2022 Volume 87 Issue 4 Pages 313-318
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.
Syngenesis leads to the production of the next generation through fertilization of a female gamete by a male gamete. It is responsible for genetic diversity in sexually reproducing organisms and continuously contributes to the prosperity of the species. In plants, gametes are formed in gametophytes evolved from meiotic products. Meiosis comprises two rounds of cell division, namely, meiosis I and II. Meiosis I reduce the number of chromosomes in half; however, without correct reduction of chromosome number fertile gametes cannot be formed. Different approaches have been used to reveal the mechanisms underlying correct chromosome reduction in a variety of plants. Specifically, genetic approaches to identify the genes related to these mechanisms have been most successful in Arabidopsis thaliana, whereby numerous genes have been identified as, for example, synapsis-related genes, ASY1, SDS, ZYP1 (Caryl et al. 2000, Azumi et al. 2002, Higgins et al. 2005) and those associated with recombination between homologous chromosomes (HCs), AtDMC1, AtSPO11-1, PTD (Klimyuk and Jones 1997, Grelon et al. 2001, Wijeratne et al. 2006) isolated from A. thaliana. Concomitantly, a variety of techniques to analyze meiotic chromosome behavior, such as DAPI staining, immunolocalization, or FISH analysis, have been developed for plants. In contrast to the abundance of information obtained by the above methods on chromosome configuration in fixed cells, information regarding chromosome dynamics in living plant cells remains very limited. Recently, chromosome-visualizing systems in living PMCs have been developed for A. thaliana (Prusicki et al. 2019, Valuchova et al. 2020). These systems utilize fusion genes that express fluorescently labeled chromosome-associated proteins. We took this approach and constructed the pAtDMC1:H2B:GFP fusion gene using the expression regulatory region of AtDMC1 (Klimyuk and Jones 1997) as the meiosis-specific promoter, A. thaliana histone H2B gene, and sGFP (Niwa et al. 1999). Our fusion gene allowed us to successfully observe meiotic chromosome behavior in living A. thaliana PMCs.
A. thaliana (Landsberg electa) genomic DNA was prepared from leaves using the DNeasy Plant Mini kit (QIAGEN). The A. thaliana histone H2B (H2B) gene (AT3G45980) was amplified from genomic DNA by PCR using a pair of primer sets (SalI-H2B Fw; 5′-GGGGGTCGACATGGCGAAGGCAGATAAGAAACCAGCGGAG-3′, attB2 H2B Rv2; 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGAGAACTCGTAAACTTCGTAACCGCC-3′). Similarly, the AtDMC1 (AT3G22880) regulatory sequence (pAtDMC1), from −3691 to +402, relative to A of the ATG start codon, was amplified from genomic DNA by PCR using a pair of primer sets (attB1+AtDMC1−3691; 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGTTGGTTTGTGACAGCTACGCGATATTGC-3′, SalI-AtDMC1 Rv; 5′-GGGGGTCGACCTGCATCTGGCTCGTTTCTTCAGCTCT-3′). Both PCR products were digested with Sal I (TaKaRa Bio Inc.), and ligated to each other using a DNA ligation Kit 〈Mighty Mix〉 (TaKaRa Bio Inc.). The ligated PCR product (pAtDMC1:H2B) was introduced into the pDONR/zeo vector (Invitrogen) using BP Clonase II (Invitrogen). In turn, pAtDMC1:H2B was transferred into the plant-transforming binary vector pGWB4 containing the sGFP reporter gene using LR Clonase (Invitrogen). Then, the recombined pGWB4 was introduced into Agrobacterium by electroporation, and A. thaliana Columbia plants were transformed with Agrobacterium by the vacuum infiltration method (Bechtold and Pelletier 1998). A. thaliana plants were grown in soil in a growth room at 22°C under a 16 h light/8 h dark cycle. Transformants were screened on MS plates containing 50 mg mL−1 hygromycin B and 50 mg mL−1 kanamycin. The genomic DNA of the transformants was extracted using the DNeasy Plant Mini Kit. Library preparation and sequencing were performed by Novogene with the NovaSeq 6000 system (Illumina). Paired-end reads comprising 150 bp were trimmed and filtered based on quality using fastp (version 0.23.2, Chen et al. 2018) and the inserted site was detected by tDNAscan (Sun et al. 2019) followed by PCR validation. Sequenced reads were deposited in NCBI/DDBJ/EBI under accession number DRR357117.
Microscopic observationSeveral inflorescences were excised from one transformed plant, and buds of the appropriate size (approximately 0.4 mm) were selected. For chromosome observation, anthers were removed from the buds and crushed between a glass slide and a cover glass using a roller. GFP-tagged chromosomes of PMCs were observed using an inverted confocal laser scanning microscope (CLSM, Zeiss LSM700) or a multi-photon CLSM (Olympus FVMPE-RS). As for time-lapse analysis, one or two sepals were removed from the buds using fine needles, and the buds were then placed on double-sided tape attached to a cover slip. The cover slip was then submerged in water in a Petri dish.
An outline of the constructed pAtDMC1:H2B:GFP fusion gene is shown in Fig. 1. The fragment of the AtDMC1 gene from −3691 to +402 bp relative to the start codon, including the first intron, and the coding region of A. thaliana histone H2B gene extending from the start codon to the last codon before the stop codon was fused and then linked to the sGFP gene on the pGWB4 vector. This fusion gene was introduced into A. thaliana using Agrobacterium. Whole genome sequencing of the transformants determined its insertion site at 4987914 bp on chromosome 3 (Fig. 1), between AT3G14830, which encodes an unknown protein, and AT3G14840, which encodes the LIK1 protein. Almost all transformants grew and produced seeds normally. Furthermore, we did not observe any phenotypic abnormalities in the transformants.
To confirm the expression of the fusion gene in PMCs and the incorporation of the fusion gene products into meiotic chromosomes, we directly observed PMCs extruded from anthers using CLSM. As shown in Fig. 2, meiotic chromosomes of PMCs were clearly observed, and nuclei and chromosomes of somatic cells of anthers were also observed around PMCs. PMCs were enclosed by autofluorescent sheets that were excited and emitted green light similar to GFP. Debris of the sheets, which probably arose from the compression of anthers, was observed clinging to PMCs in some places.
The nuclei of PMCs at the pre-meiotic interphase appeared as regular or oval spherical shapes (Fig. 2A). In leptotene, chromosomes showed a one-sided distribution in the nuclear space, presumably caused by the presence of nucleoli on the other side (Fig. 2B). At zygotene, chromosomes were still one-sided and showed extruded portions, the ends of which always showed knobs (Fig. 2C). Meanwhile, at pachytene, chromosomes extended in random directions (Fig. 2D). Although up to metaphase II chromosome fluorescence was bright and clear (Fig. 2E–G), at telophase II and the tetrad stage, it became weak and hazy (Fig. 2H, I). Further, although the fluorescence strength of PMC chromosomes varied, most of the transformed plants that survived antibiotic screening showed GFP fluorescence to some extent. This observation was conducted within 30 min after PMCs were extruded, as fluorescence from PMCs became rapidly attenuated upon release from the anthers.
Time-lapse analysis of meiotic chromosome behaviorWe conducted a time-lapse analysis of transgenic plants using conventional CLSM and multi-photon CLSM. Based on the observation of extruded PMCs, buds of the appropriate size were selected which allowed us to observe that meiosis proceeded from premeiotic interphase through anaphase II (Fig. 3). Although we used a low level of exciting laser light to avoid damage to PMCs, occasionally, the nuclei of PMCs shrank and meiosis stopped. In our live-meiosis observation system, substages of prophase I, namely, leptotene, zygotene, pachytene, diplotene, and diakinesis, were indistinguishable, and condensation of chromosomes toward metaphase I started more suddenly than expected. In one anther, meiosis seemed to proceed synchronously (Fig. 4). Metaphase I took approximately 25 min (n=13) and bright bivalents moved back and forth near the metaphase plate. Neighboring PMCs started anaphase I together and HCs separated and moved in their respective directions. After anaphase I, the cell chromosomes decondensed and formed two nuclei. Subsequently, chromosomes condensed again and emitted a slightly stronger fluorescence. Metaphase II took approximately 26 min (n=11) to proceed to anaphase II (Fig. 5). Altogether, the interval between metaphases I and II lasted for approximately 122 min (n=10). With our system, we were able to detect up to telophase II.
Recently, several plant meiosis-visualization systems using fluorescence-labeled proteins have been developed. The REC8 promoter, the RPS5A promoter (Prusicki et al. 2019), and genes ASY1, HistoneH2B, HTA10, DR5, and PCNA (Valuchova et al. 2020) were used in these systems. In particular, REC8 (SYN1) and ASY1 are useful because their expression is specific to meiosis (Bai et al. 1999, Caryl et al. 2000). These gene constructs enabled successful visualization of PMC chromosomes. Additionally, AtDMC1 is known to be specifically expressed in meiocytes. Therefore, herein, we aimed to establish a novel meiosis-observation system using the AtDMC1 gene. Based on the GUS assay analysis of the AtDMC1 promoter region (Klimyuk and Jones 1997), we constructed fusion gene pAtDMC1:H2B:GFP, and transformed A. thaliana. When we directly observed PMCs extruded from anthers, clear meiotic chromosome images appeared, in which, for the first time, we effectively identified substages of prophase I (Fig. 2), which was previously considered an impossible task in the case of living PMCs. The cells used for direct observation were flattened between glass sides and cover slips, whereby the three-dimensional conformation of their chromosomes was probably slightly deformed. Furthermore, in the extruded PMCs, the signals did not last long. Therefore, a direct observation method cannot be adopted for time-lapse observations. However, the method developed herein clearly indicated that the histone H2B:GFP fusion protein was expressed in PMCs and incorporated into meiotic chromosomes correctly, which indicates that the AtDMC region worked as a good promoter for the of expression of the histone H2B:GFP fusion protein in PMCs. Direct observation proved a good screening tool for plants that express the fusion gene at a high rate. Indeed, in the time-lapse experiments, we observed signals as early as the premeiotic interphase as round nuclei, and overall chromosome conformational changes during meiosis I and II were recognized (Fig. 3). Further, the fusion gene was expressed in cells of other anther tissues, likely due to the effect of neighboring genes. The data from the TAIR Klepikova Atlas indicate that the protein AT3G14830 is expressed in flower tissues (Klepikova et al. 2016). Despite its expression in additional tissues, our fusion gene would be very useful for tracing chromosome conformational changes during meiosis. At present, discrimination among substages of Prophase I is difficult; however, in the future, microscopes with higher-resolution will be available. Locus-specific fluorescent protein genes are indispensable for analysis of the homologous-chromosome pairing process. Introduction of genes encoding such proteins to pAtDMC1:H2B:GFP-bearing plants will reveal how the loci behave during the pairing process.
We gratefully acknowledge Dr. Tetsuya Higashiyama and Dr. Yoshikatsu Sato for technical advice on the operation of CLSM. We also thank Olympus Corp. for allowing us to use their multi-photon CLSM and for their kind support for the operation of CLSM and analysis of the data. We would like to thank Editage (www.editage.com) for English language editing.
