Study On The Regulation Mechanism Of Nitric Oxide On Endogenous Hormones And MicroRNA Of Alfalfa Seedlings Under PEG Stress

Alfalfa?Medicago sativa L.?is a high-quality legume forage with high yield and rich nutrition,it is a forage crop with a long cultivation history and the most extensive planting area in the world,its root system is strong and can coexist with rhizobia,and it plays an important role in improving ecology environment and nitrogen fixation.However,for a long time,for arid and semi-arid areas in the northwest,water shortage severely restricted the large-scale cultivation of alfalfa,which is not compatible with the growing agricultural and animal husbandry industrialization system,therefore,enhancing the drought resistance of alfalfa has a crucial role in promoting the development of industrialization in the northwest region.Nitric oxide?Nitric oxide,NO?is a new type of gaseous signaling molecule,it plays an important role in plant growth and development and stress.In this study,alfalfa seedlings were used as test materials,polyethylene glycol?PEG-6000?was used to simulate drought stress,and nitric oxide releasing agent sodium nitroprusside?SNP?and scavenger cPTIO?carboxy-PTIO?,using physiological and biochemical theories,small RNA sequencing technology and bioinformatics methods to analyze nitric oxide on morphological characteristics of alfalfa leaves under PEG stress,changes in the content of four endogenous hormones?ABA,IAA,SA and GA₃?and miRNA regulation,study the effect of nitric oxide on endogenous hormones and miRNA regulation of alfalfa seedlings under PEG stress.The research results are as follows:?1?By analyzing the leaf morphology of alfalfa,it showed that endogenous nitric oxide has a more significant ability to induce stomatal closure than exogenous nitric oxide.Under PEG stress,the length and width of the leaf stomata decreased,and the stomatal conductance reached a minimum;exogenous nitric oxide scavenger will increase the stomatal length and width of the leaves,and its stomatal conductance is higher than exogenous nitric oxide treatment,indicating that endogenous nitric oxide in alfalfa leaves under PEG stress significantly promotes the stomatal closure effect.Nitric oxide plays an important role in improving the drought resistance of leaf tissue,as the stress treatment time prolonged,the exogenous addition of nitric oxide increased the sponge tissue thickness of alfalfa leaves,its palisade/spongy ratio decreases;when the nitric oxide in the leaves is removed,the thickness of the palisade tissue increases,and its palisade/spongy ratio reaches its maximum,therefore,in a certain extent,nitric oxide can alleviate the damage caused by drought stress through enhancing the palisade/spongy ratio of leaf tissue.?2?Analysis of four endogenous hormones in the leaves and roots of alfalfa under PEG stress,it showed that nitric oxide could regulate the growth and resistance of plants by inducing the metabolic levels of IAA,ABA,GA₃ and SA in alfalfa and mutual transformation in root Inversion,especially the regulation of ABA and SA in alfalfa is more significant.1)The application of exogenous nitric oxide under PEG stress can promote the increase of nitric oxide content in alfalfa leaves and roots,and its scavenger significantly reduces the nitric oxide content in leaves;2)With the prolongation of treatment time,the ABA and SA content gradually increased in leaves and roots,while the ABA and SA content in the roots increased later than the leaves.On the 8th day,the content of ABA in exogenous nitric oxide treatment in leaves was 6.68 times higher than SA content,while the content of SA in sprayed nitric oxide scavenger was 77.22%lower than ABA content,and ABA content in leaves was significantly higher than roots;3)Nitric oxide will induce an increase IAA and GA₃ content in the leaves and roots in the short term,on the 4th day,the content of IAA in the leaves with exogenous nitric oxide treatment was 1.63 times higher than its scavenger,however,the content of GA₃ in leaves and roots gradually decreased after increasing to the maximum on the 2nd day.?3?The differential expression and analysis of miRNA indicated that endogenous nitric oxide under PEG stress can regulate plant growth and development by enriching more biological processes and molecular functions related to PEG stress.A total of 91 known miRNAs and 176unknown miRNAs were identified in four treatments of alfalfa leaves,which were from 47 and 61miRNAs families,respectively;31 differentially expressed miRNAs were identified in PEG treatments,most of which were Up-regulated expression under drought stress;12 differentially expressed miRNAs were identified in the PEG+cPTIO treatments,most of which were down-regulated,among them,mtr-miR5213-5p had the highest expression level and down-regulated expression in PEG+cPTIO treatments.Four treatments were analyzed,and 15 co-expressed differentially expressed miRNAs were screened out to predict the target gene function of the miRNA,GO enrichment and KEGG pathway analysis showed that endogenous nitric oxide in alfalfa leaves can participate in oxidative stress response,hormone and phenylpropane metabolism related biological processes and molecular functions to respond drought stress.The joint analysis of transcriptome and miRNA showed that the removal of nitric oxide in alfalfa leaves under PEG stress caused some miRNA down-regulated.However,miR2118 is up-regulated and involved in the biosynthesis of phenylpropanes in alfalfa leaves,the target gene encoded by c103018.graph_c0₂090 can regulate the plant's tolerance to drought through abscisic acid?ABA?signaling pathway;miR156 family plays a vital role and can enhance plant stress tolerance by regulating the SPL gene;miR2199 mainly functions as a BHLH transcription factor,it involved in plant drought tolerance response from the aspects of stomata,leaf hair and root hair development and sensitivity to abscisic acid.The quantitative results of qRT-PCR confirmed that the miRNA expression was consistent with the sequencing results and showed a negatively regulated expression pattern with its target genes.