| In order to resist the harsh ecological environments,plants produce a large number of metabolites such as flavonoids for ecological defense.There is sex differentiation in the response of dioecious plants to a variety of environmental conditions.As a typical dioecious plant,Broussonetia papyrifera(Paper mulberry)has a wide range of distribution,strong adaptability,and abundant secondary metabolites.It is a pioneer species in heavy metal remediation areas.The whole genome sequencing results showed that the expansion of flavonoid synthesis genes was an important event in the evolution of paper mulberry.In the wild environment,is there sex differentiation in paper mulberry?What are the key genes involved in the synthesis of flavonoids in paper mulberry leaves?Is there a coupling between the flavonoid synthesis genes and heavy metal stress response genes?The above problems currently have no systematic research.In this study,a field investigation was conducted on the sex ratio of the population in a stressed environment(Xiangtan,XT)with composite pollution of metals such as manganese,cadmium,lead,and zinc,and a non-stressed environment(Changsha,CS)meeting the normal plant growth conditions.The contents of total flavonoids,total polyphenols,total protein and minerals of paper mulberry leaves(female and male plants)in different development stages(flowering stage,fruiting stage and defoliation stage)were measured,and the antioxidant activity of paper mulberry leaves was evaluated.The purpose of this part was to clarify the differences of different gender paper mulberry in response to stress environment and the variation characteristics of total flavonoids in leaves.To determine the key genes of flavonoid biosynthesis,from the perspective of sex differentiation,paper mulberry leaves in a non-stressed environment were used as research materials.Then,key genes and key metabolites in different sexual trees were screened using transcriptomes and metabonomics technology.Through the correlation analysis of differential genes and differential metabolites,the key genes of flavonoid synthesis in paper mulberry leaves were determined,and the expression regulation patterns and mechanisms at the transcriptional and metabolic levels in paper mulberry leaves of different genders were explored.Combined with the transcriptome data screened by leaf resistance genes of paper mulberry,the RobustRankAggreg(RRA)method was used to screen the shared genes between the key genes of flavonoid synthesis and paper mulberry resistance genes,which provided evidence for the internal molecular mechanism of environmental factors and flavonoid synthesis regulation.The main results are as follows:(1)Through field surveys of paper mulberry in two habitats,the sex ratio of in the stress environment was 1.5～2.27,there was an obvious sex ratio bias,and it was biased towards male plants(p<0.05).The flavonoids content in the leaves of paper mulberry in flowering,fruiting and deciduous stages was compared between the two environments.In general,flavonoids accumulated gradually with developmental time,and the content in female plants was higher than that in the male plants(p<0.05).The flavonoids content in paper mulberry leaves in stress environment was higher than that in non-stress environment(p<0.05).(2)The key genes and metabolites differentially expressed in the leaves of paper mulberry were screened by transcriptome and metabolome technology.The leaves of paper mulberry were analyzed by flavonoid metabolomics,and 192 flavonoid metabolites were identified.Compared with male plants,the metabolite contents of female plants were reduced by 72%,55.1%and 77.7%in flowering stage,fruiting stage and defoliation stage respectively,that is,the flavonoid content in female leaves was higher than that in male leaves.The composition of flavonoids in female and male plants was similar,and 16 kinds of flavonoids accumulated after flowering.The differentially expressed genes in the transcriptome of paper mulberry eaves of different sexes were functionally annotated,and a large number of differentially expressed genes were annotated into the "Secondary metabolites biosynthesis,transport and catabolism";KEGG pathway analysis showed that in the three development stages,the differential genes in the leaves of female and male plants were significantly enriched in the flavonoids biosynthesis pathway,indicating that the differential gene expression in the leaves of paper mulberry between different sexes was closely related to flavonoids biosynthesis.(3)A significant enrichment of differentially expressed genes and metabolites was observed in the flavonoid biosynthesis pathway.4 key structural genes involved in flavonoids accumulation during leaves development were identified(CHS2、CHS7、CHI and DFR).Most of them are upstream genes in flavonoids biosynthesis pathway,and overexpression of these genes increased the accumulation of flavonoids.Furthermore,8 reliable TFs(two HSFs,two MYBP,three AP2/ERF,one NFYA-HAP2,and two WRKY)were found.The TFs were found to regulate flavonoid biosynthesis by promoting the expression of multiple structural genes.(4)By constructing the gene datasets of B.papyrifera leaves in non-stress environment and stress environment,the gene expression of flavonoids biosynthesis pathway in the two datasets were normalized,including 16 and 20 genes related to flavonoids biosynthesis,respectively,including 9 common genes.The RRA method was used to explore the coupling relationship between flavonoids synthesis and heavy metal stress response in paper mulberry leaves.CHS,DFR and BZ1 were shared genes for flavonoids synthesis and heavy metal resistance in paper mulberry leaves.This study showed that under metal stress,the accumulation of flavonoids involved in plant defense mechanism was enhanced in paper mulberry leaves,and there was a sex difference response.The key genes for flavonoid synthesis in paper mulberry leaves were identified,and heavy metal stress affected the expression of flavonoid synthesis genes involved in plant defense mechanisms of paper mulberry leaves.This study has positive significance for correctly understanding the ecological mechanism of paper mulberry on heavy metal stress,and provides a theoretical basis for the restoration practice of paper mulberry in heavy metal polluted areas. |
