# A Comparative Bioinformatic Investigation of the Rubisco Small Subunit Gene Family in True Grasses Reveals Novel Targets for Enhanced Photosynthetic Efficiency

## TL;DR

This study explores the Rubisco small subunit gene family in grasses to find genetic targets for improving photosynthesis in crops.

## Contribution

The study identifies novel RBCS gene targets, including a C4-like mutation, for enhancing photosynthetic efficiency in crops.

## Key findings

- Triticeae/Aveneae grasses show syntenic RBCS gene duplications on chromosomes 2 and 3.
- A paired I-box promoter arrangement in RBCS genes correlates with higher expression in Triticaeae.
- A G59C mutation in RBCS increases Rubisco complex stability, suggesting a target for crop improvement.

## Abstract

Ribulose bisphosphate carboxylase (RuBisCO) is the primary regulator of carbon fixation in the plant kingdom. Although the large subunit (RBCL) is the site of catalysis, RuBisCO efficiency is also influenced by the sequence divergence of the small subunit (RBCS). This project compared the RBCS gene family in C3 and C4 grasses to identify genetic targets for improved crop photosynthesis. Triticeae/Aveneae phylogeny groups exhibited a syntenic tandem duplication array averaging 326.1 Kbp on ancestral chromosomes 2 and 3, with additional copies on other chromosomes. Promoter analysis revealed a paired I-box element promoter arrangement in chromosome 5 RBCS of H. vulgare, S. cereale, and A. tauschii. The I-box pair was associated with significantly enhanced expression, suggesting functional adaptation of specific RBCS gene copies in Triticaeae. H. vulgare-derived pan-transcriptome data showed that RBCS expression was 50.32% and 28.44% higher in winter-type accessions compared to spring types for coleoptile (p < 0.05) and shoot, respectively (p < 0.01). Molecular dynamics simulations of a mutant H. vulgare Rubisco carrying a C4-like amino acid substitution (G59C) in RBCS significantly enhanced the stability of the Rubisco complex. Given the known structural efficiency of C4 Rubisco complexes, G59C could serve as an engineering target for enhanced RBCS in economically crucial crop species which, in comparison, possess less efficient Rubisco complexes.