Anatomical Structure And Growth Physiological Characteristics Of Populus Talassica × Populus Euphratica In Response To NaCl Stress And Transcriptome And Metabolome Analysis

In order to explore the anatomical characteristics,physiological response and molecular regulat ion mechanism of Populus talassica×Populus euphratica under salt stress,the variety of P.talas sica×P.euphratica,which had been popularized and planted in saline-alkali land,was selected as the experimental material,and the basal salt content of potted soil was 0.49 g/kg as the control（CK）,and two Na Cl concentration gradients of 200 and 400 mmol/L were set up for a total of3 treatments.On the basis of growth and physiological response,transcriptome and metabolome analysis were performed.The following research results have been obtained:1.Under salt stress,the anatomical structure of the root,stem and leaf of P.talassica×P.e uphratica changed in accordance with the enviro nment.Under Na Cl treatment conditions,the seed lings maintained water in the body by increasing the thickness of root secondary phloem,secondar y xylem thickness,xylem aperture,stem cortex thickness,phloem thickness,xylem thickness,p ulp diameter and leaf thickness,fence tissue thickness,grid-to-sea ratio and leaf structural compa ctness,and further adapted to the external salt environment.2.The growth and physiological characteristics of P.talassica×P.euphratica seedlings change d significantly under Na Cl treatment,and the growth parameters such as plant height,ground di ameter,root length and biomass were significantly improved by 200 mmol/l Na Cl treatment compa red with CK,but the average leaf area decreased significantly compared with CK（P<0.05）.Wi th the increase of Na Cl concentration and the prolongation of stress time,the growth parameters of P.talassica×P.euphratica seedlings under 400 mmol/l Na Cl treatment were significantly lower than those of CK（P<0.05）.In addition,compared with CK,Na Cl treatment significantly reduce d the relative water content（RWC）and water potential of P.talassica×P.euphratica leaves,and the daily variation of water potential showed a trend of low in the morning,evening,high an d noon.Compared with CK,Na Cl treatment significantly reduced the stomatal density,chloroph yll content,net photosynthetic rate and instantaneous water use efficiency of P.talassica×P.euph ratica leaves,but significantly increased the intercellular CO₂concentration（P<0.05）。At the s ame time,under the influence of different salinities,stress intensified,relative conductivity incr eased,osmoregulation and reactive oxygen species metabolism also responded.Compared with CK,the 400 mmol/L Na Cl treatment significantly increased the contents of malondialdehyde,proline,soluble sugars and soluble protein in P.talassica×P.euphratica leaves.With the increase of Na Cl concentration,the activities of the three antioxidant enzymes showed a trend of first increasing and then decreasing.Compared with CK,Na Cl treatment significantly increased the Na⁺content a nd Na⁺/K⁺in roots,stems and leaves of P.talassica×P.euphratica,but significantly reduced the K⁺content in roots,stems and leaves.The overall performance of Na⁺content in each organ was as follows:leaf>root>stem,K⁺content distribution was generally manifested as:leaf>stem>root,Na⁺/K⁺overall manifested as:root>leaf>stem.3.Based on the study of the salt-tolerant growth,physiological characteristics and anatomical structure of P.talassica×P.euphratica,transcriptomic analysis was carried out on the seedlings of P.talassica×P.euphratica after Na Cl treatment.Taking|log₂（fold change）|≥1,padj≤0.05 as the screening criteria,a total of 41617 differentially expressed genes were identified in the roots,stems and leaves of P.talassica×P.euphratica,of which 21603 differentially expressed genes we re upregulated and 20014 differentially expressed genes were down-regulated.GO enrichment analysi s of differential genes found that the differential genes in the roots,stems and leaves of P.talassi ca×P.euphratica were mainly enriched in the"cellular carbohydrate metabolism process"and"cel lular dextran metabolism process"in biological processes,the"apoplast"and"cell wall"in cell co mponents,and the"hydrolase activity"and"transferase activity"in molecular functions.The results of KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly enriched in"carbon metabolism","a mino acid biosynthesis","plant-pathogen interaction"and othe r pathways.Combined with the results of GO enrichment and KEGG enrichment analysis,"LOC105131256,LOC1051256,LOC1051256,LOC105129452,LOC10513467,LOC10513463,LOC105134637,LOC105112947,LOC105112947"were screened from the differentially expressed genes of r oots,stems and leaves and some other high-expression candidate genes that may be associated wit h salt tolerance of P.talassica×P.euphratica.These genes are involved in P.talassica×P.euphratic a response to salt stress.4.Since the differentially expressed genes are highly expressed in the roots and stems of P.ta lassica×P.euphratica,a total of 18 samples from the roots and stems of P.talassica×P.euphrati ca seedlings were selected in this experiment and divided into 6 groups for metabolome study.Ba sed on the UPLC-MS/MS detection platform and self-built database,a total of 1298 metabolites were detected in the roots and stems of P.talassica×P.euphratica.Taking FC>2.0 or FC<0.5,a nd the P value<0.05 of VIP>1.0 and T-test as the screening criteria,a total of 583 differential me tabolites were screened in the root and stem of P.talassica×P.euphratica,of which 201 were u p-regulated and 251 were down-regulated.The enrichment results of KEGG pathway showed that t he differential metabolites in the roots and stems of P.talassica×P.euphratica were mainly enrich ed in metabolic pathways such as"biosynthesis of a mino acids","biosynthesis of secondary meta bolites","ABC transporter"and"biosynthesis of flavonoids".Differential metabolites are mainly a mino acids and their derivatives,nucleotides and their derivatives,flavonoids,organic acids,phen olic acids and alkaloids,which can be used as metabolites characteristic of P.talassica×P.euphr atica salt-tolerant.5.Based on the joint analysis of transcriptome metabolome,the pathways co-enriched by dif ferential genes and differential metabolites are mainly metabolic pathways such as"a mino acid bi osynthesis","carbon metabolism","plant hormone signal transduction","ABC transporter"and"fla vonoid biosynthesis",which are closely related to the salt tolerance of P.talassica×P.euphratica.The 10 genes with the strongest association with transcriptomics were LOC105115394,LOC105128125,LOC105115927,LOC105134725,LOC105124002,LOC105122358,LOC105112769,LOC105110065,LOC105120036,LOC105129277.The top 10 metabolites that have a greater impact on tra nscriptomics are xanthosides,citicoline,guanosine 3’,5’-cyclic monophosphate,uridine 5’-bisphos phate,jasmonyl-L-isoleucine,phosphoenolpyruvate,cytidine-5’-monophosphate,sucracyctotetrase,arachidicine（C20:0）,and stachylose.These metabolites are a mino acids and their derivatives,n ucleotides and their derivatives,lipids,and organic acids.Overall,NaCl treatment changed the anatomical characteristics of roots,stems and leaves of P.talassica×P.euphratica,which was the structural adaptation mechanism of P.talassica×P.eup hratica in response to salt stress.In addition,P.talassica×P.euphratica can adapt to the external salt enviro nment by reducing the leaf growth rate and promoting taproot growth.At the same ti me,P.talassica×P.euphratica can also enhance salt tolerance adaptability through a series of phy siological pathways such as self-osmotic substance regulation,hormone regulation,water regulati on,photosynthetic pathway,protein synthesis,ion distribution and reactive oxygen species clearanc e.Salt stress induces differences in the expression of physiological metabolism-related genes involv ed in a mino acid biosynthesis,carbon metabolism,plant hormone signaling,ABC transporter,a nd flavonoid biosynthesis.Related differential metabolites mainly include a mino acids and their de rivatives,nucleotides and their derivatives,flavonoids,organic acids,phenolic acids and alkaloid s.