Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone

Abstract

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO 2 -fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob AtL plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L 8 A S 8 t Rubisco [comprising AtL and tobacco small (S) subunits] in tob AtL-R1 leaves compared with tob AtL , despite >threefold lower steady-state Rubisco mRNA levels in tob . Accompanying twofold increases in photosynthetic CO 2 -assimilation rate and plant growth were measured for tob lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.T he increasing global demands for food supply, bioenergy production, and CO 2 -sequestration have placed a high need on improving agriculture yields and resource use (1, 2). It is now widely recognized that yield increases are possible by enhancing the light harvesting and CO 2 -fixation processes of photosynthesis (3-5). A major target for improvement is the enzyme Rubisco [ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase] whose deficiencies in CO 2 -fixing speed and efficiency pose a key limitation to photosynthetic CO 2 capture (6, 7). In plants, the complex, multistep catalytic mechanism of Rubisco to bind its 5-carbon substrate RuBP, orient its C-2 for carboxylation, and then process the 6-carbon product into two 3-phosphoglycerate (3PGA) products, limits its throughput to one to four catalytic cycles per second (8). The mechanism also makes Rubisco prone to competitive inhibition by O 2 that produces only one 3PGA and 2-phosphoglycolate (2PG). Metabolic recycling of 2PG by photorespiration requires energy and results in most plants losing 30% of their fixed CO 2 (5). To compensate for these catalytic limitations, plants like rice and wheat invest up to 50% of the leaf protein into Rubisco, which accounts for ∼25% of their leaf nitrogen (9).Natural diversity in Rubisco catalysis demonstrates that plant Rubisco is not the pinnacle of evolution (6, 7). Better-performing versions in some red algae have the potential to raise the yield of ...

Rubisco Catalytic Properties and Temperature Response in Crops

“…For instance, there are only two common residues under positive selection out of 10 between this study and methodologically similar work with a different sampling design published earlier (Galmés et al, 2014c). This fact raises questions of epistatic interactions and residue coevolution within Rubisco (Wang et al, 2011) as well as residue coevolution and complementarity between Rubisco and its chaperones (Whitney et al, 2015), which both may prevent the evolution of identical amino acid replacements because of different genetic backgrounds.…”

“…The design of future attempts at Rubisco engineering in crops should be based on surveys of Rubisco catalytic and genetic diversity with a particular stress on relatives of the crops in question. Growing knowledge of the Rubisco catalytic spectrum combined with existing engineering toolkits for Rubisco (Whitney and Sharwood 2008) and its chaperones (Whitney et al, 2015) give us hope that the Rubisco efficiency, and hence the photosynthetic capacity, of crops could be improved in the near future.…”

Surveying Rubisco diversity and temperature response to improve crop photosynthetic efficiency

“…the conversion of C 3 crops to C 4 photosynthesis (Driever and Kromdijk, 2013). Direct replacement of Rubisco will also likely necessitate coengineering of ancillary proteins to achieve maximum results, as demonstrated recently through work with the cochaperone RAF1 (Whitney et al, 2015). The recent introduction of a faster cyanobacterial Rubisco that could sustain higher photosynthetic rates, albeit at high CO 2 concentrations (Lin et al, 2014b;Occhialini et al, 2016), confirms the feasibility and potential of interspecies Rubisco substitutions.…”

“…Potential unintended effects on assembly could be a factor when mutating residues known to be involved in interactions between the large and small subunits. Careful consideration must also be given to avoiding effects on holoenzyme assembly and compatibility with ancillary proteins or assembly chaperones (Carmo-Silva et al, 2015;Whitney et al, 2015). This presents a promising avenue for future work in model systems, testing these residues either singly or in combination, with previous studies having shown strong potential for modifying Rubisco catalysis with targeted amino acid substitutions (Whitney et al, 2011b).…”

“…However, advances in engineering precise changes in model systems continue to provide important developments that are increasing our understanding of Rubisco catalysis (Spreitzer et al, 2005;Whitney et al, 2011aWhitney et al, , 2011bMorita et al, 2014;Wilson et al, 2016), regulation (Andralojc et al, 2012;Carmo-Silva and Salvucci, 2013;Bracher et al, 2015), and biogenesis (Saschenbrecker et al, 2007;Whitney and Sharwood, 2008;Lin et al, 2014b;Hauser et al, 2015;Whitney et al, 2015).…”