[3S01a] 09. GteX - Part 2

2:00 PM - 2:30 PM JST(5:00 AM - 5:30 AM UTC)

[3GteX-07-KL] Rubisco Engineering for Improving Photosynthesis and Productivity in Plants

○Hiroshi Fukayama¹ (1. Kobe University (Japan))

Keywords:

Photosynthesis,Plant,Genetic engineering,Rubisco,Agriculture

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme in photosynthetic CO₂ fixation, and its inefficient catalytic properties are a bottleneck for plant photosynthesis and productivity. Rubisco in C₄ plants (e.g., maize and sorghum) generally shows a higher catalytic rate (k_cat) than that in C₃ plants (e.g., rice and Arabidopsis thaliana). Rubisco is composed of a chloroplast-encoded large subunit (rbcL), which contains the catalytic site, and a nuclear-encoded small subunit (RbcS). First, we introduced the C₄-sorghum RbcS into rice and knocked out the rice RbcS multigene family using the CRISPR/Cas9 system. These transgenic rice expressed a hybrid Rubisco composed of rice rbcL and sorghum RbcS (CSS-Rubisco). The CSS-Rubisco showed a significantly higher k_cat, similar to that of C₄ plants. The crystal structure of CSS-Rubisco revealed a substantial structural difference in the β-hairpin (βC-βD) around Leu102 of sorghum RbcS (Ile102 in rice), which is likely to affect the flexibility of the 60s loop in the catalytic site and influence its catalytic properties. However, introducing the I102L mutation into rice RbcS had limited effect on Rubisco kinetics. In addition, the hybrid Rubisco of the Ile102 type C₄-RbcS in maize also showed a significantly higher k_cat than that in rice. These results suggest that C₄-RbcSs are useful for enhancing the k_cat of Rubisco in rice and that amino acids other than L102 also contribute to conferring C₄ plant-like catalytic properties. Since the catalytic site of Rubisco is located in rbcL, it is expected to play an important role in determining its catalytic properties. In rice, chloroplast transformation has not been established, but chloroplast genome editing has become possible through nuclear transformation using chloroplast-targeted TALECD (ptpTALECD). Thus, we knocked out the rice rbcL using ptpTALECD and introduced chloroplast-targeted C₄ plant RbcLs, successfully producing transgenic rice expressing hybrid Rubisco composed of C₄ rbcLs and rice RbcS. However, these hybrid Rubisco did not exhibit the high k_cat seen in C₄ plants. On the other hand, we also generated a range of mutated rbcLs in Arabidopsis using ptpTALECD. Among them, the M309I or D397N substitutions in rbcL resulted in increased k_cat. Cryo-electron microscopy structural analysis showed that the M309I and D397N substitutions induce significant structural alterations in the 60s loops. These results suggest that modifying the structure around the 60s loop is effective in improving the k_cat of Rubisco. In conclusion, we succeeded in enhancing Rubisco catalytic performance in plants by introducing C₄-RbcSs or point mutations into rbcL. Among these plants, some also showed enhanced photosynthesis and growth, especially under elevated CO₂ conditions. Therefore, our results highlight the potential of Rubisco engineering to improve photosynthesis, which could lead to increased yields across a variety of crops in the future.