Plant Rubisco biogenesis : a study of the requirements and processing steps during co- and post-translational modification of the large subunit

The production of chemical energy from light energy and C0{u2082} by photosynthesis is essential for life in the biosphere, and thus so is the activity of the often rate limiting CO{u2082}-fixing enzyme Rubisco (D-ribulose-l,5-bisphosphate carboxylase/oxygenase). The importance of this 'gateway' enzyme for carbon entry into the biosphere, and it's apparent inherent flaws in catalysis, have made it a key target for improvement as a key step towards enhancing photosynthesis and agricultural yield potential. For example, the diminished carboxylation efficiency and C0{u2082}/0{u2082} specificity of crop Rubisco are key targets for improvement. As a consequence of its catalytic limitations most crops allocate 20 to 50% of their total soluble protein to Rubisco. Significant attention has been, and continues to be, devoted to improving our understanding of sequence-performance relationships among various natural Rubisco isoforms in the hope of identifying solutions for bioengineering and transplanting more efficient forms into crops. Critical to such goals has been to understand the synthesis and assembly requirements of Rubisco, an area receiving increased interest of late. Of the many important processes involved in protein biogenesis and function are often a myriad of post-translational processing events. For Rubisco in plants both its chloroplast genome (plastome) encoded catalytic large subunit (LSu) and nucleus encoded, cytosolic made small subunits (SSu) undergo a number of N-terminal co- and post-translational modifications (PTMs) both prior to, during and (possibly) following their assembly into hexadecameric holoenzyme complexes (designated L{u2088}S{u2088}) within the chloroplast stroma. Consistent with the highly conserved N-terminal amino acid sequence of plant LSu's, all undergo co-translational excision of Met-1 and Ser-2 to expose a Pro-3 that is N^{u03B1}-acetylated. As well, most plant species undergo trimethylation of a highly conserved Lys-14 residue in the LSu. Despite the known pervasive role on structure and function that N-terminal PTMs have on many proteins, a functional role for the LSu processing events in higher plant Rubiscos and a complete picture of the processing enzymes involved remain unknown. The homologous recombination route for transforming the plastome provides a versatile means for engineering specific changes to its nucleotide sequence. For engineering Rubisco in tobacco chloroplasts a tailor made transforming master-line called ^{cm}trL allows for the efficient transplantation of foreign and mutated tobacco LSu genes (rbcL) for functional study. In this study ^{cm}trL was used to introduce recombinant tobacco rbcL genes incorporating both residue altering and 'silent' nucleotide changes to codons Ser-2, Pro-3 and Lys-14 in the LSu. The amino acid changes to Ser-2 and Pro-3 were found to perturb normal processing of the nascent LSu with all mutations of Pro-3 producing a novel acetyl-Ser-2 LSu N-termini and a Ser-2-Arg substitution preventing excision of Met-1 that was also N-acetylated. These findings provide new insights into the putative series of LSu processing events in plastids and suggest the involvement of other candidate processing enzymes for future consideration and testing, in particular with regard to possibly stimulating recombinant protein expression in plastids. From the ten tobacco variant mutant rbcL transplastomic lines produced in this study it was observed that changes to the highly conserved 5'coding region of rbcL reduced Rubisco synthesis by as much as 2-fold, even in lines coding a synonymous codon change at Ser-2. In general the varying restrictions to Rubisco content in each tobacco line appeared mostly correlated with the positioning and number of the nucleotide mutations introduced into the 5' coding region of the rbcL mRNA and their predicted perturbations to mRNA secondary structure around regions associated with translation initiation. This suggests limitations to the translational processing of the rbcL mRNA may be imposing greater restrictive constraints on selection of nucleotide changes to the 5'rbcL coding sequence, possibly explaining the complete conservation of the MSP N-terminal amino acid sequence in plant LSu's. It was interesting to note that Rubisco biogenesis and catalysis were unaffected by substitutions to Lys-14. Consistent with the high substrate specificity of the large subunit methyltransferase that usually trimethylates the highly conserved Lys-14 in most plant LSu's, the substituted amino acids were non-methylated. The high resource and energy costs associated with methylation reactions intuitively suggests it likely plays a functional role. Among the possibilities discussed is a protective role against Rubisco proteolysis during leaf senescence associated with age or exposure to abiotic stress (e.g. temperature, drought). Future analyses of variations in LSu translation rate and stability in different aged leaves from the mutant Rubisco transplastomic lines (and proposed new ones) pose important starting points to pinpointing the cause(s) for any varied changes in Rubisco biogenesis and resolving functional roles for the LSu co- and post-translational modifications