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.
