玉米C4PPDK转录调控机制解析

Increasing radiation use efficiency in C3 crops by introducing C4 photosynthetic mechanisms is considered an efficient way to increase yield in C3 crops such as rice and wheat. To efficiently bio-engineer C3 to C4, we will first need to better understand the biochemistry and also regulatory mechanisms of C4 photosynthesis. C4PPDK is essential for normal functioning of C4 photosynthesis in maize, therefore, it is important to understand its regulatory mechanisms. In this study, we propose to investigate the transcription factors ZmBBX7 and ZmBBX14 that regulate the expression of C4PPDK, construct the transcriptional regulatory pathway and test the influence of light quality/quantity and photoperiod on their regulation. We will also examine the cis-elements in PPDK promoter and identify other transcription factors that are involved in the transcriptional regulation of C4PPDK. In addition, we will examine the cis and trans regulatory elements of the homologs of maize C4PPDK in other C3 and C4 monocots, in order to uncover the evolutionary origin of their different expression in C3 and C4 species. The proposed study will lay the foundation for understanding the regulatory mechanism of C4 photosynthesis, which could help effectively engineer C4 photosynthesis into C3 crops.

通过C4改造提高C3作物的光合效率被认为是进一步提高C3作物，如水稻、小麦产量的一个重要途径。要想有效的对C3作物进行C4改造，首先必须理解C4光合调控机理。C4PPDK是玉米C4光合所必需的关键酶，因而，研究C4PPDK表达调控机制对深入理解C4光合调控机理具有重要意义。本项目将通过系统分析调控C4PPDK转录的转录因子ZmBBX7和ZmBBX14，重点构建BBX-C4PPDK转录调控通路并分析外界光强、光质、光周期对此通路的影响，确定调控C4PPDK表达的启动子顺式作用元件及转录因子功能结构域；筛选验证其他调控C4PPDK转录的转录因子，进一步构建C4PPDK转录调控网络系统；解析造成C4玉米和C3水稻BBX-PPDK转录调控通路差异的原因，分析其进化基础，为深入理解C4光合调控机理，有效进行C3作物的C4改造奠定理论基础。

C4PPDK是玉米C4光合所必需的关键酶，C4PPDK功能缺失造成玉米幼苗黄化死亡。因而，研究C4PPDK表达调控机制，挖掘影响C4PPDK表达的转录因子和顺式作用元件，解析C3和C4作物C4PPDK表达调控机制差异对深入理解C4光合调控机理具有重要意义。本项目首先利用酵母单杂、ChIP-seq、EMSA、Dual-luciferase等技术，明确玉米ZmBBX7/14转录因子DNA结合结构域为CCT结构域，ZmBBX7/14转录因子通过结合到C4PPDK启动子区域CCAC元件激活C4PPDK基因表达；转录因子HY5、bHLH105、COL4、STH和STO等与BBX互作，共同调控C4PPDK基因的转录。对ZmBBX CRISPR/Cas9以及过表达突变体的分析进一步证实玉米ZmBBX7/14-C4PPDK调控通路在玉米生长发育中发挥重要作用；其次，利用原生质体瞬时转化技术，本项目初步确定玉米和水稻PPDK基因启动子的差异是造成PPDK基因表达差异的主要因素，玉米和水稻来源的BBX转录因子均可激活玉米PPDK基因表达。此结果在C3、C4作物中具有普遍性；此外，本项目利用玉米叶片维管束鞘细胞及叶肉细胞进行了RNA-seq，H3K4me3和H3K27me3 ChIP-seq，ATAC-seq及BS-seq测序，发现C4PPDK在叶肉细胞特异性高表达可能受到染色质开放程度以及H3K27me3的调控；利用酵母单杂和大规模ChIP-seq测序技术，共筛选到31个调控玉米C4PPDK基因表达的转录因子，初步了构建玉米C4PPDK基因的转录调控网络。这些结果将极大丰富人们对玉米C4PPDK基因表达调控的认识，为深入理解C4光合调控机理，有效进行C3作物的C4改造奠定了基础。