Knockdown of Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) Dehydrin genes, Reveals their potential role in enhancing osmotic and salt tolerance in cotton.

In this investigation, whole-genome identification and functional characterization of the cotton dehydrin genes was carried out. A total of 16, 7, and 7 dehydrin proteins were identified in G. hirsutum, G. arboreum and G. raimondii, respectively. Through RNA sequence data and RT-qPCR validation, Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) were highly upregulated, and knockdown of the two genes, significantly reduced the ability of the plants to tolerate the effects of osmotic and salt stress. The VIGS-plants recorded significantly higher concentration levels of oxidants, hydrogen peroxide (H2O2) and malondialdehyde (MDA), furthermore, the four stress responsive genes GhLEA2, Gh_D12G2017 (CDKF4), Gh_A07G0747 (GPCR) and a transcription factor, trihelix, Gh_A05G2067, were significantly downregulated in VIGS-plants, but upregulated in wild types under osmotic and salt stress condition. The result indicated that dehydrin proteins are vital for plants and can be exploited in developing a more osmotic and salt stress-resilient germplasm to boost and improve cotton production.

Genome-wide analysis of the cotton G-coupled receptor proteins (GPCR) and functional analysis of GTOM1, a novel cotton GPCR gene under drought and cold stress.

BACKGROUND: The efficient detection and initiation of appropriate response to abiotic stresses are important to plants survival. The plant G-protein coupled receptors (GPCRs) are diverse membranous proteins that are responsible for signal transduction. RESULTS: In this research work, we identified a novel gene of the GPCR domain, transformed and carried out the functional analysis in Arabidopsis under drought and cold stresses. The transgenic lines exposed to drought and cold stress conditions showed higher germination rate, increased root length and higher fresh biomass accumulation. Besides, the levels of antioxidant enzymes, glutathione (GSH) and ascorbate peroxidase (APX) exhibited continuously increasing trends, with approximately threefold higher than the control, implying that these ROS-scavenging enzymes were responsible for the detoxification of ROS induced by drought and cold stresses. Similarly, the transgenic lines exhibited stable cell membrane stability (CMS), reduced water loss rate in the detached leaves and significant values for the saturated leaves compared to the wild types. Highly stress-responsive miRNAs were found to be targeted by the novel gene and based on GO analysis; the protein encoded by the gene was responsible for maintaining an integral component of membrane. In cotton, the virus-induced gene silencing (VIGS) plants exhibited a higher susceptibility to drought and cold stresses compared to the wild types. CONCLUSION: The novel GPCR gene enhanced drought and cold stress tolerance in transgenic Arabidopsis plants by promoting root growth and induction of ROS scavenging enzymes. The outcome showed that the gene had a role in enhancing drought and cold stress tolerance, and can be further exploited in breeding for more stress-resilient and tolerant crops.

Functional characterization of Gh_A08G1120 (GH3.5) gene reveal their significant role in enhancing drought and salt stress tolerance in cotton.

BACKGROUND: Auxins play an important role in plant growth and development; the auxins responsive gene; auxin/indole-3-acetic acid (Aux/IAA), small auxin-up RNAs (SAUR) and Gretchen Hagen3 (GH3) control their mechanisms. The GH3 genes function in homeostasis by the catalytic activities in auxin conjugation and bounding free indole-3-acetic acid (IAA) to amino acids. RESULTS: In our study, we identified the GH3 genes in three cotton species; Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii, analyzed their chromosomal distribution, phylogenetic relationships, cis-regulatory element function and performed virus induced gene silencing of the novel Gh_A08G1120 (GH3.5) gene. The phylogenetic tree showed four clusters of genes with clade 1, 3 and 4 having mainly members of the GH3 of the cotton species while clade 2 was mainly members belonging to Arabidopsis. There were no paralogous genes, and few orthologous genes were observed between Gossypium and other species. All the GO terms were detected, but only 14 genes were found to have described GO terms in upland cotton, more biological functions were detected, as compared to the other functions. The GH3.17 subfamily harbored the highest number of the cis-regulatory elements, most having promoters towards dehydration-responsiveness. The RNA expression analysis revealed that 10 and 8 genes in drought and salinity stress conditions respectively were upregulated in G. hirsutum. All the genes that were upregulated in plants under salt stress conditions were also upregulated in drought stress; moreover, Gh_A08G1120 (GH3.5) exhibited a significant upregulation across the two stress factors. Functional characterization of Gh_A08G1120 (GH3.5) through virus-induced gene silencing (VIGS) revealed that the VIGS plants ability to tolerate drought and salt stresses was significantly reduced compared to the wild types. The chlorophyll content, relative leaf water content (RLWC), and superoxide dismutase (SOD) concentration level were reduced significantly while malondialdehyde concentration and ion leakage as a measure of cell membrane stability (CMS) increased in VIGS plants under drought and salt stress conditions. CONCLUSION: This study revealed the significance of the GH3 genes in enabling the plant's adaptation to drought and salt stress conditions as evidenced by the VIGS results and RT-qPCR analysis.

Map-Based Functional Analysis of the GhNLP Genes Reveals Their Roles in Enhancing Tolerance to N-Deficiency in Cotton.

Nitrogen is a key macronutrient needed by plants to boost their production, but the development of cotton genotypes through conventional approaches has hit a bottleneck due to the narrow genetic base of the elite cotton cultivars, due to intensive selection and inbreeding. Based on our previous research, in which the BC2F2 generations developed from two upland cotton genotypes, an abiotic stress-tolerant genotype, G. tomentosum (donor parent) and a highly-susceptible, and a highly-susceptible, but very productive, G. hirsutum (recurrent parent), were profiled under drought stress conditions. The phenotypic and the genotypic data generated through genotyping by sequencing (GBS) were integrated to map drought-tolerant quantitative trait loci (QTLs). Within the stable QTLs region for the various drought tolerance traits, a nodule-inception-like protein (NLP) gene was identified. We performed a phylogenetic analysis of the NLP proteins, mapped their chromosomal positions, intron-exon structures and conducted ds/dn analysis, which showed that most NLP genes underwent negative or purifying selection. Moreover, the functions of one of the highly upregulated genes, Gh_A05G3286 (Gh NLP5), were evaluated using the virus gene silencing (VIGS) mechanism. A total of 226 proteins encoded by the NLP genes were identified, with 105, 61, and 60 in Gossypium hirsutum, G. raimondii, and G. arboreum, respectively. Comprehensive Insilico analysis revealed that the proteins encoded by the NLP genes had varying molecular weights, protein lengths, isoelectric points (pI), and grand hydropathy values (GRAVY). The GRAVY values ranged from a negative one to zero, showing that proteins were hydrophilic. Moreover, various cis-regulatory elements that are the binding sites for stress-associated transcription factors were found in the promoters of various NLP genes. In addition, many miRNAs were predicted to target NLP genes, notably miR167a, miR167b, miR160, and miR167 that were previously shown to target five NAC genes, including NAC1 and CUC1, under N-limited conditions. The real-time quantitative polymerase chain reaction (RT-qPCR) analysis, revealed that five genes, Gh_D02G2018, Gh_A12G0439, Gh_A03G0493, Gh_A03G1178, and Gh_A05G3286 were significantly upregulated and perhaps could be the key NLP genes regulating plant response under N-limited conditions. Furthermore, the knockdown of the Gh_A05G3286 (GhNLP5) gene by virus-induced silencing (VIGS) significantly reduced the ability of these plants to the knockdown of the Gh_A05G3286 (GhNLP5) gene by virus-induced gene silencing (VIGS) significantly reduced the ability of the VIGS-plants to tolerate N-limited conditions compared to the wild types (WT). The VIGS-plants registered lower chlorophyll content, fresh shoot biomass, and fresh root biomass, addition to higher levels of malondialdehyde (MDA) and significantly reduced levels of proline, and superoxide dismutase (SOD) compared to the WT under N-limited conditions. Subsequently, the expression levels of the Nitrogen-stress responsive genes, GhTap46, GhRPL18A, and GhKLU were shown to be significantly downregulated in VIGS-plants compared to their WT under N-limited conditions. The downregulation of the nitrogen-stress responsive genes provided evidence that the silenced gene had an integral role in enhancing cotton plant tolerance to N-limited conditions.

Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton.

BACKGROUND: Late embryogenesis abundant (LEA) proteins are large groups of hydrophilic proteins with major role in drought and other abiotic stresses tolerance in plants. In-depth study and characterization of LEA protein families have been carried out in other plants, but not in upland cotton. The main aim of this research work was to characterize the late embryogenesis abundant (LEA) protein families and to carry out gene expression analysis to determine their potential role in drought stress tolerance in upland cotton. Increased cotton production in the face of declining precipitation and availability of fresh water for agriculture use is the focus for breeders, cotton being the backbone of textile industries and a cash crop for many countries globally. RESULTS: In this work, a total of 242, 136 and 142 LEA genes were identified in G. hirsutum, G. arboreum and G. raimondii respectively. The identified genes were classified into eight groups based on their conserved domain and phylogenetic tree analysis. LEA 2 were the most abundant, this could be attributed to their hydrophobic character. Upland cotton LEA genes have fewer introns and are distributed in all chromosomes. Majority of the duplicated LEA genes were segmental. Syntenic analysis showed that greater percentages of LEA genes are conserved. Segmental gene duplication played a key role in the expansion of LEA genes. Sixty three miRNAs were found to target 89 genes, such as miR164, ghr-miR394 among others. Gene ontology analysis revealed that LEA genes are involved in desiccation and defense responses. Almost all the LEA genes in their promoters contained ABRE, MBS, W-Box and TAC-elements, functionally known to be involved in drought stress and other stress responses. Majority of the LEA genes were involved in secretory pathways. Expression profile analysis indicated that most of the LEA genes were highly expressed in drought tolerant cultivars Gossypium tomentosum as opposed to drought susceptible, G. hirsutum. The tolerant genotypes have a greater ability to modulate genes under drought stress than the more susceptible upland cotton cultivars. CONCLUSION: The finding provides comprehensive information on LEA genes in upland cotton, G. hirsutum and possible function in plants under drought stress.

QTL Mapping of Fiber Quality and Yield-Related Traits in an Intra-Specific Upland Cotton Using Genotype by Sequencing (GBS).

Fiber quality and yield improvement are crucial for cotton domestication and breeding. With the transformation in spinning techniques and multiplicity needs, the development of cotton fiber quality and yield is of great importance. A genetic map of 5178 Single Nucleotide Polymorphism (SNP) markers were generated using 277 F2:3 population, from an intra-specific cross between two upland cotton accessions, CCRI35 a high fiber quality as female and Nan Dan Ba Di Da Hua (NH), with good yield properties as male parent. The map spanned 4768.098 cM with an average distance of 0.92 cM. A total of 110 Quantitative Traits Loci (QTLs) were identified for 11 traits, but only 30 QTLs were consistent in at least two environments. The highest percentage of phenotypic variance explained by a single QTL was 15.45%. Two major cluster regions were found, cluster 1 (chromosome17-D03) and cluster 2 (chromosome26-D12). Five candidate genes were identified in the two QTL cluster regions. Based on GO functional annotation, all the genes were highly correlated with fiber development, with functions such as protein kinase and phosphorylation. The five genes were associated with various fiber traits as follows: Gh_D03G0889 linked to qFM-D03_cb, Gh_D12G0093, Gh_D12G0410, Gh_D12G0435 associated with qFS-D12_cb and Gh_D12G0969 linked to qFY-D12_cb. Further structural annotation and fine mapping is needed to determine the specific role played by the five identified genes in fiber quality and yield related pathway.

GBS Mapping and Analysis of Genes Conserved between Gossypium tomentosum and Gossypium hirsutum Cotton Cultivars that Respond to Drought Stress at the Seedling Stage of the BC2F2 Generation.

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.

Whole Genome Analysis of Cyclin Dependent Kinase (CDK) Gene Family in Cotton and Functional Evaluation of the Role of CDKF4 Gene in Drought and Salt Stress Tolerance in Plants.

Cotton (Gossypium spp.) is the number one crop cultivated for fiber production and the cornerstone of the textile industry. Drought and salt stress are the major abiotic stresses, which can have a huge economic impact on cotton production; this has been aggravated with continued climate change, and compounded by pollution. Various survival strategies evolved by plants include the induction of various stress responsive genes, such as cyclin dependent kinases (CDKs). In this study, we performed a whole-genome identification and analysis of the CDK gene family in cotton. We identified 31, 12, and 15 CDK genes in G. hirsutum, G. arboreum, and G. raimondii respectively, and they were classified into 6 groups. CDK genes were distributed in 15, 10, and 9 linkage groups of AD, D, and A genomes, respectively. Evolutionary analysis revealed that segmental types of gene duplication were the primary force underlying CDK genes expansion. RNA sequence and RT-qPCR validation revealed that Gh_D12G2017 (CDKF4) was strongly induced by drought and salt stresses. The transient expression of Gh_D12G2017-GFP fusion protein in the protoplast showed that Gh_D12G2017 was localized in the nucleus. The transgenic Arabidopsis lines exhibited higher concentration levels of the antioxidant enzymes measured, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) concentrations under drought and salt stress conditions with very low levels of oxidants. Moreover, cell membrane stability (CMS), excised leaf water loss (ELWL), saturated leaf weight (SLW), and chlorophyll content measurements showed that the transgenic Arabidopsis lines were highly tolerant to either of the stress factors compared to their wild types. Moreover, the expression of the stress-related genes was also significantly up-regulated in Gh_D12G2017(CDKF4) transgenic Arabidopsis plants under drought and salt conditions. We infer that CDKF-4s and CDKG-2s might be the primary regulators of salt and drought responses in cotton.

Simple Sequence Repeat (SSR) Genetic Linkage Map of D Genome Diploid Cotton Derived from an Interspecific Cross between Gossypium davidsonii and Gossypium klotzschianum.

The challenge in tetraploid cotton cultivars is the narrow genetic base and therefore, the bottleneck is how to obtain interspecific hybrids and introduce the germplasm directly from wild cotton to elite cultivars. Construction of genetic maps has provided insight into understanding the genome structure, interrelationships between organisms in relation to evolution, and discovery of genes that carry important agronomic traits in plants. In this study, we generated an interspecific hybrid between two wild diploid cottons, Gossypium davidsonii and Gossypium klotzschianum, and genotyped 188 F2:3 populations in order to develop a genetic map. We screened 12,560 SWU Simple Sequence Repeat (SSR) primers and obtained 1000 polymorphic markers which accounted for only 8%. A total of 928 polymorphic primers were successfully scored and only 728 were effectively linked across the 13 chromosomes, but with an asymmetrical distribution. The map length was 1480.23 cM, with an average length of 2.182 cM between adjacent markers. A high percentage of the markers on the map developed, and for the physical map of G. raimondii, exhibited highly significant collinearity, with two types of duplication. High level of segregation distortion was observed. A total of 27 key genes were identified with diverse roles in plant hormone signaling, development, and defense reactions. The achievement of developing the F2:3 population and its genetic map constructions may be a landmark in establishing a new tool for the genetic improvement of cultivars from wild plants in cotton. Our map had an increased recombination length compared to other maps developed from other D genome cotton species.

High-Density Linkage Map Construction and Mapping of Salt-Tolerant QTLs at Seedling Stage in Upland Cotton Using Genotyping by Sequencing (GBS).

Over 6% of agricultural land is affected by salinity. It is becoming obligatory to use saline soils, so growing salt-tolerant plants is a priority. To gain an understanding of the genetic basis of upland cotton tolerance to salinity at seedling stage, an intra-specific cross was developed from CCRI35, tolerant to salinity, as female with Nan Dan (NH), sensitive to salinity, as the male. A genetic map of 5178 SNP markers was developed from 277 F2:3 populations. The map spanned 4768.098 cM, with an average distance of 0.92 cM. A total of 66 QTLs for 10 traits related to salinity were detected in three environments (0, 110, and 150 mM salt treatment). Only 14 QTLs were consistent, accounting for 2.72% to 9.87% of phenotypic variation. Parental contributions were found to be in the ratio of 3:1, 10 QTLs from the sensitive and four QTLs from the resistant parent. Five QTLs were located in At and nine QTLs in the Dt sub-genome. Moreover, eight clusters were identified, in which 12 putative key genes were found to be related to salinity. The GBS-SNPs-based genetic map developed is the first high-density genetic map that has the potential to provide deeper insights into upland cotton salinity tolerance. The 12 key genes found in this study could be used for QTL fine mapping and cloning for further studies.

Late embryogenesis abundant gene LEA3 (Gh_A08G0694) enhances drought and salt stress tolerance in cotton.

Plants have evolved a complex and organized response to abiotic stress that involves physiological and metabolic reprogramming, transcription control, epigenetic regulation, and expressions of thousand interacting genes for instance the late embryogenesis abundant (LEA) proteins are expressed in multiple environmental variables during the plant developmental period, and thus play critical role in enhancing drought and salt stress tolerance. A comprehensive molecular and functional characterization of the LEA3 gene was carried out in cotton under abiotic stress conditions in order to elucidate their functions. Seventy eight genes were identified in cotton, and were clustered into six clades moreover; the LEA genes were more upregulated in the tissues of the tetraploid cotton compared to the diploid type. A key gene, Gh_A08G0694 was the most upregulated, and was knocked in tetraploid cotton, the knocked out significantly increased the susceptibility of cotton plants to salinity and drought stresses, moreover, several ABA/stress-associated genes were down regulated. Similarly, overexpression of the key gene, significantly increased tolerance of the overexpressed plants to drought and salinity stress. The key gene is homologous to GhLEA3 protein, found to have strong interaction to key abiotic stress tolerance genes, voltage-dependent anion channel 1 (VDAC1) and glyceraldehyde-3-phosphate dehydrogenase A (gapA).

High seroprevalence of Immunoglobulin G (IgG) and IgM antibodies to SARS-CoV-2 in asymptomatic and symptomatic individuals amidst vaccination roll-out in western Kenya.

The population's antibody response is a key factor in comprehending SARS-CoV-2 epidemiology. This is especially important in African settings where COVID-19 impact, and vaccination rates are relatively low. This study aimed at characterizing the Immunoglobulin G (IgG) and Immunoglobulin M (IgM) in both SARS-CoV-2 asymptomatic and symptomatic individuals in Kisumu and Siaya counties in western Kenya using enzyme linked immunosorbent assays. The IgG and IgM overall seroprevalence in 98 symptomatic and asymptomatic individuals in western Kenya between December 2021-March 2022 was 76.5% (95% CI = 66.9-84.5) and 29.6% (95% CI = 20.8-39.7) respectively. In terms of gender, males had slightly higher IgG positivity 87.5% (35/40) than females 68.9% (40/58). Amidst the ongoing vaccination roll-out during the study period, over half of the study participants (55.1%, 95% CI = 44.7-65.2) had not received any vaccine. About one third, (31.6%, 95% CI = 22.6-41.8) of the study participants had been fully vaccinated, with close to a quarter (13.3% 95% CI = 7.26-21.6) partially vaccinated. When considering the vaccination status and seroprevalence, out of the 31 fully vaccinated individuals, IgG seropositivity was 81.1% (95% CI = 70.2-96.3) and IgM seropositivity was 35.5% (95% CI = 19.22-54.6). Out of the participants that had not been vaccinated at all, IgG seroprevalence was 70.4% (95% CI 56.4-82.0) with 20.4% (95% CI 10.6-33.5) seropositivity for IgM antibodies. On PCR testing, 33.7% were positive, with 66.3% negative. The 32 positive individuals included 12(37.5%) fully vaccinated, 8(25%) partially vaccinated and 12(37.5%) unvaccinated. SARs-CoV-2 PCR positivity did not significantly predict IgG (p = 0.469 [95% CI 0.514-4.230]) and IgM (p = 0.964 [95% CI 0.380-2.516]) positivity. These data indicate a high seroprevalence of antibodies to SARS-CoV-2 in western Kenya. This suggests that a larger fraction of the population was infected with SARS-CoV-2 within the defined period than what PCR testing could cover.

Functional Characterization of Cotton C-Repeat Binding Factor Genes Reveal Their Potential Role in Cold Stress Tolerance.

Low temperature is a common biological abiotic stress in major cotton-growing areas. Cold stress significantly affects the growth, yield, and yield quality of cotton. Therefore, it is important to develop more robust and cold stress-resilient cotton germplasms. In response to climate change and erratic weather conditions, plants have evolved various survival mechanisms, one of which involves the induction of various stress responsive transcript factors, of which the C-repeat-binding factors (CBFs) have a positive effect in enhancing plants response to cold stress. In this study, genomewide identification and functional characterization of the cotton CBFs were carried out. A total of 29, 28, 25, 21, 30, 26, and 15 proteins encoded by the CBF genes were identified in seven Gossypium species. A phylogenetic evaluation revealed seven clades, with Clades 1 and 6 being the largest. Moreover, the majority of the proteins encoded by the genes were predicted to be located within the nucleus, while some were distributed in other parts of the cell. Based on the transcriptome and RT-qPCR analysis, Gthu17439 (GthCBF4) was highly upregulated and was further validated through forward genetics. The Gthu17439 (GthCBF4) overexpressed plants exhibited significantly higher tolerance to cold stress, as evidenced by the higher germination rate, increased root growth, and high-induction levels of stress-responsive genes. Furthermore, the overexpressed plants under cold stress had significantly reduced oxidative damage due to a reduction in hydrogen peroxide (H2O2) production. Moreover, the overexpressed plants under cold stress had minimal cell damage compared to the wild types, as evidenced by the Trypan and 3,3'-Diaminobenzidine (DAB) staining effect. The results showed that the Gthu17439 (GthCBF4) could be playing a significant role in enhancing cold stress tolerance in cotton and can be further exploited in developing cotton germplasm with improved cold-stress tolerance.

Functional Characterization of GhACX3 Gene Reveals Its Significant Role in Enhancing Drought and Salt Stress Tolerance in Cotton.

The acyl-coenzyme A oxidase 3 (ACX3) gene involved in the ß-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 ß-oxidation limited to fatty acid breakdown. However, ACX3 peroxisomal ß-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.

Knockdown of Cytochrome P450 Genes Gh_D07G1197 and Gh_A13G2057 on Chromosomes D07 and A13 Reveals Their Putative Role in Enhancing Drought and Salt Stress Tolerance in Gossypium hirsutum.

We identified 672, 374, and 379 CYPs proteins encoded by the CYPs genes in Gossypiumhirsutum, Gossypiumraimondii, and Gossypiumarboreum, respectively. The genes were found to be distributed in all 26 chromosomes of the tetraploid cotton, with chrA05, chrA12, and their homeolog chromosomes harboring the highest number of genes. The physiochemical properties of the proteins encoded by the CYP450 genes varied in terms of their protein lengths, molecular weight, isoelectric points (pI), and even grand hydropathy values (GRAVY). However, over 99% of the cotton proteins had GRAVY values below 0, which indicated that the majority of the proteins encoded by the CYP450 genes were hydrophilic in nature, a common property of proteins encoded by stress-responsive genes. Moreover, through the RNA interference (RNAi) technique, the expression levels of Gh_D07G1197 and Gh_A13G2057 were suppressed, and the silenced plants showed a higher concentration of hydrogen peroxide (H2O2) with a significant reduction in the concentration levels of glutathione (GSH), ascorbate peroxidase (APX), and proline compared to the wild types under drought and salt stress conditions. Furthermore, the stress-responsive genes 1-Pyrroline⁻5-Carboxylate Synthetase (GhP5CS), superoxide dismutase (GhSOD), and myeloblastosis (GhMYB) were downregulated in VIGS plants, but showed upregulation in the leaf tissues of the wild types under drought and salt stress conditions. In addition, CYP450-silenced cotton plants exhibited a high level of oxidative injury due to high levels of oxidant enzymes, in addition to negative effects on CMS, ELWL, RLWC, and chlorophyll content The results provide the basic foundation for future exploration of the proteins encoded by the CYP450 genes in order to understand the physiological and biochemical mechanisms in enhancing drought and salt stress tolerance in plants.

Overexpression of Cotton a DTX/MATE Gene Enhances Drought, Salt, and Cold Stress Tolerance in Transgenic Arabidopsis.

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.

Knockdown of ghAlba_4 and ghAlba_5 Proteins in Cotton Inhibits Root Growth and Increases Sensitivity to Drought and Salt Stresses.

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.

Knockdown of GhIQD31 and GhIQD32 increases drought and salt stress sensitivity in Gossypium hirsutum.

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.

A Novel G-Protein-Coupled Receptors Gene from Upland Cotton Enhances Salt Stress Tolerance in Transgenic Arabidopsis.

Plants have developed a number of survival strategies which are significant for enhancing their adaptation to various biotic and abiotic stress factors. At the transcriptome level, G-protein-coupled receptors (GPCRs) are of great significance, enabling the plants to detect a wide range of endogenous and exogenous signals which are employed by the plants in regulating various responses in development and adaptation. In this research work, we carried out genome-wide analysis of target of Myb1 (TOM1), a member of the GPCR gene family. The functional role of TOM1 in salt stress tolerance was studied using a transgenic Arabidopsis plants over-expressing the gene. By the use of the functional domain PF06454, we obtained 16 TOM genes members in Gossypium hirsutum, 9 in Gossypium arboreum, and 11 in Gossypium raimondii. The genes had varying physiochemical properties, and it is significant to note that all the grand average of hydropathy (GRAVY) values were less than one, indicating that all are hydrophobic in nature. In all the genes analysed here, both the exonic and intronic regions were found. The expression level of Gh_A07G0747 (GhTOM) was significantly high in the transgenic lines as compared to the wild type; a similar trend in expression was observed in all the salt-related genes tested in this study. The study in epidermal cells confirmed the localization of the protein coded by the gene TOM1 in the plasma membrane. Analysis of anti-oxidant enzymes showed higher concentrations of antioxidants in transgenic lines and relatively lower levels of oxidant substances such as H2O2. The low malondialdehyde (MDA) level in transgenic lines indicated that the transgenic lines had relatively low level of oxidative damage compared to the wild types. The results obtained indicate that Gh_A07G0747 (GhTOM) can be a putative target gene for enhancing salt stress tolerance in plants and could be exploited in the future for the development of salt stress-tolerant cotton cultivars.

Genome-Wide Analysis of Multidrug and Toxic Compound Extrusion (MATE) Family in Gossypium raimondii and Gossypium arboreum and Its Expression Analysis Under Salt, Cadmium, and Drought 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.