Identification of NHXs in Gossypium species and the positive role of GhNHX1 in salt tolerance.
ABSTRACT: BACKGROUND:Plant Na+/H+ antiporters (NHXs) are membrane-localized proteins that maintain cellular Na+/K+ and pH homeostasis. Considerable evidence highlighted the critical roles of NHX family in plant development and salt response; however, NHXs in cotton are rarely studied. RESULTS:The comprehensive and systematic comparative study of NHXs in three Gossypium species was performed. We identified 12, 12, and 23 putative NHX proteins from G. arboreum, G. raimondii, and G. hirsutum, respectively. Phylogenetic study revealed that repeated polyploidization of Gossypium spp. contributed to the expansion of NHX family. Gene structure analysis showed that cotton NHXs contain many introns, which will lead to alternative splicing and help plants to adapt to high salt concentrations in soil. The expression changes of NHXs indicate the possible differences in the roles of distinct NHXs in salt response. GhNHX1 was proved to be located in the vacuolar system and intensively induced by salt stress in cotton. Silencing of GhNHX1 resulted in enhanced sensitivity of cotton seedlings to high salt concentrations, which suggests that GhNHX1 positively regulates cotton tolerance to salt stress. CONCLUSION:We characterized the gene structure, phylogenetic relationship, chromosomal location, and expression pattern of NHX genes from G. arboreum, G. raimondii, and G. hirsutum. Our findings indicated that the cotton NHX genes are regulated meticulously and differently at the transcription level with possible alternative splicing. The tolerance of plants to salt stress may rely on the expression level of a particular NHX, rather than the number of NHXs in the genome. This study could provide significant insights into the function of plant NHXs, as well as propose promising candidate genes for breeding salt-resistant cotton cultivars.
Project description:Soil salinization, which is primarily due to excessive Na+ levels, is a major abiotic stress adversely affecting plant growth and development. The Na+/H+ antiporter (NHX) is a transmembrane protein mediating the transport of Na+ or K+ and H+ across the membrane to modulate the ionic balance of plants in response to salt stress. Research regarding NHXs has mainly focused on the vacuolar-type NHX family members. However, the biological functions of the endosomal-type NHXs remain relatively uncharacterized. In this study, 22 NHX family members were identified in Gossypium hirsutum. A phylogenetic analysis divided the GhNHX genes into two categories, with 18 and 4 in the vacuolar and endosomal groups, respectively. The chromosomal distribution of the NHX genes revealed the significant impact of genome-wide duplication during the polyploidization process on the number of GhNHX genes. Analyses of gene structures and conserved motifs indicated that GhNHX genes in the same phylogenetic cluster are conserved. Additionally, the salt-induced expression patterns confirmed that the expression levels of most of the GhNHX genes are affected by salinity. Specifically, in the endosomal group, GhNHX4A expression was substantially up-regulated by salt stress. A yeast functional complementation test proved that GhNHX4A can partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. Silencing GhNHX4A expression decreased the resistance of cotton to salt stress because of an increase in the accumulation of Na+ in stems and a decrease in the accumulation of K+ in roots. The results of this study may provide the basis for an in-depth characterization of the regulatory functions of NHX genes related to cotton salt tolerance, especially the endosomal-type GhNHX4A. Furthermore, the presented data may be useful for selecting appropriate candidate genes for the breeding of new salt-tolerant cotton varieties.
Project description:The sodium/hydrogen antiporter (NHX) gene family with the Na+/H+ exchange protein domain is a transporter of sodium and hydrogen ions and plays an important role in the response of plants to salt stress. Studying the response of cotton to salt stress through comprehensive identification and analysis of NHX genes in several species and their roles in salt tolerance mechanisms is of great significance. In this study, 23, 24, 12, and 12 NHX genes were identified from Gossypium hirsutum (Gh), G. barbadense, G. arboreum and G. raimondii, respectively. Phylogenetic analysis showed that these genes were mainly divided into three clades with significant subcellular localization, namely, endosome (Endo-class), plasma membrane (PM-class) and vacuole (Vac-class). By analyzing the structure of NHX genes and proteins, each branch of the NHX gene family was found to be structurally conserved, and collinearity analysis showed that NHX genes were mainly expressed through whole genome and segmental duplication. The non-synonymous (Ka)/synonymous (Ks) values showed that the NHX gene family experienced strong purifying selection during long-term evolution. Cis-acting element analysis showed that the NHX gene family may be related to the regulation of abscisic acid (ABA) and methyl jasmonate (MeJA) hormones. Additionally, transcriptomic data analysis and qRT-PCR showed that GhNHXs exhibited different expression patterns in each tissue and under different salinities. These results provide an important reference for us to further understand and analyze the molecular regulation mechanism of cotton NHX genes.
Project description:BACKGROUND:Cotton (Gossypium spp.) is the most important world-wide fiber crop but salt stress limits cotton production in coastal and other areas. Growth regulation factors (GRFs) play regulatory roles in response to salt stress, but their roles have not been studied in cotton under salt stress. RESULTS:We identified 19 GRF genes in G. raimondii, 18 in G. arboreum, 34 in G. hirsutum and 45 in G. barbadense, respectively. These GRF genes were phylogenetically analyzed leading to the recognition of seven GRF clades. GRF genes from diploid cottons (G. raimondii and G. arboreum) were largely retained in allopolyploid cotton, with subsequent gene expansion in G. barbadense relative to G. hirsutum. Most G. hirsutum GRF (GhGRF) genes are preferentially expressed in young and growing tissues. To explore their possible role in salt stress, we used qRT-PCR to study expression responses to NaCl treatment, showing that five GhGRF genes were down-regulated in leaves. RNA-seq experiments showed that seven GhGRF genes exhibited decreased expression in leaves under NaCl treatment, three of which (GhGRF3, GhGRF4, and GhGRF16) were identified by both RNA-seq and qRT-PCR. We also identified six and three GRF genes that exhibit decreased expression under salt stress in G. arboreum and G. barbadense, respectively. Consistent with its lack of leaf withering or yellowing under the salt treatment conditions, G. arboreum had better salt tolerance than G. hirsutum and G. barbadense. Our results suggest that GRF genes are involved in salt stress responses in Gossypium. CONCLUSION:In summary, we identified candidate GRF genes that were involved in salt stress responses in cotton.
Project description:Nucleotide binding site (NBS) genes encode a large family of disease resistance (R) proteins in plants. The availability of genomic data of the two diploid cotton species, Gossypium arboreum and Gossypium raimondii, and the two allotetraploid cotton species, Gossypium hirsutum (TM-1) and Gossypium barbadense allow for a more comprehensive and systematic comparative study of NBS-encoding genes to elucidate the mechanisms of cotton disease resistance.Based on the genome assembly data, 246, 365, 588 and 682 NBS-encoding genes were identified in G. arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. The distribution of NBS-encoding genes among the chromosomes was nonrandom and uneven, and was tended to form clusters. Gene structure analysis showed that G. arboreum and G. hirsutum possessed a greater proportion of CN, CNL, and N genes and a lower proportion of NL, TN and TNL genes compared to that of G. raimondii and G. barbadense, while the percentages of RN and RNL genes remained relatively unchanged. The percentage changes among them were largest for TNL genes, about 7 times. Exon statistics showed that the average exon numbers per NBS gene in G. raimondii and G. barbadense were all greater than that in G. arboretum and G. hirsutum. Phylogenetic analysis revealed that the TIR-NBS genes of G. barbadense were closely related with that of G. raimondii. Sequence similarity analysis showed that diploid cotton G. arboreum possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. hirsutum, while diploid G. raimondii possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. barbadense. The synteny analysis showed that more NBS genes in G. raimondii and G. arboreum were syntenic with that in G. barbadense and G. hirsutum, respectively.The structural architectures, amino acid sequence similarities and synteny of NBS-encoding genes between G. arboreum and G. hirsutum, and between G. raimondii and G. barbadense were the highest among comparisons between the diploid and allotetraploid genomes, indicating that G. hirsutum inherited more NBS-encoding genes from G. arboreum, while G. barbadense inherited more NBS-encoding genes from G. raimondii. This asymmetric evolution of NBS-encoding genes may help to explain why G. raimondii and G. barbadense are more resistant to Verticillium wilt, whereas G. arboreum and G. hirsutum are more susceptible to Verticillium wilt. The disease resistances of the allotetraploid cotton were related to their NBS-encoding genes especially in regard from which diploid progenitor they were derived, and the TNL genes may have a significant role in disease resistance to Verticillium wilt in G. raimondii and G. barbadense.
Project description:Reactive oxygen species (ROS) are important molecules in the plant, which are involved in many biological processes, including fiber development and adaptation to abiotic stress in cotton. We carried out transcription analysis to determine the evolution of the ROS genes and analyzed their expression levels in various tissues of cotton plant under abiotic stress conditions. There were 515, 260, and 261 genes of ROS network that were identified in Gossypium hirsutum (AD? genome), G. arboreum (A genome), and G. raimondii (D genome), respectively. The ROS network genes were found to be distributed in all the cotton chromosomes, but with a tendency of aggregating on either the lower or upper arms of the chromosomes. Moreover, all the cotton ROS network genes were grouped into 17 families as per the phylogenetic tress analysis. A total of 243 gene pairs were orthologous in G. arboreum and G. raimondii. There were 240 gene pairs that were orthologous in G. arboreum, G. raimondii, and G. hirsutum. The synonymous substitution value (Ks) peaks of orthologous gene pairs between the At subgenome and the A progenitor genome (G. arboreum), D subgenome and D progenitor genome (G. raimondii) were 0.004 and 0.015, respectively. The Ks peaks of ROS network orthologous gene pairs between the two progenitor genomes (A and D genomes) and two subgenomes (At and Dt subgenome) were 0.045. The majority of Ka/Ks value of orthologous gene pairs between the A, D genomes and two subgenomes of TM-1 were lower than 1.0. RNA seq. analysis and RT-qPCR validation, showed that, CSD1,2,3,5,6; FSD1,2; MSD1,2; APX3,11; FRO5.6; and RBOH6 played a major role in fiber development while CSD1, APX1, APX2, MDAR1, GPX4-6-7, FER2, RBOH6, RBOH11, and FRO5 were integral for enhancing salt stress in cotton. ROS network-mediated signal pathway enhances the mechanism of fiber development and regulation of abiotic stress in Gossypium. This study will enhance the understanding of ROS network and form the basic foundation in exploring the mechanism of ROS network-involving the fiber development and regulation of abiotic stress in cotton.
Project description:Acetyl-CoA carboxylase is an important enzyme, which catalyzes acetyl-CoA's carboxylation to produce malonyl-CoA and to serve as a committed step for de novo fatty acid biosynthesis in plastids. In this study, 24 putative cotton BCCP genes were identified based on the lately published genome data in Gossypium. Among them, 4, 4, 8, and 8 BCCP homologs were identified in Gossypium raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. These genes were divided into two classes based on a phylogenetic analysis. In each class, these homologs were relatively conserved in gene structure and motifs. The chromosomal distribution pattern revealed that all the BCCP genes were distributed equally on corresponding chromosomes or scaffold in the four cotton species. Segmental duplication was a predominant duplication event in both of G. hirsutum and G. barbadense. The analysis of the expression profile showed that 8 GhBCCP genes expressed in all the tested tissues with changed expression levels, and GhBCCP genes belonging to class II were predominantly expressed in developing ovules. Meanwhile, the expression analysis for the 16 cotton BCCP genes from G. raimondii, G. arboreum and G. hirsutum showed that they were induced or suppressed by cold or salt stress, and their expression patterns varied among different tissues. These findings will help to determine the functional and evolutionary characteristics of the BCCP genes in Gossypium species.
Project description:Plant P-type H?-ATPase (P-ATPase) is a membrane protein existing in the plasma membrane that plays an important role in the transmembrane transport of plant cells. To understand the variety and quantity of P-ATPase proteins in different cotton species, we combined four databases from two diploid cotton species (Gossypium raimondii and G. arboreum) and two tetraploid cotton species (G. hirsutum and G. barbadense) to screen the P-ATPase gene family and resolved the evolutionary relationships between the former cotton species. We identified 53, 51, 99 and 98 P-ATPase genes from G. arboretum, G. raimondii, G. barbadense and G. hirsutum, respectively. The structural and phylogenetic analyses revealed that the gene structure was consistent between P-ATPase genes, with a close evolutionary relationship. The expression analysis of P-ATPase genes showed that many P-ATPase genes were highly expressed in various tissues and at different fiber developmental stages in G. hirsutum, suggesting that they have potential functions during growth and fiber development in cotton.
Project description:Plant non-specific lipid transfer proteins (nsLTPs) are involved in many biological processes. In this study, 51, 47 and 91 nsLTPs were identified in Gossypium arboreum, G. raimondii and their descendant allotetraploid G. hirsutum, respectively. All the nsLTPs were phylogenetically divided into 8 distinct subfamilies. Besides, the recent duplication, which is considered cotton-specific whole genome duplication, may have led to nsLTP expansion in Gossypium. Both tandem and segmental duplication contributed to nsLTP expansion in G. arboreum and G. hirsutum, while tandem duplication was the dominant pattern in G. raimondii. Additionally, the interspecific orthologous gene pairs in Gossypium were identified. Some GaLTPs and GrLTPs lost their orthologs in the At and Dt subgenomes, respectively, of G. hirsutum. The distribution of these GrLTPs and GaLTPs within each subfamily was complementary, suggesting that the loss and retention of nsLTPs in G. hirsutum might not be random. Moreover, the nsLTPs in the At and Dt subgenomes might have evolved symmetrically. Furthermore, both intraspecific and interspecific orthologous genes showed considerable expression variation, suggesting that their functions were strongly differentiated. Our results lay an important foundation for expansion and evolutionary analysis of the nsLTP family in Gossypium, and advance nsLTP studies in other plants, especially polyploid plants.
Project description:Intron length polymorphisms (ILPs), a type of gene-based functional marker, could themselves be related to the particular traits. Here, we developed a genome-wide cotton ILPs based on orthologs annotation from two sequenced diploid species, A-genome Gossypium arboreum and D-genome G. raimondii. We identified 10,180 putative ILP markers from 5,021 orthologous genes. Among these, 535 ILP markers from 9 gene families related to stress were selected for experimental verification. Polymorphic rates were 72.71% between G. arboreum and G. raimondii and 36.45% between G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. Furthermore, 14 polymorphic ILP markers were detected in 264 G. hirsutum accessions. Coupled with previous simple sequence repeats (SSRs) evaluations and salt tolerance assays from the same individuals, we found a total of 25 marker-trait associations involved in nine ILPs. The nine genes, temporally named as C1 to C9, showed the various expressions in different organs and tissues, and five genes (C3, C4, C5, C7 and C9) were significantly upregulated after salt treatment. We verified that the five genes play important roles in salt tolerance. Particularly, silencing of C4 (encodes WRKY DNA-binding protein) and C9 (encodes Mitogen-activated protein kinase) can significantly enhance cotton susceptibility to salt stress.
Project description:The protein phosphatase (PP2C) gene family, known to participate in cellular processes, is one of the momentous and conserved plant-specific gene families that regulate signal transduction in eukaryotic organisms. Recently, PP2Cs were identified in Arabidopsis and various other crop species, but analysis of PP2C in cotton is yet to be reported. In the current research, we found 87 (Gossypium arboreum), 147 (Gossypium barbadense), 181 (Gossypium hirsutum), and 99 (Gossypium raimondii) PP2C-encoding genes in total from the cotton genome. Herein, we provide a comprehensive analysis of the PP2C gene family in cotton, such as gene structure organization, gene duplications, expression profiling, chromosomal mapping, protein motif organization, and phylogenetic relationships of each species. Phylogenetic analysis further categorized PP2C genes into 12 subgroups based on conserved domain composition analysis. Moreover, we observed a strong signature of purifying selection among duplicated pairs (i.e., segmental and dispersed) of Gossypium hirsutum. We also observed the tissue-specific response of GhPP2C genes in organ and fiber development by comparing the RNA-sequence (RNA-seq) data reported on different organs. The qRT-PCR validation of 30 GhPP2C genes suggested their critical role in cotton by exposure to heat, cold, drought, and salt stress treatments. Hence, our findings provide an overview of the PP2C gene family in cotton based on various bioinformatic tools that demonstrated their critical role in organ and fiber development, and abiotic stress tolerance, thereby contributing to the genetic improvement of cotton for the resistant cultivar.