In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.

Meiosis in angiosperm plants is followed by mitotic divisions to form multicellular haploid gametophytes. Termination of meiosis and transition to gametophytic development is, in Arabidopsis, governed by a dedicated mechanism that involves SMG7 and TDM1 proteins. Mutants carrying the smg7-6 allele are semi-fertile due to reduced pollen production. We found that instead of forming tetrads, smg7-6 pollen mother cells undergo multiple rounds of chromosome condensation and spindle assembly at the end of meiosis, resembling aberrant attempts to undergo additional meiotic divisions. A suppressor screen uncovered a mutation in centromeric histone H3 (CENH3) that increased fertility and promoted meiotic exit in smg7-6 plants. The mutation led to inefficient splicing of the CENH3 mRNA and a substantial decrease of CENH3, resulting in smaller centromeres. The reduced level of CENH3 delayed formation of the mitotic spindle but did not have an apparent effect on plant growth and development. We suggest that impaired spindle re-assembly at the end of meiosis limits aberrant divisions in smg7-6 plants and promotes formation of tetrads and viable pollen. Furthermore, the mutant with reduced level of CENH3 was very inefficient haploid inducer indicating that differences in centromere size is not the key determinant of centromere-mediate genome elimination.

Meiosis, at the transition between diploid and haploid life cycle phases, is accompanied by reprograming of cell division machinery and followed by a transition back to mitosis. We show that, in Arabidopsis , this transition is driven by inhibition of translation, achieved by a mechanism that involves processing bodies (P-bodies). During the second meiotic division, the meiosis-specific protein THREE-DIVISION MUTANT 1 (TDM1) is incorporated into P-bodies through interaction with SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA 7 (SMG7). TDM1 attracts eIF4F, the main translation initiation complex, temporarily sequestering it in P-bodies and inhibiting translation. The failure of tdm1 mutants to terminate meiosis can be overcome by chemical inhibition of translation. We propose that TDM1-containing P-bodies down-regulate expression of meiotic transcripts to facilitate transition of cell fates to postmeiotic gametophyte differentiation.