Effects Of Salt Stress On Nut Quality And Seedlings' Physiological And Molecular Regulation Of Carya Illinoensis

China has a long coastline with large amount of saline soil.Therefore,the screening of plants suitable for growth in saline-alkali land is of great significance to improve the efficiency of saline-alkali land utilization,control soil salinization and strengthen the construction of coastal shelterbelt system.In recent years,Carya illinoensis as one of nut fruit tree species with good economic value has been widely planted in China due to its excellent characteristics such as barren tolerance,drought tolerance and disease resistance.At present,the research works for C.illinoensis to resist various stresses are mainly focused on physiological and biochemical aspects,and scarcely involve its molecular mechanism.In this study,the main cultivated thin-shell pecan,C.illinoensis'Pawnee',was used as the target of investigation,the effects of Na Cl concentrations on the quality of'Pawnee'nuts and the physiological and biochemical characteristics of its seedlings were analyzed,and the molecular mechanism of'Pawnee'in response to salt stress was revealed by analytic techniques of metabolome and transcriptome.The main research results are presented as follows:(1)Different concentrations of NaCl(0,0.3 and 0.6%)affected the nut morphological characteristics and nutritional composition of'Pawnee'growing for 10 years.With the increase of Na Cl concentrations,the nut length,single-nut weight,seed shell thickness,protein and reducing sugar in the nut showed an upward tendency,but the contents of fat and soluble sugar showed a trend to slowly decrease,and also the relative contents of palmitic acid,?-linolenic acid and cis-11-eicosaenoic acid were slightly varied with Na Cl treatment.(2)NaCl stress significantly inhibited the development of'Pawnee'seedlings at later growth stages,and the inhibitory effect became more serious with increasing Na Cl concentration.Na Cl stress significantly increased the Na~+/K~+ratios and proline contents,but significantly reduced photosynthesis,decreased the chlorophyll contents and damaged the chloroplast ultrastructure,and such changes became more significant with increasing Na Cl concentrations and treatment times.Moreover,the content of malondialdehyde(MDA)and the activities of three antioxidant enzymes(SOD,POD and CAT)were significantly increased with increasing Na Cl concentration after treatment for 10 or 30 days,and in the later treatment period,however,they showed a downward trend with increasing treatment time,especially at the high Na Cl concentrations(0.6%and 0.9%).(3)UHPLC-MS analysis indicated that both the treatment time and concentration of Na Cl significantly affected the metabolome of'Pawnee'seedling leaves,while the treatment time showed more significant effect on the metabolome.A total of 5,880 known metabolites were detected and analyzed by weighted gene co-expression network analysis(WGCNA),and then classified into three modules related to salt stress.Metabolites in modules Plum1 and Lightgreen were positively correlated with Na Cl treatment,mainly belonging to tolipids and lipid-like molecules,phenylpropanoids and polyketides.However,metabolites in module Lightcyan were negatively correlated with Na Cl treatment,which mainly belonged to lipids and lipid-like molecules,organic acids and derivatives,organoheterocyclic compounds and benzenoids.Combined with the differential analysis of the key metabolites of co-expression module and Deseq2,seven key metabolites with significant differences were identified,including four significantly up-regulated metabolites of Melleolide L,Eupolauridine,Amygdalin and Methicillin,and three significantly down-regulated metabolites of Ethyl 9-hexadecenoate,Limonene and Elaidic acid.These metabolites may play an important role in the response of C.illinoensis to salt stress.(4)Illumina HiSeq 4000 sequencing platform was used to analyze the transcriptome of'Pawnee'seedlings.After the treatment with 0.3%and 0.6%Na Cl for 8,24 and 48 h,a total of 7,913 DEGs were identified to include 4,566 up-regulated DEGs and 3,347 down-regulated DEGs,respectively.Functional enrichment analysis showed that these DEGs were mainly involved in biological processes such as hormone metabolism,transcriptional regulation,ATPase transport and defense response.Furthermore,it was found that several key DEGs such as ATPase10,ceria-binding protein CML19 and NAC transcription factor 72 may play an important role in regulating the tolerance of C.illinoensis to salt stress.(5)The gene family of Cil GDSL-lipase in C.illinoensis was identified and analyzed by bioinformatics technique.A total of 87 Cil GDSL genes were identified and then divided into 12 sub-families according to their structure and motif composition.Most of the Cil GDSL genes had four introns consisting of?helices and?chains respectively,which were located outside the cells(25 Cil GDSLs)and in the chloroplasts(22Cil GDSLs).The q RT-PCR results verified that some Cil GDSL genes were induced by salt treatment to present high expression levels,indicating that these genes played an important role in the response of C.illinoensis to salt stress.(6)By integrating the analyses on physiological properties,genetic and metabolic profiles of C.illinoensis after stressed with different concentrations of salt for 24 h,it was found that there was a very stronger correlation between the physiological parameters of C.illinoensis and its metabolites,while the DEGs may participate in regulating the physiological properties of this plant mainly through key metabolites.In particular,a NAC transcription factor gene(Ci NAC72)was found to play an important role in salt stress of this thin-shell pecan,and it may enhance the tolerance of C.illinoensis to salt stress by regulating amygdalin compounds.The above results suggested that the thin-shell C.illinoensis has the physiological and biological characteristics suitable for growth in the saline soil and a molecular regulation mechanism in response to salt stress.Overall,this study not only establishes a solid foundation for it as the candidate of new tree species with high salt resistance,but also provides a practical certification for it as an excellent fruit tree species to develop ecological and high-value horticulture industry in salt-soil area of China coastal zone.