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Drought, salinity and cold stresses have a major impact on cotton production, thus identification and utilization of plant genes vital for plant improvement Whole-genome identification and functional characterizations of the IQ67-domain (IQD) protein family was carried out in which 148, 77, and 79 IQD genes were identified in Gossypium hirsutum, G. raimondii, and G. arboreum. The entire IQD proteins had varied physiochemical properties, however; their grand hydropathy values were negative, which demonstrated that the proteins were hydrophilic, a property common among the proteins encoded by various stresses responsive genes, such as the late embryogenesis abundant (LEA) proteins. The IQD proteins were predicted to be majorly sublocalized in the nucleus; moreover, various cis-regulatory elements with higher role in enhancing abiotic stress tolerance were detected. RNA-seq and RT-qPCR analysis revealed two key genes, Gh_D06G0014 and Gh_A09G1608 with significantly higher upregulation across the various tissues under drought, salt and cold stress. Knockdown of the two genes negatively affected the ability of G. hirsutum to tolerate the effects of the three stress factors, being all the antioxidant assayed were significantly low concentrations compared to the oxidizing enzymes in VIGS plants under stress, furthermore, morphological and physiological traits were all negatively affected in VIGS plants. Expression levels of GhLEA2, GhCDK_F4, GPCR (TOM1) and Gh_A05G2067 (TH), the stress responsive genes were all downregulated in the VIGS plants, but significantly upregulated in WT and positively controlled plants. The results demonstrated that the IQD genes could be responsible for enhancing drought, salt and cold stress tolerance in cotton.

Background Cotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally. The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions. However, plants have evolved a number of survival strategies, among them is the induction of various stress-responsive genes such as the ribosomal protein large (RPL) gene. The RPL gene families encode critical proteins, which alleviate the effects of drought and salt stress in plants. In this study, comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses. Results Based on the genome-wide evaluation, 26, 8, and 5 proteins containing the RPL14B domain were identified in Gossypium hirsutum, G. raimondii, and G. arboreum, respectively. Furthermore, through bioinformatics analysis, key cis-regulatory elements related to RPL14B genes were discovered. The Myb binding sites (MBS), abscisic acid-responsive element (ABRE), CAAT-box, TATA box, TGACG-motif, and CGTCA-motif responsive to methyl jasmonate, as well as the TCA-motif responsive to salicylic acid, were identified. Expression analysis revealed a key gene, Gh_D01G0234 (RPL14B), with significantly higher induction levels was further evaluated through a reverse genetic approach. The knockdown of Gh_D01G0234 (RPL14B) significantly affected the performance of cotton seedlings under drought/salt stress conditions, as evidenced by a substantial reduction in various morphological and physiological traits. Moreover, the level of the antioxidant enzyme was significantly reduced in VIGS-plants, while oxidant enzyme levels increased significantly, as demonstrated by the higher malondialdehyde concentration level. Conclusion The results revealed the potential role of the RPL14B gene in promoting the induction of antioxidant enzymes, which are key in oxidizing the various oxidants. The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.

The acyl-coenzyme A oxidase 3 (ACX3) gene involved in the β-oxidation pathway plays a critical role in plant growth and development as well as stress response. Earlier on, studies focused primarily on the role of β-oxidation limited to fatty acid breakdown. However, ACX3 peroxisomal β-oxidation pathways result in a downstream cascade of events that act as a transduction of biochemical and physiological responses to stress. A role that is yet to be studied extensively. In this study, we identified 20, 18, 22, 23, 20, 11, and 9 proteins in Gossypium hirsutum, G. barbadense, G. tomentosum, G. mustelinum, G. darwinii, G. arboretum, and G. raimondii genomes, respectively. The tetraploid cotton genome had protein ranging between 18 and 22, while diploids had between 9 and 11. After analyzing the gene family evolution or selection pressure, we found that this gene family undergoes purely segmental duplication both in diploids and tetraploids. W-Box (WRKY-binding site), ABRE, CAAT–Box, TATA-box, MYB, MBS, LTR, TGACG, and CGTCA-motif are abiotic stress cis-regulatory elements identified in this gene family. All these are the binding sites for abiotic stress transcription factors, indicating that this gene is essential. Genes found in G. hirsutum showed a clear response to drought and salinity stress, with higher expression under drought and salt stress, particularly in the leaf and root, according to expression analysis. We selected Gh_DO1GO186, one of the highly expressed genes, for functional characterization. We functionally characterized the GhACX3 gene through overexpression and virus-induced gene silencing (VIGS). Overexpression of this gene enhanced tolerance under stress, which was exhibited by the germination assay. The overexpressed seed growth rate was faster relative to control under drought and salt stress conditions. The survival rate was also higher in overexpressed plants relative to control plants under stress. In contrast, the silencing of the GhACX3 gene in cotton plants resulted in plants showing the stress susceptibility phenotype and reduced root length compared to control. Biochemical analysis also demonstrated that GhACX3-silenced plants experienced oxidative stress while the overexpressed plants did not. This study has revealed the importance of the ACX3 family during stress tolerance and can breed stress-resilient cultivar.

Background Cotton is an important commercial crop for being a valuable source of natural fiber. Its production has undergone a sharp decline because of abiotic stresses, etc. Drought is one of the major abiotic stress causing significant yield losses in cotton. However, plants have evolved self-defense mechanisms to cope abiotic factors like drought, salt, cold, etc. The evolution of stress responsive transcription factors such as the trihelix, a nodule-inception-like protein (NLP), and the late embryogenesis abundant proteins have shown positive response in the resistance improvement to several abiotic stresses. Results Genome wide identification and characterization of the effects of Light-Harvesting Chloro a/b binding (LHC) genes were carried out in cotton under drought stress conditions. A hundred and nine proteins encoded by the LHC genes were found in the cotton genome, with 55, 27, and 27 genes found to be distributed in Gossypium hirsutum, G. arboreum, and G. raimondii, respectively. The proteins encoded by the genes were unevenly distributed on various chromosomes. The Ka/Ks (Non-synonymous substitution rate/Synonymous substitution rate) values were less than one, an indication of negative selection of the gene family. Differential expressions of genes showed that majority of the genes are being highly upregulated in the roots as compared with leaves and stem tissues. Most genes were found to be highly expressed in MR-85, a relative drought tolerant germplasm. Conclusion The results provide proofs of the possible role of the LHC genes in improving drought stress tolerance, and can be explored by cotton breeders in releasing a more drought tolerant cotton varieties.

Abiotic stress is an important bottleneck in crop yield around the world. The main purpose of this study is to describe the function and expression patterns of Fructose-1, 6-bisphosphate Aldolase (FBA) gene family in Gossypium species under salt stress. FBA genes are implicated in plant growth and development as well as salt stress tolerance. In this study, 19 FBA genes were found in G. hirsutum, while 9 genes each in case of G. arboreum and G. raimondii. Based on protein domains and phylogenetic analysis, FBA genes were categorized into two classes I and II. Gene structure analysis and motifs division of FBA genes in three Gossypium species showed that numerous genes of FBA have unequal distribution of introns, suggesting new functions in the gene family. Combined syntenic blocks of FBA genes revealed 31duplicated genes among the three Gossypium species. Three kinds of duplications (segmental, tandem and dispersed) were observed with most of the FBA genes duplicated in segmental pattern and remaineds conserved, which could be a possible basis of expansion of FBA genes in cotton (Gossypium). Gene ontology results showed that FBA genes were involved in a couple of mechanisms such as vacuole compartmentalization, glycolysis process and zinc ion binding. The expression analysis depicted that most of class I genes were upregulated in salt tolerant species, G. darwinii as compared to salt susceptible species G. hirsutum. These finding shows comprehensive information on structure and promising function of FBA gene family in Gossypium plant under salt stress.

The extrusion of toxins and substances at a cellular level is a vital life process in plants under abiotic stress. The multidrug and toxic compound extrusion (MATE) gene family plays a large role in the exportation of toxins and other substrates. We carried out a genome-wide analysis of MATE gene families in Gossypium raimondii and Gossypium arboreum and assessed their expression levels under salt, cadmium and drought stresses. We identified 70 and 68 MATE genes in G. raimondii and G. arboreum, respectively. The majority of the genes were predicted to be localized within the plasma membrane, with some distributed in other cell parts. Based on phylogenetic analysis, the genes were subdivided into three subfamilies, designated as M1, M2 and M3. Closely related members shared similar gene structures, and thus were highly conserved in nature and have mainly evolved through purifying selection. The genes were distributed in all chromosomes. Twenty-nine gene duplication events were detected, with segmental being the dominant type. GO annotation revealed a link to salt, drought and cadmium stresses. The genes exhibited differential expression, with GrMATE18, GrMATE34, GaMATE41 and GaMATE51 significantly upregulated under drought, salt and cadmium stress, and these could possibly be the candidate genes. Our results provide the first data on the genome-wide and functional characterization of MATE genes in diploid cotton, and are important for breeders of more stress-tolerant cotton genotypes.

We found 33, 17, and 20 Alba genes in Gossypium hirsutum, Gossypium arboretum, and Gossypium raimondii, respectively. The Alba protein lengths ranged from 62 to 312 aa, the molecular weight (MW) from 7.003 to 34.55 kDa, grand average hydropathy values of −1.012 to 0.609 and isoelectric (pI) values of −3 to 11. Moreover, miRNAs such as gra-miR8770 targeted four genes, gra-miR8752 and gra-miR8666 targeted three genes, and each and gra-miR8657 a, b, c, d, e targeted 10 genes each, while the rests targeted 1 to 2 genes each. Similarly, various cis-regulatory elements were detected with significant roles in enhancing abiotic stress tolerance, such as CBFHV (RYCGAC) with a role in cold stress acclimation among others. Two genes, Gh_D01G0884 and Gh_D01G0922, were found to be highly induced under water deficit and salt stress conditions. Through virus-induced gene silencing (VIGS), the VIGS cotton plants were found to be highly susceptible to both water deficit and salt stresses; the VIGS plants exhibited a significant reduction in root growth, low cell membrane stability (CMS), saturated leaf weight (SLW), chlorophyll content levels, and higher excised leaf water loss (ELWL). Furthermore, the stress-responsive genes and ROS scavenging enzymes were significantly reduced in the VIGS plants compared to either the wild type (WT) and or the positively controlled plants. The VIGS plants registered higher concentration levels of hydrogen peroxide and malondialdehyde, with significantly lower levels of the various antioxidants evaluated an indication that the VIGS plants were highly affected by salt and drought stresses. This result provides a key foundation for future exploration of the Alba proteins in relation to abiotic stress.

In plants, aldehyde dehydrogenases (ALDHs) function as 'aldehyde scavengers' by removing reactive aldehydes and thus play important roles in stress responses. To date, 30 ALDHs have been identified in Gossypium raimondii, whereas ALDHs have not been studied in Gossypium arboreum or in tetraploid cotton. In this study, we identified 30, 59 and 59 aldehyde dehydrogenase (ALDH) genes from G. arboreum, G. hirsutum and G. barbadense, respectively. Gene structure analysis revealed that members of the same family exhibit similar exon-intron structures and structural domains, and all members of the ALDH18 family possess a distinct AA-kinase domain. Synteny analysis showed that segmental and tandem duplications have played an important role in the expansion and evolution of ALDHs in cotton. Phylogenetic and synteny analysis between G. arboreum and G. raimondii demonstrated that all GaALDHs and GrALDHs are orthologous and that most GaALDHs are located in syntenic blocks corresponding to those of G. raimondii, implying that these genes appeared before the divergence of G. arboreum and G. raimondii and that no expansion of the ALDH superfamily has occurred in these two cotton species. Quantitative real-time PCR analysis revealed that the majority of GaALDHs and GhALDHs are up-regulated under conditions of high salinity and drought, indicating that these genes may be stress responsive. The findings of this study, based on genome-wide identification of ALDHs in Gossypium and analysis of their evolution and expression, provide a foundation for further analysis of ALDHs and suggest potential target genes for improving stress resistance in cotton.

Abiotic stress is an important limiting factor in crop growth and yield around the world. Owing to the continued genetic erosion of the upland cotton germplasm due to intense selection and inbreeding, attention has shifted towards wild cotton progenitors which offer unique traits that can be introgressed into the cultivated cotton to improve their genetic performance. The purpose of this study was to characterize the Pkinase gene family in a previously developed genetic map of the F2 population derived from a cross between two cotton species: Gossypium hirsutum (CCRI 12-4) and Gossypium darwinii (5-7). Based on phylogenetic analysis, Pkinase (PF00069) was found to be the dominant domain with 151 genes in three cotton species, categorized into 13 subfamilies. Structure analysis of G. hirsutum genes showed that a greater percentage of genes and their exons were highly conserved within the group. Syntenic analysis of gene blocks revealed 99 duplicated genes among G. hirsutum, Gossypium arboreum and Gossypium raimondii. Most of the genes were duplicated in segmental pattern. Expression pattern analysis showed that the Pkinase gene family possessed species-level variation in induction to salinity and G. darwinii had higher expression levels as compared to G. hirsutum. Based on RNA sequence analysis and preliminary RT-qPCR verification, we hypothesized that the Pkinase gene family, regulated by transcription factors (TFs) and miRNAs, might play key roles in salt stress tolerance. These findings inferred comprehensive information on possible structure and function of Pkinase gene family in cotton under salt stress.

Background Cotton (Gossypium hirsutum) provides the largest natural fiber for the textile manufacturing industries, but its production is on the decline due to the effects of salinity. Soil salt-alkalization leads to damage in cotton growth and a decrease in yields. Hyperosmolality-gated calcium-permeable channels (OSCA) have been found to be involved in the detection of extracellular changes which trigger an increase in cytosolic free calcium concentration. Hyperosmolality-induced calcium ion increases have been widely speculated to be playing a role in osmosensing in plants. However, the molecular nature of the corresponding calcium ion channels remains unclearly. In this research work, we describe the OSCA genes and their putative function in osmosensing in plants by carrying out genome-wide identification, characterization and functional analysis of the significantly up-regulated OSCA gene, GhOSCA1.1 through reverse genetics. Result A total of 35, 21 and 22 OSCA genes were identified in G. hirsutum, G. arboreum, and G. raimondii genomes, respectively, and were classified into four different clades according to their gene structure and phylogenetic relationship. Gene and protein structure analysis indicated that 35 GhOSCA genes contained a conserved RSN1_7TM (PF02714) domain. Moreover, the cis-regulatory element analysis indicated that the OSCA genes were involved in response to abiotic stress. Furthermore, the knockdown of one of the highly up-regulated genes, Gh_OSCA1.1 showed that the virus-induced gene silenced (VIGS) plants were highly sensitive to dehydration and salinity stresses compared with the none VIGS plants as evident with higher concentration levels of oxidant enzymes compared with the antioxidant enzymes on the leaves of the stressed plants. Conclusion This study provides the first systematic analysis of the OSCA gene family and will be important for understanding the putative functions of the proteins encoded by the OSCA genes in cotton. These results provide a new insight of defense responses in general and lay the foundation for further investigation of the molecular role played by the OSCA genes, thereby providing suitable approaches to improve crop performance under salinity and drought stress conditions.

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 (AD1 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.

Abiotic stresses have negative effects on plants growth and development. Plants, being sessile, have developed specific adaptive strategies that allow them to rapidly detect and respond to abiotic stress factors. The detoxification efflux carriers (DTX)/multidrug and toxic compound extrusion (MATE) transporters are of significance in the translocation of abscisic acid (ABA), a phytohormone with profound role in plants under various abiotic stress conditions. The ABA signaling cascades are the core regulators of abiotic stress responses in plants, triggering major changes in gene expression and adaptive physiological responses. We therefore carried out genome-wide analysis of the DTX/MATE gene family, transformed a DTX/MATE gene in Arabidopsis and carried out functional analysis under drought, salt, and cold stress conditions. We identified 128, 70, and 72 DTX/MATE genes in Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii, respectively. The proteins encoded by the DTX/MATE genes showed varied physiochemical properties but they all were hydrophobic. The Gh_D06G0281 (DTX/MATE) over-expressing Arabidopsis lines were highly tolerant under drought, salt, and cold stress with high production of antioxidant enzymes and significantly reduced levels of oxidants. Lipid peroxidation, as measured by the level of malondialdehyde concentrations was relatively low in transgenic lines compared to wild types, an indication of reduced oxidative stress levels in the transgenic plants. Based on physiological measurements, the transgenic plants exhibited significantly higher relative leaf water content, reduced excised leaf water loss and a significant reduction in ion leakage as a measure of the cell membrane stability compared to the wild types. Abiotic stress responsive genes, ABF4, CBL1, SOS1, and RD29B were highly expressed in the transgenic lines compared to the non-transformed wild type plants. The protein encoded by the Gh_D06G0281 (DTX/MATE) gene was predicted to be located within the plasma membrane. Since signals from extracellular stimuli are transmitted through the plasma membrane most of which are conducted by plasma membrane proteins it is possible the Gh_D06G0281 (DTX/MATE) gene product could be important for this process.

Abiotic stresses have negative effects on plants growth and development. Plants, being sessile, have developed specific adaptive strategies that allow them to rapidly detect and respond to abiotic stress factors. The detoxification efflux carriers (DTX)/multidrug and toxic compound extrusion (MATE) transporters are of significance in the translocation of abscisic acid (ABA), a phytohormone with profound role in plants under various abiotic stress conditions. The ABA signaling cascades are the core regulators of abiotic stress responses in plants, triggering major changes in gene expression and adaptive physiological responses. We therefore carried out genome-wide analysis of the DTX/MATE gene family, transformed a DTX/MATE gene in Arabidopsis and carried out functional analysis under drought, salt, and cold stress conditions. We identified 128, 70, and 72 DTX/MATE genes in Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii, respectively. The proteins encoded by the DTX/MATE genes showed varied physiochemical properties but they all were hydrophobic. The Gh_D06G0281 (DTX/MATE) over-expressing Arabidopsis lines were highly tolerant under drought, salt, and cold stress with high production of antioxidant enzymes and significantly reduced levels of oxidants. Lipid peroxidation, as measured by the level of malondialdehyde concentrations was relatively low in transgenic lines compared to wild types, an indication of reduced oxidative stress levels in the transgenic plants. Based on physiological measurements, the transgenic plants exhibited significantly higher relative leaf water content, reduced excised leaf water loss and a significant reduction in ion leakage as a measure of the cell membrane stability compared to the wild types. Abiotic stress responsive genes, ABF4, CBL1, SOS1, and RD29B were highly expressed in the transgenic lines compared to the non-transformed wild type plants. The protein encoded by the Gh_D06G0281 (DTX/MATE) gene was predicted to be located within the plasma membrane. Since signals from extracellular stimuli are transmitted through the plasma membrane most of which are conducted by plasma membrane proteins it is possible the Gh_D06G0281 (DTX/MATE) gene product could be important for this process.

Cotton production is on the decline due to ever-changing environmental conditions. Drought and salinity stress contribute to over 30% of total loss in cotton production, the situation has worsened more due to the narrow genetic base of the cultivated upland cotton. The genetic diversity of upland cotton has been eroded over the years due to intense selection and inbreeding. To break the bottleneck, the wild cotton progenitors offer unique traits which can be introgressed into the cultivated cotton, thereby improving their performance. In this research, we developed a BC2F2 population between wild male parent, G. tomentosum as the donor, known for its high tolerance to drought and the elite female parent, G. hirsutum as the recurrent parent, which is high yielding but sensitive to drought stress. The population was genotyped through the genotyping by sequencing (GBS) method, in which 10,888 single-nucleotide polymorphism (SNP) s were generated and used to construct a genetic map. The map spanned 4191.3 cM, with average marker distance of 0.3849 cM. The map size of the two sub genomes had a narrow range, 2149 cM and 2042.3 cM for At and Dt_sub genomes respectively. A total of 66,434 genes were mined, with 32,032 (48.2%) and 34,402 (51.8%) genes being obtained within the At and Dt_sub genomes respectively. Pkinase (PF00069) was found to be the dominant domain, with 1069 genes. Analysis of the main sub family, serine threonine protein kinases through gene ontology (GO), cis element and miRNA targets analysis revealed that most of the genes were involved in various functions aimed at enhancing abiotic stress tolerance. Further analysis of the RNA sequence data and qRT-PCR validation revealed 16 putative genes, which were highly up regulated under drought stress condition, and were found to be targeted by ghr-miR169a and ghr-miR164, previously associated with NAC(NAM, ATAF1/2 and CUC2) and myeloblastosis (MYB), the top rank drought stress tolerance genes. These genes can be exploited further to aid in development of more drought tolerant cotton genotypes.