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

苋菜和黄花是双子叶植物，它们利用高效的C4光合作用途径。本实验室以前已经证明，在C4双子叶植物中，编码二氧化碳固定酶1，5-二磷酸核酮糖(Rubisco)的大亚基(LSU，由叶绿体编码的rbcL基因产生)和小亚基(SSU，由核编码的红细胞基因产生)的基因受到光和发育过程的强烈调控。RbcL基因表达的快速增加是对光照的响应，这些增加是由于翻译水平的调节。此外，rbcL mRNAs的翻译、加工和稳定性的变化被发现发生在几个C4特有的过程中，包括LSU多肽对束鞘细胞的特异性定位，叶片光合作用能力的变化，以及叶片发育的不同阶段。为了确定C4植物中rbcL表达的转录后调控因子，我们的研究集中在光介导的翻译诱导上，这是涉及转录后调控的植物过程中最具可操作性的过程。我们已经证明，在光照生长但没有黄化的植物中，多种蛋白质与苋菜红rbcL基因的5‘端区域相互作用。最有趣的是，p47rbcL mRNA结合蛋白显示出与其结合活性相关的几个独特的性质，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结合只发生在从光生长植物中制备的叶绿体提取物中；在暗生长植物提取物中没有P47结合活性。P47对rbcL mRNA的5‘端区域(UTR和编码区)具有高度的特异性，并且只与成熟的处理过的rbcL转录本对应的RNA结合(5’UTR终止于-66)。在体外，5‘UTRs较长的rbcL转录本与P47无关。RbcL 5‘非编码区长度的光相关变异在体内被发现，这些不同的5’末端可能阻止或增强P47的结合，也可能影响rbcL的表达。该项目将阐明P47与rbcL mRNA结合的光和加工相关差异的调节机制。实验室将确定P47是否在光介导的多聚体联合、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，研究人员将进一步鉴定P47rbcL 5‘UTRmRNA结合蛋白，确定其可能的亚基结构和mRNA结合性质。他们将映射p47结合所需的rbcL 5‘RNA内的序列，并确定5’UTR内发生的光相关处理差异如何影响这种结合活性。最后，他们将通过在转基因C4黄曲霉植物中结构性或诱导性表达(正义和反义)P47来确定P47的生物学功能。这些研究将提供关于mRNA结合蛋白在特定的C4植物中，也可能在其他植物中，在转录后水平决定光合作用基因表达的新的见解。这个项目将描述与光合作用活动控制相关的RNA-蛋白质相互作用，并可能为研究物种高光合作用生产力的过程提供见解。通过了解这些过程，科学家可能最终能够提高作物物种的光合作用生产率，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.