The RNA-seq transcriptomic analysis reveals genes mediating salt tolerance through rapid triggering of ion transporters in a mutant barley.
ABSTRACT: Considering the complex nature of salinity tolerance mechanisms, the use of isogenic lines or mutants possessing the same genetic background albeit different tolerance to salinity is a suitable method for reduction of analytical complexity to study these mechanisms. In the present study, whole transcriptome analysis was evaluated using RNA-seq method between a salt-tolerant mutant line "M4-73-30" and its wild-type "Zarjou" cultivar at seedling stage after six hours of exposure to salt stress (300 mM NaCl). Transcriptome sequencing yielded 20 million reads for each genotype. A total number of 7116 transcripts with differential expression were identified, 1586 and 1479 of which were obtained with significantly increased expression in the mutant and the wild-type, respectively. In addition, the families of WRKY, ERF, AP2/EREBP, NAC, CTR/DRE, AP2/ERF, MAD, MIKC, HSF, and bZIP were identified as the important transcription factors with specific expression in the mutant genotype. The RNA-seq results were confirmed at several time points using qRT-PCR for some important salt-responsive genes. In general, the results revealed that the mutant accumulated higher levels of sodium ion in the root and decreased its transfer to the shoot. Also, the mutant increased the amount of potassium ion leading to the maintenance a high ratio [K+]/[Na+] in the shoot compared to its wild-type via fast stomata closure and consequently transpiration reduction under the salt stress. Moreover, a reduction in photosynthesis and respiration was observed in the mutant, resulting in utilization of the stored energy and the carbon for maintaining the plant tissues, which is considered as a mechanism of salt tolerance in plants. Up-regulation of catalase, peroxidase, and ascorbate peroxidase genes has resulted in higher accumulation of H2O2 in the wild-type compared to the mutant. Therefore, the wild-type initiated rapid ROS signals which led to less oxidative scavenging in comparison with the mutant. The mutant increased expression in the ion transporters and the channels related to the salinity to maintain the ion homeostasis. In overall, the results demonstrated that the mutant responded better to the salt stress under both osmotic and ionic stress phases and lower damage was observed in the mutant compared to its wild-type under the salt stress.
Project description:The AP2/ERF transcription factors play crucial roles in plant growth, development and responses to biotic and abiotic stresses. A total of 119 AP2/ERF genes (JcAP2/ERFs) have been identified in the physic nut genome; they include 16 AP2, 4 RAV, 1 Soloist, and 98 ERF genes. Phylogenetic analysis indicated that physic nut AP2 genes could be divided into 3 subgroups, while ERF genes could be classed into 11 groups or 43 subgroups. The AP2/ERF genes are non-randomly distributed across the 11 linkage groups of the physic nut genome and retain many duplicates which arose from ancient duplication events. The expression patterns of several JcAP2/ERF duplicates in the physic nut showed differences among four tissues (root, stem, leaf, and seed), and 38 JcAP2/ERF genes responded to at least one abiotic stressor (drought, salinity, phosphate starvation, and nitrogen starvation) in leaves and/or roots according to analysis of digital gene expression tag data. The expression of JcERF011 was downregulated by salinity stress in physic nut roots. Overexpression of the JcERF011 gene in rice plants increased its sensitivity to salinity stress. The increased expression levels of several salt tolerance-related genes were impaired in the JcERF011-overexpressing plants under salinity stress.
Project description:AP2/ERF transcription factors (TFs) play indispensable roles in plant growth, development, and especially in various abiotic stresses responses. The AP2/ERF TF family has been discovered and classified in more than 50 species. However, little is known about the <i>AP2/ERF</i> gene family of Chinese willow (<i>Salix matsudana</i>), which is a tetraploid ornamental tree species that is widely planted and is also considered as a species that can improve the soil salinity of coastal beaches. In this study, 364 <i>AP2/ERF</i> genes of <i>Salix matsudana</i> (<i>SmAP2/ERF</i>) were identified depending on the recently produced whole genome sequencing data of <i>Salix matsudana</i>. These genes were renamed according to the chromosomal location of the <i>SmAP2/ERF</i> genes. The <i>SmAP2/ERF</i> genes included three major subfamilies: AP2 (55 members), ERF (301 members), and RAV (six members) and two Soloist genes. Genes' structure and conserved motifs were analyzed in SmAP2/ERF family members, and introns were not found in most genes of the ERF subfamily, some unique motifs were found to be important for the function of <i>SmAP2/ERF</i> genes. Syntenic relationships between the <i>SmAP2/ERF</i> genes and <i>AP2/ERF</i> genes from <i>Populus trichocarpa</i> and <i>Salix purpurea</i> showed that <i>Salix matsudana</i> is genetically more closely related to <i>Populus trichocarpa</i> than to <i>Salix purpurea</i>. Evolution analysis on paralog gene pairs suggested that progenitor of <i>S. matsudana</i> originated from hybridization between two different diploid salix germplasms and underwent genome duplication not more than 10 Mya. RNA sequencing results demonstrated the differential expression patterns of some <i>SmAP2/ERF</i> genes under salt stress and this information can help reveal the mechanism of salt tolerance regulation in <i>Salix matsudana</i>.
Project description:The AP2/ERF transcription factors (TFs) comprise one of the largest gene superfamilies in plants. These TFs perform vital roles in plant growth, development, and responses to biotic and abiotic stresses. In this study, 171 AP2/ERF TFs were identified in cauliflower (Brassica oleracea L. var. botrytis), one of the most important horticultural crops in Brassica. Among these TFs, 15, 9, and 1 TFs were classified into the AP2, RAV, and Soloist family, respectively. The other 146 TFs belong to ERF family, which were further divided into the ERF and DREB subfamilies. The ERF subfamily contained 91 TFs, while the DREB subfamily contained 55 TFs. Phylogenetic analysis results indicated that the AP2/ERF TFs can be classified into 13 groups, in which 25 conserved motifs were confirmed. Some motifs were group- or subgroup- specific, implying that they are significant to the functions of the AP2/ERF TFs of these clades. In addition, 35 AP2/ERF TFs from the 13 groups were selected randomly and then used for expression pattern analysis under salt and drought stresses. The majority of these AP2/ERF TFs exhibited positive responses to these stress conditions. In specific, Bra-botrytis-ERF054a, Bra-botrytis-ERF056, and Bra-botrytis-CRF2a demonstrated rapid responses. By contrast, six AP2/ERF TFs were showed to delay responses to both stresses. The AP2/ERF TFs exhibiting specific expression patterns under salt or drought stresses were also confirmed. Further functional analysis indicated that ectopic overexpression of Bra-botrytis-ERF056 could increase tolerance to both salt and drought treatments. These findings provide new insights into the AP2/ERF TFs present in cauliflower, and offer candidate AP2/ERF TFs for further studies on their roles in salt and drought stress tolerance.
Project description:The APETALA2/ethylene-responsive element binding factor (AP2/ERF) family is one of the largest transcription factor (TF) families in plants that includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and RAV (Related to ABI3/VP). AP2/ERFs are known to play significant roles in various plant processes including growth and development and biotic and abiotic stress responses. Considering this, a comprehensive genome-wide study was conducted in foxtail millet (Setaria italica L.). A total of 171 AP2/ERF genes were identified by systematic sequence analysis and were physically mapped onto nine chromosomes. Phylogenetic analysis grouped AP2/ERF genes into six classes (I to VI). Duplication analysis revealed that 12 (?7%) SiAP2/ERF genes were tandem repeated and 22 (?13%) were segmentally duplicated. Comparative physical mapping between foxtail millet AP2/ERF genes and its orthologs of sorghum (18 genes), maize (14 genes), rice (9 genes) and Brachypodium (6 genes) showed the evolutionary insights of AP2/ERF gene family and also the decrease in orthology with increase in phylogenetic distance. The evolutionary significance in terms of gene-duplication and divergence was analyzed by estimating synonymous and non-synonymous substitution rates. Expression profiling of candidate AP2/ERF genes against drought, salt and phytohormones revealed insights into their precise and/or overlapping expression patterns which could be responsible for their functional divergence in foxtail millet. The study showed that the genes SiAP2/ERF-069, SiAP2/ERF-103 and SiAP2/ERF-120 may be considered as potential candidate genes for further functional validation as well for utilization in crop improvement programs for stress resistance since these genes were up-regulated under drought and salinity stresses in ABA dependent manner. Altogether the present study provides new insights into evolution, divergence and systematic functional analysis of AP2/ERF gene family at genome level in foxtail millet which may be utilized for improving stress adaptation and tolerance in millets, cereals and bioenergy grasses.
Project description:Transcription factors of the AP2/ERF family play important roles in plant growth, development, and responses to biotic and abiotic stresses. In this study, a physic nut AP2/ERF gene, JcDREB2, was functionally characterized. Real-time PCR analysis revealed that JcDREB2 was expressed mainly in the leaf and could be induced by abscisic acid but suppressed by gibberellin (GA) and salt. Transient expression of a JcDREB2-YFP fusion protein in Arabidopsis protoplasts cells suggested that JcDREB2 is localized in the nucleus. Rice plants overexpressing JcDREB2 exhibited dwarf and GA-deficient phenotypes with shorter shoots and roots than those of wild-type plants. The dwarfism phenotype could be rescued by the application of exogenous GA3. The expression levels of GA biosynthetic genes including OsGA20ox1, OsGA20ox2, OsGA20ox4, OsGA3ox2, OsCPS1, OsKO2, and OsKAO were significantly reduced in plants overexpressing JcDREB2. Overexpression of JcDREB2 in rice increased sensitivity to salt stress. Increases in the expression levels of several salt-tolerance-related genes in response to salt stress were impaired in JcDREB2-overexpressing plants. These results demonstrated not only that JcDREB2 influences GA metabolism, but also that it can participate in the regulation of the salt stress response in rice.
Project description:A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.
Project description:The APETALA2 (AP2)/ethylene response factor plays vital functions in response to environmental stimulus. The ethylene response factor (ERF) subfamily B3 group belongs to the AP2/ERF superfamily and contains a single AP2/ERF domain. Phylogenetic analysis of the ERF subfamily B3 group genes from <i>Arabdiposis thaliana, Gossypium arboreum, Gossypium hirsutum</i>, and <i>Gossypium raimondii</i> made it possible to divide them into three groups and showed that the ERF subfamily B3 group genes are conserved in cotton. Collinearity analysis identified172 orthologous/paralogous gene pairs between <i>G. arboreum</i> and <i>G. hirsutum</i>; 178 between <i>G. hirsutum</i> and <i>G. raimondii</i>; and 1,392 in <i>G. hirsutum</i>. The <i>GhERF</i> subfamily B3 group gene family experienced massive gene family expansion through either segmental or whole genome duplication events, with most genes showing signature compatible with the action of purifying selection during evolution. Most <i>G. hirsutum</i> ERF subfamily B3 group genes are responsive to salt stress. <i>GhERF13.12</i> transgenic Arabidopsis showed enhanced salt stress tolerance and exhibited regulation of related biochemical parameters and enhanced expression of genes participating in ABA signaling, proline biosynthesis, and ROS scavenging. In addition, the silencing of the <i>GhERF13.12</i> gene leads to increased sensitivity to salt stress in cotton. These results indicate that the ERF subfamily B3 group had remained conserved during evolution and that <i>GhERF13.12</i> induces salt stress tolerance in Arabidopsis and cotton.
Project description:The APETALA 2/Ethylene-responsive element binding factor (AP2/ERF) transcription factor gene family is widely involved in the biotic and abiotic stress regulation. Haynaldia villosa (VV, 2n = 14), a wild species of wheat, is a potential gene pool for wheat improvement. H. villosa confers high resistance to several wheat diseases and high tolerance to some abiotic stress. In this study, ERF1-V, an ethylene-responsive element-binding factor gene of the AP2/ERF transcription factor gene family from wild H. villosa, was cloned and characterized. Sequence and phylogenetic analysis showed that ERF1-V is a deduced B2 type ERF gene. ERF1-V was first identified as a Blumeria graminis f. sp. tritici (Bgt) up-regulated gene, and later found to be induced by drought, salt and cold stresses. In responses to hormones, ERF1-V was up-regulated by ethylene and abscisic acid, but down-regulated by salicylic acid and jasmonic acid. Over expression of ERF1-V in wheat could improve resistance to powdery mildew, salt and drought stress. Chlorophyll content, malondialdehyde content, superoxide dismutase and peroxidase activity were significantly differences between the recipient Yangmai158 and the transgenic plants following salt treatment. Furthermore, the expression levels of some stress responsive genes were differences after drought or salt treatments. Although ERF1-V was activated by the constitutive promoter, the agronomic traits, including flowering time, plant height, effective tiller number, spikelet number per spike and grain size, did not changed significantly. ERF1-V is a valuable gene for wheat improvement by genetic engineering.
Project description:Salinity stress has become an increasing threat to food security worldwide and elucidation of the mechanism for salinity tolerance is of great significance. Induced mutation, especially spaceflight mutagenesis, is one important method for crop breeding. In this study, we show that a spaceflight-induced wheat mutant, named salinity tolerance 1 (st1), is a salinity-tolerant line. We report the characteristics of transcriptomic sequence variation induced by spaceflight, and show that mutations in genes associated with sodium ion transport may directly contribute to salinity tolerance in st1. Furthermore, GO and KEGG enrichment analysis of differentially expressed genes (DEGs) between salinity-treated st1 and wild type suggested that the homeostasis of oxidation-reduction process is important for salt tolerance in st1. Through KEGG pathway analysis, "Butanoate metabolism" was identified as a new pathway for salinity responses. Additionally, key genes for salinity tolerance, such as genes encoding arginine decarboxylase, polyamine oxidase, hormones-related, were not only salt-induced in st1 but also showed higher expression in salt-treated st1 compared with salt-treated WT, indicating that these genes may play important roles in salinity tolerance in st1. This study presents valuable genetic resources for studies on transcriptome variation caused by induced mutation and the identification of salt tolerance genes in crops.
Project description:The receptor for activated C kinase 1 (RACK1) is a WD40 type protein that is involved in multiple signaling pathways and is conserved from prokaryotes to eukaryotes. Here we report that rice RACK1A (OsRACK1A) is regulated by circadian clocks and plays an important role in the salt stress response. OsRACK1A was found to follow a rhythmic expression profile under circadian conditions at both the transcription and the translation levels, although the expression was arrhythmic under salt stress. Analysis of plant survival rates, fresh weight, proline content, malondialdehyde, and chlorophyll showed that suppression of OsRACK1A enhanced tolerance to salt stress. The ion concentration in both roots and leaves revealed that OsRACK1A-suppressed transgenic rice could maintain low Na+ and high K+ concentrations. Furthermore, OsRACK1A-suppressed transgenic rice accumulated significantly more abscisic acid (ABA) and more transcripts of ABA- and stress-inducible genes compared with the wild-type plants. Real-time quantitative polymerase chain reaction analysis revealed that many stress-related genes, including APETALA 2/Ethylene Responsive Factor (AP2/ERF) transcription factors, were upregulated in the OsRACK1A-suppressed transgenic rice line. We identified putative interactors of OsRACK1A, and found that OsRACK1A interacted with many salt stress-responsive proteins directly. These results suggest that OsRACK1A is regulated by circadian rhythm, and involved in the regulation of salt stress responses.