Hybrid Rubisco with Complete Replacement of Rice Rubisco Small Subunits by Sorghum Counterparts Confers C4 Plant-like High Catalytic Activity

Abstract

Photosynthetic rate at the present atmospheric condition is limited by the CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) because of its extremely low catalytic rate (k_cat) and poor affinity for CO₂ (K_c) and specificity for CO₂ (S_c/o). Rubisco in C₄ plants generally shows higher k_cat than that in C₃ plants. Rubisco consists of eight large subunits and eight small subunits (RbcS). Previously, the chimeric incorporation of sorghum C₄-type RbcS significantly increased the k_cat of Rubisco in a C₃ plant, rice. In this study, we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco. Obtained hybrid Rubisco showed almost C₄ plant-like catalytic properties, i.e., higher k_cat, higher K_c, and lower S_c/o. Transgenic lines expressing the hybrid Rubisco accumulated reduced levels of Rubisco, whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high CO₂ condition compared with wild-type rice. High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the βC-βD hairpin in RbcS. We propose that such differences, particularly in the βC-βD hairpin, may impact the flexibility of Rubisco catalytic site and change its catalytic properties.

Keywords: C4 plant; CRISPR-Cas9; Rubisco; photosynthesis; rice.