Multi-Omics Analysis And Functional Validation Of Key Gene Networks Linked To Drought Tolerance In Cotton

Cotton encounters long-term drought stress problems resulting in major yield losses.The tissues of Marie galante-85,Latifolium-40,and CRI-12 were subjected to transcriptome sequencing,metabolome,and functional analysis.The number of clean reads were 2.66 Gb,which was then compared to the reference genome in which 64,617 genes were obtained.Analysis of differentially expressed genes at different time points revealed that up-regulated genes were significantly higher than the down-regulated genes under drought stress,suggesting that drought stress tolerant cotton genotypes mainly respond to drought stress through positive regulation.By analyzing the metabolomes of the selected materials,471 metabolites were detected.The association analysis of RNA-Seq and metabolite synthesis indicates that not only a single gene or metabolite,but a multifaceted regulation and signaling mechanisms engage in the role against drought stress.We used transcriptome profiles to investigate the coexpression patterns of gene networks associated with drought stress tolerance.We identified three gene modules containing 3567 genes highly associated with drought stress tolerance.Within these modules,based on intramodular significance we identified 13 hub genes for further validation.Yellow module has five hub genes(Gh?A07G0563,Gh?D05G0221,Gh?A05G3716,Gh?D12G1438,and Gh?D05G0697),brown module contains three hub genes belonging to the ALDH family(Gh?A06G1257,Gh?A06G1256,and Gh?D06G1578),and pink module has five hub genes(Gh?A02G1616,Gh?D12G2599,Gh?D07G2232,Gh?A02G0527,and Gh?D07G0629).Based on RT-q PCR results Gh?A06G1257 gene has the highest expression under drought stress in different plant tissues and it might be the true candidate gene linked to drought stress tolerance in cotton.Silencing of Gh?A06G1257 in cotton leaves conferred significant sensitivity in response to drought stress treatments.Overexpression of Gh?A06G1257 in Arabidopsis also confirms its role in drought stress tolerance.L-valine,Glutaric acid,L-proline,L-Glutamic acid,and L-Tryptophan were found to be the most significant metabolites playing roles in drought stress tolerance.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-q PCR 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(Wildtype)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-q PCR analysis.Overexpression of Gh?D01G0514 in Arabidopsis also confirms its role in drought stress tolerance.Thus,we inferred that,this candidate gene might be responsible for drought stress tolerance in cotton.These findings add 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.