Study On The Physiological Response To Betaine Under Osmotic Stress And Signal Pathway Of Betaine Synthesis In Musa Spp.

Banana(Musa spp.)is one of internationally competitive agricultural products in China,but drought stress and salt stress have become the main factors limiting the development of the banana industry.Plant drought stress and salt stress can cause osmotic stress.Betaine,one of the important osmotic adjustment substances,plays a key role in the regulation of abiotic stress.However,studies on the effects of exogenous betaine on the stress resistance of banana under osmotic stress and the regulation mechanism of banana synthetic betaine have not been reported.In this study,leaf surface was sprayed with 10 mmol/L betaine for 24 h and the roots were incubated with 100 mmol/L mannitol to simulate the osmotic stress to determine physiological indexes such as the MDA content,antioxidant enzyme activity,osmotic adjustment substance content,photosynthetic characteristics and other physiology of leaves and roots of Brazilian banana(M.Paradisiaca.).The key enzymes of betaine synthesis,CMO(choline monooxygenase)and BADH(betaine aldehyde dehydrogenase)gene sequences and protein sequences were obtained from the banana A genome database for bioinformatics analysis to predict their function and possible hormone signaling substances regulating the synthesis of betaine.Brazilian banana seedlings were treated with osmotic stress(100 mmol/L),exogenous betaine(10 mmol/L)and hormone signal substance(100 μmol/L).And the relative expression of CMO and BADH genes was detected by qRT-PCR and the accumulation of betaine was measured in Brazilian banana seedlings.The relationship between the expression of betaine synthetic gene and the amount of betaine synthesis accumulation was analyzed,and the main hormone signal material for regulating betaine synthesis was selected.The overall transcriptional level of Brazilian banana seedlings was further analyzed by RNA-seq technology under the induction of the selected hormone signal substances,and the hormone signaling pathway regulating betaine synthesis was explored.The research results are as follows:(1)Exogenous betaine significantly decreased MDA accumulation in leaves and roots of banana seedlings under osmotic stress,which was 31.52%and 40.99%lower than osmotic stress at 48 h,and significantly increased the activities of SOD,POD and CAT in leaves and roots of banana seedlings.After pretreatment with exogenous betaine for 24 h,the chlorophyll content and carotenoid content of banana leaves increased significantly,increasing by 16.50%and 23.71%respectively,slowed down the chlorophyll and carotenoid content under osmotic stress,significantly increased chlorophyll fluorescence parameters FV/FM,YII and ETR under osmotic stress,slowed the changes of chlorophyll fluorescence parameters such as FV/FM,YII,ETR,Y(NPQ)and Y(NO)and significantly increased the synthetic accumulation of soluble sugar,soluble protein and betaine in osmotic adjustment substances.The soluble protein content in leaves and roots of banana seedlings reached the highest at 24 h and 48 h,which were 107.44%and 99.10%higher than osmotic stress,respectively.The accumulation of betaine in leaves and roots was 19.69%and 12.29%higher than that of osmotic stress at 48 h.(2)Two banana betaine synthetic genes,MaCMO(Ma07_g21070)and MaBADH(Ma09_g15520)were screened from the banana A genome database,and bioinformatics analysis was carried out.The gene promoter regions of MaCMO and MaBADH have 12 and 15 plant hormones(MeJA,Eth,SA,ABA,and GA)and cis-regulatory elements associated with stress,respectively.Protein domain,GO functional analysis and multiple sequence alignment revealed that MaCMO has two typical conserved domains,Rieske type[2Fe-2S]and iron ion binding region,which is a class of proteins with iron ion binding and oxidoreductase activity;MaBADH has a betaine aldehyde dehydrogenase domain,which is a class of oxidoreductase-reactive proteins that acts on aldehyde or carbonyl with NAD or NADP as receptors.The constructed phylogenetic tree showed that both of them were closely related to pineapple,corn and rice in the grass family.When the CMO and BADH genes were systematically evolved,the monocotyledonous plants had obvious branches.(3)The expression of MaCMO and MaBADH was increased by osmotic stress,while the exogenous betaine inhibited the expression of MaCMO and MaBADH under osmotic stress,but both of them could significantly increase the synthesis and accumulation of betaine in banana seedlings.After 48 h of osmotic stress,the accumulation of betaine in leaves and roots increased by 12.93%and 15.78%,and the accumulation of betaine in leaves and roots increased by 4.79%and 7.17%.(4)Hormone signal substance(ABA,MeJA,ethephon,SA)can induce the expression of MaCMO and MaBADH and the accumulation of betaine in banana seedlings,but the degrees of response is different.The gene expression and the accumulation of betaine induced by MeJA are the highest.Under MeJA induction,MaCMO reached the highest of 3.795 and 2.479 in leaves and roots at 8 h,respectively.MaBADH reached a maximum of 1.326 at 12 h in the leaves and reached a maximum of 1.686 at 8 h in the roots.The induction of betaine synthesis in leaves and roots was significantly increased by 29.97%and 26.85%at 48 h respectively after MeJA induction.(5)When treated with MeJA for 8 h,the differential genes related to jasmonic acid signaling pathway were mostly up-regulated in leaves(43 genes up-regulated),and only a few down-regulated in roots(62 genes up-regulated,8 genes down-regulated).Ethylene signaling pathway-related differentially expressed genes were up-regulated in both leaves and roots(16 genes up-regulated and 8 down-regulated in leaves,47 genes up-regulated and 18 genes down-regulated in roots).The differential genes related to gibberellin signaling pathway were few and down-regulated in leaves and roots.The differential genes related to salicylic acid and abscisic acid signaling pathways were up-regulated in leaves and roots.There are also a large number of differentially expressed genes related to stress resistance(13 1 and 289 stress-resistant candidate genes were screened in leaves and roots).(6)When treated with MeJA for 8 h,there are two differentially expressed genes of jasmonic acid responsive transcription factor MYC2 in leaves and roots,and both of them were up-regulated.2 and 4 differentially expressed genes of ethylene-responsive transcription factor ERF 1/2 were up-regulated in leaves and roots,respectively.Asmonic acid and ethylene signals are activated.Besides the promoter regions of the MaCMO and MaBADH genes were predicted to have cis-acting elements G-box and ERF that interact with the transcription factors MYC2 and ERF 1/2.In summary,exogenous betaine can effectively alleviate banana osmotic stress and improve physiological response.There is a positive correlation between MaCMO and MaBADH gene expression and betaine synthesis accumulation.Under osmotic stress,banana can activate the molecular protective mechanism of betaine.The synthesis and accumulation of endogenous betaine in banana is closely related to the signal of plant hormone,which may be dominated by jasmonic acid signal and supplemented by ethylene signal synergistically regulating betaine synthesis.