Epigenetic Modification Under Abiotic Stress And Identification And Functional Analysis Of PcG Family Members In Medicago Truncatula

Epigenetic phenomena have important impacts on various life processes,such as plant growth and development,stress responses,senescence,and death,and are often observed in conjunction with environmental changes where they may modify environmental impact on biological functions.Medicago truncatula is a model species for legume forage.At present,epigenetic studies on the growth and development of M.truncatula have been carried out,but there are few reports on epigenetic regulation of its response mechanism under abiotic stress.In this study,Medicago truncatula was used as the material to study its epigenetic characteristics under salt,drought,high and low temperature stresses,and screened epigenetically related biological processes and regulatory genes involved in stress.In addition,we performed the genome-wide identification,protein-protein interaction analysis,phylogenetic analysis,synteny analysis and expression pattern analysis of the Polycomb group(PcG)family involved in epigenetic modification.Based on the identification results,the key member MtBMI1 was subjected to subcellular location and GUS chemical staining analysis of Arabidopsis tissues,and further functional analysis was carried out in transformed Arabidopsis by overexpression.The main results are as follows:1.Four abiotic stresses altered the levels of DNA methylation and histone modifications in M.truncatula.Among them,DNA methylation levels were elevated in M.truncatula under all four stresses.However,the methylated histone content increased only under salt stress and decreased under the other three stresses.The ubiquitinated histone content increased under salt and low temperature stresses,decreased under high temperature stress and did not change significantly under drought stress.The acetylated histone content decreased under all four abiotic stresses.These results suggested that epigenetics was involved in the regulation process of M.truncatula under abiotic stress,but there were significant differences in the regulatory patterns of different epigenetic modifications.Further correlation analysis showed that6-methyladenine and acetylated histone were significantly and positively correlated with leaf morphological indicators,while they were highly significantly and negatively correlated with stress-related physiological indicators.2.We screened and obtained 234 core genes and 193 differentially expressed genes involved in epigenetic regulation.Gene set enrichment analysis(GSEA)showed that the largest regulatory networks under salt and low temperature stresses were histone deacetylation and histone acetylation,respectively.The maximum regulatory network under drought and high temperature stresses was histone methylation.GO enrichment analysis showed that 77,88,30 and 76 differentially expressed genes were enriched in52,49,42 and 48 epigenetically related biological processes under salt,drought,low temperature and high temperature stresses,respectively.3.For the first time,64 PcG members involved in epigenetic modifications were identified in M.truncatula.All of the PcG genes were distributed unevenly over eight chromosomes.Among them,34 PcG proteins exhibited protein-protein interactions,and 37 PcG genes targeted 113 mi RNAs.PcG proteins had the highest number of colinear genes(36)with Glycine max and the lowest number(8)with the three monocotyledons,Oryza sativa,Brachypodium distachyon and Sorghum bicolor.Meanwhile,the PcG genes of M.truncatula were induced by different stresses and hormones,and each member of the PcG family contained various cis-acting elements related to environmental stimuli and stress responses.4.MtBMI1 is the only BMI1 member of the PcG family identified in M.truncatula.The gene sequence is 5386 bp in length,with seven exons and six introns.The CDS is1290 bp in length and encodes a total of 429 amino acid sequences.The MtBMI1 gene has two functional domains,zf-C3HC4 and RAWUL.MtBMI1 of M.truncatula is more closely related to Pv BMI1-1 of Phaseolus vulgaris and Gm BMI1-1 of Glycine max.The subcellular location analysis of tobacco epidermal cells showed that MtBMI1 protein was localized to the nucleus.Simultaneously,the GUS chemical staining results showed strong MtBMI1 promoter expression activity in the mature inflorescence,stigma,flower,stem and stalk of Arabidopsis.5.Furthermore,MtBMI1 was overexpressed in Arabidopsis for functional analysis.The results revealed that the histone H2AK119 ub content was significantly higher in transgenic Arabidopsis compared to the wild type.In addition,the transgenic lines under drought stress showed obvious water-loss phenotypes compared to the wild type.The root length and fresh weight of root were significantly lower than those of the wild type,and the malondialdehyde and proline content were significantly higher than those of the wild type.Additionally,the expression levels of 12 genes related to epigenetic modifications were generally up-regulated in transgenic Arabidopsis and further increased after drought treatment.These results illustrated that MtBMI1 played a negative regulatory role for in response to drought stress of M.truncatula,and this gene could have an effect on other epigenetic modifications in Arabidopsis.