Abstract：
The leaf is a determinate organ with a final size under genetic control. Numerous factors that regulate final leaf size have been identified in Arabidopsis thaliana; although most of these factors play their roles during the growth of leaf primordia, much less is known about leaf initiation and its effects on final leaf size. In this study, we characterized oligocellula6-D (oli6-D), a semidominant mutant of Arabidopsis thaliana with smaller leaves than the wild type due to its reduced leaf cell numbers. A time-course analysis showed that oli6-D had approximately 50% fewer leaf cells, even immediately after leaf initiation; this difference was maintained throughout leaf development. Next-generation sequencing showed that oli6-D had chromosomal duplication involving 2-kbp and 3-Mbp regions of chromosomes 2 and 4, respectively. Several duplicated genes examined had approximately twofold higher expression levels, and at least one gene acquired a new intron/exon structure due to a chromosome fusion event. oli6-D showed reduced auxin responses in leaf primordia, primary roots, and embryos as well as reduced apical dominance and partial auxin-resistant root growth. CRISPR/Cas9-mediated genome editing enabled the removal of a 3-Mbp duplicated segment, the largest targeted deletion in plants thus far. As a result, oli6-D restored the wild-type leaf phenotypes, demonstrating that oli6-D is a gain-of-function mutant. Our results suggest a new regulatory point of leaf size determination that functions at a very early stage of leaf development and is negatively regulated by one or more genes located in the duplicated chromosomal segments.
摘要：
叶子是在遗传控制下具有最终尺寸的决定性器官。在拟南芥中已经确定了许多调节最终叶片大小的因素;尽管这些因素中的大多数在叶片原基的生长过程中发挥了作用，关于叶片起始及其对最终叶片大小的影响知之甚少。在这项研究中,我们表征了寡纤维素6-D(oli6-D)，拟南芥的半显性突变体，由于其叶片细胞数量减少，叶片比野生型小。时程分析表明，oli6-D的叶细胞减少了约50％，甚至在叶开始后立即；这种差异在整个叶片发育过程中一直保持。下一代测序显示，oli6-D具有染色体重复，分别涉及染色体2和4的2-kbp和3-Mbp区域。检查的几个重复基因具有大约两倍的高表达水平，并且至少一个基因由于染色体融合事件而获得了新的内含子/外显子结构。oli6-D显示叶片原基中生长素反应降低，主根,和胚胎以及降低的顶端优势和部分抗生长素的根生长。CRISPR/Cas9介导的基因组编辑能够去除3-Mbp的重复片段，迄今为止植物中最大的靶向缺失。因此,oli6-D恢复了野生型叶片表型，证明oli6-D是功能获得突变体。我们的结果表明，叶片大小确定的新调控点在叶片发育的早期阶段起作用，并受到位于重复染色体片段中的一个或多个基因的负调控。
