Going Against the Grain: Investigating the C4 Wheat Hypothesis with Spatial Transcriptomics

The possibility of a C4 photosynthetic pathway present in the developing grain of wheat, a C3 plant, has been the source of scientific debate. Wheat is critical to food security and may benefit greatly from the biological advantages conferred by C4 photosynthesis under heat and drought stress. Therefore, significant research has gone towards engineering wheat to use C4 biochemistry, resulting in the discovery of a unique photosynthetic pathway in the grain that has been suggested to be C4 specific. Here, we employed a spatial transcriptomics analysis of the developing wheat grain to further investigate the spatial expression patterns of C4 specific genes. Our results showed that most of the genes related to C4 photosynthesis were expressed in the grain in the theorised tissue locations, including phosphoenolpyruvate carboxylase (ppc) and pyruvate orthophosphate dikinase (ppdk). The photosynthetic pericarp cells were the site of ppc synthesis while the endosperm was the site of ppc carboxylation activity. Notably, isoforms of aspartate aminotransferase, alanine aminotransferase and malate dehydrogenase exhibited spatially distinct expression patterns, with tissue specificity, possibly linked to the unique functions of individual isoforms. As wheat performance under stress has been associated with the levels of expression of these C4 genes, confirmation of an active C4 pathway in the grain would have significant agronomic implications. Our results provide novel gene expression data for key genes related to photosynthesis, which could contribute to future development of highly productive, climate change resilient wheat varieties.