The Mechanism Of Drought Tolerance Regulated By Chitosan In White Clover

White clover(Trifolium repens)is an excellent perennial legume species belonging to the genus Trifolium.Due to its characteristics of high yield,good forage quality and strong biological nitrogen fixation ability,it has been used all over the world,it also applied to ornamental lawn.However,white clover prefers cold,cool and humid climate,and has lower drought and heat resistance,so,drought is the main stress factor limiting its production and utilization.Chitosan(CTS)is a non-toxic and pollution-free animal-derived hormone,which is widely used in agricultural production due to its environmental friendly.Its improvement of plant disease resistance is related to the promotion of the synthesis of endogenous abscisic acid.Polyamine(PA)is a widely existing growth regulator in plants.It can improve drought resistance by interacting with hormones,enhancing antioxidant protection and photosynthesis.The objectives of this study were to understand the physiological mechanism of CTS regulated drought tolerance of white clover by determining the effects of CTS and spermine(Spm)on endogenous PA metabolism,hormone levels,and the antioxidant system under drought stress;to reveal the metabolic mechanism of CTS regulated drought tolerance of white clover by analyzing the effects of exogenous CTS on the differentially expressed metabolites(DEMs)of white clover using GC-MS and LC-MS techniques,which also provided metabolomic data for combined analysis;to explain the molecular mechanism of CTS regulated drought tolerance of white clover by analyzing the effects of exogenous CTS on the differentially expressed genes(DEGs)and the metabolic pathway using transcriptome technology,which also provided transcriptome data for combined analysis;to mine the possible metabolites and genes related to the CTS improved drought tolerance of white clover by combining analysis of metabolome and transcriptome data;preliminary test the role of Tr SAMS which regulated by CTS and related to polyamine synthesis in drought stress.The main research results are as follows:1.Exogenous CTS and Spm could enhance PA accumulation through upregulating ADC and SAMDC,downregulating Cu AO and PAO activity and gene expression,and altered ABA,CTK,GA and IAA accumulation under drought conditions.The variations in endogenous PAs and hormones enhanced antioxidant defense,total phenols and flavonoids,which has a relationship with abating drought-caused membrane lipid peroxidation as manifested by lower O₂.^-,H₂O₂ and MDA content.All variations played a vital role in mitigating oxidative damage,increasing cell membrane stability,suppressing leaf senescence,and maintaining plant growth in response to drought stress.Co-application of CTS and Spm was more effective than the use of either CTS and Spm alone in the mitigation of drought stress.2.Based on GC-MS and LC-MS technology,metabolic patterns in white clover were changed by exogenous application of CTS under drought conditions,and GC-MS mainly identified carbohydrates,amino acids,organic acids and some other metabolites,while LC-MS mainly identified amino acids,organic acids,secondary metabolites and some other metabolites.These results showed that exogenous CTS induced upregulation of drought-related metabolites,thereby improving osmotic regulation under stress and maintaining energy metabolism,enhancing antioxidant defense to mitigate the oxidative damage induced by stress and stabilizing cell membrane.CTS plays an important role in improving drought tolerance of white clover.3.The number of Unigene annotated by transcriptome is 51932.From the species distribution map annotated by NR,we knew that more than half transcripts(57.9%)were annotated to Medicago truncatula and 21.1%to Glycine Max.Transcription,translation,ribosome structure,biological origin,replication,recombination and repair account for the largest proportion in COG functional annotation.(P+CTS)/P showed 2460 and 1124 DEGs were up-regulated and down-regulated,respectively,of which 748 and 818 genes were annotated.The effects of exogenous CTS on DEGs in white clover under drought condition involved many metabolic pathways,and a large number of DEGs were up-regulated in flavonoid,ASA and GSH synthesis,photosynthetic metabolic and ribosomal protein pathway.4.We found that there is a strong correlation between Unigene and metabolites by using the Co-inertia analysis(CIA),the total variation of two common components of CIA was82.6%,while four groups can be divided obviously,and associated with 29 major metabolic pathways,among which,metabolism of carbohydrates and amino acids play an important role in CTS adjusted drought tolerance of white clover.Under drought condition,exogenous chitosan regulates some of the key genes in these metabolism,such as?-glucosidase,OTC and GABA-T as well as SPDS,SAMS and ODC which related to polyamine synthesis.CTS regulated drought tolerance of white clover is closely related to the above pathways or genes.5.Via homology clone and RACE amplification,the full-length sequence of SAMS of white clover was obtained.Tr SAMS is highly conservative with three conservative structural domains,it was stable and hydrophilic protein,no transmembrane structure,the fluorescence of the target protein was found in the nucleus by subcellular localization.By analyzing the expression pattern of Tr SAMS,it was found that SAMS can be induced by abiotic stress,hormones and signals,and the expressions in roots and leaves were different.Compared with wild type,Arabidopsis thaliana with overexpression of Tr SAMS had higher endogenous polyamines,growth and drought tolerance.In conclusion,(1)exogenous CTS and Spm could enhance the accumulation of PA and hormones,improve antioxidant defense,leading to the alleviation of oxidative damage caused by drought stress,in addition,co-application of CTS and Spm was more effective than the use of either CTS and Spm alone in the mitigation of drought stress;(2)based on GC-MS and LC-MS technology,exogenous application of CTS could induced the accumulation of carbohydrates,amino acids,organic acids,secondary metabolites and some other metabolites,thereby improving osmotic adjustment ability,antioxidant metabolism,energy metabolism,carbon balance and utilization,and mitochondrial respiration to drive ATP synthesis,thus playing an important role in drought resistance;(3)transcriptome analysis showed that CTS regulate drought tolerance by regulating key genes of many metabolic pathways,such as flavonoids pathway,ASA and GSH synthesis pathway,photosynthetic metabolic pathways,ribosomal protein pathways,then improve the osmotic regulation ability,maintain energy metabolism,enhance the antioxidant defense,reduce drought stress induced oxidative damage,so,CTS could improve drought tolerance of white clover;(4)there was a strong correlation between Unigene and metabolites using CIA analysis,among which carbohydrate and amino acid metabolism played an important role in CTS regulated drought tolerance of white clover by.In addition,SPDS,SAMS and ODC related to polyamine synthesis were significantly regulated;(5)Tr SAMS have three conserved domains,was a stable and hydrophilic protein,no transmembrane structures and located in the nucleus.Tr SAMS can be induced by abiotic stresses,hormones and signals.Compared with wild type,Arabidopsis thaliana with overexpression of Tr SAMS had higher endogenous polyamines,growth and drought tolerance.