A spontaneous mutation in a key C4 pathway gene significantly alters leaf δ13C, uncoupling its relationship with water use efficiency and photosynthetic performance in <i>Zea mays</i>

Increases in global temperature and drought are negatively impacting the yields of major crops. Therefore, targeted improvements to intrinsic water use efficiency (WUEi) are needed to reduce the water required for agricultural production. While it is very time-consuming to directly measure WUEi, stable carbon isotope ratios (δ13C) are a reliable high throughput proxy trait for quantifying WUEi in C3 species. While genetic studies have improved our understanding of the relationship between WUEi and δ13C in C4 species, the knowledge needed to implement δ13C in breeding schemes is incomplete. Using a maize line with an extremely negative δ13C value, a quantitative genetics approach was used to identify a large deletion in carbonic anhydrase1 (cah1). Carbonic anhydrase is the first enzymatic step of the C4 photosynthetic pathway and is known to affect δ13C. Surprisingly, the line with the mutant allele had significantly higher carbonic anhydrase activity with a concurrent reduction in δ13C, the opposite of what would be expected based on C4 carbon isotope fractionation theory. These observations decouple δ13C and WUEi, which calls for further investigation into carbon isotope discrimination in C4 species.