Post-transcriptional Control of rbcL Gene Expression in the C4 dicots Amaranth and Flaveria Bidentis

阿马兰斯和黄顶菊是双子叶植物，利用高效的C4光合作用途径。 该实验室先前已经表明，编码CO2固定酶1，5-二磷酸核酮糖（Rubisco）的大亚基（LSU，由质体编码的rbcL基因产生）和小亚基（SSU，由核编码的rbcS基因产生）的基因在C4双子叶植物中受到光和发育过程的强烈调控。 非常快速的增加rbcL基因表达诱导响应照明，这些增加是由于在翻译水平上的调节。 此外，已发现rbcL mRNA的翻译、加工和稳定性的改变发生在几个C4特异性过程中，包括LSU多肽在束鞘细胞的特异性定位、叶光合能力的变化以及叶发育的各个阶段。 为了确定C4植物中rbcL表达的转录后调节因子，我们将我们的调查集中在光介导的翻译诱导，这是最可操纵的植物过程中，转录后控制已被牵连。 我们已经证明，多个蛋白质相互作用的5'区阿马兰斯rbcL mRNA在光生长，但不黄化植物。 最有趣的是，p47 rbcL mRNA结合蛋白显示出与其结合活性相关的几种独特性质，并且是参与光介导的rbcL翻译激活的反式作用调节因子的潜在候选者。 p47结合仅发生在从光照生长的植物制备的质体提取物中;在黑暗生长的植物的提取物中没有p47结合活性。 p47对rbcL mRNA的5'区（UTR和编码区）具有高度特异性，并且仅与对应于成熟加工的rbcL转录物（5' UTR终止于-66）的RNA结合。 具有较长5 'UTR的rbcL转录物在体外不与p47结合。 发现rbcL 5' UTR长度的光相关变化在体内发生，并且这些不同的5'末端可能阻止或增强p47结合，也可能影响rbcL表达。 该项目将阐明光和加工相关的p47与rbcL mRNA结合的差异介导的机制。 实验室将确定p47是否在光介导的多聚体缔合、光介导的5'加工或翻译激活中发挥作用。 为了实现这些目标，研究人员将进一步表征p47 rbcL 5' UTR mRNA结合蛋白，确定其可能的亚基结构和mRNA结合特性。 他们将绘制p47结合所必需的rbcL 5 'RNA内的序列，并确定5' UTR内发生的光依赖性加工差异如何影响这种结合活性。 最后，他们将通过p47在转基因C4 F中的组成型或诱导表达（正义和反义）来确定p47的生物学功能。黄顶菊属植物 这些调查将提供新的见解mRNA结合蛋白的功能，在确定光合基因的表达在转录后水平在专门的C4植物，并可能在其他植物物种以及。 该项目将表征与光合活性控制相关的RNA-蛋白质相互作用，并可能提供对正在研究的物种的高光合生产力负责的过程的见解。 通过了解这些过程，科学家们最终可能能够提高作物物种的光合生产力，生产出更有效地吸收二氧化碳的植物。

Amaranth and Flaveria bidentis are dicotyledonous plants that use the highly efficient C4 pathway of photosynthesis. This laboratory has previously shown that genes encoding the large subunit (LSU, produced from the plastid-encoded rbcL gene) and small subunit (SSU, produced from nuclear-encoded rbcS genes) of the CO2 fixation enzyme ribulose 1,5-bisphosphate (Rubisco) are strongly regulated by light and by developmental processes in a C4 dicot. Very rapid increases in rbcL gene expression are induced in response to illumination, and these increases are due to regulation at the translational level. In addition, alterations in the translation, processing, and stability of rbcL mRNAs have been found to occur during several C4-specific processes, including specific localization of the LSU polypeptide to bundle sheath cells, changes in leaf photosynthetic capacity, and during various stages of leaf development. To identify post-transcriptional regulators of rbcL expression in C4 plants, we have centered our investigations around the light-mediated induction of translation, which is the most manipulatable of the plant processes where post-transcriptional control has been implicated. We have demonstrated that multiple proteins interact with 5' regions of amaranth rbcL mRNA in light-grown, but not etiolated plants. Most interestingly, the p47 rbcL mRNA binding protein shows several unique properties associated with its binding activities, and is a potential candidate for a trans-acting regulatory factor involved in the light-mediated activation of rbcL translation. p47 binding occurs only in plastid extracts prepared from light-grown plants; there is no p47 binding activity in extracts from dark-grown plants. p47 is highly specific for 5' regions (UTR and coding regions) of rbcL mRNA, and binding occurs only with RNAs corresponding to mature processed rbcL transcripts (5' UTR terminating at -66). rbcL transcripts with longer 5'UTRs do not associate with p47 in vitro. Light-associated variations in the length of the rbcL 5' UTR were found to occur in vivo, and these different 5' termini may prevent or enhance p47 binding, possibly affecting rbcL expression as well. This project will elucidate the mechanisms by which light- and processing-associated differences in p47 binding to rbcL mRNA are mediated. The laboratory will determine if p47 plays a role in light-mediated polysome-association, light-mediated 5' processing, or translational activation. To accomplish these goals, the investigators will further characterize the p47 rbcL 5' UTR mRNA binding protein, determine its possible subunit structure, and mRNA binding properties. They will map sequences within rbcL 5'RNA that are necessary for p47 binding, and determine how light-dependent processing differences that occur within the 5'UTR affect this binding activity. Finally, they will determine the biological function of p47 by constitutive or induced expression (sense and antisense) of p47 in transgenic C4 F. bidentis plants. These investigations will provide novel insights about the function of mRNA binding proteins in determining photosynthetic gene expression at post-transcriptional levels in specialized C4 plants, and possibly in other plant species as well. This project will characterize RNA-protein interactions associated with control of photosynthetic activity, and may provide insights into processes responsible for the high photosynthetic productivity of the species being studied. By understanding these processes, scientists may ultimately be able to increase the photosynthetic productivity of crop species, producing plants that are more efficient at assimilating CO2.