Conserved CDC20 Cell Cycle Functions Are Carried out by Two of the Five Isoforms in Arabidopsis thaliana

BackgroundThe CDC20 and Cdh1/CCS52 proteins are substrate determinants and activators of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase and as such they control the mitotic cell cycle by targeting the degradation of various cell cycle regulators. In yeasts and animals the main CDC20 function is the destruction of securin and mitotic cyclins. Plants have multiple CDC20 gene copies whose functions have not been explored yet. In Arabidopsis thaliana there are five CDC20 isoforms and here we aimed at defining their contribution to cell cycle regulation, substrate selectivity and plant development. Methodology/Principal FindingsStudying the gene structure and phylogeny of plant CDC20s, the expression of the five AtCDC20 gene copies and their interactions with the APC/C subunit APC10, the CCS52 proteins, components of the mitotic checkpoint complex (MCC) and mitotic cyclin substrates, conserved CDC20 functions could be assigned for AtCDC20.1 and AtCDC20.2. The other three intron-less genes were silent and specific for Arabidopsis. We show that AtCDC20.1 and AtCDC20.2 are components of the MCC and interact with mitotic cyclins with unexpected specificity. AtCDC20.1 and AtCDC20.2 are expressed in meristems, organ primordia and AtCDC20.1 also in pollen grains and developing seeds. Knocking down both genes simultaneously by RNAi resulted in severe delay in plant development and male sterility. In these lines, the meristem size was reduced while the cell size and ploidy levels were unaffected indicating that the lower cell number and likely slowdown of the cell cycle are the cause of reduced plant growth. Conclusions/SignificanceThe intron-containing CDC20 gene copies provide conserved and redundant functions for cell cycle progression in plants and are required for meristem maintenance, plant growth and male gametophyte formation. The Arabidopsis-specific intron-less genes are possibly “retrogenes” and have hitherto undefined functions or are pseudogenes.研究背景：CDC20与Cdh1/CCS52蛋白是后期促进复合物/环体（Anaphase Promoting Complex/Cyclosome，APC/C）E3泛素连接酶的底物决定因子与激活因子，二者通过靶向降解多种细胞周期调控因子，进而调控有丝分裂细胞周期。在酵母与动物中，CDC20的主要功能是降解分离酶抑制蛋白（securin）与有丝分裂周期蛋白。植物拥有多份CDC20基因拷贝，但其功能尚未得到阐明。拟南芥（Arabidopsis thaliana）中存在5种CDC20同工型，本研究旨在明确它们在细胞周期调控、底物选择性以及植物发育中的作用。 材料与方法/主要研究结果：本研究通过分析植物CDC20的基因结构与系统发育，检测5种拟南芥CDC20（AtCDC20）基因拷贝的表达情况，以及它们与APC/C亚基APC10、CCS52蛋白、有丝分裂检验点复合物（Mitotic Checkpoint Complex，MCC）组分以及有丝分裂周期蛋白底物的互作关系，确定了AtCDC20.1与AtCDC20.2的保守CDC20功能。其余3个无内含子的基因为拟南芥所特有且表达沉默。研究表明，AtCDC20.1与AtCDC20.2是MCC的组成成分，并以意外的特异性与有丝分裂周期蛋白发生互作。AtCDC20.1与AtCDC20.2在分生组织、器官原基中表达，其中AtCDC20.1还在花粉粒与发育中的种子中表达。通过RNA干扰（RNAi）同时敲低这两个基因，会导致植物发育严重迟滞与雄性不育。在该转基因株系中，分生组织大小减小，但细胞大小与倍性水平未受影响，这表明细胞数量减少与细胞周期进程放缓是植物生长受抑的原因。 结论与意义：含有内含子的CDC20基因拷贝可为植物细胞周期进程提供保守且冗余的功能，且对于分生组织维持、植物生长与雄配子体形成不可或缺。拟南芥特有的无内含子基因可能为“逆转录基因（retrogenes）”，其功能迄今尚未明确，或为假基因。