Genome-wide Analysis Revealed Plant Regeneration Capacity And Drought Tolerance In Gossypium Hirsutum

Cotton is an important natural fiber crop and its seeds are the main oil source.The establishment of highly effective gene transformation system in cotton is a prerequisite for the genetic improvements.Callus induction,as the initial step in most in vitro plant regeneration system,is a process that differentiated cells dedifferentiate to acquire pluripotency.Regeneration is the ability of multicellular organisms to regenerate or grow new cells,tissues,or even whole organs in response to injury.However,the molecular mechanism of callus induction and the relationship between callus induction,regeneration capacity,and cotton leaf regeneration still unclear.Identifying the candidate genes that regulate callus induction rate will optimize the plant transformation system and speed up the regeneration process.In this study,cotton leaf regeneration level for 21 accessions in the field(new leaves)was observed after the first harvest.Callus induction and regeneration capacity of roots and shoots for hypocotyl,cotyledons and shoot tip tissues were used to validate field leaf regeneration level.We found a substantial relationship between field regeneration capability and callus induction with its regeneration capacity for the hypocotyl,cotyledons,and shoot tip.A significant correlation between physiological parameters and callus induction was discovered.All our result provides a clue for checking the regeneration capacity through leaf regeneration level in the field.In the current study,21 traits including callus induction rate,callus mass,callus color,shoot number,and root number of a core collection of 384 upland cotton accessions we analyzed and used for GWAS.A total of 47 significantly associated SNPs for callus induction traits were identified.Sixteen genes associated with callus induction rate were selected among two regions chromosomes A08 and A04.The WUSCHEL-related Homeobox(WOX)genes were involved in callus induction.However,the functions of WOX genes are less known in cotton.We identified 39,40,21,and 20 WOX genes in G.hirsutum,G.barbadense,G.arboreum,and G.raimondii,respectively.All the WOX genes in four cotton species could be classified into three clades,consistent with previous research.The expressions of WOX genes in germinating hypocotyls and callus were characterized and found that most genes were up-regulated.One candidate gene Gh＿A01G127500 was selected to perform the virus-induced gene silencing(VIGS)experiment and found that the growth of the silenced plant(p CLCr VA: Gh WOX4＿A01)was significantly inhibited compared with the wild-type.In the silenced plant,there is an increase in antioxidant activities and a decrease in oxidant activities compared with the control plant.In physiological analysis,the relative electrolyte leakage level and the excised leaf water loss of the silenced plant were increased.This study proved that WOX genes play important roles in drought stress and callus induction,but more work must be done to address the molecular functions of WOX genes.Drought is one of the most significant abiotic stresses,resulting in considerable yield losses in cotton.ORP(Oxysterol-binding protein-related proteins)genes are involved in non-vesicular sterol transport,vesicular signaling,and lipid metabolism.Previous studies about ORP genes in Arabidopsis,soybean,and petunia have also demonstrated its significant role in abiotic stress.Yet,there have been no reports of the systematic identification and study of ORP genes in cotton.This work found that G.hirsutum,G.barbadense,G.arboreum,and G.raimondii have 14,14,7,and 7 ORP genes.Phylogenetic analysis showed that all ORP genes could be classified into four groups.Gene structure and conserved motif analysis suggest that the function of this gene family was conserved.The Ka/Ks study demonstrated that this gene family was subjected to purifying selection during evolution.Four ORP genes,in particular Gh ORP＿A02,were shown to be upregulated by abiotic stress treatment by transcriptome data.The cis-acting elements in the ORP promoters were responsive to phytohormone and various abiotic stresses.The silenced plants of Gh ORP＿A02 were more sensitive to drought stress when compared to the control.A wide family of proteins known as the oxysterol-binding protein(OSBP)and OSBP-related proteins(ORPs)primarily function in lipid transport and sensing.Its evidence that ORP5 encourages cell division and proliferation.Plant lipoxygenase(LOX)genes catalyze the oxidation of polyunsaturated fatty acids into a variety of functional oxylipins.However,knowledge of their functions in drought stress in cotton is still scarce.We identified 34,38,23,and 20 LOX genes in G.hirsutum,G.barbadense,G.arboreum,and G.raimondii,respectively.All LOX genes could be divided into three main categories according to the phylogenetic tree analysis and each category containing 39,37,and 43 LOX genes.The majority of LOX genes were up-regulated in germinating hypocotyls and calluses.One candidate gene Gh＿A02G037000 was silenced by VIGS and we discovered that the growth of the silenced plant(p CLCr VA: Gh LOX5＿A02)was greatly decreased compared to the wild type.The infected plant’s excised leaf water loss and relative electrolyte leakage level both increased.The silenced plant has higher antioxidant activity than the control.