Genome-wide Identification And Functional Characterization Of PP2C Gene Family In Response To Drought Stress In Cotton

Plants are continuously exposed to abiotic stresses which affect plant growth and productivity.Among them,drought is the most significant factor which limits crop production.Cotton is an important fiber and oil crop,commercially grown worldwide.Drought stress particularly causes significant losses in cotton production,as more than half of the global cotton is grown in high water shortage areas.Therefore,it is imperative to understand the molecular and genetic basis underlying cotton drought response for developing the cultivars with improved tolerance.Thus,we explored the Gossypium hirsutum genome for protein phosphatases(PP2C)gene family that is largely known to participate in various stress responses.In the present study,we found 87(G.arboreum),147(G.barbadense),181(G.hirsutum),and 99(G.raimondii)PP2C-encoding genes in total from the cotton genome.We provided a comprehensive analysis of the PP2 C gene family in cotton,such as gene structure organization,gene duplications,expression profiling,chromosomal mapping,protein motif organization and phylogenetic relationships of each specie.PP2 C genes were further categorized into 12 subgroups based on their phylogenetic relationships.We employed previously reported RNA-seq data to observe the tissue-specific response of Gh PP2 C genes in organ and fiber development.The q RT-PCR validation of 30 Gh PP2 C genes suggested their critical role in cotton by exposure to heat,cold,drought,and salt stress treatments.We characterized three strongly induced PP2 C genes in response to drought stress.GhHAI2,GhAHG3 and GhABI2 proteins belong to clade A of cotton PP2 C and share high sequence similarity with Arabidopsis HAI2,AHG3 and ABI2,respectively.The expression analysis revealed that GhHAI2,GhAHG3 and GhABI2 genes were significantly up-regulated after exposure to drought and ABA.The virus-induced gene silencing(VIGS)experiment was conducted to investigate the role of these genes in drought tolerance.Silencing of GhHAI2,GhAHG3 and GhABI2 genes enhanced the resistance of cotton plants to drought stress.The phenotype of silenced plants under normal growth conditions had no significant difference compared with the control plants.After drought stress,the control plants displayed severe leaf wilting than silenced plants accompanied by high transpiration loss and less relative water content.The silenced plants showed significant induction in photosynthetic rates,intracellular CO2 concentration,stomatal conductance and transpiration rate.In addition,higher antioxidant enzyme activity and decreased MDA accumulation was found in silenced plants compare with the control.The root phenotype analysis showed that silencing of genes promoted the root thickness,volume,dry weight and diameter which further improved the drought tolerance.These results indicated that GhHAI2,GhAHG3 and GhABI2 genes are negatively involved in drought stress tolerance in cotton.The yeast two-hybrid(Y2H)and luciferase complementation imaging assays(LCI)were performed to verify the interaction between GhHAI2,GhAHG3 and GhABI2 and ABA receptors Gh PYLs.The results revealed that Gh PYLs interact with and inhibit the phosphatase activity of GhHAI2,GhAHG3 and GhABI2 thereby activating the downstream target genes involved in ABA signaling pathway.Furthermore,q RT-PCR analysis was performed to confirm the expression of drought responsive genes of ABA pathway.The results showed that silencing of GhHAI2,GhAHG3,and GhABI2 genes significantly increased the expression of ABA-dependent marker genes as compared to ABA-independent marker gene.Consistently,the expression of ABA biosynthesis genes Gh NCEDa and Gh NCEDc was significantly higher in silenced plants compared to the control.These results showed that GhHAI2,GhAHG3 and GhABI2 are involved in cotton drought tolerance via the ABA-dependent signaling pathway.Hence,our findings provide a comprehensive overview of the PP2 C gene family in cotton based on various bioinformatics tools that demonstrated their evolution and critical role in organ and fiber development.The functional characterization of three candidate genes involved in drought tolerance will aid future research by leading to cotton genetic improvement for resistant cultivars.