Physiological Mechanisms Of Allantoin In Regulating Saline-Alkaline Tolerance Of Sugar Beet(Beta Vulgaris L.)

Saline–alkaline land as the major resource of reserve arable land in China,exploiting it contributes to sustainable development of agriculture.Sugar beet is a major sugar crop as well as an energy crop with excellent saline–alkaline tolerance,which may act as a pioneer crop for exploiting resources of saline–alkaline land.The relatively weak saline–alkaline tolerance at seedling stage is the primary bottleneck for high yield and sugar of sugar beet in saline–alkaline regions.Therefore,investigating the mechanism of saline–alkaline tolerance of sugar beet,exploring its potential of saline–alkaline tolerance,and developing regulatory techniques for high yield and sugar of sugar beet on saline–alkaline land are important for exploiting saline–alkaline land and promoting sustainable development of agriculture.Integrating the saline–alkaline soil conditions of the Songnen Plain and the saline–alkaline tolerance of sugar beet,this study firstly conducted out proteomic and metabolomic analyses of sugar beet under saline–alkaline stress(irrigated with Hoagland’s nutrient solution containing Na Cl,Na₂SO₄,Na HCO₃,and Na₂CO₃ in a molar ratio of 4:2:2:1,EC 10.48 ds·m^-1 and p H 9.42)and captured the core metabolite‘allantoin’by exploring the metabolites in the key pathways of sugar beet adaptation to saline–alkaline stress.Secondly,allantoin was applied in the form of seed soaking,foliar spray,and seed soaking+foliar spray to study the regulatory effects of exogenous allantoin on growth,photosynthetic system,osmoregulation,ion homeostasis,antioxidant system,sucrose metabolism,organic acid metabolism,nitrogen metabolism,polyamine metabolism,hormone,and endogenous allantoin metabolism in sugar beet seedlings under saline–alkaline stress and compare the regulatory effects of different application methods.Meanwhile,the role of allantoin in regulating saline–alkaline tolerance of sugar beet was verified by using potassium oxonate（PO,an inhibitor of allantoin biosynthesis）.In addition,transcriptome analysis was performed on seedlings treated with allantoin in the form of seed soaking+foliar spray under saline–alkaline stress,based on the results of it,sugar beet was treated with jasmonic acid（JA）,diethyldithiocarbamic acid（DIECA,an inhibitor of JA biosynthesis）,sodium nitroprusside（SNP,a donor of nitric oxide）,2-（4-carboxyphenyl）-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide（c PTIO,a scavenger of NO）,allantoin+DIECA,allantoin+c PTIO,JA+c PTIO,and SNP+DIECA,respectively,with the form of seed soaking+foliar spraying to investigate the role of JA and NO in allantoin regulating saline–alkaline tolerance of sugar beet and preliminarily reveal the signaling pathwa y of allantoin in regulating saline–alkaline tolerance of sugar beet.Finally,the effects of different allantoin application methods on the yield and quality of sugar beet was investigated by field experiment in saline–alkaline land.The present study provided theoretical basis and technical reference for further application of exogenous allantoin.The results of this study were as follows:（1）The key proteins of sugar beet adapted to saline–alkaline stress were mainly involved in biological processes,such as response to oxidative stress,response to salt stress,purine nucleoside catabolism,and allantoin catabolism metabolism;constituted cellular components,such as chloroplasts,thylakoid,and PSII;performed molecular functions,such as redox and nucleotide catabolism.The key metabolites of sugar beet adapted to saline–alkaline stress were mainly distributed in purine metabolism,caffeine metabolism,zeatin biosynthesis,tricarboxylic acid cycle,glutathione metabolism,and pyrimidine metabolism.The combined proteomic and metabolomic profiling indicated that purine metabolism was the core pathway of sugar beet adaptation to saline–alkaline stress.During saline–alkaline stress,the protein abundance of enzymes related to allantoin biosynthesis（PRGFT,5’-Nase,ADD,XDH,and OHCUD）and the accumulation of allantoin and its precursors（GMP,AMP,guanosine,xanthosine,inosine,adenosine,guanine,xanthine,hypoxanthine,and adenine）were consistently up-regulated,while the protein abundance of enzymes related to allantoin catabolism（ALLase,ALLD,and UAH）and the accumulation of allantoic acid（intermediate metabolite of allantoin）were consistently down-regulated,and the abundance of proteins related to purine salvage（HGPRT and APRT）were also significantly down-regulated in the purine metabolic pathway.Importantly,allantoin in the center of an interplay network constructed by key proteins and metabolites.（2）Exogenous allantoin enhanced the germination rate of seeds and promoted seedling growth under saline–alkaline stress.Meanwhile,exogenous allantoin enhanced net photosynthetic rate and photosynthetic pigment content,improved actual photochemical efficiency,photochemical quenching coefficient,and degree of xanthophyll de-epoxidation,and enhanced ribulose 1,5-bisphosphate carboxylase activity under saline–alkaline conditions.（3）Exogenous allantoin increased the contents of soluble sugars,soluble proteins,proline,free amino acids,and betaine and reduced water potential and osmotic potential in seedlings under saline–alkaline stress.Meanwhile,exogenous allantoin enhanced activities of plasma membrane H⁺-ATPase in seedling roots under saline–alkaline conditions,promoted H⁺and Na⁺efflux,and reduced Na⁺/K⁺.（4）Exogenous allantoin enhanced the activity of antioxidant enzymes（superoxide dismutase,peroxidase,and catalase）and reduced the contents of reactive oxygen,malondialdehyde,and carbonyl protein in seedlings under saline–alkaline stress.Meanwhile,exogenous allantoin elevated the activities of monodehydroascorbate reductase,dehydroascorbate reductase,and glutathione reductase in the ascorbate-glutathione cycle of seedlings under saline–alkaline stress and increased the content of reduced ascorbate and glutathione.In addition,exogenous allantoin enhanced the activities of phenylalanine ammonia-lyase and polyphenol oxidase in seedlings under saline–alkaline conditions and increased the polyphenols contents.（5）Exogenous allantoin enhanced the activities of sucrose phosphate synthase,sucrose synthase,and sucrose invertase（acid and neutral）,increased sucrose content,and decreased contents of fructose and glucose in seedlings under saline–alkaline stress.Meanwhile,exogenous allantoin enhanced phosphoenolpyruvate carboxylase activity in seedlings under saline–alkaline stress,but its role in regulating the organic acid content was different between leaves and roots.The conte nts of citric and succinic acid were increased and lactic acid content was decreased in leaves,while the pattern of changes in the contents of them in the root were opposite to that in leaves.In addition,exogenous allantoin increased the ntrogen content in seedlings under saline–alkaline conditions,elevated the activities of nitrate reductase,glutamine synthetase,and amiontransferase,and weakened glutamate dehydrogenase activity.（6）Exogenous allantoin increased the activities of arginine decarboxylase,ornithine decarboxylase,S-adenosylmethionine decarboxylase,and diamine oxidase and increased the ployamines content in seedlings under saline–alkaline stress.Meanwhile,exogenous allantoin increased the contents of auxin,cytokinin,gibberellin,and brassinosteroid and decreased ethylene content under saline–alkaline stress conditions,but it had no significant effect on abscisic acid content.In addition,exogenous allantoin decreased activities of xanthine dehydrogenase,urate oxidase,and allantoin synthase and uric acid content and increased allantoinase activity and allantoate content in seedlings under saline–alkaline stress,but it increased endogenous allantoin content significantly.（7）A total of 147 critical genes responding to allantoin under saline–alkaline stress were identified by transcriptome analysis,which were mainly involved in hormone signal transduction,α-linolenic acid metabolism,arginine biosynthesis,arginine and proline metabolism,MAPK signaling pathway,and purine metabolism.Genes activating the JA signaling pathway（lipoxygenase,acyl-Co A oxidase,jasmonic acid-amino synthetase,coronatine-insensitive protein,and transcription factor MYC2）and involved in NO biosynthesis（nitric oxide synthase and ornithine carbamoyltransferase）were up-regulated,while genes inhibiting the JA signaling pathway（mitogen-activated protein kinase kinase 3,mitogen-activated protein kinase 6,and jasmonate ZIM domain-containing protein）and involved in the competitive pathway for NO biosynthesis（urease and arginase）were down-regulated.Exogenous allantoin promoted the biosynthesis of JA and NO,while PO treatment inhibited their biosynthesis.Both exogenous JA and NO enhanced seedling saline–alkaline tolerance（decreased superoxide anion content,hydrogen peroxide conten t,and electrolyte leakage and increased maximal photochemical efficiency）,while DIECA and c PTIO reduced saline–alkaline tolerance and weakened allantoin-induced saline–alkaline tolerance,and none of the four had significant effects on allantoin biosynthesis.Meanwhile,exogenous JA and NO promoted each other’s biosynthesis,while DIECA and c PTIO inhibited NO and JA biosynthesis and attenuated exogenous NO-and JA-induced saline–alkaline tolerance,respectively.In addition,c PTIO slightly attenuated exogenous allantoin-induced JA biosynthesis,whereas DIECA completely eliminated exogenous allantoin-induced NO biosynthesis.（8）In the saline–alkaline land experiment,exogenous allantoin enhanced leaf area index,net photosynthetic rate,and dry matter accumulation of sugar beet.At the same time,it significantly increased the yield and sugar content of tubers.Seed soaking coupled with foliar spraying with 0.1m M allantoin was optimal.