Foxtail Millet NF-Y Families: Genome-Wide Survey and Evolution Analyses Identified Two Functional Genes Important in Abiotic Stresses.
ABSTRACT: It was reported that Nuclear Factor Y (NF-Y) genes were involved in abiotic stress in plants. Foxtail millet (Setaria italica), an elite stress tolerant crop, provided an impetus for the investigation of the NF-Y families in abiotic responses. In the present study, a total of 39 NF-Y genes were identified in foxtail millet. Synteny analyses suggested that foxtail millet NF-Y genes had experienced rapid expansion and strong purifying selection during the process of plant evolution. De novo transcriptome assembly of foxtail millet revealed 11 drought up-regulated NF-Y genes. SiNF-YA1 and SiNF-YB8 were highly activated in leaves and/or roots by drought and salt stresses. Abscisic acid (ABA) and H2O2 played positive roles in the induction of SiNF-YA1 and SiNF-YB8 under stress treatments. Transient luciferase (LUC) expression assays revealed that SiNF-YA1 and SiNF-YB8 could activate the LUC gene driven by the tobacco (Nicotiana tobacam) NtERD10, NtLEA5, NtCAT, NtSOD, or NtPOD promoter under normal or stress conditions. Overexpression of SiNF-YA1 enhanced drought and salt tolerance by activating stress-related genes NtERD10 and NtCAT1 and by maintaining relatively stable relative water content (RWC) and contents of chlorophyll, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) in transgenic lines under stresses. SiNF-YB8 regulated expression of NtSOD, NtPOD, NtLEA5, and NtERD10 and conferred relatively high RWC and chlorophyll contents and low MDA content, resulting in drought and osmotic tolerance in transgenic lines under stresses. Therefore, SiNF-YA1 and SiNF-YB8 could activate stress-related genes and improve physiological traits, resulting in tolerance to abiotic stresses in plants. All these results will facilitate functional characterization of foxtail millet NF-Ys in future studies.
Project description:Plant calcium-dependent protein kinases (CDPKs) were reported to play important roles in plant resistance to abiotic stress. Foxtail millet cultivation "H138" was used for RNA-seq analysis. The data from drought-induced <i>de novo</i> transcriptomic sequences of foxtail millet showed that CDPKs were up- or down-regulated by drought to different degrees. In this study, 29 foxtail millet CDPKs were classified into four subgroups. These genes were unevenly distributed on nine foxtail millet chromosomes, and chromosomes 2, 3, and 9 contained the most SiCDPK members. Analysis of putative <i>cis</i>-acting elements showed that most foxtail millet CDPK genes contained the ABRE, LTR, HSE, MYB, MYC, DRE, CGTCA-motif, and TGACG-motif <i>cis</i>-acting elements, which could be activated by abiotic stresses. Real-time PCR analysis indicated that 29 SiCDPK genes experienced different degrees of induction under drought and ABA stresses. <i>SiCDPK24</i> had the highest expression levels at 6 and 12 h of drought treatment and was chosen for further analysis. SiCDPK24 localized to the cell membrane and the nucleus of <i>Arabidopsis</i> mesophyll protoplasts. Western blot analysis showed that SiCDPK24 protein had autophosphorylation activity. Overexpression of <i>SiCDPK24</i> in <i>Arabidopsis</i> enhanced drought resistance and improved the survival rate under drought stress. It also activated the expressions of nine stress-related genes, namely <i>RD29A</i>, <i>RD29B</i>, <i>RD22</i>, <i>KIN1</i>, <i>COR15</i>, <i>COR47</i>, <i>LEA14</i>, <i>CBF3/DREB1A</i>, and <i>DREB2A</i>. These genes are involved in resistance to abiotic stresses in <i>Arabidopsis</i>. These results indicate that foxtail millet CDPK genes play important roles in resisting drought stress.
Project description:HD-Zip proteins represent the major transcription factors in higher plants, playing essential roles in plant development and stress responses. Foxtail millet is a crop to investigate the systems biology of millet and biofuel grasses and the HD-Zip gene family has not been studied in foxtail millet. For further investigation of the expression profile of the HD-Zip gene family in foxtail millet, a comprehensive genome-wide expression analysis was conducted in this study. We found 47 protein-encoding genes in foxtail millet using BLAST search tools; the putative proteins were classified into four subfamilies, namely, subfamilies I, II, III, and IV. Gene structure and motif analysis indicate that the genes in one subfamily were conserved. Promotor analysis showed that HD-Zip gene was involved in abiotic stress. Duplication analysis revealed that 8 (~17%) hdz genes were tandemly duplicated and 28 (58%) were segmentally duplicated; purifying duplication plays important roles in gene expansion. Microsynteny analysis revealed the maximum relationship in foxtail millet-sorghum and foxtail millet-rice. Expression profiling upon the abiotic stresses of drought and high salinity and the biotic stress of ABA revealed that some genes regulated responses to drought and salinity stresses via an ABA-dependent process, especially sihdz29 and sihdz45. Our study provides new insight into evolutionary and functional analyses of HD-Zip genes involved in environmental stress responses in foxtail millet.
Project description:Foxtail millet is very a drought-tolerant crop. Basic helix-loop-helix (bHLH) transcription factors are involved in many drought-stress responses, but foxtail millet bHLH genes have been scarcely examined. We identified 149 foxtail millet bHLH genes in a genome-wide analysis and performed Swiss-Prot, GO, and KEGG pathway analyses for these genes. Phylogenetic analyses placed the genes into 25 clades, with some remaining orphans. We identified homologs based on gene trees and Swiss-Prot annotation. We also inferred that some homologs underwent positive selection in foxtail millet ancestors, and selected motifs differed among homologs. Expression of eight foxtail millet bHLH genes varied with drought stress. One of these genes was localized to a QTL that contributes to drought tolerance in foxtail millet. We also perform a cis-acting regulatory element analysis on foxtail millet bHLH genes and some drought-induced genes. Foxtail millet bHLH genes were inferred to have a possible key role in drought tolerance. This study clarifies both the function of foxtail millet bHLH genes and drought tolerance in foxtail millet.
Project description:Late embryogenesis abundant (LEA) proteins are involved in protecting higher plants from damage caused by environmental stresses. Foxtail millet (Setaria italica) is an important cereal crop for food and feed in semi-arid areas. However, the molecular mechanisms underlying tolerance to these conditions are not well defined.Here, we characterized a novel atypical LEA gene named SiLEA14 from foxtail millet. It contains two exons separated by one intron. SiLEA14 was expressed in roots, stems, leaves, inflorescences and seeds at different levels under normal growth conditions. In addition, SiLEA14 was dramatically induced by osmotic stress, NaCl and exogenous abscisic acid. The SiLEA14 protein was localized in the nucleus and the cytoplasm. Overexpression of SiLEA14 improved Escherichia coli growth performance compared with the control under salt stress. To further assess the function of SiLEA14 in plants, transgenic Arabidopsis and foxtail millet plants that overexpressed SiLEA14 were obtained. The transgenic Arabidopsis seedlings showed higher tolerance to salt and osmotic stress than the wild type (WT). Similarly, the transgenic foxtail millet showed improved growth under salt and drought stresses compared with the WT. Taken together, our results indicated that SiLEA14 is a novel atypical LEA protein and plays important roles in resistance to abiotic stresses in plants.We characterized a novel atypical LEA gene SiLEA14 from foxtail millet, which plays important roles in plant abiotic stress resistance. Modification of SiLEA14 expression may improve abiotic stress resistance in agricultural crops.
Project description:Foxtail millet (<i>Setaria italica</i> (L.) P. Beauv) is an important food and forage crop because of its health benefits and adaptation to drought stress; however, reports of transcriptomic analysis of genes responding to re-watering after drought stress in foxtail millet are rare. The present study evaluated physiological parameters, such as proline content, p5cs enzyme activity, anti-oxidation enzyme activities, and investigated gene expression patterns using RNA sequencing of the drought-tolerant foxtail millet variety (Jigu 16) treated with drought stress and rehydration. The results indicated that drought stress-responsive genes were related to many multiple metabolic processes, such as photosynthesis, signal transduction, phenylpropanoid biosynthesis, starch and sucrose metabolism, and osmotic adjustment. Furthermore, the ?1-pyrroline-5-carboxylate synthetase genes, <i>SiP5CS1</i> and <i>SiP5CS2</i>, were remarkably upregulated in foxtail millet under drought stress conditions. Foxtail millet can also recover well on rehydration after drought stress through gene regulation. Our data demonstrate that recovery on rehydration primarily involves proline metabolism, sugar metabolism, hormone signal transduction, water transport, and detoxification, plus reversal of the expression direction of most drought-responsive genes. Our results provided a detailed description of the comparative transcriptome response of foxtail millet variety Jigu 16 under drought and rehydration environments. Furthermore, we identify <i>SiP5CS2</i> as an important gene likely involved in the drought tolerance of foxtail millet.
Project description:Plant lipoxygenases (LOXs), a kind of non-heme iron-containing dioxygenases, participate plant physiological activities (especially in response to biotic and abiotic stresses) through oxidizing various lipids. However, there was few investigations on LOXs in foxtail millet (<i>Setaria italica</i>). In this study, we identified the <i>LOX</i> gene family in foxtail millet, and divided the total 12 members into three sub-families on the basis of their phylogenetic relationships. Under salt and drought stress, <i>LOX</i> genes showed different expression patterns. Among them, only <i>SiLOX7</i> showed up-regulated expression in Yugu1 (YG1) and Qinhuang2 (QH2), two stress-tolerant varieties, indicating that <i>SiLOX7</i> may play an important role in responses to abiotic stress. Our research provides a basis for further investigation of the role of <i>LOX</i> genes in the adaptation to abiotic stresses and other possible biological functions in foxtail millet.
Project description:MADS-box transcription factors play vital roles in multiple biological processes in plants. At present, a comprehensive investigation into the genome-wide identification and classification of <i>MADS-box</i> genes in foxtail millet (<i>Setaria italica</i> L.) has not been reported. In this study, we identified 72 <i>MADS-box</i> genes in the foxtail millet genome and give an overview of the phylogeny, chromosomal location, gene structures, and potential functions of the proteins encoded by these genes. We also found that the expression of 10 MIKC-type <i>MADS-box</i> genes was induced by abiotic stresses (PEG-6000 and NaCl) and exogenous hormones (ABA and GA), which suggests that these genes may play important regulatory roles in response to different stresses. Further studies showed that transgenic <i>Arabidopsis</i> and rice (<i>Oryza sativa</i> L.) plants overexpressing <i>SiMADS51</i> had reduced drought stress tolerance as revealed by lower survival rates and poorer growth performance under drought stress conditions, which demonstrated that <i>SiMADS51</i> is a negative regulator of drought stress tolerance in plants. Moreover, expression of some stress-related genes were down-regulated in the <i>SiMADS51</i>-overexpressing plants. The results of our study provide an overall picture of the <i>MADS-box</i> gene family in foxtail millet and establish a foundation for further research on the mechanisms of action of MADS-box proteins with respect to abiotic stresses.
Project description:The DREB (dehydration-responsive element binding)-type transcription factors regulate the expression of stress-inducible genes by binding the DRE/CRT cis-elements in promoter regions. The upstream transcription factors that regulate the transcription of DREB transcription factors have not been clearly defined, although the function of DREB transcription factors in abiotic stress is known. In this study, an abscisic acid (ABA)-responsive DREB-binding protein gene (SiARDP) was cloned from foxtail millet (Setaria italica). The transcript level of SiARDP increased not only after drought, high salt, and low temperature stresses, but also after an ABA treatment in foxtail millet seedlings. Two ABA-responsive elements (ABRE1: ACGTGTC; ABRE2: ACGTGGC) exist in the promoter of SiARDP. Further analyses showed that two ABA-responsive element binding (AREB)-type transcription factors, SiAREB1 and SiAREB2, could physically bind to the ABRE core element in vitro and in vivo. The constitutive expression of SiARDP in Arabidopsis thaliana enhanced drought and salt tolerance during seed germination and seedling development, and overexpression of SiARDP in foxtail millet improved drought tolerance. The expression levels of target genes of SiARDP were upregulated in transgenic Arabidopsis and foxtail millet. These results reveal that SiARDP, one of the target genes of SiAREB, is involved in ABA-dependent signal pathways and plays a critical role in the abiotic stress response in plants.
Project description:LIM proteins have been found to play important roles in many life activities, including the regulation of gene expression, construction of the cytoskeleton, signal transduction and metabolic regulation. Because of their important roles in many aspects of plant development, LIM genes have been studied in many plant species. However, the LIM gene family has not yet been characterized in foxtail millet. In this study, we analyzed the whole genome of foxtail millet and identified 10 LIM genes. All LIM gene promoters contain MYB and MYC cis-acting elements that are related to drought stress. Based on the presence of multiple abiotic stress-related cis-elements in the promoter of SiWLIM2b, we chose this gene for further study. We analyzed SiWLIM2b expression under abiotic stress and hormone treatments using qRT-PCR. We found that SiWLIM2b was induced by various abiotic stresses and hormones. Under drought conditions, transgenic rice of SiWLIM2b-overexpression had a higher survival rate, higher relative water content and less cell damage than wild type (WT) rice. These results indicate that overexpression of the foxtail millet SiWLIM2b gene enhances drought tolerance in transgenic rice, and the SiWLIM2b gene can potentially be used for molecular breeding of crops with increased resistance to abiotic stress.
Project description:Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene (SiLTP) was isolated from a foxtail millet (Setaria italica) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis. Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP-OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo, respectively. Moreover, the SiLTP expression levels were higher in SiARDP-OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought tolerance levels.