The Mechanism Of Selenium Regulating The Growth,development And Lignin Metabolism Of Alfalfa(Medicago Sativa L.)

Selenium(Se)is an essential trace element for humans and animals.It has physiological functions such as enhancing immunity,anti-aging and improving animal production performance.Moreover,an appropriate amount of Se can promote plant growth and development,improve crop yield and quality,and enhance crop stress resistance.Therefore,the appropriate supply of Se is of great significance to animals,plants and humans.Alfalfa(Medicago sativa L.),as a high-quality legume forage,has a strong ability to absorb and enrich Se.It is an important carrier for converting inorganic Se into organic Se,and it is also a direct source of Se intake for animals and humans.In this study,different Se sources and Se application methods were used through germination experiment,pot experiment and field experiment,respectively,in order to explore the regulation effect and mechanism of Se biofortification on photosynthetic characteristics,lignocellulose deposition and quality of alfalfa,and lay a scientific foundation for functional alfalfa product development and selenium-rich industry.The main results are as follows:1.The effect of Se application on seed germination and morphological development of alfalfa was studied through germination experiment.The results showed that:(1)The optimum germination concentrations of sodium selenite,methionine Se and nano-Se were 0.2,4 and 0.2 mg·L-1,respectively,and the optimum root development concentrations were 0.2,4 and 0.4 mg·L-1,respectively.A comprehensive comparison of the Se application treatments revealed that the 0.2 mg·L-1 nano-Se treatment showed the best germination index and was significantly higher than the methionine Se treatment(P<0.05),and the 0.8 mg·L-1 nano-Se treatment showed significantly higher epicotyl length than the other two Se application treatments(P<0.05).The dry weight percentage of the 8 mg·L-1 methionine Se treatment and the Se content of the 6 mg·L-1 methionine Se treatment were significantly higher than the other two Se application treatments,and the nitrate reductase activity of the 2 mg·L-1 methionine Se treatment was significantly higher than that of the sodium selenite treatment(P<0.05).The vigor index and hypocoty length of 0.2 mg·L-1 sodium selenite treatment were significantly higher than those of methionine Se treatment(P<0.05).The total amino acid content of 0.2 mg·L-1 sodium selenite treatment was significantly higher than that of the other two Se treatments(P<0.05).2.Pot experiment(I)was conducted to study the effects of Se application on photosynthetic characteristics,selenium transport and quality of alfalfa;(1)In this experiment,a total of 13 treatments were set up,including sodium selenite(S),methionine Se(SS)and nano-Se(SSS)soaking(0.2,4,0.2 mg·L-1),foliar spraying(20,40,60 mg·L-1)and control(deionized water foliar spraying),which were recorded as SJ,SSJ,SSSJ,SL,SM,SH,SSL,SSM,SSH,SSSL,SSSM and SSSH,respectively.SM,SSM and SSSM treatments all significantly increased the net photosynthetic rate of the upper and lower layers leaves;SJ,SM and SSL,SSSM treatments significantly increased Y(II)of the upper and lower layers leaves,respectively(P<0.05),while SSSH treatment significantly decreased Y(II)of the upper and lower layers leaves(P<0.05).The SSSJ,SM,SSSM and SL,SSL treatments significantly improved the ETR of the upper and lower layers leaves,respectively(P<0.05);the SSJ,SL,SM,SSM,SSSM and SSSJ,SM,SSSM treatments significantly improved the q P and Yield of the upper and lower layers leaves,respectively(P<0.05).(2)The foliar spraying of the three Se sources significantly increased the Se content and Se accumulation in the shoots and roots(P<0.05),and the overall performance was that the Se content and Se accumulation in the shoots of alfalfa treated with nano-Se were the largest,while the Se content and Se accumulation in the roots of alfalfa treated with sodium selenite were the largest.Except for SSSJ,SL and SSSL treatments,all Se treatments significantly increased the respiration rate of leaves(P<0.05).The application of Se also increased the stem diameter,fresh(dry)grass yield,root fresh weight(except SSSH),root dry weight(except SSSL and SSSH),root volume,root activity(except SH and SSH),lateral root number(except SSJ and SSSH),leaf area(except SH,SSH and SSSH),and decreased root shoot ratio(except SL,SSM and SSSM).(3)Except for SH,SSH and SSSH treatments,all Se treatments significantly increased the carotenoid content in leaves(P<0.05).Se application increased the total flavonoids,anthocyanins(except SH,SSH and SSSH),tea polyphenols,reducing sugar,soluble sugar(except SH),N(except SH and SSSH),Mn,Fe,Zn,reduced glutathione(except SH and SSH),vitamin C(except SSSH),auxin content,nitrate reductase(except SH and SSSH),catalase(except SH,SSH and SSSH)and phenylalanine ammonia-lyase activity of alfalfa,However,the contents of C(except SM),hydrogen peroxide(except SH,SSH and SSSH)and superoxide anion free radicals(except SH,SSH and SSSH)were reduced;In addition,Se application significantly reduced crude fiber,neutral detergent fiber(except SM,SSL,SSM,and SSSM),acid detergent fiber(except SM and SSSM),leaf lignin(except SJ),aboveground lignin(except SJ,SSJ,and SH),cellulose(except SSJ,SM,and SSSM)content,caffeic acid coenzyme A-3-O-methyltransferase(except SH)and caffeic acid-3-O-methyltransferase activity(except SJ,SSSJ,SH,SSH,and SSSH)(P<0.05).(4)The intermembrane cavity of mitochondria in SSSM-treated alfalfa leaves increased,and the mitochondrial basal grains and folds of the inner membrane increased,In contrast,SSSH treatment caused the mitochondrial membrane of alfalfa leaves to become blurred,the mitochondrial matrix to become thinner,the intermembrane lumen of the inner membrane to be reduced,and the mitochondrial cristae to become blurred and the inner mitochondrial membrane folds to be broken and not obvious;both SSSM and SSSH treatments significantly reduced the wall thickness of alfalfa leaf pulp cells(P<0.05).3.The effects of Se application on the growth and development,physiological characteristics and lignin deposition of alfalfa were studied through field experiments;(1)A field experiment conducted in 2020 and 2021 was divided into eight treatments.which were root irrigation with 0.2,4 and 0.4 mg·L-1 sodium selenite(S-RI),Methionine Se(SS-RI),nano-Se(SSS-RI)and deionized water(CK-RI),respectively.The above three Se fertilizers(S-FS,SS-FS and SSS-FS)of 40 mg·L-1 and deionized water(CK-FS)were used for foliar spraying.Foliar spraying and irrigation roots on the upper and lower layers leaves were marked as US,LS,UI and LI,respectively.The results showed that:The net photosynthetic rate(Pn),photochemical quenching(q P),and nonphotochemical quenching(NPQ)of the upper-layer leaves of alfalfa treated with Se root irrigation and foliar spray increased by 1.81-23.65%,2.50-7.69%,16.95-71.88%,7.15-22.08%,13.40-88.89%,and 29.63-81.48%,respectively,compared with the control during the 2 years.The lower-layer leaves increased by 1.28-47.51%,2.86-20.00%,10.00-23.33%;4.77-34.38%,5.00-172.73%,and 3.45-110.00%,respectively,compared with the control.In alfalfa,Se treatment increased the total flavonoid,anthocyanin,soluble sugar,and reducing sugar contents and phenylalanine ammonia-lyase activity;however,it decreased caffeic acid 3-O-methyltransferase and caffeioyl-Co A-3-O-methyltransferase activities and cellulose and hemicellulose contents.S-RI and SSS-FS treatments in 2020 and SS-FS(RI)treatments in 2021 significantly reduced lignin content in alfalfa leaves(P<0.05).SSS-RI and SS-FS treatments in 2020 and 2021,respectively,significantly reduced the above ground lignin content in alfalfa compared with the control,and SS-RI and SSS-FS treatments over the 2 years(P<0.05).Se application increased Se,Fe,Mn,N content and forage yield of alfalfa,but decreased C content and C:N in both years,especially the C:N reduction rate of SSS-FS treatment reached a significant level(P<0.05).(2)All Se treatments reduced the deposition of S lignin in the first internode of alfalfa stem.SS-FS and SSS-FS treatments mainly reduced the deposition of S lignin and G lignin in the sixth internode of alfalfa stem,respectively.S-FS treatment only slightly reduced the deposition of G lignin,while each root irrigation treatmentmainly reduced the deposition of G lignin.Se application increased plant height,stem diameter,epidermis thickness,cortex thickness,primary lignin vessel number and pith diameter of alfalfa,but decreased primary xylem area and pith parenchyma cell wall thickness of the first internode,and SS(SSS)-FS treatment reduced the mechanical strength of alfalfa stems.4.Pot experiment(Ⅱ)was conducted to study the molecular regulation of Se application on lignin metabolism of alfalfa.The same Se source,treatment concentration and alfalfa material were used as in the field trial leaf spray treatment,after 90 days of alfalfa emergence,spray Se on the leaves,and spray deionized water as the control.After 72 hours of spraying,take fresh samples from the middle of the plant(stems and leaves)for transcriptomics and real-time fluorescence quantitative(q PCR)analysis.The results show that:The number of differentially expressed genes in sodium selenite vs control treatment was the least,and compared with that in methionine Se vs control treatment,the overall differential multiple of up-regulated genes was greater,while the overall differential multiple of down-regulated genes in nano-Se vs control treatment was greater;Se application regulates the photosynthetic carbon fixation efficiency of plants by regulating the expression of genes related to photosynthesis and carbon metabolism;Regulate the energy conversion efficiency of plants by regulating the expression of related genes such as glycolysis/gluconeogenesis and pentose phosphate pathway;Regulate plant growth by regulating the expression of genes related to hormone signal transduction and amino acid metabolism;The accumulation of flavonoids secondary metabolites was promoted by regulating the phenylpropane metabolism pathway,and the deposition of lignin was reduced;Through the analysis of gene expression results determined by KEGG pathway map and q RT-PCR,sodium selenite treatment mainly negatively regulates the expression of lignin-specific synthesis pathway-related genes COMT,CCo AOMT and CAD,methionine Se treatment mainly negatively regulates the expression of lignin-specific synthesis pathway-related genes COMT,and nano-Se treatment mainly negatively regulates the expression of phenylpropionic acid synthesis pathway-related genes 4CL and CSE,and the expression of lignin polymerization gene POX.