Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize

Abstract

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process. INTRODUCTIONRibulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) is the major enzyme by which green plants, algae, cyanobacteria, and other autotrophic organisms sequester CO 2 into organic compounds via the Calvin-Benson pathway (Andersson and Backlund, 2008). Rubisco catalyzes the photosynthetic carbon reduction and the photorespiratory carbon oxidation reactions of the substrate ribulose-1,5-bisphosphate with CO 2 and O 2 , respectively. The inefficiency of Rubisco in fixing CO 2 has a limiting impact on agricultural productivity and in compensation, Rubisco accounts for as much as 20 to 30% and 4 to 9% of total nitrogen compounds in C 3 and C 4 higher plants, respectively (Feller et al., 2008).Attempts to improve the catalytic properties of plant Rubisco (reviewed in Parry et al., 2003;Mueller-Cajar and Whitney, 2008) have met with only modest success, which can be traced in part to the lack of a comprehensive knowledge of its biogenesis and the absence of an in vitro reconstitution system. Form I Rubisco, found in higher plants, algae, and cyanobacteria, is a hexadecamer composed of eight large (50-kD) and eight small (13-15 kD) subunits, denoted here as LS and SS, respectively. The genes encoding LS (rbcL) and SS (RBCS) are located in the chloroplast and nuclear genomes, respectively. SS is expressed as a preprotein that is translocated into the chloroplast, where its signal peptide is cleaved prior to its assembly with LS (Nishimura et al., 2008). The two subunits accumulate stoichiometrically in the chloroplast, a phenomenon that is mediated by feedback inhibi...

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells

“…However, the results showed that rbcL transcripts were not increased relative to the wild type in M cell RNA prepared from RBCS-BSD2, signifying that the increased rbcL transcript phenotype of bsd2 is likely a pleiotropic effect of the lack of Rubisco and not the effect of missing BSD2 in the M. The same phenomenon is seen in the maize raf1 mutant, which is blocked at a similar step in Rubisco assembly to bsd2 (Feiz et al, 2012). The function of BSD2 in the M remains unclear.…”

“…Based on available evidence, BSD2 was proposed to interact with the large subunit (LS) of Rubisco to facilitate its translation and/or folding (Brutnell et al, 1999). Subsequent work in maize showed that BSD2 likely interacts with nascent LS and/or newly imported small subunit as well as other assembly factors (RAF1 and RAF2) to assemble the Rubisco holoenzyme (Feiz et al, 2012(Feiz et al, , 2014. More limited data are consistent with analogous roles in Nicotiana benthamiana (Wostrikoff and Stern, 2007) and Chlamydomonas reinhardtii (Doron et al, 2014).…”

Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis

“…Although the OR proteins carry a DnaJ cysteine-rich zinc figure domain, they are not molecular chaperones due to the lack of the DnaJ-like defining J domain. A member of the specific OR group proteins is BUNDLE SHEATH DE-FECTIVE2 (BSD2), which affects Rubisco accumulation and is believed to function via direct interaction with the polypeptide substrates for Rubisco assembly (35,36). Accordingly, the physical association of OR with PSY might promote the proper folding of PSY to enhance its stability and activity.…”

The BSD2 ortholog in Chlamydomonas reinhardtii is a polysome‐associated chaperone that co‐migrates on sucrose gradients with the rbcL transcript encoding the Rubisco large subunit

“…For example, the cyanobacterial RbcX gene, which is co-transcribed with the rbcL and rbcS genes in the rbcLXS operon (Saschenbrecker et al, 2007), stabilizes the RbcL as a dimer and facilitates the core assembly of an octagonal structure (Liu et al, 2010). Similarly, in the photosynthetic eukaryote Zea mays, Raf1 is an important specific factor that is required for post-translational holoenzyme assembly (Feiz et al, 2012). Another potential factor that is required for Rubisco expression is the bundle sheath defective-2 (BSD2) protein, which was initially identified in maize (Brutnell et al, 1999).…”