A spontaneous mutation in a key C ₄ pathway gene significantly alters leaf δ ¹³ C, uncoupling its relationship with WUE and photosynthetic performance in Zea mays

Increases in global temperature and drought are negatively impacting the yields of major crops. Therefore, targeted improvements to intrinsic water use efficiency ( WUE _i ) are needed to reduce the water required for agricultural production. While it is very time-consuming to directly measure WUE _i , stable carbon isotope ratios (δ ¹³ C) are a reliable high throughput proxy trait for quantifying WUE _i in C ₃ species. While genetic studies have improved our understanding of the relationship between WUE _i and δ ¹³ C in C ₄ species, the knowledge needed to implement δ ¹³ C in breeding schemes is incomplete. Using a Zea mays line with an extremely negative δ ¹³ C 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 C ₄ photosynthetic pathway and is known to affect δ ¹³ C. Surprisingly, the line with the mutant allele has significantly higher carbonic anhydrase activity with a concurrent reduction in δ ¹³ C, opposite of what would be expected based on C ₄ carbon isotope fractionation theory. These observed decouple δ ¹³ C and WUE _i , which calls for further investigation into carbon isotope discrimination in C ₄ species.