Genome-wide Identification And Functional Characterization Of GhLEA3、GhACX3 And GhRPL14B Genes In Cotton Under Drought And Salt Stress

Cotton（Gossypium spp.）is a cash crop grown all over the world.It is the primary source of natural fiber as represented by cotton lint,which accounts for 90%of the overall economic value of cotton.However,cotton is a versatile crop that is highly sensitive to abiotic stress.These abiotic stresses impede early cotton establishment as well as the entire growth cycle,affecting the plant’s adaptive capacity and restricting the quality and yield of the fiber at an early point.As a result,elucidating the mechanism by which cotton respond to abiotic stress is important for both fiber production and cotton breeding programs.The acclimation of cotton to abiotic stress is a complicated and coordinated response involving thousands of genes and their interactions with multiple environmental variables throughout the plant developmental cycle.Once the plant is subjected to abiotic stress,the stress stimuli are perceived through alterations in turgor pressure or activities of membrane receptors.These extracellular signals are then transformed into intracellular signals by generating secondary messengers like Ca²⁺that activate transcription factors（TFs）or protein kinases（PKs）,resulting in differential expression of unique genes such as osmolytes（proline,glutamate,glycine betaine and sugars）to protect protoplasm from dehydration and enzyme inactivation,protective protein（Late embryogenesis abundance（LEA））and antioxidant systems that scavenge reactive oxygen species（ROS）which attack cellular membrane and organelle through peroxidation damages.This master research study focuses on the molecular response of the group 3 LEA,ACX3 and RPL14B proteins to tolerant and sensitive cotton genotypes under abiotic stress conditions（drought and salinity）.Group 3 Late Embryogenesis Abundant（LEA3）are hydrophilic,intrinsically disordered proteins that play an important role of ensuring membrane stability,calcium and metallic ion binding,DNA-RNA interaction,and protecting other proteins from aggregation,in response to abiotic stresses.In this study,we identified 13,14,14,17,16,7 and 8 LEA genes corresponding to G.hirsutum,G.barbadense,G.tomentosum,G.mustelinum,G.arboreum,and G.raimondii respectively that were clustered into six clades.Genes identified in G.hirsutum showed a clear response to drought and salinity stress,with higher expression under drought and salt stress,particularly in the leaves and roots,according to expression analysis.We selected the highly expressed genes,Gh＿A08G0694 and Gh＿A10G1465,for functional characterization by constructing an overexpression vector and silencing vector and transforming them into Arabidopsis and Gossypium hirsutum,respectively.We profiled the cloned genes under drought and salt stress conditions.Silencing of Gh LEA3（Gh＿A08G0694 and Gh＿A10G1465）increased susceptibility of cotton plants to salinity and drought stresses and caused the downregulation in the expression of several ABA/stress-associated genes.In comparison,over-expression of Gh LEA3 enhanced transgenic Arabidopsis plant tolerance and upregulated the expression of stress-associated genes.Biochemical analysis demonstrated that Gh LEA3 silenced plants experienced oxidative stress as compared to the overexpressed plants.Subcellular localization analysis predicted these genes to be localized in the nucleus,chloroplast,mitochondrion,and cytoplasm.Further analysis revealed that these genes are homologous to Gh LEA3 protein,which interacts with mitochondrial outer membrane protein porin,voltage-dependent anion channels（VDAC1）,and Glyceraldehyde-3-phosphate dehydrogenase（GAPA）.Collectively,our study highlights that Gh LEA3genes regulates drought and salinity stress tolerance through interactions with Gh VDAC1,Gh GAPA,and other regulatory genes.Moreover,they are also involved in the signaling pathway by modulating the transcription of stress-associated genes.As the initiator for fatty acidβ-oxidation,the Acyl-coenzyme A oxidase 3,peroxisomal（ACX3）regulates plants lipid metabolism and production of important plant phytohormones like jasmonic acid（JA）and indole-3-acetic acid（IAA）produced viaβ-oxidation pathway thereby regulating plant growth and abiotic stress response.In this study,we identified 20,18,22,23,20,11 and 9 proteins in G.hirsutum,G.barbadense,G.tomentosum,G.mustelinum,G.darwinii,G.arboreum and G.raimondii genomes.The tetraploid cotton genome had protein ranging between 18 and 22,while diploid were 9and 11.Expression profiling of genes identified in G.hirsutum indicated that,this gene family is highly induced in the roots and leaves under drought and salt stress conditions.We selected Gh＿D01G0186one of the highly expressed genes and which was previously identified in the drought QTL map for functional analysis.We functionally characterized the Gh＿D01G0186 gene through overexpression and VIGS.Under drought and salt stress,Gh＿D01G0186 transgenic plants had increased tolerance to stress and the seeds exhibited higher germination rate than the wild type.In contrast,the silencing of the Gh＿D01G0186 gene in cotton plants results in plants showing stress susceptibility phenotype and reduced root length as compared to wild type.RPL proteins participate in plant abiotic stress（drought/salinity）tolerance responses but the mechanism involving this protein is not fully understood.In the present study,the 60S Ribosomal Protein L14-2 was identified in cotton and functionally characterized.26,6 and 9 RPL14B genes were identified in G.hirsutum,G.arboreum and G.raimondii cotton species respectively.Genes identified in G.hirsutum were profiled in salt/drought stress conditions and found to be differentially expressed.We selected the Gh＿D01G0234 gene for functional analysis via VIGS assay.Knock down of Gh＿D01G0234 had a direct impact on cotton seedling performance under drought/salt stress.We did observe significant differences in morphological and physiological traits between knockdown and control plants.Moreover,antioxidant enzyme levels were significantly decreased in VIGS-plants,while oxidant enzyme levels were significantly higher,as demonstrated by the higher Malondialdehyde concentration level（MDA）.The silenced plants were highly susceptible to abiotic stress as compared to the control plants.In conclusion,functional characterization of the LEA3,ACX3 and RPL14B genes through overexpression and VIGS revealed their potential role in enhancing drought and salt stress tolerance.These genes were highly expressed under both stress conditions and transgenic plants enhanced stress tolerance.Therefore,these genes can be considered for genetic modification of cotton germplasm and engineering climate smart cotton cultivar resistant to drought/salt stress.