Rubisco large-subunit translation is autoregulated in response to its assembly state in tobacco chloroplasts

Abstract

Plants rely on ribulose bisphosphate carboxylase/oxygenase (Rubisco) for carbon fixation. Higher plant Rubisco possesses an L8S8 structure, with the large subunit (LS) encoded in the chloroplast by rbcL and the small subunit encoded by the nuclear RBCS gene family. Because its components accumulate stoichiometrically but are encoded in two genetic compartments, rbcL and RBCS expression must be tightly coordinated. Although this coordination has been observed, the underlying mechanisms have not been defined. Here, we use tobacco to understand how LS translation is related to its assembly status. To do so, two transgenic lines deficient in LS biogenesis were created: a chloroplast transformant expressing a truncated and unstable LS polypeptide, and a line where a homolog of the maize Rubisco-specific chaperone, BSD2, was repressed by RNAi. We found that in both lines, LS translation is no longer regulated by the availability of small subunit (SS), indicating that LS translation is not activated by the presence of its assembly partner but, rather, undergoes an autoregulation of translation. Pulse labeling experiments indicate that LS is synthesized but not accumulated in the transgenic lines, suggesting that accumulation of a repressor motif is required for LS assemblydependent translational regulation.autoregulation ͉ gene expression ͉ synthesis ͉ plant M acromolecular organellar energetic complexes are essential and require assembly in defined stoichiometric ratios. Their biogenesis is complicated by their dual genetic origins, with subunits encoded both in the organelle and the nucleus. Prior studies led to the notion of concerted accumulation: the absence of one core subunit is accompanied by the loss of its assembly partners, as exemplified by mitochondrial mutants in yeast (1) or chloroplast mutants in Chlamydomonas (2). Two main mechanisms responsible for unassembled subunits' fate have been identified. The most common is rapid proteolytic degradation, and, in certain cases, substrates for specific chloroplast proteases have been identified (3-7). Alternatively, some chloroplastencoded proteins are subject to assembly-dependent translational regulation or control by epistasy of synthesis (CES) (8). The chloroplast uses CES in a hierarchical manner, whereas proteolysis is a relatively nonspecific mechanism for disposing of excess or incorrectly folded subunits.Insights into CES were first described for Chlamydomonas cytf, which is encoded by petA. Unassembled cytf could be shown to repress petA translation initiation, acting through the 5Ј untranslated region (9) and the cytf C-terminal domain (10). Analogous regulatory features were subsequently uncovered for the PS I subunits PsaA and PsaC (11) and the PS II subunits D1 and CP47 (12). Together, these results suggest that assemblydependent CES is a general feature of chloroplast gene expression in Chlamydomonas. However, its prevalence in higher plants is unclear.Here, we have used Rubisco assembly as a model to investigate whether CES occurs in higher plant...

Functional Incorporation of Sorghum Small Subunit Increases the Catalytic Turnover Rate of Rubisco in Transgenic Rice

“…In particular, the Rubisco content in SS5, a high expressing line, was 24% higher than that in nontransgenic rice. Rubisco content is not always regulated by the transcript abundance of RbcS when part of RbcL was truncated or Rubisco-specific chaperone was knocked down in tobacco (Wostrikoff and Stern, 2007). In this report, RNAi knockdown of RbcS did not influence the expression of RbcL.…”

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

“…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).…”

“…One possibility is that BSD2 is involved in repressing LS translation in M chloroplasts through a phenomenon known as control by epistasy of synthesis (Choquet and Wollman, 2009). In the case of Rubisco, unassembled LS undergoes translational repression through direct or indirect interaction with the rbcL transcript (Wostrikoff and Stern, 2007). Thus, a potential role of BSD2 in M chloroplasts could be to promote the folding of enough LS to exert a negative autoregulatory effect.…”

Setaria viridis: A Model for C4 Photosynthesis

“…These screens have led to the identification of a few genes that affect the accumulation of C 4 shuttle enzymes and Rubisco. Examples include Golden 2 (Hall et al, 1998a;Fitter et al, 2002;Waters et al, 2009) and Bundle Sheath Defective 2 (Roth et al, 1996;Brutnell et al, 1999;Wostrikoff and Stern, 2007). These mutants display BS cell-specific defects, but this is because the process or protein that is regulated by the gene products is localized to that cell type; therefore, these genes should not be regarded as genes regulating C 4 differentiation.…”

“…For instance, BSD2 is required for the assembly of Rubisco holoenzyme (Brutnell et al, 1999) and displays a BS-specific defect because Rubisco is localized to the BS chloroplast. However, the Bsd2 gene is expressed in both BS and M cells and plays an essential role in regulating Rubisco accumulation in both C 3 and C 4 plants (Roth et al, 1996;Brutnell et al, 1999;Wostrikoff and Stern, 2007). This localization of a general C 3 function to either BS or M cells is likely to be prevalent in C 4 grasses, as over 18% of the maize transcriptome is differentially expressed between these two cell types (Sawers et al, 2007), including a sizable fraction of the plastid proteome (Majeran et al, 2005(Majeran et al, , 2008Friso et al, 2010).…”