DataSheet_2_ZmCCA1a on Chromosome 10 of Maize Delays Flowering of Arabidopsis thaliana.pdf

Maize (Zea mays) is a major cereal crop that originated at low latitudes, and thus photoperiod sensitivity is an important barrier to the use of tropical/subtropical germplasm in temperate regions. However, studies of the mechanisms underlying circadian regulation in maize are at an early stage. In this study we cloned ZmCCA1a on chromosome 10 of maize by map-based cloning. The gene is homologous to the Myb transcription factor genes AtCCA1/AtLHY in Arabidopsis thaliana; the deduced Myb domain of ZmCCA1a showed high similarity with that of AtCCA1/AtLHY and ZmCCA1b. Transiently or constitutively expressed ZmCCA1a-YFPs were localized to nuclei of Arabidopsis mesophyll protoplasts, agroinfiltrated tobacco leaves, and leaf and root cells of transgenic seedlings of Arabidopsis thaliana. Unlike AtCCA1/AtLHY, ZmCCA1a did not form homodimers nor interact with ZmCCA1b. Transcripts of ZmCCA1a showed circadian rhythm with peak expression around sunrise in maize inbred lines CML288 (photoperiod sensitive) and Huangzao 4 (HZ4; photoperiod insensitive). Under short days, transcription of ZmCCA1a in CML288 and HZ4 was repressed compared with that under long days, whereas the effect of photoperiod on ZmCCA1a expression was moderate in HZ4. In ZmCCA1a-overexpressing A. thaliana (ZmCCA1a-ox) lines, the circadian rhythm was disrupted under constant light and flowering was delayed under long days, but the hypocotyl length was not affected. In addition, expression of endogenous AtCCA1/AtLHY and the downstream genes AtGI, AtCO, and AtFt was repressed in ZmCCA1a-ox seedlings. The present results suggest that the function of ZmCCA1a is similar, at least in part, to that of AtCCA1/AtLHY and ZmCCA1b, implying that ZmCCA1a is likely to be an important component of the circadian clock pathway in maize.

玉蜀黍（Zea mays，俗称玉米）是起源于低纬度区域的主要谷类作物，因此光周期敏感性成为热带/亚热带种质资源在温带地区推广应用的关键限制因素。然而，当前针对玉米生物钟调控机制的研究仍处于起步阶段。本研究通过图位克隆技术，成功克隆了玉米第10号染色体上的ZmCCA1a基因。该基因与拟南芥（Arabidopsis thaliana）中的MYB类转录因子基因AtCCA1/AtLHY同源；其推导所得的MYB结构域与AtCCA1/AtLHY及ZmCCA1b的对应结构域具有高度序列相似性。瞬时或组成型表达的ZmCCA1a-YFP融合蛋白可定位于拟南芥叶肉原生质体、农杆菌浸润处理的烟草叶片，以及转基因拟南芥幼苗的叶肉与根细胞的细胞核内。与AtCCA1/AtLHY不同，ZmCCA1a既无法形成同源二聚体，也不能与ZmCCA1b发生蛋白相互作用。在玉米光周期敏感自交系CML288与光周期不敏感自交系黄早四（HZ4）中，ZmCCA1a的转录本呈现典型的昼夜节律性表达，其表达峰值均出现在日出前后。与长日照条件相比，短日照处理下CML288与HZ4中ZmCCA1a的转录水平均受到抑制，但光周期对HZ4中ZmCCA1a表达的调控效应相对温和。在过表达ZmCCA1a的拟南芥（ZmCCA1a-ox）转基因株系中，恒定光照条件下的生物钟节律被显著扰乱，长日照条件下植株开花时间延迟，但下胚轴长度未受明显影响。此外，ZmCCA1a-ox幼苗体内内源AtCCA1/AtLHY及其下游靶基因AtGI、AtCO与AtFt的转录表达均受到显著抑制。本研究结果表明，ZmCCA1a的功能至少在部分生物学过程中与AtCCA1/AtLHY及ZmCCA1b高度相似，这提示ZmCCA1a极有可能是玉米生物钟调控通路中的关键组成元件。