| Genomic structural variations and abundant genetic diversity are resulting from polyploidy,the increasing genetic variation may influence physiological and morphological characteristics of newly formed autopolyploid significantly.The molecular mechanisms of plant resist to drought between different species have been reported.However,less study is reported from perennial woody plants.Polyploids have a broader environmental tolerance and habitat distribution than their diploid parents.However,relatively little is known about the detailed physiological and molecular mechanisms underlying polyploid plants to drought stress.Plants of root induced by asexual propagation genetic background were used which avoided the influence of different genetic information aboveground and belowground parts of plant on research results.In order to understand the molecular model of how mulberry trees resist drought stress,in this study we compared stress tolerance,transcriptome,and flavonoid metabolome between own-rooted stem cuttings of mulberry cultivar Shinichinose（2n=2X=28）and its autotriploid Shaansang-305（2n=3X=42）.The plant response variables investigated consisted of leaf relative water content,antioxidant activity,stress tolerance,gene expression levels,the secondary metabolite flavonoid,and epidermis wax content.The main results obtained can be described,as follows:1.Physiological indexes of plant resistance were measured.The results showed that triploid possessed higher intercellular CO2 and lower stomatal conductance when compared with diploid under the same treatment.The MDA content increased at first,then decreased in both diploid and triploid and SOD activity showed a continually increasing.There is significant difference in RWC at the drought day 9 time point between triploid and triploid.In addition,drought stress induces the production of endogenous hormone and especially ABA.2.To reveal the mechanisms underlying the drought resistance in triploid mulberry,we performed RNA-seq using both diploid and triploid leaves before and after drought stress.Differential expression analysis for annotated genes was conducted.GO and KEGG term enrichment analysis of differentially expressed genes was performed.The GO analysis indicated that the main enrichment of triploid was in―metabolic process‖which was differed with diploid that the main enrichment was―catalytic activity‖.KEGG pathway enrichment analysis revealed that hormone signal transduction is the common pathway with diploid and triploid that resists drought stress.The cutin and wax biosynthesis is always the pathway for differential gene enrichment in triploid.WGCNA analysis further revealed that ABA signaling pathway is drought response pathway in mulberry,in addition,the expression of genes related to the ABA signaling pathway was significantly enhanced.3.Changes of flavonoid metabolites which possess property of oxidation-reduction in their leaves at 15 days of drought were analyzed.The content of isoscutellarein increased more in triploid.The levels of some reducing compounds,such as dihydroflavonols and anthocyanins,were elevated in driploid but not in triploid.Association analysis across omics data showed that differential metabolites induced by drought have significant correlation with corresponding genes in diploid and triploid,separately.We speculate that the different performance in two mulberry cytotypes may be associated with a distinct drought susceptibility threshold in the diploid vis-à-vis the triploid.4.The stomata closure was induced in mulberry leaves and their palisade tissue-spongy tissue ratio increased in both cultivars under drought stress.Moreover,an increase in wax content in the epidermis was induced by drought,with more wax content on the triploid than diploid leaves.Further assays showed that the water loss rate（45%）in the triploid was lower than that of its diploid progenitors（59%）,as well as,the triploid leached chlorophyll less rapidly from intact leaves only up to 25%,versus diploid up to 32%.Consequently,wax accumulation is favor to reduce leaf permeability of triploid mulberry,there by avoid excessive water loss.5.Tsp O genes was filtered out from transcriptomics data,the protein encoded by the Tsp O gene in mulberry and the protein was hydrophobic and had multiple glycosylation and phosphorylation sites.Ma Tsp O promoter regions were found to contain a variety of cis-acting elements that associated with stress and plant hormone response.These results provided the reference for further functional exploration of Ma Tsp O protein.In conclusion,in this study we compared physiological index,transcriptome,and flavonoid metabolome between own-rooted stem cuttings of mulberry cultivar Shinichinose（2n=2X=28）and its autotriploid Shaansang-305（2n=3X=42）,to reveal drought-tolerance mechanism in triploid mulberry from plant response variable such as RWC,antioxidant activity,stress tolerance,gene expression levels,flavonoids and cuticular wax content.The results showed that the drought tolerance of mulberry autotriploid was enhanced when compared with diploid.This study provides a comprehensive research in triploid mulberry response to drought,the methods and results presented here could be an important reference for further studies on mechanism for polyploids to resist various environmental stresses. |
