A Conserved Sequence from Heat-Adapted Species Improves Rubisco Activase Thermostability in Wheat1[OPEN]

Abstract

Eleven amino acids conserved in Rubisco activase (Rca) variants present in warm-adapted species increase the thermostability of wheat Rca by 7°C. 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.