Complementation of a Setaria Rubisco activase mutant with Agave Rubisco activase restores growth and photosynthesis

Photosynthetic carbon assimilation is sensitive to heat stress, with a key component being the thermal sensitivity of Rubisco activase (RCA). One approach to increasing heat tolerance of photosynthesis is to increase the thermotolerance of RCA. Here, we have used a transgenic approach to express RCAβ from Agave tequilana (AtRCA) in Setaria viridis . A second line was created where the endogenous SvRCAβ was substituted for AtRCAβ through complementation of a null mutant, ΔrcaB (AtRCA[bKO]). In vitro assays showed that Agave RCA readily activates Setaria Rubisco, and that its thermostability is higher than that of Setaria RCA. AtRCA[bKO] plants exhibited higher CO ₂ assimilation at 25°C compared to WT and AtRCA plants, although Rubisco content and activation did not change. After six hours at 44°C, AtRCA[bKO] plants retained higher CO ₂ assimilation rates than AtRCA, however, after 24 hours CO ₂ assimilation declined to similar levels in all lines, suggesting that RCA may not limit carbon assimilation at the conditions tested. Taken together, our results show that substitution of RCA from a CAM plant into a C ₄ model allows normal growth and supports slightly increased carbon assimilation under control and short-term heat conditions.