| Phellodendron amurense Rupr.,a species of Rutaceae,is a dioecious plant as recorded.However,monoecious P.amurense was first discovered during previous research.The top of monoecious P.amurense died when it was young,and the lateral buds grew and developed into branches of different sexes,which suggested that the sex formation of P.amurense occurred after the formation of branches,and there was different sex expression in the same genetic background.This phenomenon has led the sex differentiation mechanism of P.amurense to epigenetic.DNA methylation is one of the most common and intensively studied modes of epigenetic regulation and we attempted to make a breakthrough in DNA methylation in this study.The expression characteristics of sex-related genes in P.amurense was analyzed,and its epigenetic sexual regulation mode was preliminarily explored in this study.As a whole,the sex formation of plants should be the result of signal transmission and coordination among various organs.In order to get the information of the overall sex characteristics and better understand its sexual formation process,we analyzed the expression characteristics of sex-related genes in the annual branch bark,leaves,and flower organs of both female and male plants of P.amurense.The results are as follows:(1)The number of sex-related genes expressed in each part of P.amurense was different,and regardless of the female or male,the most sex-related genes were expressed in flower organs,followed by annual branch bark and the least in leaves.The specifically expressed genes were most in flower organs,most of which were related to flower development;the genes specifically expressed in annual branch bark are mostly related to embryonic development and plant sexual differentiation;there are very few genes specifically expressed in leaves(only two genes:a hormone-related gene and a female fertility gene).(2)A total of 611 sex-related genes were obtained from the female,while 612 sex-related genes were obtained from the male.The genes specifically expressed in the female were CCR4-NOT,60s ribosomal protein,ESR2,ERF114,while the genes specifically expressed in the male plants were ARR17,ABORTED MICROSPORES,X10A,CDPKO,V-type proton ATPase subunit G1;(3)There are most differentially expressed genes(DEGs)in floral organs between female and male,followed by annual branch bark,and fewest differentially expressed genes in leaves.The DEGs in floral organs between male and female included sex-determining candidate genes and a large number of flower development genes.The DEGs in the annual branch bark between female and male involved some sex-determining candidate genes.such as SRS5 and MCM6,and relatively few genes related to flower development.The number of DEGs between male and female leaves is extremely small,and key genes for female flowers development were not involved.We speculate that annual branch bark might be used as a good material for studying P.amurense sex according to its expression characteristics of sex-related genes.In addition,through analysis of the DEGs between the bark of different stages,it was found that there were a large number of sex-related genes differentially expressed in annual branch bark compared to trunk bark,and many sex-determining candidate genes are upregulated.As to sexual epigenetic regulation in P.amurense,we attempted to make a breakthrough in DNA methylation.DNA methylation is one of the most common and intensively studied modes of epigenetic regulation.The effect of DNA methylation on P.amurense was studied by a precise demethylation method,in order to prove epigenetic involvement in sex differentiation of P.amurense.After treatment with 5azaC,some male flowers were induced to transform into female flowers with complete structure and function,and there were no other variation types.Studies on the morphology and function of induced female flowers have found that they are completely consistent with natural female flowers in terms of morphology,structure,and function,characterized in that stamens degenerated to different degrees and ovaries could develop into fruits with seeds.Male flowers of P.amurense could be induced into female flowers with complete structure and function after 5-azaC treatment,demonstrating that epigenetics is involved in the sex regulation of P.amurense,with DNA methylation as one of its regulatory modes.Some male flowers could be induced into female flowers with no other variation types after treatment with 5-azaC,during which key genes related to female flower development were involved.Genes related to the development of P.amurense female flowers were explored in this study using the induced female flowers.Differential expression analysis was conducted between female and male flowers to acquire information on DEGs between the two samples.A total of 332 sex-related DEGs were identified between female flowers and male flowers,which involved sex regulatory genes(including RPP0W,PAL3,MCM6,MCM2,ABA2,GPAT3,GAST1,TOZ19),genes related to floral initiation and development(including PIN1,PHB,AGL11,AINTEGUMENTA,AIL6,SUP,ESR2,SRS5,YABBY2,SEUSS,RL1,RL2,BAM2,FD,ZAT3 etc.),biochemical pathway related genes(including MET1,AGO16,BASS2 etc.)and hormone related genes(including LAX3,RGA1,GI etc.).In order to explore genes related to the development of female flowers,296 DEGs were obtained by the differential analysis between the induced female flowers and male flowers,among which 216 genes also showed differences in female and male flowers.Compared with male flowers,these genes showed differences in both the female and the induced female flowers,which were more likely to be the genes related to the development of female flowers.DEGs between male flowers with no change after demethylation and male flowers were less likely to be key genes and could be regarded as not linked with sex formation.After removing such genes,188 candidate genes were screened out finally.Combining with their functions,genes associated with sex regulation and floral initiation,including RPP0W,PAL3,MCM2,MCM6,SUP,ANT,AIL6,AGL11,SEUSS,SRS5,and ESR2,were preliminarily considered to be the key genes for female flower development.Moreover,genes related to male flower development were explored using DEGs between female and male flowers of P.amurense.Through the differential characteristics between female and male flowers and combining with gene functions reported in literature,it was preliminarily speculated that the sex-determining genes TDF1 and ARR17,and early male flower development genes PI,AP3,AG,FD,TGA10,and MS1,may play a crucial role in the development of male flowers in P.amurense.This study has established an experimental platform suitable for studying sex determination of P.amurense female flowers,confirmed the sexual epigenetic regulation of P.amurense,and identified some genes related to female and male flower development.It has laid a preliminary foundation for understanding P.amurense sex formation process and studying sex differentiation mechanism,and has reference value for the study of plant sex differentiation. |
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