Study On The Molecular Mechanism Of MYB97, MYB101and MYB120in Controlling Pollen Tube Reception

In pollination and fertilization of flowering plants, the mature pollen is delivered to the stigma of pistil. It hydrates and germinates to produce a pollen tube when the pollen-stigma interaction is compatible. The pollen tube invades into the stigma cells and grows towards to the embryo sac through the transmitting tract and funicular. Finally the pollen tube enters the embryo sac and discharges the two sperm cells for double fertilization. The process that the pollen tube interacts with the synergid cell and discharges the two sperm cells is called pollen tube reception, which is an important step in sexual plants reproduction. It has been reported that components from both pollen tube and synergid cells are required in pollen tube reception. However, little is known about the regulatory mechanisms underlying in pollen tube reception. Our previous study found that the mutations in the three MYB genes, MYB97, MYB101and MYB120, caused that the myb97myb101mybl20triple mutant pollen tubes could not stop growing and discharge the sperm cells, leading to failure of double fertilization, which suggested that MYB97, MYB101and MYB120play important roles in pollen tube reception. This thesis further studied the molecular mechanisms that MYB97, MYB101and MYB120are involved in pollen tube reception by identification and characterization of the downstream genes of MYB97, MYB101and MYB120using the methods of genetics, gene expression profile, protein-DNA and protein-protein interactions.Microarray assay of pollen expression profile indicated that compared to wild type, the expressional levels of24genes were significantly changed in the myb97myb101myb120triple mutant, where eight genes were downregulated and sixteen genes were upregulated. Among them, four upregulated genes and four downregulated genes were further confirmed using Real-Time PCR. The promoters of these genes contain MYB-binding motifs (MYBGAHV). As demonstrated by EMSA analysis, MYB101could bind to the MYBGAHV motifs in the NAC105, HIR2and CAP1promoters, respectively, suggesting that NAC105, H1R2and CAP1could be the direct target genes of MYB101. Genetic analysis showed that nac105, hir2and cap1mutants did not exhibit any phenotype like the myb97myb101myb120triple mutant, implying the possibility that these genes could be functionally redundant to other unknown genes. Nevertheless, further study is required to address this question.CAP1belongs to the CAP subfamily which has22members. Phylogenetic tree and gene expression analyses indicated that CAP1shares a high amino acids similarity with CAP3and CAP4. CAP1, CAP3and CAP4all are expressed in pollen. Furthermore, the expressional levels of CAP3and CAP4were significantly downregulated in the myb97myb101myb120triple mutant, by comparison with those in wild type pollen. EMSA analysis further demonstrated that MYB101could bind to the MYBGAHV motifs in the CAP3and CAP4promoters, suggesting that CAP3and CAP4could be the targets of MYB101. Complemention experiments showed that the expression of CAP3and CAP4driven by the MYB101promoter was able to partially restore the fertility of the myb97myb101myb120triple mutant, which suggested that CAP3and CAP4genes could be involved in pollen tube reception. CAP1, CAP3and CAP4are cysteine-rich secretory proteins. In GFP-protein fusion analysis, CAP3-GFP fusion protein could be detected in cell wall of the plasmolyzed onion cells, indicating that CAP3-GFP fusion could be secreted into extracellular matrix. In FRAP analysis, CAP3-GFP fusion protein could be transported to the apical area of pollen tube, suggesting that CAP3may function in extracellular matrix of pollen tube.Furthermore, in vitro MST analysis showed that the CAPs could interact with plasma membrane receptor-like protein kinase FERONIA which is involved in pollen tube reception. Yeast two hybrid analysis also demonstrated that the CAPs could interact with the ectodomain of FERONIA. These results imply that the CAPs had the potentials to function as signal molecules and participate in pollen tube-synergid cell interaction required for pollen tube reception.In summary, based on the previous progress, this study further characterized the downstream genes of MYB97, MYB101and MYB120transcription factors, and identified the candidate proteins CAP3and CAP4for the signal molecules involved in pollen tube reception. The results shed a light on the molecular mechanism that MYB transcription factors control pollen tube reception as well as the molecular mechanism of FERONIA signaling pathway.