Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis.

Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

A complex role of Arabidopsis CDKD;3 in meiotic progression and cytokinesis.

Meiosis is a specialized cell division that halves the number of chromosomes in two consecutive rounds of chromosome segregation. In angiosperm plants is meiosis followed by mitotic divisions to form rudimentary haploid gametophytes. In Arabidopsis, termination of meiosis and transition to gametophytic development are governed by TDM1 and SMG7 that mediate inhibition of translation. Mutants deficient in this mechanism do not form tetrads but instead undergo multiple cycles of aberrant nuclear divisions that are likely caused by the failure to downregulate cyclin dependent kinases during meiotic exit. A suppressor screen to identify genes that contribute to meiotic exit uncovered a mutation in cyclin-dependent kinase D;3 (CDKD;3) that alleviates meiotic defects in smg7 deficient plants. The CDKD;3 deficiency prevents aberrant meiotic divisions observed in smg7 mutants or delays their onset after initiation of cytokinesis, which permits formation of functional microspores. Although CDKD;3 acts as an activator of cyclin-dependent kinase A;1 (CDKA;1), the main cyclin dependent kinase that regulates meiosis, cdkd;3 mutation appears to promote meiotic exit independently of CDKA;1. Furthermore, analysis of CDKD;3 interactome revealed enrichment for proteins implicated in cytokinesis, suggesting a more complex function of CDKD;3 in cell cycle regulation.

Meiotic exit in Arabidopsis is driven by P-body-mediated inhibition of translation.

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.

Application of Chemical Inhibitors in Live Cell Imaging of Plant Meiosis Using Light Sheet Fluorescence Microscopy.

Live imaging combined with the application of chemical inhibitors is a powerful research tool that enables researchers to precisely time the inhibition of cellular processes and study the consequences of these perturbations. This approach is usually applied to in vitro cultivated cells that are easily accessible to chemical treatments and microscopic observations. Here we describe a method for live cell imaging of Arabidopsis meiocytes embedded within floral organs combined with the application of a chemical drug at desired timepoints during meiosis. We describe a customized solution for the Zeiss Z.1 light sheet microscope, including sample preparation and data processing, and demonstrate its utility for the analysis of meiotic progression upon spindle inhibition.

A CENH3 mutation promotes meiotic exit and restores fertility in SMG7-deficient Arabidopsis.

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-mediated genome elimination.

Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy.

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.