Impact Of Water Deficiency On Leaf Cuticle Wax And Cutin And Gene Expression Networks In Cotton (Gossypium Hirsutum L.)

Object: Water deficit(WD)has big influence on the productivity of crops.Formation of cuticular layer with increased content of wax and cutin on leaf surfaces is closely related to drought tolerance.Identification of drought tolerance associated wax components and cutin monomers and the genes responsible for their biosynthesis is essential for understanding the physiological and genetic mechanisms underlying drought tolerance and improving crop drought resistance.Methods: In this experiment,two varieties,Xinluzao 22(XL22,drought-tolerant)and Xinluzao 17(XL17,drought-sensitive)were used as materials.The plant height,leaf area,relative water content,stomatal density and stomatal diameter of XL22 and XL17 at seedling and bud stages were analyzed under well-watered(WW)conditions and WD conditions,respectively.Using GC-MS technology,the effects of water deficit on the changes of wax and cutin content,components and wax structure of cotton varieties with different drought tolerance levels were determined.RNA-seq was used to analyze gene expression patterns at the transcriptome level in cotton during drought stress.GO,KEGG and WGCNA analyses were performed on the differential genes to understand the gene regulatory network of cotton leaves in response to drought.Results:(1)XL17 and XL22 decreased significantly in plant height and leaf area and relative water content under the WD conditions.The results suggested that the effect of WD on plant height growth of XL22 was45.4%,and that of XL17 was 68.1% from seedling stage to bud stage.Compared with the WW conditions,WD reduced the leaf area of XL22 by 43.6% and 38.2% at seedling stage and bud stage.WD reduced the leaf area of XL22 by 53.7% and 51.1% at seedling stage and bud stage.After the WD treatment,the RWC of XL22 leaves was reduced by 15.51% from the seedling stage to the bud stage,while that of XL17 leaves was decreased by 20.8%.These results also show that,XL17 is more sensitive to WD induction.WD resulted in an increase in leaf surface stomatal density of XL22 and XL17,and a decrease in the stomatal aperture.But there is no significant difference between the two varieties.Compared with the WW conditions,due to the reduction of leaf area under WD conditions,although the stomatal density per unit leaf area was significantly increased in both varieties,but the total stomatal density of XL22 and XL17 decreased by 32 % and 40 %,respectively.WD stress significantly induced the accumulation of wax and cutin in cotton leaf cuticle,but there was no significant difference among varieties.Under the WD conditions,the total wax content of XL22 increased by 58.76 % and 59.52 % at the seedling stage and bud stage,respectively.Correspondingly,it increased by 56.92 % and 56.56 % for XL17,respectively.C29 and C31 ones had a higher basal level than other four and were also the most significantly induced by WD.This is the main reason for the increase in wax content induced by WD.WD significantly repressed accumulation of the major cutin monomers(linolenic acid(C18:3n-3)and linoleic acid(C18:2n-6))in leaves of both XL22 and XL17.Reduction of primary alcohols at the bud stage seemed to be more significant in XL17 than in XL22 under the WD conditions.WD-induced increase of C16:0 DCA and C18:0 DCA was significant only in drought sensitive XL17 but not in drought tolerant XL22.In addition,although WD-induced reduction of hexacosanol(C26-OH)was observed in both XL22 and XL17 at both developmental stages,a significant reduction of C26-OH was observed only in the drought sensitive XL17 at the bud stage.(2)Discovered by transcriptome analysis,we identified a total of 22,241 genes to be differentially expressed between the WW and WD plants of XL22 or XL17.Of those differentially expressed genes(DEGs),11,196 were up-regulated and 11,045 were down-regulated.Through GO and KEGG enrichment analysis,it was found that DEGs of XL22 was mainly enriched in metabolism of alpha-linolenic acid,linoleic acid,galactose,starch and sucrose,biosynthesis of unsaturated fatty acids,and biosynthesis of steroid,cutin,suberine and wax.XL17 is mainly associated with fatty acid,alpha-linolenic acid metabolism.The results of RNA-seq analysis showed that under WW and WD conditions,the changes of leaf wax,cutin and its components at the seedling stage and bud stage of XL22 and XL17 were consistent with the expression levels of related genes.Analysis by co-expression network,among the genes that were commonly induced by WD were the Gh CER1 genes involved in biosynthesis of alkanes,consistent with the observation of enhanced accumulation of alkanes in cotton leaves under the WD conditions.Interestingly,under the WD conditions,several Gh CYP86 genes(GH＿A08G1579,GH＿D08G1598,GH＿A11G0933 and GH＿D11G0960),which encode enzymes catalyzing the omega-hydroxylation of fatty acids and were identified to be the hub genes of turquoise module of the co-expression gene modules.This is consistent with the transcript abundance of the Gh CYP86 genes(GH＿A08G1579,GH＿D08G1598,GH＿A11G0933 and GH＿D11G0960).Under the WD conditions,a more pronounced reduction in fatty alcohol content was observed in XL17 than in XL22.Whether the differential expression changes of these genes in XL22 and XL17 might be the intrinsic reason for the different drought susceptibility of these two varieties remains to be further investigated.Conclusion: In this study,we investigated the relationship between wax components and cutin monomers of cotton leaves and WD stress response and the gene regulatory network involved in the response to WD stress in cotton leaves.Our results provide clues for the further study of the physiological significance of leaf epidermal lipids in plant growth and development,and also provide a certain research basis for the breeding of drought-tolerant varieties by molecular means,and also provide an idea and possible solution for the difference of drought resistance of different varieties.