The Mechanism Of GhWOX4 Regulating Drought Tolerance In Arabidopsis And Cotton

Cotton is a paramount cash crop around the globe with an interesting history,known for its unique fiber.Cotton possesses an exceptional role in the world economy.Being a biggest source of natural fiber,it also provides edible oil,protein meal and biofuel in a huge aggregate.Amid its life-span cotton experiences a lot of biotic as well as abiotic stresses(drought,salinity,high temperature etc.),that affect its growth and production.In global climate-change scenario among all abiotic stresses,drought is a leading cause of cotton growth and yield loss.Drought not only causes oxidative stress but also osmotic stress,that severely affects biochemical,physiological and morphological mechanisms.These changes trigger the protective measures in plants.The drought stress response is a complex phenomenon.To better understand this phenomenon,study at molecular level is more effective.The WUSCHEL Related homeobox(WOX)gene family is well known for its central role in plant growth and development.But there is not enough work available regarding the role of WOX genes in abiotic stress response.Alongside,the molecular link between drought stress and vascular growth and development is relatively uncharted.In present work,functional genomic study of GhWOX4 was performed,that belongs to WUS-clade of the WOX gene family.The primary structure of GhWOX4 protein showed high sequence and domain resemblance withWOX4 proteins of other plant species.To assess the protein structure and function based upon protein secondary structure,a protein structure and homology modeling analysis was executed.GhWOX4 protein was found out clasping helix-loop-helix-turn-helix structure and signature sequence of WUS-clade.On the basis of subcellular-localization assay GhWOX4 was detected to be localized in the nucleus.Here,validated a crucial role of GhWOX4,a transcription factor,modulating drought stress tolerance with that of vasculature growth in cotton.Knock-down of GhWOX4 decreased the stem width and severely compromised vascular growth and drought tolerance in cotton.The proline,chlorophyll and relative water contents(RWC)were significantly declined,while electrolyte leakage(EL %)and malondialdehyde(MDA)contents were increased in VIGS(virus induced gene silencing)plants.On the other hand,GhWOX4-gene was transferred to Arabidopsis,this ectopic expression enhanced the tolerance to drought stress.The proline,RWC % and survival-rate were higher,but EL %,(water loss)WL % and MDA contents were decreased in transgenic lines as compared to wild type(WT)plants.Overexpression of GhWOX4 in Arabidopsis increased the stem width proving that GhWOX4 may positively regulates the vascular growth and plant development.Plant stem cross-sections revealed reduced vascular region and cambial activity in VIGS plants as compared to control.Furthermore,next generation sequencing(RNAseq)was employed to understand the molecular dynamics.Comparative RNA-seq analysis revealed important phytohormone and transcription factors(TFs)related to plant growth,development and stress response were repressed in GhWOX4-VIGS plants as compared to control plants.Auxin responsive protein(AUX/IAA),abscisic acid(ABA),and ethylene were significantly induced in TRV:156-0h/TRV:156-1h plants in comparison to GhWOX4-VIGS plants.Additionally,MYC-b HLH,NAC,MYB,WRKY,homeodomain,and heat-shock transcription factors(HSF)were differentially expressed in control plants as compared to GhWOX4-silenced plants.Gene ontology(GO)analysis categorized the differentially expressed genes(DEGs)into biological processes,cellular components and molecular functions.DEGs related to stress,water deprivation,and desiccation response were repressed in drought treated GhWOX4-VIGS plants as compared to control.KEGG analysis categorized DEGs in the form of different pathways resulted in;pathways linked to MAPK-signaling,hormonal-signaling,phenylpropanoid biosynthesis,flavone and flavonoid biosynthesis,starch and sucrose metabolism,gluconeogenesis and circadian rhythm,were over-induced in TRV: 156-0 h/TRV: 156-1 h(control)in comparison to TRV: VIGS-0 h/TRV: VIGS-1 h(GhWOX4-silenced)plants.These results backed up the insilico promotor analysis.The promotor-zone of GhWOX4 was found congested with plant growth,light,and stress response related cis-regulatory-elements.To extend the domain of our study,a system-biology tool,weighted gene correlation network analysis(WGCNA)was employed.This research paradigm proposed a comprehensive insight of sequenced data,by providing highly correlated gene-clusters.The 17 gene-modules were obtained with diverse number of genes,but four modules were found to be highly correlated with stress tolerance,plant growth and development.Modules were riddled through KEGG and GO enrichment analyses,the three highly correlated modules were found to be involved in oxidation-reduction process,stress response,transcription factor activity,sequence specific DNA-binding,defense response,developmental process,carbohydrate metabolism etc.In each module hub genes were obtained by using node and edges plot,WGCNA output files.In two significant modules,16 hub genes were identified on the basis of degree method.Hub genes were mainly involved in stress response,growth and development,transcription factor activity,metabolism and kinase activity.Out of these hub genes,two were already reported having protein-protein interaction with that of GhWOX4.Some unique genes were also found,as some hub genes were resulted out to be uncharacterized.These central genes may play a pivotal role in stress response,metabolic processes,plant growth and development.This study improves our context for elucidating the pivotal role of GhWOX4 transcription factor(TF),which mediates drought tolerance,plays a decisive role in plant growth and development,and is likely involved in different regulatory pathways in cotton.