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Comparative Study
. 2007 Apr 10;104(15):6466-71.
doi: 10.1073/pnas.0610586104. Epub 2007 Apr 2.

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

Katia Wostrikoff  1 , David Stern
Affiliations
Comparative Study

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

Katia Wostrikoff et al. Proc Natl Acad Sci U S A. .

Abstract

Plants rely on ribulose bisphosphate carboxylase/oxygenase (Rubisco) for carbon fixation. Higher plant Rubisco possesses an L(8)S(8) 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 assembly-dependent translational regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of RBCS RNAi transformants. (A) Silencing construct targeting RBCS genes. An RBCS inverted repeat, separated by the chalcone synthase intron (CHSA), is under the control of the CaMV 35S promoter and octopine synthase (ocs) 3′ UTR. (B) RNA and protein analysis from a representative RBCS RNAi transformant (siSS). Total leaf RNA (5 μg or the indicated dilution of WT) was hybridized by using probes directed against rbcL or RBCS transcripts. The ethidium bromide stain is provided as a loading control. Total leaf proteins (30 μg or the indicated dilution of WT) were separated as described in Methods, and analyzed by using a Rubisco antibody. Cytf is provided as a loading control. The controls were a vector transformant for RNA analysis and the WT for protein analysis. (C) For polysome analysis, total leaf extract from siSS or WT were fractionated on sucrose gradients. An equal proportion of RNA from each fraction was analyzed by gel blot.
Fig. 2.
Fig. 2.
Creation of an LS/SS double mutant. (A) The relevant region of the chloroplast transformation vector is shown beneath the chloroplast rbcL–accD genomic region. The black arrowhead indicates the 4-bp insertion discussed in the text. In the gray box below the diagram, the mutant and WT LS sequences are compared, with altered amino acids italicized. (B) The four strains were photographed under similar lighting and magnification. (C) RT-PCR (Upper) and protein (Lower) analyses were performed, with RBCS transcript accumulation assessed by RT-PCR with actin as a control and immunoblotting with antisera shown at the right.
Fig. 3.
Fig. 3.
Transcript accumulation and polysome loading in transformed lines. (A) RNA gel blot for the lines shown across the top hybridized with rbcL, aadA, and psaA (top to bottom). rbcL-aadA is a dicistronic transcript present in LS* and aadA; that part of the gel has been cropped for the aadA probe. rRNA stained by ethidium bromide is shown as a loading control. (B) Polysomes were prepared and analyzed as described in the Fig. 1 legend for the lines shown at left. The discistronic rbcL–aadA mRNA is indicated by a diamond shape.
Fig. 4.
Fig. 4.
VIGS analysis of LS regulation. (A) Leaf phenotypes observed 3 weeks after inoculation for the lines shown. (B) RT-PCR was performed as described in Methods, with dilutions of cDNA from the control TRV2 line used to estimate the degree of silencing for the BSD2 and RBCS transcripts. Actin was used as a loading control. (C) Immunoblot analysis for the lines shown across the top and antibodies shown at right. Dilutions of protein from the control TRV2 were used to help estimate residual LS accumulation. Cytf was a loading control.
Fig. 5.
Fig. 5.
Polysome association of the rbcL transcript in VIGS-silenced lines. Polysomes were prepared as described in the Fig. 1 legend for the lines shown at left. RNA gel blots were probed simultaneously for rbcL and psaA.
Fig. 6.
Fig. 6.
Pulse labeling of chloroplast proteins. (A) Equal amounts of radiolabeled translation products for the transplastomic lines shown across the top were analyzed as described in Methods. LS and LS* indicate full-length and truncated LS, respectively, and a protein presumed to be D1 is indicated. (B) Labeled proteins from VIGS lines were analyzed in the same way. Two dilutions of control proteins were used to estimate residual translation levels in experimental lines.
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