Overexpression and knockdown of cotton GhdadD gene reveals its drought and salt stress tolerance role.

The 5'-deoxyadenosine deaminase (DADD), a member of the amidohydrolase family regulates biological purine metabolism. In this study, bioinformatic analysis, overexpression and knockdown of GhdadD gene were detected to identify its potential role in drought and salt stress tolerance. The results revealed that GhdadD was induced by ABA, Auxin, MBS and light responsive elements. In transgenic Arabidopsis, seed germination rate and root length were increased under drought or salt stress. GhdadD overexpressed seedlings resulted in higher plant height, less leaf damage and lower ion permeability. The expression of osmotic stress and ABA-responsive genes were up regulated. While in GhdadD-silenced cotton seedlings, CAT, SOD activity and soluble sugar content were reduced, MDA content was increased, and the stoma opening was depressed under drought or salt stress. Some osmics stress marker genes were also up regulated. These data indicating that GhdadD enhanced plant resistance to drought and salt stress through ABA pathways.

Comparative analysis of SARS-CoV-2 detection methods using stool, blood, and nasopharyngeal swab samples.

Introduction: as a public health policy, the ongoing global coronavirus disease 2019 vaccination drives require continuous tracking, tracing, and testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic testing is important in virus detection and understanding its spread for timely intervention. This is especially important for low-income settings where the majority of the population remains untested. This is well supported by the fact that of about 9% of the Kenyan population had been tested for the virus. Methods: this was a cross-sectional study conducted at the Kisumu and Siaya Referral Hospitals in Kenya. Here we report on the sensitivity and specificity of the rapid antigen detection test (Ag-RDT) of SARS-CoV-2 compared with the quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) using stool and nasopharyngeal swab samples. Further, the mean Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibody levels among symptomatic and asymptomatic individuals in western Kenya were evaluated. Results: the sensitivity and specificity of Ag-RDT were 76.3% (95% CI, 59.8-88.6%) and 96.3% (95% CI, 87.3-99.5%) with a negative and positive predictive value of 85% (95% CI, 73.8%-93.0%) and 93% (95% CI, 78.6%-99.2%) respectively. There was substantial agreement of 88% (Kappa value of 0.75, 95% CI, 0.74-0.77) between Ag-RDT and nasopharyngeal swab RT-qPCR, and between stool and nasopharyngeal swab RT-qPCR results (83.7% agreement, Kapa value 0.62, 95% CI 0.45-0.80). The mean IgM and IgG antibody response to SARS-CoV-2 were not different in asymptomatic individuals, 1.11 (95% CI, 0.78-1.44) and 0.88 (95% CI, 0.65-1.11) compared to symptomatic individuals 4.30 (95% CI 3.30-5.31) and 4.16 (95% CI 3.32 -5.00). Conclusion: the choice of an appropriate SARS-CoV-2 diagnostic, screening, and surveillance test should be guided by the specific study needs and a rational approach for optimal results.

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.

Transcriptomics for Drought Stress Mediated by Biological Processes in-relation to Key Regulated Pathways in Gossypium darwinii.

BACKGROUND: Wild cotton Gossypium darwinii, an allotetraploid harbours important traits useful for tolerating abiotic stress, i.e., drought, salt and good genetic stability, hence these characteristics can be transferred to cultivated cotton for genetic improvement. MATERIALS AND METHODS: In this study, we analyzed the RNA-seq transcriptomes from leaves of G. darwinii seedlings with and without drought stress. A total of 86.7 million valid reads with an average length of 95.79 bp were generated from the two samples and 58,960 transcripts with a length of more than 500 bp were assembled. We searched the known proteins on the strength of sequence similarity; these transcripts were annotated with COG, KEGG and GO functional categories. According to gene expression abundance RPKM value, we carried out RT-qPCR analysis to determine the expression pattern of the obtained transcription factors. RESULTS: A total of 58,960 genes was differentially expressed (DEG), with 32,693 and 25,919 genes found to be upregulated and downregulated, respectively. Through gene ontology and KEGG pathways, the upregulated genes were found to associate with all the GO terms, molecular functions (MF), biological process (BP) and cellular components (CC), which are highly linked to enhancing drought stress tolerance. CONCLUSION: The study provides an in-depth knowledge of regulation of pathways and genes involved in photosynthesis during drought stress in G. darwinii. These pathways and genes were found to be significantly downregulated and this information could be further utilized by cotton breeders in developing a more drought tolerant cotton germplasm.

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).

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.

Identification and functional characterization of Gh_D01G0514 (GhNAC072) transcription factor in response to drought stress tolerance in cotton.

Cotton encounters long-term drought stress problems resulting in major yield losses. Transcription factors (TFs) plays an important role in response to biotic and abiotic stresses. The coexpression patterns of gene networks associated with drought stress tolerance were investigated using transcriptome profiles. Applying a weighted gene coexpression network analysis, we discovered a salmon module with 144 genes strongly linked to drought stress tolerance. Based on coexpression and RT-qPCR analysis GH_D01G0514 was selected as the candidate gene, as it was also identified as a hub gene in both roots and leaves with a consistent expression in response to drought stress in both tissues. For validation of GH_D01G0514, Virus Induced Gene Silencing was performed and VIGS plants showed significantly higher excised leaf water loss and ion leakage, while lower relative water and chlorophyll contents as compared to WT (Wild type) and positive control plants. Furthermore, the WT and positive control seedlings showed higher CAT and SOD activities, and lower activities of hydrogen peroxide and MDA enzymes as compared to the VIGS plants. Gh_D01G0514 (GhNAC072) was localized in the nucleus and cytoplasm. Y2H assay demonstrates that Gh_D01G0514 has a potential of auto activation. It was observed that the Gh_D01G0514 was highly upregulated in both tissues based on RNA Seq and RT-qPCR analysis. Thus, we inferred that, this candidate gene might be responsible for drought stress tolerance in cotton. This finding adds significantly to the existing knowledge of drought stress tolerance in cotton and deep molecular analysis are required to understand the molecular mechanisms underlying drought stress tolerance in cotton.

Transcriptomic and proteomic analyses of a new cytoplasmic male sterile line with a wild Gossypium bickii genetic background.

BACKGROUND: Cotton is an important fiber crop but has serious heterosis effects, and cytoplasmic male sterility (CMS) is the major cause of heterosis in plants. However, to the best of our knowledge, no studies have investigated CMS Yamian A in cotton with the genetic background of Australian wild Gossypium bickii. Conjoint transcriptomic and proteomic analysis was first performed between Yamian A and its maintainer Yamian B. RESULTS: We detected 550 differentially expressed transcript-derived fragments (TDFs) and at least 1013 proteins in anthers at various developmental stages. Forty-two TDFs and 11 differentially expressed proteins (DEPs) were annotated by analysis in the genomic databases of G. austral, G. arboreum and G. hirsutum. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to better understand the functions of these TDFs and DEPs. Transcriptomic and proteomic results showed that UDP-glucuronosyl/UDP-glucosyltransferase, 60S ribosomal protein L13a-4-like, and glutathione S-transferase were upregulated; while heat shock protein Hsp20, ATPase, F0 complex, and subunit D were downregulated at the microspore abortion stage of Yamian A. In addition, several TDFs from the transcriptome and several DEPs from the proteome were detected and confirmed by quantitative real-time PCR as being expressed in the buds of seven different periods of development. We established the databases of differentially expressed genes and proteins between Yamian A and its maintainer Yamian B in the anthers at various developmental stages and constructed an interaction network based on the databases for a comprehensive understanding of the mechanism underlying CMS with a wild cotton genetic background. CONCLUSION: We first analyzed the molecular mechanism of CMS Yamian A from the perspective of omics, thereby providing an experimental basis and theoretical foundation for future research attempting to analyze the abortion mechanism of new CMS with a wild Gossypium bickii background and to realize three-line matching.

Comparative transcriptome analysis reveals evolutionary divergence and shared network of cold and salt stress response in diploid D-genome cotton.

BACKGROUND: Wild species of cotton are excellent resistance to abiotic stress. Diploid D-genome cotton showed abundant phenotypic diversity and was the putative donor species of allotetraploid cotton which produce the largest textile natural fiber. RESULTS: A total of 41,053 genes were expressed in all samples by mapping RNA-seq Illumina reads of G. thurberi (D1), G. klotzschianum (D3-k), G. raimondii (D5) and G. trilobum (D8) to reference genome. The numbers of differently expressed genes (DEGs) were significantly higher under cold stress than salt stress. However, 34.1% DEGs under salt stress were overlapped with cold stress in four species. Notably, a potential shared network (cold and salt response, including 16 genes) was mined out by gene co-expression analysis. A total of 47,180-55,548 unique genes were identified in four diploid species by De novo assembly. Furthermore, 163, 344, 330, and 161 positively selected genes (PSGs) were detected in thurberi, G. klotzschianum, G. raimondii and G. trilobum by evolutionary analysis, respectively, and 9.5-17% PSGs of four species were DEGs in corresponding species under cold or salt stress. What's more, most of PSGs were enriched GO term related to response to stimulation. G. klotzschianum showed the best tolerance under both cold and salt stress. Interestingly, we found that a RALF-like protein coding gene not only is PSGs of G. klotzschianum, but also belongs to the potential shared network. CONCLUSION: Our study provided new evidence that gene expression variations of evolution by natural selection were essential drivers of the morphological variations related to environmental adaptation during evolution. Additionally, there exist shared regulated networks under cold and salt stress, such as Ca2+ signal transduction and oxidation-reduction mechanisms. Our work establishes a transcriptomic selection mechanism for altering gene expression of the four diploid D-genome cotton and provides available gene resource underlying multi-abiotic resistant cotton breeding strategy.

Identification of a genome-specific repetitive element in the Gossypium D genome.

The activity of genome-specific repetitive sequences is the main cause of genome variation between Gossypium A and D genomes. Through comparative analysis of the two genomes, we retrieved a repetitive element termed ICRd motif, which appears frequently in the diploid Gossypium raimondii (D5) genome but rarely in the diploid Gossypium arboreum (A2) genome. We further explored the existence of the ICRd motif in chromosomes of G. raimondii, G. arboreum, and two tetraploid (AADD) cotton species, Gossypium hirsutum and Gossypium barbadense, by fluorescence in situ hybridization (FISH), and observed that the ICRd motif exists in the D5 and D-subgenomes but not in the A2 and A-subgenomes. The ICRd motif comprises two components, a variable tandem repeat (TR) region and a conservative sequence (CS). The two constituents each have hundreds of repeats that evenly distribute across 13 chromosomes of the D5genome. The ICRd motif (and its repeats) was revealed as the common conservative region harbored by ancient Long Terminal Repeat Retrotransposons. Identification and investigation of the ICRd motif promotes the study of A and D genome differences, facilitates research on Gossypium genome evolution, and provides assistance to subgenome identification and genome assembling.

Genetic regulatory networks for salt-alkali stress in Gossypium hirsutum with differing morphological characteristics.

BACKGROUND: Cotton grows in altering environments that are often unfavorable or stressful for its growth and development. Consequently, the plant must cope with abiotic stresses such as soil salinity, drought, and excessive temperatures. Alkali-salt stress response remains a cumbersome biological process and is regulated via a multifaceted transcriptional regulatory network in cotton. RESULTS: To discover the molecular mechanisms of alkali-salt stress response in cotton, a comprehensive transcriptome analysis was carried out after alkali-salt stress treatment in three accessions of Gossypium hirsutum with contrasting phenotype. Expression level analysis proved that alkali-salt stress response presented significant stage-specific and tissue-specific. GO enrichment analysis typically suggested that signal transduction process involved in salt-alkali stress response at SS3 and SS12 stages in leaf; carbohydrate metabolic process and oxidation-reduction process involved in SS48 stages in leaf; the oxidation-reduction process involved at all three phases in the root. The Co-expression analysis suggested a potential GhSOS3/GhCBL10-SOS2 network was involved in salt-alkali stress response. Furthermore, Salt-alkali sensitivity was increased in GhSOS3 and GhCBL10 Virus-induced Gene Silencing (VIGS) plants. CONCLUSION: The findings may facilitate to elucidate the underlying mechanisms of alkali-salt stress response and provide an available resource to scrutinize the role of candidate genes and signaling pathway governing alkali-salt stress response.

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.

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.

Comparative transcriptome, physiological and biochemical analyses reveal response mechanism mediated by CBF4 and ICE2 in enhancing cold stress tolerance in Gossypium thurberi.

Low temperature is one of the key environmental stresses that impair plant growth and significantly restricts the productivity and spatial distribution of crop plants. Gossypium thurberi, a wild diploid cotton species, has adapted to a wide range of temperatures and exhibits a better tolerance to chilling stress. Here, we compared phenotypes and physiochemical changes in G. thurberi under cold stress and found this species indeed showed better cold tolerance. Therefore, to understand the molecular mechanisms of the cold tolerance in G. thurberi, we compared transcription changes in leaves of G. thurberi under cold stress by high-throughput transcriptome sequencing. In total, 35 617 unigenes were identified in the whole-genome transcription profile, and 4226 differentially expressed genes (DEGs) were discovered in the leaves upon cold treatment. Gene Ontology (GO) classification analyses showed that the majority of DEGs belonged to categories of signal transduction, transcription factors (TFs) and carbohydrate transport and metabolism. The expression of several cold-responsive genes such as ICE1, CBF4, RAP2-7 and abscisic acid (ABA) biosynthesis genes involved in different signalling pathways were induced after G. thurberi seedlings were exposed to cold stress. Furthermore, cold sensitivity was increased in CBF4 and ICE2 virus-induced gene silencing (VIGS) plants, and high level of malondialdehyde (MDA) showed that the CBF4 and ICE2 silenced plants were under oxidative stress compared to their wild types, which relatively had higher levels of antioxidant enzyme activity, as evident by high levels of proline and superoxide dismutase (SOD) content. In conclusion, our findings reveal a new regulatory network of cold stress response in G. thurberi and broaden our understanding of the cold tolerance mechanism in cotton, which might accelerate functional genomics studies and genetic improvement for cold stress tolerance in cultivated cotton.

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.

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.

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.

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.