Heat Stress Transcription Factors Research Articles

Ectopic Overexpression of Maize Heat Stress Transcription Factor ZmHsf05 Confers Drought Tolerance in Transgenic Rice.

Drought is a key factor affecting plant growth and development. Heat shock transcription factors (Hsfs) have been reported to respond to diverse abiotic stresses, including drought stress. In the present study, functional characterization of maize heat shock transcription factor 05 (ZmHsf05) gene was conducted. Homologous analysis showed that ZmHsf05 belongs to Class A2 Hsfs. The mRNA expression level of ZmHsf05 can be affected by drought, high temperature, salt, and abscisic acid (ABA) treatment. Ectopic overexpression of ZmHsf05 in rice (Oryza sativa) could significantly enhance the drought tolerance. Faced with drought stress, transgenic rice exhibited better phenotypic performance, higher survival rate, higher proline content, and lower leaf water loss rate, compared with wild-type plant Zhonghua11. Additionally, we assessed the agronomic traits of seven transgenic rice lines overexpressing ZmHsf05 and found that ZmHsf05 altered agronomical traits in the field trials. Moreover, rice overexpressing ZmHsf05 was more sensitive to ABA and had either a lower germination rate or shorter shoot length under ABA treatment. The transcription level of key genes in the ABA synthesis and drought-related pathway were significantly improved in transgenic rice after drought stress. Collectively, our results showed that ZmHsf05 could improve drought tolerance in rice, likely in an ABA-dependent manner.

Transcriptomics Analysis of Heat Stress-Induced Genes in Pepper (Capsicum annuum L.) Seedlings

Pepper (Capsicum annuum L.) is one of the most economically important crops worldwide. Heat stress (HS) can significantly reduce pepper yield and quality. However, changes at a molecular level in response to HS and the subsequent recovery are poorly understood. In this study, 17-03 and H1023 were identified as heat-tolerant and heat-sensitive varieties, respectively. Their leaves’ transcript abundance was quantified using RNA sequencing to elucidate the effect of HS and subsequent recovery on gene expression. A total of 11,633 differentially expressed genes (DEGs) were identified, and the differential expression of 14 randomly selected DEGs was validated using reverse-transcription polymerase chain reaction. Functional enrichment analysis revealed that the most enriched pathways were metabolic processes under stress and photosynthesis and light harvesting during HS and after recovery from HS. The most significantly enriched pathways of 17-03 and H1023 were the same under HS, but differed during recovery. Furthermore, we identified 38 heat shock factors (Hsps), 17 HS transcription factors (Hsfs) and 38 NAC (NAM, ATAF1/2, and CUC2), and 35 WRKY proteins that were responsive to HS or recovery. These findings facilitate a better understanding of the molecular mechanisms underlying HS and recovery in different pepper genotypes.

Exploring the master regulator heat stress transcription factor HSFA1a-mediated transcriptional cascade of HSFs in the heat stress response of tomato

Exploring the master regulator heat stress transcription factor HSFA1a-mediated transcriptional cascade of HSFs in the heat stress response of tomato

Nitric oxide induced thermotolerance in strawberry plants by activation of antioxidant systems and transcriptional regulation of heat shock proteins

ABSTRACT An experiment was conducted to examine whether application of sodium nitroprusside (SNP), a donor of NO, can improve thermo-tolerance of strawberry (Fragaria × ananassa) plants through inducing antioxidant system, and up-regulating heat stress transcription factors (HSFs) and heat shock proteins (HSPs) genes. Ventana strawberry plants were exposed to various temperatures (25, 35, and 40°C) for 24 h after pre-treatment with 0, 50, and 100 μM SNP. Heat stress significantly induced malondialdehyde and hydrogen peroxide (H2O2) contents, and increased accumulation of proline, whereas reduced relative water content (RWC), leaf chlorophyll fluorescence, and carotenoid content. In addition, heat stress enhanced superoxide dismutase and guaiacol peroxidase activities, increased glutathione and ascorbic acid contents, and reduced catalase and ascorbate peroxidase activities. Pre-treatment with SNP, especially at 100 μM, ameliorated heat injury by controlling the overaccumulation of H2O2, reducing lipid peroxidation, improvement of RWC, and increasing the enzymatic and non-enzymatic antioxidants. Transcriptomic profiling analysis showed that the expression of FaTHSFA2a, FaTHSFB1a, HSP70, and HSP90 in SNP pre-treated plants was significantly higher than non-treated plants after 2 h of heat stress at 40°C. These results suggested that NO alleviates heat-induced oxidative damage by modulating antioxidant pathways and fast inducing the expression of heat-stress related genes.

Identification and Regulation of Tomato Serine/Arginine-Rich Proteins Under High Temperatures.

Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.

Reprogramming of Tomato Leaf Metabolome by the Activity of Heat Stress Transcription Factor HsfB1.

Plants respond to high temperatures with global changes of the transcriptome, proteome, and metabolome. Heat stress transcription factors (Hsfs) are the core regulators of transcriptome responses as they control the reprogramming of expression of hundreds of genes. The thermotolerance-related function of Hsfs is mainly based on the regulation of many heat shock proteins (HSPs). Instead, the Hsf-dependent reprogramming of metabolic pathways and their contribution to thermotolerance are not well described. In tomato (Solanum lycopersicum), manipulation of HsfB1, either by suppression or overexpression (OE) leads to enhanced thermotolerance and coincides with distinct profile of metabolic routes based on a metabolome profiling of wild-type (WT) and HsfB1 transgenic plants. Leaves of HsfB1 knock-down plants show an accumulation of metabolites with a positive effect on thermotolerance such as the sugars sucrose and glucose and the polyamine putrescine. OE of HsfB1 leads to the accumulation of products of the phenylpropanoid and flavonoid pathways, including several caffeoyl quinic acid isomers. The latter is due to the enhanced transcription of genes coding key enzymes in both pathways, in some cases in both non-stressed and stressed plants. Our results show that beyond the control of the expression of Hsfs and HSPs, HsfB1 has a wider activity range by regulating important metabolic pathways providing an important link between stress response and physiological tomato development.

Overexpression of wheat transcription factor (TaHsfA6b) provides thermotolerance in barley.

Overexpressing a heat shock factor gene (TaHsfA6bT) from wheat provides thermotolerance in barley by constitutive expression of heat and other abiotic stress-response genes. Temperature is one of the most crucial abiotic factors defining the yield potential of temperate cereal crops, such as barley. The regulators of heat shock response (HSR), heat stress transcription factors (Hsfs), modulate the transcription level of heat-responsive genes to protect the plants from heat stress. In this study, an Hsf from wheat (TaHsfA6b) is overexpressed in barley for providing thermotolerance. Transgenic barley lines overexpressing TaHsfA6b showed improvement in thermotolerance. The constitutive overexpression of a TaHsfA6b gene upregulated the expression of major heat shock proteins and other abiotic stress-responsive genes. RNA-seq and qRT-PCR analysis confirmed the upregulation of Hsps, chaperonins, DNAJ, LEA protein genes, and genes related to anti-oxidative enzymes in transgenic lines. Excessive generation and accumulation of reactive oxygen species (ROS) occurred in wild-type (WT) plants during heat stress; however, the transgenic lines reflected improved ROS homeostasis mechanisms, showing lesser ROS accumulation under high temperature. No negative phenotypic changes were observed in overexpression lines. These results suggest that TaHsfA6b is a regulator of HSR and its overexpression altered the expression patterns of some main stress-related genes and enhanced the thermotolerance of this cereal crop.

Transcriptome profiling and gene expression analyses of eggplant (Solanum melongena L.) under heat stress.

Global warming induces heat stress in eggplant, seriously affecting its quality and yield. The response to heat stress is a complex regulatory process; however, the exact mechanism in eggplant is unknown. We analyzed the transcriptome of eggplant under different high-temperature treatments using RNA-Seq technology. Three libraries treated at high temperatures were generated and sequenced. There were 40,733,667, 40,833,852, and 40,301,285 clean reads with 83.98%, 79.69%, and 84.42% of sequences mapped to the eggplant reference genome in groups exposed to 28°C (CK), 38°C (T38), and 43°C (T43), respectively. There were 3,067 and 1,456 DEGs in T38 vs CK and T43 vs CK groups, respectively. In these two DEG groups, 315 and 342 genes were up- and down-regulated, respectively, in common. Differential expression patterns of DEGs in antioxidant enzyme systems, detoxication, phytohormones, and transcription factors under heat stress were investigated. We screened heat stress-related genes for further validation by qRT-PCR. Regulation mechanisms may differ under different temperature treatments, in which heat shock proteins and heat stress transcription factors play vital roles. These results provide insight into the molecular mechanisms of the heat stress response in eggplant and may be useful in crop breeding.

Transcriptomic insights into the roles of HSP70-16 in sepal’s responses to developmental and mild heat stress signals

Plant heat stress response (HSR) involves many heat shock proteins (HSPs), each might has distinct function. Thus, understanding of molecular bases of corresponding responses of each HSP in plant is essential for both basic and applied researches. While previous work focused dominantly on high temperature heat stress, studies on mild heat stress (about 5 °C over the optimal growth temperature) are rare. HSP70-16 is required for normal function of lateral sepals during flower opening at normal and mild heat stress, however, its underlying mechanism remains obscure. In this study, we compared transcriptomes of wild type and hsp70-16 sepals grown under normal (22 °C) and mild heat stress (27 °C) temperatures. Combined with weighted gene co-expression network analysis (WGCNA), we uncovered a HSP70-16 mediated molecular network that integrates both developmental and mild heat stress signals in Arabidopsis sepals, functioning to maintain protein homeostasis and metabolite homeostasis through a serial of network and hub genes including those encoding protein kinases, transcription factors, heat stress transcription factors, HSPs, hormones, reactive oxygen species, lipid/cuticle and cell wall components. Overall, the output of this study will provide an excellent resource for future characterization of HSPs to understand plant and heat stress interaction and obtain acquired thermos-tolerant plants.

Genomic analyses of heat stress transcription factors (HSFs) in simulated drought stress response and storage root deterioration after harvest in cassava.

Heat shock factors (HSFs) play crucial roles in various plant stress responses. However, the current knowledge about HSFs in cassava, an important crop, is still insufficient. In this research, we identified 32 cassava HSF genes (MeHSFs) and clustered them into three groups (A, B, C) based on phylogenetic analysis and structural characteristics. Conserved motif analyses showed that MeHSFs display domains characteristic to HSF transcription factors. Gene structure analyses suggested that 29 MeHSFs contained only two exons. All identified 32 cassava MeHSFs were distributed on 13 chromosomes. Their expression profiles revealed that the different MeHSFs were expressed differentially in different tissues, most high expression genes belonged to group A. The similar MeHSFs were up-regulated after treatment with both PEG and abscisic acid (ABA), which implied that these MeHSFs may participate in resistance to simulated drought stress associated with the ABA signaling pathway. In addition, several MeHSFs were induced during postharvest physiological deterioration (PPD) in cassava. Our results provided basic but important knowledge for future gene function analysis of MeHSFs toward efforts in improving tolerance to abiotic stress and PPD in cassava.

Genome-wide identification and transcript profiles of walnut heat stress transcription factor involved in abiotic stress

BackgroundWalnut (Juglans regia) is an important tree cultivated worldwide and is exposed to a series of both abiotic and biotic stress during their life-cycles. The heat stress transcription factors (HSFs) play a crucial role in plant response to various stresses by regulating the expression of stress-responsive genes. HSF genes are classified into 3 classes: HSFA, HSFB, and HSFC. HSFA gene has transcriptional activation function and is the main regulator of high temperature-induced gene expression. HSFB gene negatively regulates plant resistance to drought and NaCl. And HSFC gene may be involved in plant response to various stresses. There are some reports about the HSF family in herbaceous plants, however, there are no reports about the HSFs in walnut.ResultIn this study, based on the complete genome sequencing of walnut, the bioinformatics method was used and 29 HSF genes were identified. These HSFs covered 18 HSFA, 9 HSFB, and 2 HSFC genes. Phylogenetic analysis of these HSF proteins along with those from Arabidopsis thaliana showed that the HSFs in the two species are closely related to each other and have different evolutionary processes. The distribution of conserved motifs and the sequence analysis of HSF genes family indicated that the members of the walnut HSFs are highly conserved. Quantitative Real-Time PCR (qRT-PCR) analysis revealed that the most of walnut HSFs were expressed in the walnut varieties of ‘Qingxiang’ and ‘Xiangling’ under high temperature (HT), high salt and drought stress, and some JrHSFs expression pattern are different between the two varieties.ConclusionThe complex HSF genes family from walnut was confirmed by genome-wide identification, evolutionary exploration, sequence characterization and expression analysis. This research provides useful information for future studies on the function of the HSF genes and molecular mechanism in plant stress response.

MiRNAs involved in transcriptome remodeling during pollen development and heat stress response in Solanum lycopersicum

Cellular transitions during development and stress response depend on coordinated transcriptomic and proteomic alterations. Pollen is particular because its development is a complex process that includes meiotic and mitotic divisions which causes a high heat sensitivity of these cells. Development and stress response are accompanied by a reprogramming of the transcriptome, e.g. by post-transcriptional regulation via miRNAs. We identified known and potentially novel miRNAs in the transcriptome of developing and heat-stressed pollen of Solanum lycopersicum (tomato). The prediction of target mRNAs yielded an equal number of predicted target-sites in CDS and 3′UTR regions of target mRNAs. The result enabled the postulation of a possible link between miRNAs and a fine-tuning of transcription factor abundance during pollen development. miRNAs seem to play a role in the pollen heat stress response as well. We identified several heat stress transcription factors and heat shock proteins as putative targets of miRNAs in response to heat stress, thereby placing these miRNAs as important elements of thermotolerance. Moreover, for members of the AP2, SBP and ARF family members we could predict a miRNA-mediated regulation during development via the miR172, mir156 and mir160-family strengthening the current concept of a cross-connection between development and stress response in plants.

Transcriptomic analysis of short-term heat stress response in Pinellia ternata provided novel insights into the improved thermotolerance by spermidine and melatonin

Heat stress has been a major environmental factor limiting the growth and development of Pinellia ternata which is an important Chinese traditional medicine. It has been reported that spermidine (SPD) and melatonin (MLT) play pivotal roles in modulating heat stress response (HSR). However, the roles of SPD and MLT in HSR of P. ternata, and the potential mechanism is still unknown. Here, exogenous SPD and MLT treatments alleviated heat-induced damages in P. ternata, which was supported by the increased chlorophyll content, OJIP curve, and relative water content, and the decreased malondialdehyde and electrolyte leakage. Then, RNA sequencing between CK (control) and Heat (1 h of heat treatment) was conducted to analyze how genes were in response to short-term heat stress in P. ternata. A total of 14,243 (7870 up- and 6373 down-regulated) unigenes were differentially expressed after 1 h of heat treatment. Bioinformatics analysis revealed heat-responsive genes mainly included heat shock proteins (HSPs), ribosomal proteins, ROS-scavenging enzymes, genes involved in calcium signaling, hormone signaling transduction, photosynthesis, pathogen resistance, and transcription factors such as heat stress transcription factors (HSFs), NACs, WRKYs, and bZIPs. Among them, PtABI5, PtNAC042, PtZIP17, PtSOD1, PtHSF30, PtHSFB2b, PtERF095, PtWRKY75, PtGST1, PtHSP23.2, PtHSP70, and PtLHC1 were significantly regulated by SPD or MLT treatment with same or different trends under heat stress condition, indicating that exogenous application of MLT and SPD might enhance heat tolerance in P. ternata through regulating these genes but may with different regulatory patterns. These findings contributed to the identification of potential genes involved in short-term HSR and the improved thermotolerance by MLT and SPD in P. ternata, which provided important clues for improving thermotolerance of P. ternata.

Transcriptional Basis for Differential Thermosensitivity of Seedlings of Various Tomato Genotypes.

Transcriptional reprograming after the exposure of plants to elevated temperatures is a hallmark of stress response which is required for the manifestation of thermotolerance. Central transcription factors regulate the stress survival and recovery mechanisms and many of the core responses controlled by these factors are well described. In turn, pathways and specific genes contributing to variations in the thermotolerance capacity even among closely related plant genotypes are not well defined. A seedling-based assay was developed to directly compare the growth and transcriptome response to heat stress in four tomato genotypes with contrasting thermotolerance. The conserved and the genotype-specific alterations of mRNA abundance in response to heat stress were monitored after exposure to three different temperatures. The transcripts of the majority of genes behave similarly in all genotypes, including the majority of heat stress transcription factors and heat shock proteins, but also genes involved in photosynthesis and mitochondrial ATP production. In turn, genes involved in hormone and RNA-based regulation, such as auxin- and ethylene-related genes, or transcription factors like HsfA6b, show a differential regulation that associates with the thermotolerance pattern. Our results provide an inventory of genes likely involved in core and genotype-dependent heat stress response mechanisms with putative role in thermotolerance in tomato seedlings.

Natural variation in HsfA2 pre-mRNA splicing is associated with changes in thermotolerance during tomato domestication.

Wild relatives of crops thrive in habitats where environmental conditions can be restrictive for productivity and survival of cultivated species. The genetic basis of this variability, particularly for tolerance to high temperatures, is not well understood. We examined the capacity of wild and cultivated accessions to acclimate to rapid temperature elevations that cause heat stress (HS). We investigated genotypic variation in thermotolerance of seedlings of wild and cultivated accessions. The contribution of polymorphisms associated with thermotolerance variation was examined regarding alterations in function of the identified gene. We show that tomato germplasm underwent a progressive loss of acclimation to strong temperature elevations. Sensitivity is associated with intronic polymorphisms in the HS transcription factor HsfA2 which affect the splicing efficiency of its pre-mRNA. Intron splicing in wild species results in increased synthesis of isoform HsfA2-II, implicated in the early stress response, at the expense of HsfA2-I which is involved in establishing short-term acclimation and thermotolerance. We propose that the selection for modern HsfA2 haplotypes reduced the ability of cultivated tomatoes to rapidly acclimate to temperature elevations, but enhanced their short-term acclimation capacity. Hence, we provide evidence that alternative splicing has a central role in the definition of plant fitness plasticity to stressful conditions.

Alternative Splicing Provides a Mechanism to Regulate LlHSFA3 Function in Response to Heat Stress in Lily.

Heat stress transcription factors (HSFs) are central regulators of plant responses to heat stress. Their heat-induced transcriptional regulation has been extensively studied; however, their posttranscriptional and posttranslational regulation is poorly understood. In a previous study, we established that there were at least two HSFA3 homologs, LlHSFA3A and LlHSFA3B, in lily (Lilium spp.) and that these genes played distinct roles in thermotolerance. Here, we demonstrate that LlHSFA3B is alternatively spliced under heat stress to produce the heat-inducible splice variant LlHSFA3B-III We further show that LlHSFA3B-III protein localizes in the cytoplasm and nucleus, has no transcriptional activity, and specifically disturbs the protein interactions of intact HSFA3 orthologs LlHSFA3A-I and LlHSFA3B-I. Heterologous expression of LlHSFA3B-III in Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana increased plant tolerance of salt and prolonged heat at 40°C, yet reduced plant tolerance of acute heat shock at 45°C. Conversely, heterologous expression of LlHSFA3A-I caused opposing phenotypes, which were substantially ameliorated by coexpression of LlHSFA3B-III LlHSFA3B-III interacted with LlHSFA3A-I to limit its transactivation function and temper the function of LlHSFA3A-I, thus reducing the adverse effects of excessive LlHSFA3A-I accumulation. Based on these observations, we propose a regulatory mechanism of HSFs involving heat-inducible alternative splicing and protein interaction, which might be used in strategies to promote thermotolerance and attenuate the heat stress response in crop plants.

Heat stress transcription factor OsSPL7 plays a critical role in reactive oxygen species balance and stress responses in rice

The rice spotted leaf gene, OsSPL7, induces lesion mimic (LM) spots under heat stress. Herein, we provide several lines of evidence elucidating the importance of OsSPL7 in maintaining reactive oxygen species (ROS) balance via the regulation of downstream gene expression. osspl7 knockout (spl7ko) mutants showed LM and growth retardation. Transgenic rice lines strongly overexpressing OsSPL7 (SPL7OX-S) exhibited LM accompanied by accumulated H2O2, whereas moderate expressers of OsSPL7 (SPL7OX-M) did not, and neither of them exhibited severe growth defects. Transient expression of OsSPL7-GFP in rice protoplasts indicated that OsSPL7 localizes predominantly in the nucleus. Transcriptional activity assay suggested its function as a transcriptional activator in rice. Disease evaluation showed that both SPL7OX and spl7ko enhanced resistance to Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, the causal agents of blast and blight diseases in rice, respectively. Additionally, SPL7OX enhanced tolerance to cold stress, whereas spl7ko showed a phenotype opposite to the overexpression lines. RNA sequencing analyses identified four major groups of differentially expressed genes associated with LM, pathogen resistance, LM-pathogen resistance, and potential direct targets of OsSPL7. Collectively, our results suggest that OsSPL7 plays a critical role in plant growth and balancing ROS during biotic and abiotic stress.

Transcriptomic profiling of sorghum leaves and roots responsive to drought stress at the seedling stage

Drought stress affects the growth and productivity of crop plants including sorghum. To study the molecular basis of drought tolerance in sorghum, we conducted the transcriptomic profiling of sorghum leaves and roots under drought stress using RNA-Seq method. A total of 510, 559, and 3 687 differentially expressed genes (DEGs) in leaves, 3 368, 5 093, and 4 635 DEGs in roots responding to mild drought, severe drought, and re-watering treatments were identified, respectively. Among them, 190 common DEGs in leaves and 1 644 common DEGs in roots were responsive to mild drought, severe drought, and re-watering environment. Gene Ontology (GO) enrichment analysis revealed that the GO categories related to drought tolerance include terms related to response to stimulus especially response to water deprivation, abscisic acid stimulus, and reactive oxygen species. The major transcription factor genes responsive to drought stress include heat stress transcription factor (HSF), ethylene-responsive transcription factor (ERF), Petunia NAM, Arabidopsis ATAF1/2 and CUC2 (NAC), WRKY transcription factor (WRKY), homeodomain leucine zipper transcription factor (HD-ZIP), basic helix-loop-helix transcription factor (bHLH), and V-myb myeloblastosis viral oncogene homolog transcription facotr (MYB). Functional protein genes for heat shock protein (HSPs), late-embryogenesis-abundant protein (LEAs), chaperones, aquaporins, and expansins might play important roles in sorghum drought tolerance. Moreover, the genomic regions enriched with HSP, expansin, and aquaporin genes responsive to drought stress could be used as powerful targets for improvement of drought tolerance in sorghum and other cereals. Overall, our results provide a genome-wide analysis of DEGs in sorghum leaves and roots under mild drought, severe drought, and re-watering environments. This study contributes to a better understanding of the molecular basis of drought tolerance of sorghum and can be useful for crop improvement.

Knockout of SlMAPK3 enhances tolerance to heat stress involving ROS homeostasis in tomato plants

BackgroundHigh temperature is a major environmental stress that limits plant growth and agriculture productivity. Mitogen-activated protein kinases (MAPKs) are highly conserved serine and threonine protein kinases that participate in response to diverse environmental stresses in plants. A total of 16 putative SlMAPK genes are identified in tomato, and SlMAPK3 is one of the most extensively studied SlMAPKs. However, the role of SlMAPK3 in response to heat stress is not clearly understood in tomato plants. In this study, we performed functional analysis of SlMAPK3 for its possible role in response to heat stress.ResultsqRT-PCR analyses revealed that SlMAPK3 relative expression was depressed by heat stress. Here, wild-type (WT) tomato plants and CRISPR/Cas9-mediated slmapk3 mutant lines (L8 and L13) were used to investigate the function of SlMAPK3 in response to heat stress. Compared with WT plants, slmapk3 mutants exhibited less severe wilting and less membrane damage, showed lower reactive oxygen species (ROS) contents, and presented higher both activities and transcript levels of antioxidant enzymes, as well as elevated expressions of genes encoding heat stress transcription factors (HSFs) and heat shock proteins (HSPs).ConclusionsCRISPR/Cas9-mediated slmapk3 mutants exhibited more tolerance to heat stress than WT plants, suggesting that SlMAPK3 was a negative regulator of thermotolerance. Moreover, antioxidant enzymes and HSPs/HSFs genes expression were involved in SlMAPK3-mediated heat stress response in tomato plants.

Functional diversification of tomato HsfA1 factors is based on DNA binding domain properties

In all eukaryotes, the response to heat stress (HS) is dependent on the activity of HS transcription factors (Hsfs). Plants contain a large number of Hsfs, however, only members of the HsfA1 subfamily are considered as master regulators of stress response and thermotolerance. In Solanum lycopersicum, among the four HsfA1 members, only HsfA1a has been proposed to possess a master regulator function. We performed a comparative analysis of HsfA1a, HsfA1b, HsfA1c and HsfA1e at different levels of regulation and function. HsfA1a is constitutively expressed under control and stress conditions, while the other members are induced in specific tissues and stages of HS response. Despite that all members are localized in the nucleus when expressed in protoplasts, only HsfA1a shows a wide range of basal activity on several HS-induced genes. In contrast, HsfA1b, HsfA1c, and HsfA1e show only high activity for specific subsets of genes. Domain swapping mutants between HsfA1a and HsfA1c revealed that the variation in that transcriptional transactivation activity is due to differences in the DNA binding domain (DBD). Specifically, we identified a conserved arginine (R107) residue in the turn of β3 and β4 sheet in the C-terminus of the DBD of HsfA1a that is highly conserved in plant HsfA1 proteins, but is replaced by leucine and cysteine in tomato HsfA1c and HsfA1e, respectively. Although not directly involved in DNA interaction, R107 contributes to DNA binding and consequently the activity of HsfA1a. Thus, we demonstrate that this variation in DBD in part explains the functional diversification of tomato HsfA1 members.