| Pollination is a key process of plant sexual reproduction, which is the basis of producing seeds and fruits. The recognition between pollen grain and stigma cell is the key procedure for plant pollination. After pollen grain falling on the stigma, it undergoes adhension, hydration and germination to creat a pollen tube. Pollen tube carrying sperm cells is guided to the female gametes. In our previous study, we identified an Arabidopsis mutant kinβγ+ that showed ungerminated pollen on the stigma while it germinates normally in vitro. The adhension of mutant pollen on the stigma is abnormal. KINPy is a regulatory subunit of plant Sucrose Nonfermenting Related Protein Kinase 1 complex (SnRKl complex), a key factor for sugar signal transduction in plant involved in the metabolism regulation and hormone/development signal transduction.We analyzed the molecular mechanism of KINβγ regulating pollen germination on stigma using T-DNA insertion mutant kinfβγ/+ in this study. At first, we made a detailed observation of KINβγ expression pattern in pollen using pKINfβγ::KINfiy-YFP transgenic lines. The KINβγ expression was detected during meiosis and tetrad of pollen, in mature pollen grain and pollen tube, and the protein was located mainly in vegetative nucleus while a relatively weak expression was detected in cytoplasm of pollen and pollen tube.The mutant pollen of kinfβγ exhibits a concave shape observed by SEM. TEM results showed that the mutant pollen has more and larger vesicles, and the mitochondria show destructed outer membrane and cristaes. DAB staining revealed the abnormal structure of peroxisomes in mutant pollen pgrains. By using the fluorescence marker labelling mitochondions and peroxisomes, we found that the quantities of mitochondions and peroxisomes in mutant pollen were decreased compared with that in the wild-type pollen. RNAseq analysis showed the espressions of many genes involved in regulating the biogenesis and fission of mitochondrion and peroxisome were changed. The lower ROS level in mutant pollen was observed after CM-H2DCFDA labelling. The mutant pollen of kinβγ exhibits delayed hydration on the stigma compared with the wild-type pollen, and the compromised hydration ability might lead to the pollen abnormal germination on stigma. However, hydration ability of mutant pollen could be compensated partialy by applying hydrogen peroxide in vitro, indicating that ROS plays a critical role in pollen hydration. Our results suggest that KINβγ functions in pollen adhesion, hydration and germination on stigma by regulating pollen ROS level and and the biogenesis of mitochondria and peroxisomes.To verify whether KINβγ plays roles via SnRK1 complex, we used artificial microRNA interference strategy to construct a vector which was transformed into plants to decrease the expression of KIN10 and KIN11 that encode the a subunit of SnRK1 complex. The pollen of the transgenic lines showed similar phenotypes to kinfβγ mutant, such as the abnormal pollen shape, less mitochondrions and so on. We suggest that the KINβγ plays its function via SnRKl complex to regulate pollen adhesion, hydration and germination on stigma. |
