De Storme Nico, Keçeli Burcu Nur, Zamariola Linda, Angenon Geert, Geelen Danny
In vitro Biology and Horticulture, Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000, Ghent, Belgium.
Institute for Molecular Biology and Biotechnology, VUB, Pleinlaan 2, B-1050, Brussels, Belgium.
BMC Plant Biol. 2016 Jan 5;16:1. doi: 10.1186/s12870-015-0700-5.
The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell's chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo.
By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events.
This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.
细胞特异性染色体数目的体内测定在植物研究的多个方面提供了一种有价值的工具。然而，目前用于确定内系统倍性水平的技术不允许进行非破坏性的、细胞特异性的染色体定量。特别是在配子体细胞谱系中，它们被物理包裹在生殖器官结构中，直接进行体内倍性测定已被证明极具挑战性。以拟南芥为模型，我们在此评估重组CENH3 - GFP报告基因用于标记细胞染色中心以及监测体内配子体和体细胞染色体数目的适用性。
通过使用不同启动子调节CENH3 - GFP报告基因盒的表达，我们分离出两个报告株系，分别允许在体细胞和配子体细胞中对着丝粒染色体区域进行清晰且高度特异性的标记。利用多倍体植物系列和生殖突变体，我们证明pWOX2 - CENH3 - GFP重组融合蛋白能够确定雄配子体和雌配子体细胞中的配子体染色体数目，并且在早期胚胎发育中还能标记着丝粒区域。通过p35S - CENH3 - GFP进行的体细胞着丝粒标记在年轻的分裂组织中显示最多十个着丝粒点，反映出二倍体染色体数目（2x = 10），并揭示了在植物发育过程中GFP焦点频率的逐渐降低。此外，利用化学和遗传诱导的核内有丝分裂，我们证明CENH3介导的染色体标记为检测和表征核内有丝分裂多倍化事件提供了一种简单且有价值的工具。
本研究表明，将pWOX2 - CENH3 - GFP报告基因构建体导入拟南芥提供了一种简单可靠的方法，用于确定发育中的雄配子和雌配子以及早期胚胎发育过程中的染色体数目。另一方面，体细胞表达的CENH3 - GFP报告基因构成了一种有价值的工具，可在个体细胞水平快速确定幼苗的基本体细胞倍性水平，并以非破坏性的、基于显微镜的方式检测和量化核内有丝分裂多倍化事件。
