Effects Of Exogenous Nitric Oxide On Phenylpropanoid Metabolism And Regulation Of Mirnas In Alfalfa Seedlings Under Drought Stress

Nitric oxide(NO),as a signaling molecule in plants,plays an important role in the stress response of plants.Medicago sativa is a kind of perennial high-quality leguminous grass,which plays an important role in all kinds of artificial grassland cultivation.However,drought has always been one of the main factors restricting the further development of alfalfa industry.Therefore,researches on the drought resistance of alfalfa seedlings are always decisive for the sustainable development of the alfalfa industry.The effects of exogenous nitric oxide on non-structural carbohydrate metabolism,Phenolic substances metabolism in Phenylpropanoid Metabolism and regulation of miRNA of alfalfa seedlings under drought stress by 10% PEG were investigated.Solutions with:(i)osmotic potentials obtained using polyethylene-glycol-6000(PEG-6000);(ii)a nitric oxide donor sodium nirorpusside(SNP);(iii)a NO scavenger(carboxy-PTIO,cPTIO);were sprayed onto the leaves of alfalfa seedlings in pot experiment at different combinations.Finally,the dynamic changes of non-structural carbohydrate content,phenolic substance content and related enzyme activities in phenylpropanoid metabolism of alfalfa seedlings under drought stress and the changes of miRNA regulation were analyzed,to explore the effect of exogenous NO on phenylpropane metabolism and miRNA regulation of alfalfa seedlings under drought stress.The study obtained the following results:(1)The effects of exogenous NO on different non-structural carbohydrates and metabolic key enzymes in leaves and roots of alfalfa under drought stress were studied.The results showed that exogenous nitric oxide promoted the starch decomposition of leaves,the accumulation of starch in roots and increased the content of soluble sugar(sucrose,fructose and glucose)in leaves and roots in Medicago sativa seedlings under drought stress,which decreased the osmotic potential,promoted cell water absorption and alleviated damage caused by drought.Furthermore,under drought stress exogenous nitric oxide can increase the activity of sucrose synthetase(SS),acid invertase(AI),neatral invertase(NI),reduce the activity of Sucrose phosphate synthase(SPS)in leaves and can increase the activity of SS,SPS,invertase in roots,so that made the synthesis and decomposition of sucrose at a high level of dynamic balance.It enhanced the drought resistance of alfalfa.The metabolism of NSC and related enzymes were inhibited by NO scavenger cPTIO.(2)Exogenous NO affected the metabolism and distribution of different phenolic substances in leaves and roots of alfalfa under drought stress.The results showed exogenous NO increased total polyphenols,flavonoids and lignin content in the alfalfa leaf and root under drought stress,the content of lignin in leaves and roots increased first and then decreased,which increased by 7.52% and 17.66% respectively compared with drought stress.The content of lignin in root system was higher than that in leaf system,and it was more sensitive to exogenous NO under drought stress.Exogenous NO added the anthocyanin content in alfalfa leaves by 13.53% compared with drought stress.In addition,exogenous NO increased the activity of PAL and PPO in alfalfa under drought stress,which could make lignin and flavonoids maintain a higher metabolic level in the later stage of treatment.The NO inhibitor cPTIO could affect the synthesis of total phenols,flavonoids,lignin and anthocyanins and the activities of key enzymes in alfalfa seedlings under the drought stress,that indicated exogenous NO participates in the metabolism of phenolic substances.(3)Exogenous NO affected the miRNA expression of alfalfa under drought stress.We identified 90 conserved miRNAs belonging to the 46 family and 177 novel miRNAs using highthroughput sequencing.32 differentially expressed miRNAs were identified in PEG samples,further complementing the drought-responsive miRNAs,and 55 differentially expressed miRNAs were identified in PEG+SNP samples.In addition,the differentially expressed miRNAs were targeted with 86 and 157 potential target genes in PEG and PEG+SNP,respectively.GO and KEGG analysis have shown that many GO terminology related to plant stress tolerance wasenriched.Exogenous nitric oxide played an important role in carbohydrate metabolism under drought stress,except for carbohydrate transport,exogenous nitric oxide also regulated the cellulose biosynthetic process,starch biosynthetic process,galactolipid biosynthetic process,amylopectin biosynthetic process and carbohydrate transmembrane transportto response to drought stress.Exogenous NO may also regulate the expression of miRNA related to anthocyanin metabolism to participate in the drought resistance of alfalfa.qRT-PCR results verified that the miRNA profile was consistent with sequencing results,and putative target genes presented opposite expression patterns.