A heat-shock 20 protein isolated from watermelon (ClHSP22.8) negatively regulates the response of Arabidopsis to salt stress via multiple signaling pathways.
ABSTRACT: Heat-shock protein 20s (HSP20) were initially shown to play a role during heat shock stress; however, recent data indicated that HSP20 proteins are also involved in abiotic stress in plants. Watermelon is known to be vulnerable to various stressors; however, HSP20 proteins have yet to be investigated and characterized in the watermelon. In a previous study, we identified a negative regulator of salt stress response from watermelon: ClHSP22.8, a member of the HSP20 family. Quantitative real-time PCR (qRT-PCR) and promoter::?-glucuronidase (GUS) analysis revealed that ClHSP22.8 was expressed widely in a range of different tissues from the watermelon, but particularly in the roots of 7-day-old seedlings and flowers. Furthermore, qRT-PCR and GUS staining showed that the expression of ClHSP22.8 was significantly repressed by exogenous abscisic acid (ABA) and salt stress. The over-expression of ClHSP22.8 in Arabidopsis lines resulted in hypersensitivity to ABA and reduced tolerance to salt stress. Furthermore, the expression patterns of key regulators associated with ABA-dependent and independent pathways, and other stress-responsive signaling pathways, were also repressed in transgenic lines that over-expressed ClHSP22.8. These results indicated that ClHSP22.8 is a negative regulator in plant response to salt stress and occurs via ABA-dependent and independent, and other stress-responsive signaling pathways.
Project description:Watermelon (Citrullus lanatus L.), which is an economically important cucurbit crop that is cultivated worldwide, is vulnerable to various adverse environmental conditions. Small heat shock protein 20s (HSP20s) are the most abundant plant HSPs and they play important roles in various biotic and abiotic stress responses. However, they have not been systematically investigated in watermelon. In this study, we identified 44 watermelon HSP20 genes and analyzed their gene structures, conserved domains, phylogenetic relationships, chromosomal distributions, and expression profiles. All of the watermelon HSP20 proteins have a conserved the ?-crystallin (ACD) domain. Half of the ClHSP20s arose through gene duplication events. Plant HSP20s were grouped into 18 subfamiles and a new subfamily, nucleo-cytoplasmic XIII (CXIII), was identified in this study. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter regions of the watermelon HSP20 genes. Different from that in other species, half of the watermelon HSP20s were repressed by heat stress. Plant HSP20s displayed diverse responses to different virus infections and most of the ClHSP20s were generally repressed by Cucumber green mottle mosaic virus (CGMMV). Some ClHSP20s exhibited similar transcriptional responses to abscisic acid, melatonin, and CGMMV. Subcellular localization analyses of six selected HSP20- green fluorescence protein fusion proteins revealed diverse subcellular targeting. Some ClHSP20 proteins were affected by CGMMV, as reflected by changes in the size, number, and distribution of fluorescent granules. These systematic analyses provide a foundation for elucidating the physiological functions and biological roles of the watermelon HSP20 gene family.
Project description:The plant DNA-binding with one finger (Dof) gene family is a class of plant-specific transcription factors that play vital roles in many biological processes and stress responses. In the present study, a total of 36 ClDof genes were identified in the watermelon genome, which were unevenly distributed on 10 chromosomes. Phylogenetic analysis showed that the ClDof proteins could be divided into nine groups, and the members in a particular group had similar motif arrangement and exon-intron structure. Synteny analysis indicated the presence of a large number of syntenic relationship events between watermelon and cucumber. In promoter analysis, five kinds of stress-related and nine kinds of hormone-related cis-elements were identified in the promoter regions of ClDof genes. We then analyzed the expression patterns of nine selected ClDof genes in eight specific tissues by qRT-PCR, and the results showed that they have tissue-specific expression patterns. We also evaluated the expression levels of 12 selected ClDof genes under salt stress and ABA treatments using qRT-PCR. As a result, they showed differential expression under these treatments, suggesting their important roles in stress response. Taken together, our results provide a basis for future research on the biological functions of Dof genes in watermelon.
Project description:To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant, brassinosteroid insensitive1-9 (bri1-9). To identify genes that are antagonistically regulated by ABA and BRs, we examined published Arabidopsis microarray data sets. Of the list of genes identified, those upregulated by ABA but downregulated by BRs were enriched with a BRRE motif in their promoter sequences. After validating the microarray data using quantitative RT-PCR, we focused on RD26, which is induced by salt stress. Histochemical analysis of transgenic Arabidopsis plants expressing RD26pro:GUS revealed that the induction of GUS expression after NaCl treatment was suppressed by co-treatment with BRs, but enhanced by co-treatment with propiconazole, a BR biosynthetic inhibitor. Similarly, treatment with bikinin, an inhibitor of BIN2 kinase, not only inhibited RD26 expression, but also reduced the survival rate of the plant following exposure to salt stress. Our results suggest that ABA and BRs act antagonistically on their target genes at or after the BIN2 step in BR signaling pathways, and suggest a mechanism by which plants fine-tune their growth, particularly when stress responses and growth compete for resources.
Project description:Apple (Malus domestica Borkh.), an economically important tree fruit worldwide, frequently suffers from temperature stress during growth and development, which strongly affects the yield and quality. Heat shock protein 20 (HSP20) genes play crucial roles in protecting plants against abiotic stresses. However, they have not been systematically investigated in apple. In this study, we identified 41 HSP20 genes in the apple 'Golden Delicious' genome. These genes were unequally distributed on 15 different chromosomes and were classified into 10 subfamilies based on phylogenetic analysis and predicted subcellular localization. Chromosome mapping and synteny analysis indicated that three pairs of apple HSP20 genes were tandemly duplicated. Sequence analysis revealed that all apple HSP20 proteins reflected high structure conservation and most apple HSP20 genes (92.6%) possessed no introns, or only one intron. Numerous apple HSP20 gene promoter sequences contained stress and hormone response cis-elements. Transcriptome analysis revealed that 35 of 41 apple HSP20 genes were nearly unchanged or downregulated under normal temperature and cold stress, whereas these genes exhibited high-expression levels under heat stress. Subsequent qRT-PCR results showed that 12 of 29 selected apple HSP20 genes were extremely up-regulated (more than 1,000-fold) after 4 h of heat stress. However, the heat-upregulated genes were barely expressed or downregulated in response to cold stress, which indicated their potential function in mediating the response of apple to heat stress. Taken together, these findings lay the foundation to functionally characterize HSP20 genes to unravel their exact role in heat defense response in apple.
Project description:Glutathione S-transferases (GSTs) constitute a large family of enzymes with a wide range of cellular functions. Recently, plant GSTs have gained a great deal of attention due to their involvement in the detoxification of electrophilic xenobiotics and peroxides under adverse environmental conditions, such as salt, cold, UV-B and drought stress. A previous study reported that a GST gene (<i>CsGSTU8</i>) in tea plant was distinctly induced in response to drought, suggesting this gene plays a critical role in the drought stress response. In this study, by using quantitative real-time PCR (qRT-PCR) and β-glucuronidase (GUS) reporter lines, we further demonstrated that <i>CsGSTU8</i> was upregulated in response to drought stress and exogenous abscisic acid (ABA) treatments. Overexpression of <i>CsGSTU8</i> in <i>Arabidopsis</i> resulted in enhanced drought tolerance as indicated by the improved scavenging of excess amounts of reactive oxygen species (ROS) under drought conditions. Furthermore, we found that CsWRKY48 acts as a transcriptional activator and that its expression is induced in response to drought stress and ABA treatment. Electrophoretic mobility shift assays (EMSAs), dual-luciferase (LUC) assays and transient expression assays in tea plant leaves revealed that CsWRKY48 directly binds to the W-box elements in the promoter of <i>CsGSTU8</i> and activates its expression. Taken together, our results provide additional knowledge of drought stress responses in tea plant.
Project description:Heat shock proteins (Hsps) are common molecular chaperones present in all plants that accumulate in response to abiotic stress. Small heat shock proteins (sHsps) play important roles in alleviating diverse abiotic stresses, especially heat stress. However, very little is known about the MsHsp20 gene family in the wild apple Malus sieversii, a precious germplasm resource with excellent resistance characteristics. In this study, 12 putative M. sieversii Hsp20 genes were identified from RNA-Seq data and analyzed in terms of gene structure and phylogenetic relationships. A new Hsp20 gene, MsHsp16.9, was cloned and its function studied in response to stress. MsHsp16.9 expression was strongly induced by heat, and transgenic Arabidopsis plants overexpressing MsHsp16.9 displayed improved heat resistance, enhanced antioxidant enzyme activity, and decreased peroxide content. Overexpression of MsHsp16.9 did not alter the growth or development under normal conditions, or the hypersensitivity to exogenous ABA. Gene expression analysis indicated that MsHsp16.9 mainly modulates the expression of proteins involved in antioxidant enzyme synthesis, as well as ABA-independent stress signaling in 35S:MsHsp16.9-L11. However, MsHsp16.9 could activate ABA-dependent signaling pathways in all transgenic plants. Additionally, MsHsp16.9 may function alongside AtHsp70 to maintain protein homeostasis and protect against cell damage. Our results suggest that MsHsp16.9 is a protein chaperone that positively regulates antioxidant enzyme activity and ABA-dependent and independent signaling pathway to attenuate plant responses to severe stress. Transgenic plants exhibited luxuriant growth in high temperature environments.
Project description:MiR399f plays a crucial role in maintaining phosphate homeostasis in Arabidopsis thaliana. Under phosphate starvation conditions, AtMYB2, which plays a role in plant salt and drought stress responses, directly regulates the expression of miR399f. In this study, we found that miR399f also participates in plant responses to abscisic acid (ABA), and to abiotic stresses including salt and drought. Salt and ABA treatment induced the expression of miR399f, as confirmed by histochemical analysis of promoter-GUS fusions. Transgenic Arabidopsis plants overexpressing miR399f (miR399f-OE) exhibited enhanced tolerance to salt stress and exogenous ABA, but hypersensitivity to drought. Our in silico analysis identified ABF3 and CSP41b as putative target genes of miR399f, and expression analysis revealed that mRNA levels of ABF3 and CSP41b decreased remarkably in miR399f-OE plants under salt stress and in response to treatment with ABA. Moreover, we showed that activation of stress-responsive gene expression in response to salt stress and ABA treatment was impaired in miR399f-OE plants. Thus, these results suggested that in addition to phosphate starvation signaling, miR399f might also modulates plant responses to salt, ABA, and drought, by regulating the expression of newly discovered target genes such as ABF3 and CSP41b.
Project description:The TIFY gene family is plant-specific and encodes proteins involved in the regulation of multiple biological processes. Here, we identified 15 TIFY genes in the watermelon genome, which were divided into four subfamilies (eight JAZs, four ZMLs, two TIFYs, and one PPD) in the phylogenetic tree. The ClTIFY genes were unevenly located on eight chromosomes, and three segmental duplication events and one tandem duplication event were identified, suggesting that gene duplication plays a vital role in the expansion of the TIFY gene family in watermelon. Further analysis of the protein architectures, conserved domains, and gene structures provided additional clues for understanding the putative functions of the TIFY family members. Analysis of qRT-PCR and RNA-seq data revealed that the detected ClTIFY genes had preferential expression in specific tissues. qRT-PCR analysis revealed that nine selected TIFY genes were responsive to jasmonic acid (JA) and abiotic stresses including salt and drought. JA activated eight genes and suppressed one gene, among which ClJAZ1 and ClJAZ7 were the most significantly induced. Salt and drought stress activated nearly all the detected genes to different degrees. These results lay a foundation for further functional characterization of TIFY family genes in Citrullus lanatus.
Project description:<h4>Background</h4> NAC (NAM, ATAF and CUC) transcription factors (TFs) play vital roles in plant development and abiotic stress tolerance. Salt stress is one of the most limiting factors for rice growth and production. However, the mechanism underlying salt tolerance in rice is still poorly understood. <h4>Results</h4> In this study, we functionally characterized a rice NAC TF OsNAC3 for its involvement in ABA response and salt tolerance. ABA and NaCl treatment induced OsNAC3 expression in roots. Immunostaining showed that OsNAC3 was localized in all root cells. OsNAC3 knockout decreased rice plants’ sensitivity to ABA but increased salt stress sensitivity, while OsNAC3 overexpression showed an opposite effect. Loss of OsNAC3 also induced Na+ accumulation in the shoots. Furthermore, qRT-PCR and transcriptomic analysis were performed to identify the key OsNAC3 regulated genes related to ABA response and salt tolerance, such as OsHKT1;4, OsHKT1;5, OsLEA3–1, OsPM-1, OsPP2C68, and OsRAB-21. <h4>Conclusions</h4> This study shows that rice OsNAC3 is an important regulatory factor in ABA signal response and salt tolerance. <h4>Supplementary Information</h4> The online version contains supplementary material available at 10.1186/s12870-021-03333-7.
Project description:The caleosin (CLO) protein family displays calcium-binding properties and plays an important role in the abiotic stress response. Here, a total of 107 CLO genes were identified in 15 plant species, while no CLO genes were detected in two green algal species. Evolutionary analysis revealed that the CLO gene family may have evolved mainly in terrestrial plants and that biological functional differentiation between species and functional expansion within species have occurred. Of these, 56 CLO genes were identified in four cotton species. Collinearity analysis showed that CLO gene family expansion mainly occurred through segmental duplication and whole-genome duplication in cotton. Sequence alignment and phylogenetic analysis showed that the CLO proteins of the four cotton species were mainly divided into two types: H-caleosins (class I) and L-caleosins (class II). Cis-acting element analysis and quantitative RT–PCR (qRT–PCR) suggested that GhCLOs might be regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). Moreover, transcriptome data and qRT–PCR results revealed that GhCLO genes responded to salt and drought stresses. Under salt stress, gene-silenced plants (TRV: GhCLO06) showed obvious yellowing and wilting, higher malondialdehyde (MDA) content accumulation, and significantly lower activities of superoxide dismutase (SOD) and peroxidase (POD), indicating that GhCLO06 plays a positive regulatory role in cotton salt tolerance. In gene-silenced plants (TRV: GhCLO06), ABA-related genes (GhABF2, GhABI5, and GhNAC4) were significantly upregulated after salt stress, suggesting that the regulation of salt tolerance may be related to the ABA signaling pathway. This research provides an important reference for further understanding and analyzing the molecular regulatory mechanism of CLOs for salt tolerance.