WUSCHEL-RELATED HOMEOBOX4 acts as a key regulator in early leaf development in rice

Rice (Oryza sativa) has long and narrow leaves with parallel veins, similar to other grasses. Relative to Arabidopsis thaliana which has oval-shaped leaves, our understanding of the mechanism of leaf development is insufficient in grasses. In this study, we show that OsWOX4, a member of the WUSCHEL-RELATED HOMEOBOX gene family, plays important roles in early leaf development in rice. Inducible downregulation of OsWOX4 resulted in severe defects in leaf development, such as an arrest of vascular differentiation, a partial defect in the early cell proliferation required for midrib formation, and a failure to maintain cellular activity in general parenchyma cells. In situ analysis showed that knockdown of OsWOX4 reduced the expression of two LONELY GUY genes, which function in the synthesis of active cytokinin, in developing vascular bundles. Consistent with this, cytokinin levels were downregulated by OsWOX4 knockdown. Transcriptome analysis further showed that OsWOX4 regulates multiple genes, including those responsible for cell cycle progression and hormone action, consistent with the effects of OsWOX4 downregulation on leaf phenotypes. Collectively, these results suggest that OsWOX4 acts as a key regulator at an early stage of leaf development. Our previous work revealed that OsWOX4 is involved in the maintenance of shoot apical meristem in rice, whereas AtWOX4 is specifically associated with the maintenance of vascular stem cells in Arabidopsis. Thus, the function of the two orthologous genes seems to be diversified between rice and Arabidopsis.水稻（Oryza sativa）具有带平行叶脉的狭长叶片，与其他禾本科植物一致。相较于叶片呈椭圆形的拟南芥（Arabidopsis thaliana），目前学界对禾本科植物叶片发育机制的认知仍存在不足。 本研究证实，WUSCHEL相关同源盒（WUSCHEL-RELATED HOMEOBOX）基因家族成员OsWOX4在水稻早期叶片发育过程中发挥关键调控作用。对OsWOX4进行诱导性下调后，水稻叶片发育出现严重缺陷，具体包括维管分化停滞、中脉形成所需的早期细胞增殖出现部分障碍，以及普通薄壁细胞无法维持正常细胞活性。 原位分析结果显示，敲低OsWOX4会降低发育中维管束内两个LONELY GUY基因（LONELY GUY）的表达水平，而这类基因的功能是合成活性细胞分裂素。与此一致的是，OsWOX4敲低会导致植株体内细胞分裂素水平下调。 转录组分析进一步表明，OsWOX4可调控多个基因的表达，其中涵盖与细胞周期进程及激素信号通路相关的基因，这与OsWOX4下调对叶片表型产生的影响相符。 综上，上述结果表明OsWOX4是水稻叶片发育早期阶段的关键调控因子。我们此前的研究表明，OsWOX4参与维持水稻的茎尖分生组织，而拟南芥中的AtWOX4则特异性参与维管干细胞的维持。因此，这两个同源基因的功能在水稻与拟南芥之间出现了分化。