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

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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.