Study On Sterols Regulating Stomatal Development And Flowering In Arabidopsis

Asymmetric cell division is important for regulating cell proliferation and fate determination during stomatal development in plants. While genes that control asymmetric division and cell differentiation in stomatal development have been documented, regulators controlling the process from asymmetric division to cell differentiation remain poorly understood.Through classical genetic approach, we screened a stomatal mutant, J3158, which shows clusters of small cells and stomata in leaf epidermis. The physical asymmetry of stomatal precusor cell divisions seemed intact in fk mutants, but the cell fate asymmetry was greatly disturbed, suggesting that J3158 links these two crucial events together in the process of asymmetric division. Through map-based cloning, we found that this gene encode Arabidopsis sterol C-14 reductase, FACKEL (FK). The defective stomatal development in J3158 was complemented by overexpression of FK cDNA. Over-expression of FK cDNA in Col-0 (35S::FK) showed normal stomatal development. We therefore renamed this FK mutant allele as fk-J3158. Two other FK alleles, fk-J79 ant fk-X224, displayed stomatal patterning defects similar to fk-J3158. Sterol profile analysis revealed that the fk-J3158 mutation blocked downstream sterol production.Further investigation indicated that cyclopropylsterol isomerasel (cpil), sterol 14a-demethylase (cyp51A2), and hydra 1 (hydl) mutants, corresponding to enzymes in the same branch of the sterol biosynthetic pathway, displayed defective stomatal development phenotypes, similar to what were observed in fk. Fenpropimorph (FEN), an inhibitor of the FK sterol C-14 reductase in Arabidopsis, could also cause these abnormal small cell and stomata phenotypes in wild-type leaves. Analyses of double mutant phenotypes from fk-J3158 and known stomatal mutants or FEN-treated known stomatal mutants demonstrated that sterol biosynthesis is required for correct stomatal patterning, which is independent of the late sterol biosynthetic pathway, BR signaling, EPF-ER/TMM-MAPK cascade, SDD1, FLP and BASL. However, this pathway still requires three bHLHs (SPCH, MUTE and FAMA) as final switches to determine the three cell-type differentiations.Detailed analyses of time-lapse stomatal cell division patterns of fk-J3158 by epidermal agarose impressions indicate that the daughter cells from asymmetric division in fk-J3158 stomatal lineage divide synchronously, which occurs after the first entry division of protodermal cells. The fate transitions of the daughter cells become random. Any of them can develop into stomata, pavement cells, or still keep stomatal stem cell activity. The expression pattern of TUB6, MUTE, TMM and CYCB1;1 in fk-J3158 stomatal lineage further demonstrate the synchronous division and random cell fate decision of two daughter cells. We quantified two daughter cell sizes from asymmetric division and observed live-imaging expression of the asymmetric cell division marker BASL, and found that the physical asymmetry of cell division is normal in stomatal development of the fk mutants. Moreover, we also found that some guard cells of the mutants in the early steps of sterol biosynthetic pathway lost their terminal specific characteristics, and then acquired renewed mitotic ability. Analyses of the cell cycle-related gene expression and DNA ploidy suggested that more cells enter into the mitotic cycle in fk-J3158 than in the wild-type. All these results suggest that sterols are required for properly restricting cell proliferation, asymmetric fate specification, cell fate commitment and maintenance in the stomatal lineage cells. These events occur after physical asymmetric division of stomatal precursor cells.We found that the fk-J3158 mutant also had a long life cycle and delayed flowering time in different photoperiod, and 35S::FK plant showed early flowering time compared with wild-type. The late flowering defect of fk-J3158 was dependent on the FLOWERING LOCUS C (FLC) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) expression. This process might be independent of the downstream BR pathway, photoperiod, and autonomous pathway. The fk-J3158 mutant was more sensitive than wild-type in reducing the blotting days and total leaf numbers under GA3 treatment. Further studies suggested that FK mutation led to endogenous GAs absence in fk-J3158 and FK gene expression was also affected under GA3 and PAC treatment. In the double-mutant analysis, GA1 would be epistatic to FK in flowering. Moreover, the flowering time of fk-J3158 could be rescued by 3-week vernalization treatment, and the FLC expression was accordingly down-regulated. All these results indicated that FK may affect the flowering in Arabidopsis mainly via GA pathway and vernalization pathway.