Functional Characterization Of Cotton GaMYB62L And GaMYB85,Novel R2R3 TFs In Transgenic Arabidopsis

MYB transcription factors(TFs)are well known for being significantly involved in stress-responsive gene crosstalk and signaling pathways.They contain a conserved DNA binding domain(DBD)at the N-terminus known as the MYB domain with 53 amino acid residues and a helix-loop-helix organization,which was recently also discovered to be located at the C-terminus.MYB proteins are further divided into four types based on their DBD repeats;MYB-1R,R2R3-MYB,R1R2R3-MYB(MYB3R),and 4-RMYB proteins R2R3-MYB are broadly vastly represented within dicots and monocots,with approximately 205 putative R2R3-MYB genes in cotton D genome,which is the highest number so far(compared with 126 in Arabidopsis).R2R3-MYBs have been found to engage in numerous stress responses,plant development and intracellular metabolic processes.R2R3-MYB TFs have been implicated in diverse plant responses to abiotic stress conditions,very few drought stress-responsive R2R3-MYB studies have been found in cotton.Furthermore,the molecular system and plant gene behavior underlying drought tolerance are still unclear.Here,we transformed a two novel cloned Gossipium arboreum R2R3-MYB transcription factor(TF)genes,designated as GaMYB62 L and GaMYB85,into Arabidopsis thaliana(Col-0).Its ectopic overexpression resulted in enhanced tolerance against drought stress,possibly because of high ABA phytohormone levels,high rates of stomatal aperture closure with lower water loss rates(WLRs),improved relative water content(RWC),and noteable enhanced proline and chlorophyll contents,and good plant growth.These results presented here suggests that GaMYB62 L GaMYB85 from G.arboreum are the potential novel candidate genes for future genetic engineering of drought stress tolerance in cotton The main results are summarized as:1.Characterization of GaMYB62 L and GaMYB85 genes for drought tolerance,cold and salt stressIn the present study,two cloned R2R3-MYB gene,designated as GaMYB62 L and GaMYB85,were transformed into Arabidopsis and functionally characterized.The GaMYB62 L protein contained two SANT while GaMYB85 domains contain a single SANT domain with a conserved R2R3 imperfect repeats respectively.The GaMYB62L c DNA was 1,017 bp with a CDS of 879 bp and expected to encode a 292-amino acid protein with a MW of 38.78 k D and a p I value of 8.91.While GaMYB85 protein bioinformatic analysis revealed to contain CDS of 771 bp and encodes 257-amino acid and protein harboring a MW of 24.91 k D with ap I of 5.58.The transgenic overexpressing GaMYB62 L Arabidopsis lines(L1,L2 and L3)and overexpressed GaMYB85 lines(L3,L4,L7)exhibited higher seed germination rates under salt and osmotic stress,also a notable increase in proline and chlorophyll contents,lower water loss rate with reduced stomatal apertures,high drought avoidance compared with the WT under water deprivation and a significant plant survival rates at low temperature respectively.Additionally,both transformed genes overexpressed lines were hypersensitive to ABA in germination and root elongation assays,thus suggests that ABA is possibly involved in root development processes.2.Expression analysis of ABA induced stress related marker genes for GaMYB62 L and GaMYB85 genesMoreover,ABA induced GaMYB62 L overexpression enhanced the expression of stressrelated marker genes such as RD22,ADH1,COR15 A and RD29 A,while GaMYB85 ectopic expression led to enhanced transcripts of stress related marker genes like RD22,ADH1,RD29 A,P5CS and ABI5.Taken together,constitutive overexpression of35S:GaMYB62 L and 35S:GaMYB85 speculated to hold improved tolerance for drought,salt and cold stresses.So,these candidate genes have potential to develop abiotic stress tolerance modification in cotton plant through genetic engineering methodology.