Nonogaki Mariko, Sall Khadidiatou, Nambara Eiji, Nonogaki Hiroyuki
Department of Horticulture, Oregon State University, Corvallis, OR, 97331, USA.
Plant J. 2014 May;78(3):527-39. doi: 10.1111/tpj.12472. Epub 2014 Mar 18.
Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA-stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE-binding factor) expression in Arabidopsis Columbia-0 seeds, which caused 9- to 73-fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non-dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre-harvest sprouting during crop production, and therefore contributes to translational biology.
脱落酸是种子休眠所必需的激素。我们之前使用植物基因开关系统（一种化学诱导基因表达系统）进行的研究表明，诱导9-顺式环氧类胡萝卜素双加氧酶（NCED，一种ABA生物合成的限速基因）足以抑制拟南芥吸胀种子的萌发。在此，我们报告了一种高效实验系统的开发，该系统可在种子成熟过程中使NCED表达扩增。该系统由一个含有ABA响应元件（ABREs）的普通小麦启动子和一个双色高粱NCED构建而成，通过正反馈机制引发ABA刺激的ABA生物合成和信号传导。嵌合基因pABRE:NCED增强了拟南芥Columbia-0种子中NCED和ABF（ABRE结合因子）的表达，使ABA水平提高了9至73倍。pABRE:NCED种子表现出异常深的休眠，持续超过3个月。有趣的是，扩增的ABA途径还导致拟南芥NCED5的表达增强，揭示了天然系统中存在正反馈。这些结果证明了正反馈机制的稳健性以及种子成熟过程中NCED表达或单一代谢变化的重要性。pABRE:NCED系统提供了一个出色的实验系统，可产生来自同一母本的休眠和非休眠种子，它们仅在合子ABA方面存在差异。pABRE:NCED种子含有一个GFP标记，可用于区分转基因和无效分离株的种子，非常适合进行比较分析。除了在基础研究中的应用外，该系统还可应用于作物生产中防止收获前发芽，因此对转化生物学有贡献。
