| Litsea cubeba(Lour.)Pers.is one of the unique natural spice tree species in China.Its essential oil is an important industrial raw material.The content of essential oil in the fruit is the highest,up to 12%.L.cubeba is a typical dioecious species in Lauraceae.In actual production,the number of female plants has an important influence on the yield of essential oils in L.cubeba.The female and male floral primordia are homologous in the early development of female flowers of L.cubeba.The stamen organs in female flowers are aborted to form unisexual flowers.To study the mechanism of degeneration of stamens in the development of female flowers is of great significance to the sex formation of the female flowers in L.cubeba.This study analyzes the morphological characteristics of the development process of male and female flowers of L.cubeba,focusing on the identification of key regulatory genes and functional identification of stamen degradation in female flowers,and provides a basis for further revealing the molecular mechanism of stamen degradation and the mechanism of sex differentiation,to provide theoretical guidance for gender identification,optimization of the sex ratio and increase of fruit yield of L.cubeba.The main results are as follows:(1)The degeneration of stamen organs in female flowers is earlier than that of microspore mother cells in L.cubeba.The morphological and anatomical studies on the flower buds of L.cubeba revealed that the development stage of the female and stamen primordia of L.cubeba has a "bisexual stage",which includes the stamen and pistil primordia.After the floral organs matures,the pistils in the male flowers have no stigma,and the styles are short,forming degenerate pistils;the stamens are short,and there is no anther chamber,forming degenerate stamens in the female flowers.it was found that the degenerate stamens in the female flower were in the lack of pollen sac and other structures by anatomical observation,.This indicates that the formation of degenerated stamens in L.cubeba occurs in the early stage of organ differentiation,which may be earlier than the formation of microspore mother cells.(2)The content of salicylic acid(SA)is higher during the formation of degenerated stamens and the difference is significant between genders and different developmental stage of L.cubeba.Hormone determination results in male and female flowers of L.cubeba show that the development of female and stamen primordia to the formation of degenerated stamen,there was no significant difference in the expression levels of methyl salicylate(Me SA)in female and male flowers.In addition to Me SA and gibberellin 4(GA4),The difference of the content of jasmonic acid(JA),gibberellin 3(GA3),trans-zeatin riboside(TZR),auxin(IAA)and salicylic acid(SA)is very significant under sex,development stage and their interaction,which indicates that these hormones may be related to the gender differentiation of L.cubeba.The levels of 1-aminocyclopropane carboxylic acid(ACC)and methyl jasmonate(Me JA)were consistent,and gibberellin 1/7(GA1/7)was not detected,indicating that these hormones are not involved in the development of degenerated stamens in female flowers of L.cubeba.The results showed that there were significant differences in the contents of TZR,IAA,JA,GA3/4 and SA from the primordium of pistil and stamen to the development period before meiosis.The overall hormone levels were SA,GA3,IAA,JA,TZR and GA4 from high to low,and the GA4 level was approximately 0.SA level was the highest,especially in the period of development of staminodes before meiosis,which was more than 10 ng/g.It suggested that SA might be involved in the development of staminodes.(3)LcTGA9/10 in the salicylic acid signal transduction pathway is a potential regulatory gene for stamen degradation of L.cubeba.The results showed that 15,248 differential expressional genes(DEGs)were screened from male flowers and 7,928 from female flowers.The enrichment results of GO and KEGG showed that plant hormone signal transduction and phenylpropane biosynthesis pathway were significantly enriched,especially in female flowers.,the signal transduction and enrichment of hormone synthesis such as SA and ethene(ETH)are significantly enriched.Therefore,hormones may play an important role in the development of staminodes in female flowers.The 459 DEGs related to plant hormone signal transduction pathway were screened.Go enrichment results showed that 15 DEGs related to SA and other hormones were identified,And LcTGA9 and LcTGA10 were the genes related to SA signal transduction.The real-time quantitative results showed that LcTGA9 was the highest in pistil and LcTGA10 was the highest in staminodes.The m RNA in situ hybridization of LcTGA10 showed the expression of staminodes in the female flowers of L.cubeba.Therefore,the results of the above indicate that LcTGA9 and LcTGA10 may be the key candidate genes for regulating the degeneration and development of the stamens in the female flowers of L.cubeba.(4)The expression level of TGA10 homologous gene in unisexual male flower plants of Lauraceae is significantly higher than that in unisexual female flowers and bisexual flower plants.By collecting 12 species of bisexual flowers and 8 species of unisexual flower buds of Lauraceae,Among the related genes of hormone signal transduction pathway,and TGA9 and TGA10 homologous genes are included in this pathway.the expression level of TGA9 homologous gene was the highest in the bracts of different sex species of Lauraceae,followed by male flowers,and finally female flowers.The expression level of TGA10 homologous gene in the bracts of Lauraceae from high to low was in the order of male flower,bisexual flower and female flower.Among the 8 dioecious species of Lauraceae,the expression level of TGA9 homologous gene was significantly different in 3 species,and the expression level of TGA10 homologous gene was significantly different in 7 species.TGA10 homologous gene is more likely to participate in the formation of Lauraceae unisexual flower species.(5)LcTGA10 is involved in regulating the degeneration of stamens in the female flowers of L.cubeba.LcTGA9 and LcTGA10 belong to the TGA transcription factor.The results of Arabidopsis thaliana overexpression showed that LcTGA9 overexpression Arabidopsis thaliana and wild-type Arabidopsis thaliana lines had similar phenotypes,and there was no effect on the development of pistils and stamens.LcTGA10 overexpressed Arabidopsis thaliana(T3 generation)lines were stunted,the type was smaller than the wild type,and the average fertility was only 3.75%.The plant height is slightly lower,the inflorescences and sepals are smaller,the number of stamens is reduced,the length is short,pollen is produced in the anthers,and the pollen has little biological activity.The stigma detection of Arabidopsis thaliana overexpression is pillinated.The pollen sterility of artificially Arabidopsis thaliana overexpression lines is reduced,and the pistil is normal.The above results indicate that LcTGA10 is involved in the development of degenerated stamens in the female flowers of L.cubeba,which inhibits the development of normal stamens,leading to the formation of degenerated stamens.(6)LcTGA10 may mediate the degradation of stamens in female flowers of L.cubeba through SA pathway.The analysis of the co-expression network in L.cubeba showed that PALs,TGAs and NPRs are the key genes for the regulation of SA biosynthesis and signal transduction,and they are closely related to the genes related to the development of stamens such as SPL8.Connectivity of CMTAs and WRKYs genes is in SA biosynthesis and signaling pathways In the WT(sa)lines to which salicylic acid(SA)was applied to Arabidopsis thaliana,the expression levels of downstream response factors At NPR1,At TGA9 and At TGA10 of the SA were up-regulated relative to the WT control group,indicating that the SA hormone can induce the downstream signaling gene At NPR1,At TGA9 and At TGA10 expression.In summary,LcTGA10 may regulate the formation of degenerated stamens of L.cubeba through the SA pathway. |
PDF Full Text Request