Control Of Male Gametophyte Development By Ubiquitin Dependent Protein Degradation In Arabidopsis Thaliana

Arabidopsis pollen mother cell produces four microspores by meiosis and the microspores undergoe two mitotic cell cycles subsequently. The first asymmetric mitosis (pollen mitosis I, PMI) produces a vegetative cell and a generative cell which was embraced by the vegetative cell cytoplasma. The generative cell can divided further (pollen mitosis II, PMII) into two sperm cells in vegetative cell. Previous work had found that the ubiquitin-dependent protein degradation system play a crucial role in this process. The FBL17 controls the PMII by degrading the cell cycle inhibitors KRP6 and KRP7. Two ring type E3 ligase, RHF1a and RHF12a, also participate in the degradation of KRP6 during the PMI and PMII.In this study, we isolated an Arabidopsis SALK T-DNA insertion mutant, half of whose pollen grains were sterile. The TAIL-PCR result revealed that there is a T-DNA insertion in F-box/LRR-repeat (FBL) gene. So the mutant was designated as fbl. Reciprocal crosses indicated that the fbl mutant can hardly transmit through the male gamete. And the homozygous mutant of fblwas hardly available from the self-pollination plants of fbl/FBL. To study the stages the FBL function, we stain the pollen of fbl/FBL qrt/qrt double mutant with DAPI. Seldom fbl pollens can go through the PMI to produce bicellular pollen, which indicates that the FBL may function as a regualtor of the PMI. RT-PCR analysis of the expression of FBL was performed with RNA extracted from flowers, seedlings and calli. We found that the highest expression position was not in flower but in callus. This result suggests that the FBL protein may be a positive regulator in cell cycle progression by degrading the inhibitory factors. To confirm the hypothesis, yeast two hybridization analysis was performed between KRP proteins as bait and FBL as well as its LRR domain as prey. The result demonstrated that the both FBL and its LRR domain can interact with the KRP6. We compared the expression levels of FBL, RHF1a and RHF2a using the Cyclebse (www.cyclbase.org), which reveals that the FBL express highly in the G1-S transition whereas the RHF1a/2a in S-G2 transition.We proposed a putative working model. During the process of Arabidopsis male gametophyte development, FBL expresses in the G1-S transition. SCFFBL complex controls the degradation of KRP6 and attenuate the inhibition to the cell cycle. The deficit of FBL results in the constant KRP6 suppression on the G1-S transition and the formation of bicellular pollen. Therefore, the FBL is an essential regulator for initiation of the first post-meiosis mitotic during Arabidopsis male gametogenesis.