Genome-wide Identification Of Lipoxygenase Gene Family In Cotton And Functional Characterization Of GbLOX5 In Response To V.dahliae Infection

As an important cash crop,cotton is regarded as the backbone in the economy of many developing countries.It not only provides the quality fiber but also cottonseed oil to the world economy.However,during its growth period it encounters various biotic and abiotic stresses.Average losses due to attack of these stress factors are tremendous every year.There is an urgent need to develop strategies for broad spectrum resistance against these stresses.Traditional breeding approaches are time consuming and only provide limited solution.Long term production practices and intensive research showed that genetic engineering of resistant genes is the most efficient and effective approach for broad spectrum resistance gainst multiple stresses.Oxylipins and their derivatives such as jasmonic acid,divinyl ethers,and volatile aldehydes play significant roles during plant responses to biotic and abiotic stresses.The synthesis of these oxylipins is catalyzed by LOXs,which are non haeme,iron-containing dioxygenases,and directly or indirectly implicated in plant defense responses.Hence,a study was designed to comprehensively and systematically investigate the LOX gene family in cotton,further functionally characterize candidate LOXs genes against various biotic and abiotic stresses.The main results of this study are as follows:1.Genome-wide identification of LOX gene family in Gossypium spp.A total of 64 putative LOX genes were identified in four cotton species(Gossypium(G.hirsutum,G.barbadense,G.arboreum,and G.raimondii)).In the phylogenetic tree,these genes were clustered into three categories(9-LOX,13_LOX type I,and 13-LOX type II).Segmental duplication of putative LOX genes was observed between homologues from A2 to At and D5 to Dt hinting at allopolyploidy in cultivated tetraploid species(G.hirsutum and G.barbadense).The structure and motif composition of GhLOX genes appears to be relatively conserved among the subfamilies.Moreover,many cis-acting elements related to growth,stresses,and phytohormone signaling were found in the promoter regions of GhLOX genes.Gene expression analysis revealed that all GhLOX genes were induced at least in two tissues and the majority of GhLOX genes were up-regulated in response to heat and salinity stress.Specific expressions of some genes in response to exogenous phytohormones suggest their potential roles in regulating growth and stress responses.Our genome-wide study provides a basis for uncovering the biological roles of LOX genes in cotton development and adaptation to stress conditions.2.Functional characterization of candidate LOX genes in response to salinity stressTwo candidate LOX genes(GhLOX12,GhLOX13)were functionally characterized through adopting virus-induced gene silencing(VIGS)approach.Silencing of target genes increased the cotton sensitivity to salinity stress.Compared with controls,target gene-silenced plants showed significantly higher chlorophyll degradation,higher H2O2,malondialdehyde(MDA)and proline accumulation but significantly reduced superoxide dismutase(SOD)activity,suggesting their reduced ability to effectively degrade accumulated reactive oxygen species(ROS).3.Functional characterization of GbLOX5 in response to V.dahliaeWe isolated and cloned the full length sequence of expressed sequence tag(EST#S42334867),which is the homolog of cotton LOX5 previously reported to induce differentially between WT and GhSSN-RNAi line during illumina sequencing.Induced expression of GbLOX5 showed that it is expressed in all the tested organs including roots,stem,leaves,petals,anthers,and ovules,with dominant expression in petals.Moreover,it significantly induced in response to V.dahliae inoculation and treatment of phytohormone JA,suggesting its participation in cotton defense system by modulating the phytohormones signaling.To explore the functional role of GbLOX5 in cotton resistance to V.dahliae inoculation,we constructed two vectors,Overexpression(35S::GbLOX5)and RNA interference(35S::RNAi GbLOXS)and subsequently transferred them into cotton.GbLOX5-overexpression transgenic lines showed enhanced susceptibility upon inoculation of V dahliae,While GbLOX5-RNAi transgenic lines showed better immunity as compared with wild type.Moreover,in response to MeJA treatment expression of JA synthesis and signaling genes were reduced in GbLOX5-overexpression line,while highly induced in GbLOX5-RNAi line as compared with wild type and corresponding mocks,suggesting that GbLOX5 is a negative regulator of JA signaling pathway.Additionally,upon V.dahliae infection,JA synthesis and signaling genes were activated more strongly in GbLOX5-RNAi line compared with wild type,while the expression of these genes were suppressed in overexpression line,suggesting that enhanced resistance observed in GbLOX5-RNAi line might be due to the accumulation of JA.Likewise,transcript abundance of lignin synthesis genes and lignin contents were also found to increase in GbLOX5-RNAi line compared with wild type,while GbLOX5-overexpression line was reduced in expression of these genes and accumulation of lignin contents.These results imply that activated JA synthesis and signaling pathway might promote the lignin synthesis in GbLOX5-RNAi line,which may result in enhanced immunity of cotton to V.dahliae.Alternatively,suppression of JA synthesis and signaling pathway in GbLOX5-overexprssion line results in reduction of lignin contents and ultimately the enhanced susceptibility of these lines.