The Floral Morphogenesis Of Double Petunia And Analysis Of PMADS3 Interacted Proteins

Double-flower is one of the most important traits in ornamental plants. The study of molecular mechanism in its formation helps us to improve the value of ornamental plants. Petunia, as a model plant of flower development, possesses many natural double-flower varieties and can be an ideal material for this research. Currently most of reports about the molecular mechanism of double-flower are focused on the mutation of C-class genes, which results in petaloid stamens and a delay of the termination of floral meristem. In Arabidopsis, the disruption of regulational loop between C-class gene AGAMOUS (AG) and floral meristem identity gene WUSCHEL (WUS) is considered to be the root cause of the formation of double-flower. Based on these researches, we explored the floral morphogenesis of double petunia and the interacted proteins of C-class protein PMADS3. The findings are as follows:1. The difference in floral morphogenesis between single and double petuniaAfter years of effort, our laboratory has obtained the near-isogenic lines of single-and double-flower petunia, which were used to analysis the difference in floral morphogenesis between single and double petunia in this study. The results indicate that plant type, leaf type, inflorescence type and sepal number in double petunia are just like that in single petunia, and most of anthers in double petunia can mature normally. However, there are still many differences between single and double petunia, such as petal lobes, vascular bundles in petals, stamens, carpels and floral whorls, the numbers of which all increase in double petunia compared with that in single petunia. In addition, the inner petals of double petunia are separated, which are considered to originate from stamens. Combined with the results of previous studies, we proposed several assumptions in the formation of double-flower in petunia:(a).the expressed areas and levels of C-class genes are changed. (b). the partial interacted proteins or target genes of C-class genes are mutated.(c). the expression patterns of the other genes controlling the area of floral meristem or the termination of floral meristem are changed2. The characteristic of PMADS3 as a transcriptional factorAccording to the subcellular localization of PMADS3 and seven C-, D-, and E-class proteins, we found that C-class proteins PMADS3, FBP6 and E-class proteins FBP2, FBP5, FBP9, PMADS12 are all localized in nucleus but D-class proteins FBP7 and FBP11 are localized in cytoplasm. The interaction analysis between PMADS3 and seven C-, D-, and E-class proteins was performed by BiFC assay, which showed that PMADS3 could interact with four E-class proteins directly but not FBP6, FBP7, FBP11, and also showed that the interaction complexes of PMADS3 with four E-class proteins only existed in nucleus. The obtained results related to PMADS3 consist with the characteristic of a transcriptional factor.3. The functional analysis of PheIF3f and PhAGO10In this study, we cloned two novel genes PheIF3f and PhAGO10, and peformed functional domain analysis and subcellular localization of the two proteins. The results indicate that PheIF3f protein belongs to eIF3f sub-family, and may have functions of translational inhibition, deubiquitinase activity and blocking editing process of pre-mRNA. In addition, PheIF3f protein is proved to localize in nucleus and cytoplasm, which consists with its suggested extensive biological functions. While PhAGO10 protein belongs to ARGONAUTE family according to its functional domain analysis, and may bind small RNAs to mediate targets repression via mRNA cleavage or translational inhibition. PhAGO10 protein is verified to localize in cytoplasm, which is also in accordance with its suggested biological function. Furthermore, PhAGO10-RNAi seedling shows that shoot apical meristem is terminated at the beginning, and after a delay new meristems arise in the axils. PhAGO10-RNAi flower exhibits enlarged ovary and petaloid stamens. These phenotypes in PhAGO10-RNAi mutant are similar to that of ago10 mutant in Arabidopsis, which implies that the two genes have similar molecular function.4. PMADS3 may participate in post-transcriptional regulationAccording to the results of screening of a Y2H cDNA library by PMADS3, we obtained 21 PMADS3-interacted candidates, most of which, including PheIF3f and PhAGO10 proteins, participate in biological processes in cytoplasm. We performed the interaction analysis of PMADS3 with PheIF3f and PhAGO10 by Y2H and BiFC assays, which indicated that the entire PheIF3f and the fragment of PhAGO10 can interact with PMADS3 in yeast system, while in BiFC assay the full length of the two proteins were also verified to interact with PMADS3. In addition, the interaction complexes of PMADS3/PheIF3f and PMADS3/PhAGO10 can be both detected in cytoplasm. Furthermore, PMADS3-RNAi flower showed the similar phenotype to PhAGO10-RNAi flower, such as enlarged ovary and petaloid stamens, which suggested that the two genes had overlapping function in the floral morphogenesis. All obtained results imply that PMADS3 not only participate in transcriptional biological processes, but also take part in post-transcriptional control.