A Conserved Sequence from Heat-Adapted Species Improves Rubisco Activase Thermostability in Wheat

Abstract

The central enzyme of photosynthesis, Rubisco, is regulated by Rubisco activase (Rca). Photosynthesis is impaired during heat stress, and this limitation is often attributed to the heat-labile nature of Rca. We characterized gene expression and protein thermostability for the three Rca isoforms present in wheat (Triticum aestivum), namely TaRca1-β, TaRca2-α, and TaRca2-β. Furthermore, we compared wheat Rca with one of the two Rca isoforms from rice (Oryza sativa; OsRca-β) and Rca from other species adapted to warm environments. The TaRca1 gene was induced, whereas TaRca2 was suppressed by heat stress. The TaRca2 isoforms were sensitive to heat degradation, with thermal midpoints of 35°C ± 0.3°C, the temperature at which Rubisco activation velocity by Rca was halved. By contrast, TaRca1-β was more thermotolerant, with a thermal midpoint of 42°C, matching that of rice OsRca-β. Mutations of the TaRca2-β isoform based on sequence alignment of the thermostable TaRca1-β from wheat, OsRca-β from rice, and a consensus sequence representing Rca from warm-adapted species enabled the identification of 11 amino acid substitutions that improved its thermostability by greater than 7°C without a reduction in catalytic velocity at a standard 25°C. Protein structure modeling and mutational analysis suggested that the thermostability of these mutational variants arises from monomeric and not oligomeric thermal stabilization. These results provide a mechanism for improving the heat stress tolerance of photosynthesis in wheat and potentially other species, which is a desirable outcome considering the likelihood that crops will face more frequent heat stress conditions over the coming decades.

Figure 1.

A comparison of gene expression of Rca isoforms extracted from wheat leaves either under control conditions (22°C day/night) or after heat treatment. A, The spring wheat ‘Fielder’ harvested during the vegetative stage with heat treatment being 38°C/22°C for two diurnal cycles. B, The winter wheat ‘Mentor’ harvested during the flowering stage with heat treatment being 36°C/22°C for seven diurnal cycles. Gene expression analysis via reverse transcription quantitative PCR (qPCR) for cv Fielder used primers specific to the wheat TaRca1-β gene and TaRca2-α and TaRca2-β spliced variants, whereas for cv Mentor, primers were nonspecific for the TaRca2 spliced variants. Expression values are means ± sd of three or more biological replicates.

Figure 2.

Analysis of Rubisco interactions with Rca extracted from wheat leaves either under control conditions or after heat treatment. A, Rubisco activation velocity by Rca at 25°C. The production of 3-phosphoglycerate (3PG) by Rubisco primed with CO₂ and Mg²⁺ (ECM), Rubisco inhibited by its substrate RuBP (ER), ER Rubisco in the presence of Rca, and Rca extracts with no Rubisco present enabled the determination of V₂₅. B, Temperature-dependent Rubisco activation by Rca. Rca was incubated for 10 min in the presence of 0.2 mm ATP at the indicated temperatures prior to assaying at a standard 25°C and normalized to the fastest velocity achieved for each sample. C, DSF for Rca. Samples were heated at 1°C min⁻¹, and fluorescence units (FU) normalized to the maximum value were recorded. Values are means ± sd of three biological replicates. Curves are the ordinary least-squares fit of a Boltzmann sigmoidal equation (Eq. 1). V₂₅ and T_m are presented in Table 1.

Figure 3.

Analysis of Rubisco interactions with variants of Rca. Included in the analyses were recombinant TaRca2-α, TaRca2-β, TaRca1-β, and OsRca-β. A, Rubisco activation velocity by Rca at 25°C. Experiments consisted of 1.6 ± 0.2 μm Rca protomer added to 0.2 ± 0.05 μm Rubisco active sites inhibited by RuBP (ER). B, Temperature-dependent Rubisco activation by Rca. Rca was incubated for 10 min in the presence of 0.2 mm ATP at the indicated temperatures prior to assaying at a standard 25°C, and values are normalized to the fastest velocity achieved. C, DSF. Samples were heated at 1°C min⁻¹, and fluorescence units (FU) normalized to the maximum value were recorded. Values are means ± sd of four experimental replicates. Curves are the ordinary least-squares fit of a Boltzmann sigmoidal equation (Eq. 1). The calculated initial V₂₅ and T_m are presented in Table 1.

Figure 4.

Rca sequence alignment. Included in the alignment are TaRca2-β, TaRca1-β, OsRca-β, the consensus sequences of warm- and cold-adapted species, and a mutated sequence of TaRca2-β with 11 amino acid substitutions (TaRca2-β-11AA), eight amino acid substitutions (TaRca2-β-8AA), and three amino acid substitutions (TaRca2-β-3AA). The consensus sequences were generated from alignments of eight and nine species endemic to warm and cold environments, respectively. The mutations made to TaRca2-β-11AA, with positions indicated by blue circles, triangles, and squares, were selected based on the TaRca1-β sequence with the criteria that TaRca1-β matched the warm-adapted species’ consensus and was different from the cold-adapted species’ consensus. The mutations made to TaRca2-β-8AA, with positions indicated by blue triangles and squares, were selected based on TaRca2-β-11AA but with the additional criterion that if any of the OsRca-β residues at these 11 amino acid mutation sites matched the corresponding TaRca2-β or the cold-adapted species’ consensus sequence, then the original TaRca2-β residue was reinstated. The mutations made to TaRca2-β-3AA, with positions indicated by blue squares, were selected based on structural modeling that showed the final three substitutions to be in close proximity to adjacent monomers. The red asterisks indicate the salt bridge formed between adjacent monomers as predicted by protein modeling of TaRca2-β-3AA. Residue differences among the sequences are highlighted in red. The chloroplast signal peptide, which was not included in the analysis, is underlined in green.

Figure 5.

Analysis of Rubisco interactions with recombinant TaRca2-β and the TaRca2-β-11AA, TaRca2-β-8AA, and TaRca2-β-3AA mutants. A, Rubisco activation velocity by Rca at 25°C. B, Temperature-dependent Rubisco activation by Rca. C, DSF. Values are means ± sd of four or more experimental replicates. Curves are the ordinary least-squares fit of a Boltzmann sigmoidal equation (Eq. 1). V₂₅ and T_m are presented in Table 1.

Figure 6.

Temperature-dependent Rubisco activation by variants of Rca without ATP nucleotide. Rca variants assayed were recombinant TaRca2-β, TaRca1-β, OsRca-β, and TaRca2-β-11AA, TaRca2-β-8AA, and TaRca2-β-3AA mutants. Rca was incubated for 10 min at the indicated temperatures in the absence of ATP prior to assaying at a standard 25°C. Values are normalized to the fastest velocity achieved. Values are means ± sd of four experimental replicates. Curves are the ordinary least-squares fit of a Boltzmann sigmoidal equation (Eq. 1). T_m are presented in Table 1.

Figure 7.

Structural analysis of Rca variants. A, Structural model of the wheat Rca monomer indicating the position of the Q409E substitution and a conserved Arg-226 residue in pore loop 2. Residues colored green indicate the nucleotide-binding region. B to D, Bottom views through the hexamer central pore of TaRca2-β wild type (WT; B), TaRca1-β (C), and TaRca2-β-3AA variants (D). B, Modeling showing that Gln-409 at the C-terminal region forms a hydrogen bond with Arg-226 in pore loop 2 of an adjacent monomer in two instances in the wild-type complex. C and D, Glu-409 forms a salt bridge with Arg-226 of an adjacent monomer in four instances in TaRca1-β and the TaRca2-β-3AA variant. Cartoon illustrations of the hydrogen bonding (gray) and the salt bridge (black) between monomers of the hexameric complex are given in the top left corners. Interacting residues forming the hydrogen or salt bridge are circled in red. The Q409E and Arg-226 positions are in relation to sequence alignments as presented in Figure 4.

Figure 8.

Enzyme interaction studies of TaRca2-β and TaRca1-β variants with the inactive TaRca2-β K157Q mutant. A, The production of 3PG by reactivated Rubisco in the presence of TaRca2-β (TaRca2-β + ER) and the inactive mutant (K157Q + ER), demonstrating that the TaRca2-β K157Q mutant is essentially inactive. Values are means ± sd of four experimental replicates. B, Gel-filtration chromatogram showing homooligomeric and heterooligomeric complexes of TaRca2-β, TaRca2-β K157Q, and when the two are mixed at a concentration of 44 μm. The chromatogram is normalized to 100 at the maximum peak height. C, Observed Rca activation velocity normalized to wild-type velocity when titrating different fractions of an inactive TaRca2-β K157Q mutant with the wild-type complex (circles) or the TaRca2-β-3AA variant (squares), measured within 1 min after mixing. Values are means ± sd of three experimental replicates. The red solid curve represents a binomial distribution model, V = (1 − X)⁽⁶⁺⁶⁾*X*(1 − X)^(6-1), predicting loss of function from incorporation of a single inactive monomer into a hexameric complex. The dashed blue line represents a decline if either no heterooligomeric complexes were formed or velocity declined proportionally to inactive monomers. D, Activation velocity of 1.6 μm TaRca1-β, 1.6 μm TaRca2-β K157Q mutant, and 1.6 μm of both mixed. ATP (0.2 mm) was added to samples before incubating for 10 min at either 25°C or 40°C. Values are means ± sd of five experimental replicates. The percentage decline in velocity relative to TaRca1-β by itself is reported above the mixed samples. **, Significant at P < 0.001 determined by a nonpaired Student’s t test.