fruit quality. In this review, we emphasize the effects of abiotic stress, especially at high temperatures, on crop plants, such as tomatoes, touching upon key processes determining plant growth and yield. Specifically, we investigated the molecular mechanisms involved in HS tolerance and the challenges of developing heat-tolerant tomato varieties. Finally, we discuss a strategy for effectively improving the heat tolerance of vegetable crops.