Proteomic Analysis Of The Stigmatic Exudate And Functional Identification Of Vesicle Trafficking-related Genes In Tobacco

In angiosperm plants, the interaction between pollen and pistil plays a key role in plant sexual reproduction, and the pollen-stigma interaction is a critical step. Stigma can provide compatible pollen with nutrients required to proceed in pollination, and inhibit the hydration, germination and invasion of incompatible pollen. Understanding the roles of stigma factors interacting with pollen during pollination will shed light on the mechanisms of pollen-stigma recognition and may facilitate crop improvement in the future. Previous studies found that proteins of tobacco stigmatic exudate may be involved in the process of pollen-stigma recognition, facilitating pollen tube growth and penetration into the stigma tissue. In this study, we analyzed the protein expression profiles of tobacco stigmatic exudate through 2-DE and MALDI-TOF MS. In addition, we analysed the function of proteins involved in vesicle trafficking through VIGS technology. The main results are as follows:Through proteomic analyses, we identified 132 proteins in the stigmatic exudate of tobacco. Proteins involved in"Defense and stress response","Cell wall remodeling and metabolism","Protein metabolism and folding","Signal transduction", and"Vesicle trafficking"were overrepresented in the stigmatic exudate of tobacco. It was known that vesicle transport is active in stigma cells under the cuticle, so proteins related to vesicle trafficking may play important roles in the process of pollination in tobacco. Quantitative real-time PCR (qRT-PCR) analyses show that most of genes are specially or predominantly expressed in the stigma. Transcript analyses support the results of our proteomic analysis, so it indicates that the proteomic analysis of stigmatic exudate is reliable.NtRab8-6 is a member of small GTPase Rab8 subfamily in tobacco. Silencing the expression of NtRab8-6 caused development defects in tobacco. The silenced plants are dwarf, have smaller leaves, and increased angle between the petiole and stem and show undeveloped roots. In addition, silenced plants have small flowers with shorter pistils and abnormal stigma. We found that NtRab8-6 is negatively regulated by ethylene. NtRab8-6 is involved in ethylene signaling transduction pathway which may regulate the development of tobacco stigma and handicap the process of pollination. NtSEC8, another protein involved in vesicle trafficking, regulate pollen germination and pollen tube growth in tobacco. The silenced plants have reduced fertility due to defective male gametophyte.In conclusion, proteins in stigmatic exudate of tobacco may participate in the process of pollination. NtRab8-6 and NtSEC8 involved in vesicle trafficking may play important roles during the reproductive process of tobacco.