Physiological Mechanism And Transcriptom Analysis Of Sugar Beet(Beta Vulgaris L.) In Adaption To Alkali-Salt Stress

Soil salt-alkanity was one of major abiotic problems that hinder yield of crops.Sugar beet,one of main sugar-yielding crops in the world,possess strong tolerance to salt-alkanity.It is of great significance to explore the mechanism in tolerance of sugar beet to salt-alkali stress for fully utilizing saline-alkaline soil and promoting maintainable development of ariculture in our country.To elucidate the mechanism in adaption of sugar beet to alkali stress,two sugar beet cultivars KWS0143 and Beta464 with contrasting tolerance were selected as tested materials and treated with mixed alkaline solutions?Na HCO₃: Na₂CO₃,2:1?,and growth indexes,photosynthetic characteristics,osmolytes,antioxidase,ion balance,polyamines and organic acid metabolism were deeply analyzed combined with the observation on cell ultrastructure,and high-throughput sequencing was conducted on the cultivar KWS0143 with stronger tolerance to identify alkali-responsive genes,lnc RNAs and mi RNAs,and finally the mechanism in the adaption of sugar beet to alkali stress was s ystematically elaborated from morphology-anatomic structure-physiology-molecular,etc.Gained genes,related to alkali tolerance,could enrich the resistance gene bank of sugar beet,and provide theoretical basis for breeding saline-alkali tolerant new sugar beet varieties by transgenic technology and further excavate the potential of tolerance of sugar beet to salt-alkalinity.Our study was of great significance for enriching the theory of sugar beet discipline.Main results were as below:?1?Increasing alkali stress led to the significant reduction in net photosynthetic rate,actual photochemical efficiency,photosynthetic pigments contents,dry matter weight per plant and root morphological indexes.Reduction in net photosynthetic rate under alkali stress occurred due to nonstomatal limitation.Besides,chloroplasts in leaves of both cultivars appeared to be longer,stacks of grana of both cultivars appeared loose and numerous osmiophilic drops were visible under the 75 m M alkaline treatment.Photosynthetic capacity,dry matter accumulation and root morphological indexes were inhibited more severely than those of KWS0143.Changes in root/shoot ratio may be a way of sugar beet to adapting to alkali stress.?2?Under alkali stress,biomembranes of leaves and roots of Beta464 were injured more severely compared with those of KWS0143,and for the two sugar beet cultivars,membrane lipid peroxidation in leaves were more severe compared to that in roots.Of osmolytes including proline,betaine and soluble sugar,proline content experienced the largest increase in exposure to alkali stress,and may play a more important role in the adaption of sugar beet to alkali stress.In the response to alkali stress,SOD and POD activities in roots of two sugar beet cultivars were higher than those in leaves,which suggested that antioxidant capacity of roots might be stronger than that of leaves.Capacities of osmotic adjustment and antioxidation of KWS0143 were totally stronger than those of Beta464.?3?Na⁺ accumulation in leaves and roots of sugar beet hindered K⁺ and Ca²⁺ uptake along with increasing alkaline stress.Sugar beet can relieve Na⁺ toxicity through Na⁺ exclusion from leaves.In the response to alkali stress,K⁺ and Na⁺ transport selection coefficient increased of KWS0143 increased,and Na⁺ compartmentation of roots was strengthed.KWS0143 exhibited stronger ability of maintaining the balance of K⁺,Na⁺ and Ca²⁺ compared with Beta464.?4?Free Put,Spm and Spd participated in the response of sugar beet to alkali stress.The increase in contents of three polyamines strengthened the activities of polyamine oxidase and diamineoxidase.Of three sorts of free polyamines under alkali stress,Spd possess the highest content and experienced the largest increase,and might play a major role in the adaption of sugar beet to alkali stress.With the increasing alkali-salt stress,contents of Spm in leaves and roots of KWS0143 were higher compared with those of Beta464.Significant positive correlation existed between the contents of three free polyamines in leaves and roots,and proline content,soluble sugar content and POD activity,etc,which indicated that free polyamines might promoted the synthesis of osmotyles including proline and strengthened the activities of antioxidase including POD.?5?Under alkali stress,sugar beet could strengthen phosphoenolpyruvate carboxylase activity to motivate tricarboxylic acid cycle,thus to promote the synthesis of organic acids,and finally to relieve p H injury.Of organic acids including tartaric acid,malic acid,lactic acid,acetic acid,citric acid and succinic acid,tartaric acid might play a more important role in the resistance of sugar beet to p H sress.Sugar beet could also participate in p H adjustment outside roots through secretion of tartaric acid,malic acid and acetic acid.Under elevated alkali-salt stress,contents of malic acid,acetic acid,citric acid and succinic acid of KWS0143 were higher but contents of tartaric acid and lactic acid lower compared with Beta464.?6?Transcriptome sequencing revealed that 573 and 261 genes were significantly upregulated,and 403 and 122 genes were siginificantly downregulated,respectively,under short-term and long-term alkaline stress.Among those differentially expressed genes,52 commonly regulated genes and 30 commonly downregulated gens were identified under short-term and long-term alkaline conditions.Under short-term alkaline stress,D-3-phosphoglycerate dehydrogenase 1 gene LOC104901403,linoleate 13S-lipoxygenase 2-1 gene LOC104889933,2-oxoisovalerate dehydrogenase subunit alpha 1 LOC104895418,fatty acid hydroperoxide lyase LOC104901567 and ethylene-insensitive protein 2 gene LOC104884677 were all significantly upregulated,whereas glutamyl-t RNA reductase 1 gene LOC104905095 were significantly downregulated;while under long-term alkaline stress,metal tolerance protein 11 gene LOC104886952 was significantly upregulated,and glutamyl-t RNA reductase 1 gene LOC104905095 and omega-6 fatty acid desaturase LOC104900797 were both significantly downregulated.Alternative splicing analysis showed that 8 and 16 diffentially express genes occurred with alternative splicing.Catagories of alternative splicing which diffentially express genes occurred with after alkaline stress were alternative 3? splice site and skipping exon.?7?8535 lnc RNAs were identified using high-throughput sequencing.The length of those lnc RNAs varies from 201-12882 nt,and the average length was 424 nt.LNC₀03498 and LNC₀03048 were both predicted as the precursors of gma-mi R4995,while LNC₀03418 as the precursor of hbr-mi R6173.54 and 37 lnc RNAs were siginificantly differentially expressed under short-term and long-term alkaline stress,respectively.Several lnc RNA and corresponding target genes were found to be problebly related to stress reponse of sugar beet.LOC104902979,target gene of LNC₀01910,participated in controlling the synthesis of superoxide dismutase,LNC₀07731 coexpressed with the gene LOC104906740 encoding peroxidase,both LNC₀00160 and corresponding target gene LOC104888423?jasmonate-induced protein homolog?were significantly upregulated under short-term alkaline stress,and LNC₀00365 coexpressed with metal ion transport gene LOC104892091.?8?268 known mi RNAs were identified in this study,and 61 novel mi RNAs were predicted.96 known mi RNAs and 45 novel mi RNA were differentially expressed after alkaline treatments such as osa-mi R166 m,us-mi R159 b,gma-mi R168 b and novel₂5.67 alkali-responsive mi RNAs were predicted to target 45 genes.GO analysis for the target genes of differentially expressed mi RNAs showed that many target genes of differentially expressed mi RNAs were enriched in “oxidoreduction process”,such as polyphenol oxidase gene LOC104900758.