Transcription Factor MYB17 Regulated Lignin And Flavonoid Synthesis In Medicago Truncatula And Medicago Sativa

Alfalfa is a crucial legume forage due to its high nutritional value,high yield,and good palatability,and it is extensively cultivated worldwide.Lignin,as a component of the plant cell wall,provides mechanical support and helps plants withstand adverse external conditions.However,lignin cannot be absorbed by livestock and reduces the digestibility of forage grass.Therefore,developing alfalfa varieties with low lignin content has always been a prominent topic in improving its quality.Molecular breeding poses challenges due to alfalfa’s highly heterozygous contract tetraploid nature,self-incompatibility,and complex genome structure.The genome of Medicago truncatula shares significant homology with that of alfalfa.Studying the gene function of Medicago truncatula can provide valuable references for understanding alfalfa.In this study,we cloned and isolated the Mt MYB17 gene from Medicago truncatula,and then analyzed its sequence,expression pattern and subcellular localization.We transferred Mt MYB17 into Medicago truncatula to obtain overexpressed lines.The molecular mechanism of inhibiting lignin and flavonoid synthesis by Mt MYB17 was analyzed through physiological determination,omics analysis,and molecular biology methods.At the same time,Ms MYB17,the homologous gene of Mt MYB17 in alfalfa was cloned and overexpressed into alfalfa for phenotypic identification to obtain the breeding materials of reduced lignin alfalfa.The main results and conclusions of this study are as follows:1.Mt MYB17 belonged to the R2R3-MYB subgroup 4 family.The tissue-specific expression patterns of Mt MYB17 were examined.The results revealed that Mt MYB17 is expressed in roots,stems,and leaves,with the highest expression level detected in young stem tissues and the lowest expression level in mature stems.Expression analysis under drought and salt stress conditions revealed that Mt MYB17 was induced by both two stresses.Subcellular localization studies confirmed that Mt MYB17 was localized in the nucleus,indicating it was a nuclear protein.2.The Mt MYB17 was transferred into Medicago truncatula by Agrobacterium-mediated genetic transformation to obtain overexpressed positive plants.Subsequent investigation on the biological function of Mt MYB17 revealed that its overexpression significantly reduced lignin content in stems and leaves.The contents of G and S lignin monomers decreased significantly.Additionally,there was a significant decrease in flavonoid content in leaf tissues.These findings collectively suggest that Mt MYB17 acts as an inhibitor for both lignin and flavonoid biosynthesis pathways in Medicago truncatula.3.The metabolome analysis targeting lignin were performed in Mt MYB17 overexpressed plants.The contents of cinnamic acid,p-coumaric acid,and ferulic acid were significantly downregulated in the overexpressed lines.The flavonoid-targeted metabolome analysis revealed changes in 19 metabolites shared by overexpressed lines,with a consistent trend observed in both two lines: 5 metabolites were significantly upregulated while 14 metabolites were significantly downregulated.These findings indicate a correlation between changes in total lignin and flavonoid content and alterations in related metabolites accumulation.4.Transcriptome analysis of Mt MYB17 overexpressed plants showed that there were totally 1278 differentially expressed genes shared by overexpressed lines,with 414 genes upregulated and 863 genes downregulated.GO analysis indicated that Mt MYB17 positively regulated the catabolism of amino acids,monosaccharides,and organic acids,while negatively regulated the biological processes including phenylpropane biosynthesis and metabolism,flavonoid biosynthesis and metabolism,as well as cell wall synthesis.KEGG analysis further revealed that Mt MYB17 positively regulated amino acid metabolism,glucose metabolism,and fatty acid metabolism;conversely,and negatively regulated flavonoid biosynthesis,secondary metabolite biosynthesis,linolenic acid metabolism,isoflavone biosynthesis,and phenylpropane biosynthesis.Notably,there were 23 co-downregulated genes enriched in the phenylpropane pathway,including12 gene families such as PAL(phenylalanine ammonialyase),4CL(4-coumarate-Co A ligase),CCo AOMT(caffeoyl-Co A O-methyltransferase),indicating that the overexpression of Mt MYB17 suppresses transcriptional expression of these key structural genes involved in the phenylpropane pathway.5.The yeast single hybridization test showed that Mt MYB17 could bind to the promoters of Mt PAL2,Mt4CL2,Mt4CL-L1KE1,and Mt CCo AOMT1.These four genes were significantly downregulated in the two overexpressed lines.These results indicated that Mt MYB17 negatively regulates lignin and flavonoid synthesis by inhibiting the expression of Mt PAL2,Mt4CL2,Mt4CL-L1KE1,and Mt CCo AOMT1.In addition,a total of 101 potential interacting proteins of Mt MYB17 were screened by yeast two-hybrid screening library.The screened proteins Mtb HLH94,Mt CCR1,and Mt NET1 A were verified by rotation.The results showed that all of them interacted with the truncated Mt MYB17 protein.6.Ms MYB17 gene of alfalfa belonged to the R2R3-MYB subgroup 4.Ms MYB17 was expressed in roots,stems,and leaves,with the highest expression in young stems.The expression level of Ms MYB17 was increased under drought and salt stress,but the response time was not sustained.The Ms MYB17 protein was localized in the nucleus.The Ms MYB17 gene in alfalfa was transformed into Arabidopsis thaliana and alfalfa through Agrobacteriamediated genetic transformation.Overexpression of Ms MYB17 led to a reduction in lignin content in both Arabidopsis and alfalfa.In Arabidopsis plants,growth inhibition occurred.However,among the four overexpressed lines of alfalfa,only one line was not inhibited in growth.Furthermore,there were significant increases observed in crude protein,crude ash,phosphorus,and calcium contents.Significant decreases were found in crude fiber,sugar,and neutral detergent fiber contents.Acid detergent fiber increased significantly in one line but remained unchanged in the other three lines.Relative feeding value showed a significant increase in one line but decreased significantly in another line.In summary,the overexpression of Mt MYB17 reduced the lignin and flavonoid content of Medicago truncatula,which was related to the decreased accumulation of lignin and flavonoid-related metabolites.Mt MYB17 reduced lignin and flavonoid synthesis by inhibiting the transcriptional expression of Mt PAL2,Mt4CL2,Mt4CL-L1KE1,and Mt CCo AOMT1 in Medicago truncatula.The overexpression of Ms MYB17 decreased the lignin content of alfalfa,and a new breeding material with low lignin contents and high relative feeding value was obtained.