Time-Lapse Analysis of Chromosome Behavior in Arabidopsis thaliana Pollen Mother Cells Using pAtDMC1:H2B:GFP Fusion Gene Showed Chromosome Movement and Conformational Change at Meiosis

Abstract

Progress in protein labeling by gene fusion with fluorescent protein-encoding genes and microscopic apparatuses, such as a confocal laser-scanning microscope, has enabled time-lapse analysis of meiotic chromosome movement in living plant cells. Here, we constructed the pAtDMC1:H2B:GFP fusion gene and introduced it into Arabidopsis thaliana. Whole-genome sequencing confirmed the insertion of the fusion gene into a locus between AT3G14830 and AT3G14840. The expression of the fusion gene was not specific to meiocytes but was significant in pollen mother cells (PMCs) within anthers; thus, when PMCs were extruded from anthers for direct observation, meiotic chromosomes (e.g., thin thread-like chromosomes at leptotene or synapsed homologous chromosomes at pachytene) were detected. Further, when intact PMCs inside the anthers were analyzed over time, we observed dynamic movement and conformational changes in the PMC chromosomes, and PMCs proceeded from the premeiotic interphase to anaphase II. GFP-tagged histone H2B controlled by the AtDMC1 promoter was incorporated into meiotic chromosomes normally and stayed at least partially in chromosomes until anaphase II.