Crosstalk Of ABC-transporter And Glutaredoxin Genes To Mediate Salt Stress In Gossypium Hirsutum

The perception of the abiotic stresses and signal transduction to switch on adaptive responses are critical steps in determining the survival and growth of plants exposed to adverse environments.Production of oxidative stress in such environments,reflect an imbalance between the systemic manifestation of reactive oxygen species(ROS)and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage.Plants have numerous adaptive tolerance or resistant mechanisms to acclimatize with the changes in the environment like drought,salinity,heat,cold,ultraviolet(UV),and heavy metal stress.They include adjustment of hormonal balance,synthesis of stress proteins,activation of antioxidant defense mechanism,reconfiguration of the metabolite accumulation,and restructuring of cellular membrane.Salt tolerance is a complex trait involving the coordinated action of many gene families that perform a variety of functions such as control of water loss through stomata,ion sequestration,metabolic adjustment,osmotic adjustment and antioxidative defense.Recent advances in molecular biology,genomics,proteomics and metabolomics have provided insight into plant gene regulatory network system,which is mainly composed of inducible-genes(environmental factors and developmental cues),expression programming and regulatory elements(cis-element and trans-element),corresponding biochemical pathways and diverse signal factors.Glutaredoxins(GRXs)are ubiquitous oxidoreductase enzymes involved in diverse cellular processes and play a key role in oxidative stress responsive mechanisms.While ATP binding cassette(ABC)transporters constitute a ubiquitous superfamily of integral membrane proteins that are responsible for the ATP powered translocation of ligands across biological membranes,a process critical for most aspects of cell physiology.GRXs are assumed to interact with ABC-transporters to translocate the variety of substrates across the concentration gradient.This study aims to explore the structure–function relationship of ABC transporters and Glutaredoxin genes to cope various abiotic stresses and to provide a framework for functional validation and biochemical characterization.Furthermore,our research also includes the complete genome wide survey and regulatory network analysis of both stress associated important gene families to get in depth insights into their structural,functional and evolutionary associations for a unified approach to cope with salt stress.More importantly,the Co-expression networking of salt stress responsive core transcriptome analysis,protein-protein interaction networking and high throughput protein modeling & docking reveal the strong interaction of genes from GRX and ABC gene families.Especially,highly significant interaction was witnessed between Gh ABCG9 and Gh GRXC9 genes in mentioned analysis which provided new trends in functional genomics.Our study revealed the presence of 127 genes encoding GRX proteins in Gossypium hirsutum.A total of 758 GRX genes from two typical monocot and nine dicot species were naturally divided into four classes based on phylogenetic analysis.The classification was supported with organization of conserved protein motifs and sequence logos comparison between cotton,rice and Arabidopsis.Cotton GRX gene family has underwent strong purifying selection with limited functional divergence.A good collinearity was observed in the synteny analysis of four Gossypium species.Majority of cotton GRXs were influenced by various phytohormones and abiotic stress conditions during expression analysis,suggesting an important role of GRX proteins in response to oxidative stress.Cis-regulatory elements,gene enrichments and co-expression network analysis also support their predicted role against various abiotic stresses,which was functionally validated by overexpression of Gh GRXC9 gene in Arabidopsis,VIGS analysis in cotton,subcellular localization in tobacco plants and GUS promotor activity in Arabidopsis.Whole genome and segmental duplication were determined to be the two major impetuses for the expansion of Gh GRX gene family during the evolution.The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provides a new avenue for in-depth characterization of selected genes of importance.Complete genome wide survey of ABC-transporters gene family results into identification of 320 ORFs in Gossypium hirsutum which were distributed into 4 groups(Full,Half,Quarter and ABC2 molecules),eight families(ABCA to ABCG and ABCI)and 12 sub families(MDR,PDR,MRP,PMP,AOH,ATH,ATM,TAP,WBC,RLI,GCN and NAP)through a complex process based on topology,orientation,protein size,similarity,and phylogenetic relationships.Dynamics of complex formation of structurally characterized ABC-transporters from each subfamily shows their kinetic and molecular mechanisms to couple the binding and hydrolysis of ATP to substrate translocation,portraying a "structure to function relationship".Analysis of protein motif and genomic organizations displays structural and functional conservation within same subfamily but diverged among the different subfamilies.Abundance of small single ABCs signifies the higher gene death/birth rate and ongoing evolution for functional advancements and environmental adaptability.Investigation of whole genome duplication(WGD)infers the role of segmental and tandem duplication in the expansion of the ABC gene family in cotton.The overall Ka/Ks ratio indicates the intense purifying selection of ABC genes in four cotton species during evolution.In addition,tissue specific expression profiling and Co-expression networking of Gh ABCs based on RNASeq data suggest their involvement in diverse range of Molecular,cellular and biological processes.Predicted Cis-elements and gene enrichments also supports their predicted roles in substrates translocation,xenobiotic detoxification,disease resistance,polar auxin transport,lipid metabolism,hormonal responses and abiotic stresses.Forward and reverse genetics,together with heterologous expression,have uncovered many novel roles for plant ABC proteins.The identification of ABC genes with differential expression in response to environmental stimuli and structural advances with protein modeling will provide much-needed clarity and qualitative molecular framework for future research.This study will further broaden our insights into the evolution and functional elucidation of ABC gene family in cotton.