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BIOENERGETICS AND PHOTOSYNTHESIS

Rubisco Oligomers Composed of Linked Small and Large Subunits Assemble in Tobacco Plastids and Have Higher Affinities for CO₂ and O₂^{1,[C],[W],[OA]}

Molecular Plant Physiology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia (S.M.W., H.J.K., R.E.S.); and Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky 40546–0312 (R.L.H.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)₈ and (S40L)₁₆ oligomers that are devoid of unlinked S subunits. While there was little or no change in CO₂/O₂ specificity or carboxylation rate of the Rubisco oligomers, their K_ms for CO₂ and O₂ were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called A^NtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in A^NtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO₂-assimilation rates in A^NtS40L at varying pCO₂ corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.

An impediment to engineering higher plant Rubisco is the enzyme's complex assembly mechanism that necessitates the coordinated expression and assembly of eight plastid-encoded large (L) and eight nucleus-encoded small (S) subunits into a form I hexadecameric (L₈S₈) enzyme (Roy and Andrews, 2000). The L subunit contains the catalytic site but the S subunits, whose precise role in the structure and function of Rubisco remains poorly understood, are essential for catalytic viability (Spreitzer, 2003). The canonical belief that there are catalytic trade offs in hybrid Rubiscos formed from heterologous L and S subunits appears valid for those comprising cyanobacterial L subunits (Read and Tabita, 1992a; Wang et al., 2001) but may not be true for those comprising heterologous higher plant Rubisco subunits (Sharwood et al., 2008). However, there is strong evidence that amino acid substitutions in the S subunits can augment changes made in the L subunit (Read and Tabita, 1992b; Spreitzer et al., 2005), indicating engineering improvements to Rubisco are likely to require complementary changes to both subunits. While genetic manipulation of the Rubisco L-subunit gene (rbcL), located in the plastome, by homologous recombination in the model plant tobacco (Nicotiana tabacum) is routine (Whitney and Sharwood, 2008), there is no appropriate means for efficiently engineering the S-subunit genes (RbcS) with comparable precision. The multiple RbcS copies in the nucleus preclude targeted mutagenic or replacement strategies and limitations on the translation and/or assembly of chloroplast-synthesized S subunits currently limit their genetic manipulation by plastome transformation in higher plants (Whitney and Andrews, 2001a; Zhang et al., 2002; Dhingra et al., 2004).

Recently we demonstrated a means by which both Rubisco subunits can be simultaneously engineered by linking them together as a single fusion peptide (Whitney and Sharwood, 2007). Using the form I Rubisco from the cyanobacterium Synechococcus PCC6301 that can functionally assemble in Escherichia coli (unlike all eukaryote form I Rubiscos; Gatenby, 1987; Cloney et al., 1993; Whitney and Sharwood, 2007), it was found that linking the S subunit to the N terminus of the L subunit with flexible 20- to 60-amino acid tethers produced SL fusions that assembled into functional Rubisco complexes of octamers [(SL)₈] and joined octamers [i.e. hexadecamers (SL)₁₆] with little catalytic impairment (Whitney and Sharwood, 2007). Here we use plastome transformation to evaluate the applicability of the SL fusion strategy to engineer Rubisco in tobacco chloroplasts by examining the stability and expression of SL fusion peptides in tobacco plastids, evaluate their capacity to fold and assemble into Rubisco complexes, and measure their catalytic prowess.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

The Transplastomic A^NtS40L Lines Produce Two Fusion-Rubisco Complexes

The plastome of the selectable marker gene (aadA)-free tobacco master line ^cmtrL1 (Whitney and Sharwood, 2008) was biolistically transformed with pA^NtS40L to direct the replacement of the dimeric Rhodospirillum rubrum Rubisco-coding gene ^cmrbcM with aadA and a synthetic rbcS40L gene coding a ^TS40L fusion peptide comprising the tobacco S and L subunits tethered together by a 40-amino acid linker (Whitney and Sharwood, 2007; Fig. 1A ). Previous use of this 40-amino acid linker with SL fusions comprising Synechococcus PCC6301 (cyanobacterial) Rubisco subunits was found to be optimal for catalytic activity and maximized the amount of Rubisco assembled in E. coli (Whitney and Sharwood, 2007). The native rbcL promoter-5'-untranslated region (UTR) was not used to regulate rbcS40L expression as the rbcL translational control region may extend into the 5' end of the rbcL coding region (Kuroda and Maliga, 2001; Maliga, 2003), necessitating inclusion of additional residues on the S-subunit N terminus. Instead the rbcS40L gene was equipped with the constitutive tobacco plastomic rrn (16S rDNA) promoter and synthetic 63-bp T7 phage gene 10 (T7g10) 5'-UTR sequence (Fig. 1A), which has been shown to enhance foreign protein expression in tobacco plastids (Maliga, 2003).

View larger version (70K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Generation and identification of transplastomic tobacco ANtS40L lines. A, Plastome sequence differences between tobacco (wt), the tobacco master line cmtrL (Whitney and Sharwood, 2008), and ANtS40L lines. The homologous sequence in plasmid pANtS40L to direct recombination into the cmtrL plastome is indicated by the dotted lines and numbering that is adjusted from the GenBank accession number Z00044 to account for the rbcL coding and 3'-UTR sequence (T, rbcL terminator and 3'-UTR region) replaced in cmtrL by the codon-modified R. rubrum Rubisco gene (cmrbcM), psbA 3'-UTR (T), and loxP sites (white triangles; GenBank no. AY827488; Whitney and Sharwood, 2008). p,16S rDNA rrn promoter/5'-UTR; t, rps16 3'-UTR; p-rbcTS40L cassette, codes a 70-kD TS40L fusion peptide comprising tobacco S and L subunits joined by a 40-amino acid linker sequence (Whitney and Sharwood, 2007). Shown are the annealing positions of primers LsA, LsB, LsE (Whitney and Andrews, 2001b), and LsH (Whitney and Sharwood, 2008), the rbcL1 and rbcL2 probes, and the size of SpeI (S) plastome fragments that hybridize with the rbcL1 probe. P, rbcL promoter and 5'-UTR. B, Nondenaturing PAGE of leaf protein from a heteroplasmic LEV1 (line 2 from Whitney and Sharwood [2008], produces L2 and tobacco L8S8 Rubisco) and two ANtS40L lines grown in selective tissue culture medium 9 weeks postbiolistic bombardment and from a soil-grown cmtrL plant. L8S8, Wild-type Rubisco hexadecamers; L2, cmtrL R. rubrum Rubisco; (TS40L)8 and (TS40L)16, octameric and hexadecameric Rubiscos produced in ANtS40L that, like L8S8 Rubisco, are recognized by the tobacco Rubisco antibody (Ab); m, marker proteins.

Growth Phenotype and CO₂ Requirement by Both A^NtS40L Lines

In soil both T₀ A^NtS40L lines were unable to grow in air without CO₂ supplementation. In air containing 0.5% (v/v) CO₂ their phenotype mimicked wild-type tobacco but grew more slowly and had paler green leaves during early exponential growth (Fig. 2A ). With further maturity the leaves became darker green and produced normal-looking fertile flowers that were backcrossed with wild-type pollen (Supplemental Fig. S1).

View larger version (51K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Comparison of Rubisco content in comparable near fully expanded leaves from wild-type tobacco (wt) and T1 ANtS40L plants of similar physiological age (approximately 18 cm high). A, Growth phenotype of plants grown in air containing 0.5% (v/v) CO2. pce, Postcotyledon emergence. B and C, Samples were taken from comparable 12-cm-diameter leaves (arrows) for DNA-blot analysis (B) and replicate blots of 5 µg total leaf RNA hybridized with the rbcL1 (Fig. 1A) or RbcS (C; Whitney and Andrews, 2003) DNA probes (transcript densitometry intensities relative to wild type are shown in brackets). D, SDS-PAGE analysis of the Rubisco TS40L (70-kD), L (52-kD), and S (14.5-kD) subunits and Rubisco activase (42 kD) contents in the soluble (sol) and insoluble (pel) protein fractions of the leaves indicated in A. m, Marker proteins; *, 54-kD non-Rubisco protein recognized by the tobacco Rubisco antibody. E, Validation of [14C]CABP binding for quantifying Rubisco active site content in ANtS40L soluble leaf protein extracts. The replicate (n = 3) soluble leaf protein samples analyzed in D were activated in 20 mM NaHCO3 and 10 mM MgCl2 for 10 min at 25°C then incubated with 5 or 15 µM [14C]CABP for 10 min and the Rubisco-[14C]CABP complexes separated from unbound [14C]CABP by chromatography immediately (black bars) or after incubation with 0.94 mM [12C]CABP a further 30 min (white bars). F, SDS-PAGE of soluble and insoluble TS40L levels in 1.8 mm2 of the second to seventh leaves of a 28-cm-high T1 ANtS40L13 plant. Rubisco content measured by [14C]CABP is shown. Densitometry measurements of insoluble TS40L relative to the total amount of TS40L (soluble + insoluble) for each leaf are shown in parentheses. [See online article for color version of this figure.]

DNA-blot analysis of the A^NtS40L T₁ progeny confirmed the plants were homoplasmic (Fig. 2B) and sequencing of the inserted DNA and surrounding plastome sequence confirmed both lines were identical. Blots of total RNA from comparable young near fully expanded leaves showed the rrn promoter-T7g10 5'-UTR maintained high steady-state rbcS40L mRNA levels in the A^NtS40L lines, exceeding rbcL mRNA levels in wild type by approximately 10% to 30% (Fig. 2C). In contrast, the corresponding level of RbcS transcripts were reduced 40% in the A^NtS40L leaves (Fig. 2C). The larger rbcS40L mRNA was not detected by the RbcS probe as it shares only 65% identity with the codon-modified S-subunit coding sequence in rbcS40L (Whitney and Sharwood, 2007).

Production of a 70-kD ^TS40L peptide in A^NtS40L leaves was readily detected by SDS-PAGE and immunoblot analysis, with no wild-type 52-kD Rubisco L subunit and only a finite amount of cytosolic synthesized Rubisco S subunits (>100-fold less than wild type) detected (Fig. 2D). In wild-type leaves no insoluble Rubisco L or S subunits were detected (data not shown), while in A^NtS40L leaves approximately 50% of the ^TS40L peptide pool was insoluble, indicating problems with the folding and/or assembly of the 70-kD peptides into oligomeric complexes in tobacco chloroplasts (Fig. 2D).

Both the (^TS40L)₈ and (^TS40L)₁₆ Rubisco complexes tightly bind the Rubisco-specific inhibitor 2-carboxyarabinitol-1,5-bisphosphate (CABP). Consistent with the levels of soluble ^TS40L detected by SDS-PAGE, [¹⁴C]CABP-binding analyses indicated the Rubisco content in the A^NtS40L leaves was approximately one-fifth that of the wild-type controls (Fig. 2E). This amount was validated by the [¹⁴C]CABP-binding/[¹²C]CABP-exchange procedure (Schloss, 1988) where less than 3% of the Rubisco-bound [¹⁴C]CABP in the A^NtS40L and wild-type leaf protein extracts were displaced when incubated with a 180-fold molar excess of [¹²C]CABP for 30 min (Fig. 2E). To compensate for the reduced Rubisco content the activation (carbamylation) status of Rubisco in the A^NtS40L leaves (72% ± 8%) was almost double that in wild type (39% ± 5%) while the content of Rubisco activase—the protein that regulates Rubisco activity by facilitating the release of bound sugar phosphate inhibitors from the active site (Portis et al., 2008)—remained unchanged (Fig. 2D).

Measurements of Rubisco content in different aged leaves from an A^NtS40L line during exponential growth (28 cm in height) showed the Rubisco levels were highest in the young fully expanded leaves (Fig. 2F), consistent with the relative variation in L₈S₈ Rubisco content in different aged tobacco leaves (Jiang and Rodermel, 1995; Rodermel, 1999). The amount of insoluble ^TS40L in the different aged leaves constituted between 30% to 50% of the total ^TS40L pool, indicating the insoluble protein can be degraded by proteolysis and its products likely reassimilated (Fig. 2F).

Stability of the (^TS40L)₈ and (^TS40L)₁₆ Rubisco Complexes

The turnover of both Rubisco complexes in A^NtS40L and the L₈S₈ Rubisco in wild-type tobacco were compared by [³⁵S]-Met pulse-chase labeling using detached leaf discs. Following separation by nondenaturing PAGE, autoradiograph analysis showed the amount of radiolabel incorporated into the (^TS40L)₈ and (^TS40L)₁₆ complexes declined slightly more rapidly than L₈S₈ (Fig. 3A ), while no change in the amount of Rubisco was evident by Coomassie staining (Supplemental Fig. S2A). Although the extent to which this higher turnover of the (^TS40L)₈ and (^TS40L)₁₆ complexes contributed to their reduced content in A^NtS40L leaves was not quantified, analysis of the soluble (assembled) and insoluble ^TS40L peptides showed their rates of turnover were similar (Fig. 3B). This is consistent with observations that the level of insoluble ^TS40L does not accumulate with leaf age (Fig. 2F).

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Rubisco turnover in ANtS40L13 and wild-type tobacco detached leaf discs. A, The discs (1 cm2) were infiltrated with [35S]Met and chased with 10 mM unlabeled Met for the times shown and the soluble protein separated by nondenaturing PAGE and loss of 35S-incoportation into wild-type L8S8 Rubisco () and the ANtS40L (TS40L)8 () or (TS40L)16 () Rubiscos measured by densitometry as described (Whitney and Sharwood, 2008). B, Relative loss of 35S-incoportated into soluble () and insoluble () TS40L in ANtS40L13 samples. Shown are the average measurements (±SD) of three separate leaf samples for each time point. See Supplemental Figure S2 for more detail.

Size exclusion chromatography of soluble leaf protein showed a single ribulose-1,5-bisphosphate (RuBP) carboxylase activity peak for wild-type L₈S₈ Rubisco and two activity peaks for A^NtS40L corresponding to the (^TS40L)₁₆ and (^TS40L)₈ complexes (Fig. 4A ). Unlike that shown by nondenaturing PAGE, the peak (^TS40L)₈ activity eluted slightly later than wild-type tobacco Rubisco, indicative of a lower molecular mass. The Rubisco content in the peak activity fractions was determined by [¹⁴C]CABP binding and showed the carboxylase turnover rates (k_c^cat) of both ^TS40L fusion Rubiscos closely matched the L₈S₈ enzyme (Fig. 4A; Table I ). Analysis of CO₂/O₂ specificity (S_c/o) in the same fractions from replicate chromatography samples showed they were comparable for the (^TS40L)₈, (^TS40L)₁₆, and L₈S₈ enzymes, while the Michaelis constants (K_m) for CO₂ (K_c) were reduced 10% to 14% for the ^TS40L complexes (Table I). Comparable values for K_c were measured using rapidly sampled leaf-soluble protein extracts, confirming the higher affinity of the ^TS40L complexes for CO₂. Likewise, the ^TS40L Rubiscos had an increased affinity for O₂ with 45% lower values of K_i for O₂ (K_o; Table I).

View larger version (61K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Kinetic and PAGE analysis of (TS40L)8, (TS40L)16, and L8S8 complexes following size exclusion chromatography. A, Soluble protein from 0.6 cm2 of an ANtS40L13 and a tobacco (wild-type) leaf (see Fig. 2A) were separated though a Superdex 200HR 10/30 column (Whitney and Sharwood, 2008) and the substrate saturated carboxylase activity (kccat) for peak fractions containing fusion-Rubisco hexadecamers [(TS40L)16; 1,120 kD], octamers [(TS40L)8; 560 kD], and wild-type L8S8 (520 kD) measured. B and C, Immunoblot analyses with an antibody to tobacco Rubisco (that strongly recognizes the S subunit) of the pooled column fractions (arrows) following nondenaturing or SDS-PAGE separation.

PAGE and immunoblot analyses with an antibody to tobacco Rubisco confirmed the RuBP-carboxylase activity peaks correlated with the elution of wild-type L₈S₈ (Fig. 4B) and the A^NtS40L (^TS40L)₁₆ and (^TS40L)₈ complexes (Fig. 4C). No S subunits were detected by SDS-PAGE and immunoblot analysis of the fractions containing the ^TS40L-Rubisco complexes (Fig. 4C), indicating the small amount of endogenous cytosolic synthesized S subunits detected in the soluble leaf protein extract (Fig. 2D) are not incorporated into Rubisco.

CO₂-Assimilation Rates in A^NtS40L Leaves Match That Predicted

The low photosynthetic CO₂-assimilation rates by young fully expanded A^NtS40L leaves (Fig. 2A) were consistent with those predicted by the model for photosynthetic gas exchange (Farquhar et al., 1980; Fig. 5 ). At an intercellular pCO₂ (C_i) of approximately 400 µbar, assimilation in the wild-type leaf at the growth illumination (350 µmol quanta m^–2 s^–1) became limited by light-dependent regeneration of RuBP while photosynthesis by A^NtS40L was still carboxylase limited at 1,600 µbar pCO₂ and had a higher CO₂ compensation point (approximately 200 µbar) relative to wild type (approximately 55 µbar). The maximum CO₂-assimilation rates by the young A^NtS40L leaves (approximately 3.2 µmol m^–2 s^–1) were 9-fold lower than wild type (approximately 27 µmol m^–2 s^–1). In contrast, little difference was observed in the maximum photochemical efficiency of PSII in the dark-adapted state (variable fluorescence [F_v]/maximum fluorescence [F_m] = 0.75) and stomatal conductance (approximately 0.16 µmol m^–2 s^–1) by A^NtS40L under growth light relative to the wild type (0.80 and 0.21 µmol m^–2 s^–1, respectively), indicating no apparent problems with photochemical efficiency or limitations to leaf gas exchange in the transformants.

View larger version (9K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Photosynthetic CO2 assimilation in ANtS40L and wild-type tobacco plants. Response of CO2-assimilation rates to intercellular pCO2 (Ci) under growth illumination (350 µmol quanta m–2 s–1). Measurements were made on a comparable leaf (12–13 cm in diameter) from a wild-type (, 24.2 µmol Rubisco sites m–2), a T1 ANtS40L13 (, 2.1 µmol Rubisco sites m–2), and a T1 ANtS40L14 (, 2.2 µmol Rubisco sites m–2) plant. The Rubisco-limited CO2 assimilation rates (A) for the ANtS40L lines (solid line) were modeled according to the equation: (Farquhar et al., 1980) using the kinetic measurements listed in Table I, a Rubisco content (B) of 2.1 µmol Rubisco sites m–2, a measured nonphotorespiratory respiration rate (Rd) of 0.8 µmol m–2 s–1, and assuming a chloroplast O2 concentration (o) of 252 µM, the solubility of CO2 in water (pc) is 0.0334 M bar–1, and that the chloroplast pCO2 (Cc) and Ci are equivalent.

N-terminal Edman micosequencing of the soluble ^TS40L peptide yielded the sequence X-K-V-W-P as expected from other studies (Grimm et al., 1997). The first residue, X, chromatographed as a large broad peak just after the Glu phenylthiohydantoin derivative, consistent with it being N-methyl-Met as seen with the spinach (Spinacia oleracea) S-subunit N terminus (Ying et al., 1999). Analysis by mass spectrometry following Asp-N digestion identified a peptide at a mass-to-charge ratio (m/z) of 2,349, consistent with the N-terminal region of ^TS40L with a mass shift of 14 m/z (Supplemental Fig. S3A). Subsequent collision-induced dissociation and postsource decay fragmentation patterns localized the 14 m/z mass shift to the N terminus of the 2,349 parent ion (Supplemental Fig. S3B) consistent with N-methylation of Met-1.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Rubisco Comprising SL Fusions Can Assemble in Tobacco Chloroplasts with Minimal Catalytic Change

Here we demonstrate the feasibility of generating autotrophic tobacco lines producing Rubisco oligomers that only comprise approximately 70-kD ^TS40L peptides that can assemble into both catalytically functional (^TS40L)₈ and (^TS40L)₁₆ complexes within tobacco plastids (Figs. 1B and 4). Covalently linking the S to the L subunits prevented inclusion of endogenous cytosolic S subunits in the Rubisco complexes and circumvents previous limitations stemming from their apparent preferential incorporation over plastid-synthesized S subunits into L₈S₈ complexes (Whitney and Andrews, 2001a; Zhang et al., 2002), which has frustrated S-subunit mutagenesis studies. Analogous to that shown previously for cyanobacterial Rubisco SL fusions expressed in E. coli the catalytic properties of both tobacco ^TS40L complexes showed beneficial improvements in CO₂ affinity (K_C is reduced 10%–20% compared with an 11% reduction for the cyanobacterial ^CS40L complexes; Whitney and Sharwood, 2007; Table I). This improvement occurs without compromise to CO₂ specificity or saturated RuBP carboxylation rate but, for the tobacco SL fusions at least, an increase in the enzymes affinity for O₂ was also observed, which reduced carboxylation efficiency under ambient O₂ levels (k_c^cat/K_C^21%O2 = 94 mM s^–1) 17% relative to wild type (113 mM s^–1). Nevertheless, the close conservation of kinetic prowess by both the ^TS40L and ^CS40L complexes, and their ability to tightly bind CABP (Fig. 2E), suggests the structural conformation of the assembled L and S peptide regions closely complement that in the L₈S₈ enzyme. This provides confidence that S40L fusions should provide a novel engineering strategy for simultaneous mutagenesis of L- and S-subunit residues in tobacco (and possibly foreign) Rubisco to explore intersubunit residue interactions, their influence on catalytic activity, and whether leaf gas-exchange analysis (Fig. 5) continues to emulate the predicted changes in photosynthetic CO₂-assimilation capacity according to the models of Farquhar et al. (1980).

Problems with the Synthesis, Folding, and Assembly of ^TS40L Limit Rubisco Production in Chloroplasts

The Rubisco content in young near fully expanded leaves of the A^NtS40L lines was reduced at least 5-fold relative to wild-type controls, which limited photosynthesis and growth rates and necessitated CO₂ supplementation during juvenile plant development. As observed previously in tobacco-producing foreign (Whitney and Andrews, 2001b) or hybrid (Sharwood et al., 2008) Rubiscos, the Rubisco activase content in A^NtS40L leaves remained unchanged. The higher carbamylation status of Rubisco in the A^NtS40L leaves suggests the activity of the S40L fusion complexes can be adequately regulated by Rubisco activase, although the efficiency of this regulation remains to be examined.

The highly transcribed rrn promoter produced steady-state rbcS40L mRNA levels slightly higher than rbcL levels in wild-type controls (Fig. 2C), indicating that ^TS40L production was primarily limited posttranscriptionally. The paucity of assembled ^TS40L Rubisco can partially be attributed to 30% to 50% of the leaf ^TS40L pool accumulating as insoluble aggregates (Fig. 2, D and F). This indicates the ^TS40L folding and assembly requirements are not fully met by the plastid molecular chaperone network and correlates with comparable impediments in the folding assembly of ^CS40L peptides in E. coli (Whitney and Sharwood, 2007). Identifying the lesion point(s) impeding ^TS40L folding and assembly in plastids is difficult given our rudimentary understanding of Rubisco assembly in plastids (Roy and Andrews, 2000). Possibly the capacity of the stromal Cpn60 chaperonins and their Cpn21 cofactors (known to be involved in Rubisco assembly) to properly fold the large 70-kD ^TS40L peptides may be impeded, resulting in their rapid proteolysis and/or accumulation as misfolded insoluble aggregates (whose proteolytic turnover is comparable to that of properly assembled ^TS40L; Fig. 3). Notably, space limitations within chaperonin GroEL-GroES complexes in E. coli make it difficult for them to encapsulate and fold peptides greater than 60 kD in size (Kerner et al., 2005). It is also possible incompatibilities with other chaperones, such as the Rubisco L-subunit-specific BSDII (Brutnell et al., 1999; Wostrikoff and Stern, 2007), may also encumber prechaperonin processing of ^TS40L also resulting in their misfolding and/or inadequate protection from stromal proteases. A strategy for identifying proteins involved in Rubisco assembly may be to introduce affinity tags within the ^TS40L linker sequence (or just the L subunit itself [Rumeau et al., 2004]) and rapidly isolate unassembled Rubisco subunit-chaperone complexes.

Translational processing of the rbcS40L transcript may also account for the reduced Rubisco levels in A^NtS40L leaves. In most plastome transformation studies the coupling of heterologous UTRs and transgene sequences perturbs mRNA folding, slows translational processing, and frequently necessitates the trialing of different UTR combinations to optimize expression (Maliga, 2003). Whether unfavorable folding of the rbcS40L mRNA impedes engagement and translation by ribosomes remains to be examined by analyzing the transcript association with polysomes (Barkan, 1998). Importantly, irrespective of the possible causes for limiting ^TS40L production, once assembled into (^TS40L)₈ or (^TS40L)₁₆ complexes the peptides are relatively stable (Fig. 3) and produced in quantities suitable for future mutagenic analyses.

Can a Plastid-Signaling Event Regulate RbcS mRNA Levels?

Biogenesis of L₈S₈ Rubisco in higher plants is complicated by the disparate location of the RbcS and rbcL genes in different genomes and their translation in different cellular locations (the cytosol and stroma, respectively). Light, hormone, and particularly carbohydrate levels have been shown to play determinant roles in leaf developmental programming by a complex signal transduction pathway whose nucleus-plastid communication circuitry remains poorly defined, particularly with regard to Rubisco production (Rodermel, 1999). However, a recent advance showed expression of the L subunit in tobacco is tightly coordinated via a classical control by epistasy of synthesis paradigm wherein a peptide motif from unassembled L subunits appears to bind to its mRNA to autoregulate its translation (Wostrikoff and Stern, 2007). Whether translation of the rbcS40L mRNA is influenced by this process is uncertain; however, according to the sink (source strength) regulation of photosynthesis hypothesis (Koch, 1996) the 40% reduction in leaf RbcS mRNA levels in A^NtS40L is contradictory to the near absence of starch and Glc in its leaves (data not shown). Indeed even in comparable carbohydrate-limited tobacco leaves producing R. rubrum Rubisco that does not require S subunits, the steady-state RbcS mRNA pool was elevated >2-fold (Whitney and Andrews, 2003) as expected in response to source-strength repression. Clearly further study is required to fully examine how changes in leaf ontogeny and source strength impact on the developmental programming in the A^NtS40L lines. In particular quantifying differences in RbcS transcription rate and/or stability is required to assess whether there exists a plastid-signaling event that can regulate RbcS mRNA levels to determine whether such a regulatory event correlates with the deletion of the rbcL promoter-5'-UTR sequence from the A^NtS40L plastome.

The Unique Posttranslational Methylation of a Plastid-Synthesized Protein

Our finding that Met-1 of ^TS40L and the native S subunits share the same posttranslational monomethylation indicates that the import and proteolytic processing of the S-subunit plastid-targeting presequence is not a prerequisite for this modification process. This covalent modification may serve to protect ^TS40L from stromal peptidases, a role proposed for the S subunit as well as the conserved acetylation of the L-subunit N-terminal Pro-3 following N-formyl-Met-1 and Ser-2 removal (Houtz et al., 2008). While the necessity of N-terminal modifications for protein stability in plastids is enigmatic, some transplastomic studies have shown that some transgene products are unmodified at Met-1 (Staub et al., 2000; Whitney et al., 2001). Our previous N-terminal analysis of plastid-synthesized precursor S subunits also showed that while the native plastid-targeting sequence was processed normally, the Met-1 of the resulting mature (unmodified) S subunit was not methylated (Whitney and Andrews, 2001a). This suggests multiple N-methyltransferases with alternative sequence specificity may exist in plastids that may be defined by the deviation in charge and size of codon two of the N-terminal sequences (for example ^TS40L codes Lys-2 while the plastid-synthesized S subunits coded for Ala-2 or Gln-2; Whitney and Andrews, 2001a). Notably six other tobacco chloroplast-translated proteins contain M-K at their N terminus (Supplemental Table S1), however, their N-terminal sequences remain undetermined. Clearly more work is needed to examine the necessity and mechanisms of posttranslational processing in plastids to decipher the extent to which the N-end rules that regulate cytosolic protein degradation pathways in eukaryotes (Meinnel et al., 2006) might also apply to organelle proteins.

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Tobacco SL fusion peptides assemble in tobacco chloroplasts into functional Rubisco complexes with minimal catalytic demise. These observations provide a useful engineering approach for examining intersubunit structure-function relationships in both tobacco Rubisco L and S subunits and other foreign analogs. Application of the fusion strategy with the kinetically more efficient Rubiscos from red algae may prove particularly useful if adjoining the cognate subunits circumvents the chaperone incompatibility problems that appear to currently limit their assembly in tobacco plastids (Whitney et al., 2001).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Transforming Plasmid Construction and Sequencing

Plasmid pA^NtS40L was constructed in pGEM T-Easy (Promega). Plastome-flanking sequence comprising 916-bp of upstream sequence and 229 bp of the psbA 3'-UTR (Fig. 1A) was used to direct homologous replacement of the rbcL-promoter-5'-UTR-^cmrbcM sequence in the tobacco (Nicotiana tabacum) master line ^cmtrL1 (Whitney and Sharwood, 2008) with the 1,220-bp loxP-Prrn-aadA-Trps16-loxP HindIII cassette from p^cmtrLA (GenBank accession no. AY827488) and a 2,079-bp Prrn-rbcS40L cassette. The latter comprised the 518-bp Prrn-^cmrbcS sequence from pP^cmSTVE linked in framed with the 1,561-bp EcoRV-XbaI fragment from pET^TS40L that coded for a 40-amino acid tether-tobacco L-subunit fusion (Whitney and Sharwood, 2007). The plasmid and all PCR products were sequenced using BigDye terminator sequencing on an ABI 3730 sequencer (Biomolecular Resource Facility, Australian National University).

Transformation and Plant Growth

pA^NtS40L was purified from Escherichia coli XL1-Blue cells (10-mL culture) using the Wizard mini-prep kit (Promega) and biolistically transformed as described (Svab and Maliga, 1993) into five sterile leaves from the T₂ progeny of ^cmtrL1 (Whitney and Sharwood, 2008). Spectinomycin-resistant plants were selected on selective tissue culture medium (agar-solidified Murashige and Skoog salts containing 3% w/v Suc, hormones, and 0.5 mg mL^–1 spectinomycin [Svab and Maliga, 1993]) and two lines (13 and 14) passed through a second round of regeneration on selective media following confirmation they were plastome transformants by nondenaturing PAGE (Whitney and Sharwood, 2008). Both lines were grown to maturity in soil in a controlled environment cabinet as described (Sharwood et al., 2008), in air supplemented with 0.5% (v/v) CO₂ and 350 µmol quanta m^–2 s^–1 illumination. The flowers were backcrossed with wild-type tobacco (cv Petit Havana [N,N]) pollen and the T₁ progeny used for all subsequent analyses.

DNA, RNA, SDS-PAGE, and ³⁵S-Met Pulse-Chase Analyses

All analyses were made on samples from the fifth leaf (13 cm in width) of wild-type and A^NtS40L plants (15–18 cm in height). Total leaf DNA and RNA was extracted, agarose gel separated and blotted onto Hybond N+ membranes, and hybridized with alkaline phosphatase or [-³²P]dATP-labeled DNA probes as described (Whitney and Sharwood, 2008). The total (lysate) and soluble leaf protein was extracted, separated by SDS-PAGE, and immunoblotted with polyclonal antisera to spinach (Spinacia oleracea) Rubisco activase (Whitney and Andrews, 2001b) and tobacco Rubisco (that strongly recognizes the S subunit and poorly the L subunit) as described (Whitney and Sharwood, 2008). Proteins in 20 leaf discs (1 cm²) were pulse-chase labeled with [³⁵S]-Met and the relative incorporation of label into the Rubisco subunits and holoenzyme complexes evaluated following SDS- and nondenaturing-PAGE as described (Whitney and Andrews, 2001a; Sharwood et al., 2008).

Rubisco Purification, Content, Carbamylation, and Kinetic Analysis

Measurement of Rubisco content and carbamylation status by [¹⁴C]CABP/[¹²C]CABP exchange, the substrate saturated turnover rate (k_c^cat), and the apparent K_m for CO₂ (K_c) under varying pO₂ were measured using rapidly extracted soluble leaf protein as described (Sharwood et al., 2008). Measurements of CO₂/O₂ specificity were determined using either Rubisco purified by Q-sepharose anion exchange (Sharwood et al., 2008) or following Superdex 200HR 10/30 column chromatography (Whitney and Sharwood, 2008).

Leaf Gas Exchange

Whole-leaf gas-exchange measurements were made in the growth chamber using a LI-6400 gas-exchange system (LI-COR) as described (Sharwood et al., 2008).

N-Terminal Sequence Analysis

The ^TS40L peptide bands in the anion-exchange-purified Rubisco sample were separated by SDS-PAGE and either blotted onto polyvinyl difluoride membrane for Edman sequencing on an Applied Biosystems 494 Procise Protein Sequencing system at the Australian Proteome Analysis Facility, or excised directly from the gel for Asp-N-digestion and analyzed by mass spectrometry at the University of Kentucky, Center for Structural Biology Protein Core Facility (Supplemental Fig. S3).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Comparative phenotype of a mature flowering T₀ A^NtS40L¹³ and wild-type tobacco plants at approximately 70 cm in height.

Supplemental Figure S2. Relative turnover of ³⁵S-labeled Rubisco in leaf discs from an A^NtS40L¹³ and a wild-type tobacco leaf.

Supplemental Figure S3. Mass spectrometry analysis of Asp-N peptides derived from ^TS40L.

Supplemental Table S1. Tobacco plastome sequences coding putative peptides with Met-Lys N termini.

	ACKNOWLEDGMENTS

 
This research was facilitated by access to the Australian Analysis Facility supported under the Australian Government's National Collaborative Research Infrastructure Strategy.

Received January 1, 2009; accepted February 15, 2009; published February 20, 2009.

	FOOTNOTES

 
¹ This work was supported by a Discovery grant (no. DP0450564) awarded to S.M.W. by the Australian Research Council. Research by R.L.H. was supported by the Department of Energy (grant no. DE–FG02–92ER20075). The University of Kentucky Center for Structural Biology Protein Core Facility is supported in part by funds from the National Institutes of Health National Center for Research Resources (grant no. P20 RR020171).

² Present address: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14850.

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Spencer Michael Whitney (spencer.whitney{at}anu.edu.au).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

^[OA] Open access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.135210

^* Corresponding author; e-mail spencer.whitney{at}anu.edu.au.

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