Biochemical and transcriptomic analysis of maize diversity to elucidate drivers of leaf carbon isotope composition

Carbon isotope discrimination is used to study CO₂ diffusion, substrate availability for photosynthesis, and leaf biochemistry, but the intraspecific drivers of leaf carbon isotope composition (δ¹³C) in C₄ species are not well understood. In this study, the role of photosynthetic enzymes and post-photosynthetic fractionation on δ¹³C (‰) was explored across diverse maize inbred lines. A significant 1.3‰ difference in δ¹³C was observed between lines but δ¹³C did not correlate with in vitro leaf carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), or ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity. RNA-sequencing was used to identify potential differences in post-photosynthetic metabolism that would influence δ¹³C however, no correlations were identified that would indicate significant differences in post-photosynthetic fractionation between lines. Variation in δ¹³C has been observed between C₄ subtypes, but differential expression of NADP-ME and PEP-CK pathways within these lines did not correlate with δ¹³C. However, co-expression network analysis provided novel evidence for isoforms of C₄ enzymes and putative transporters. Together, these data indicate that diversity in maize δ¹³C cannot be fully explained by variation in CA, PEPC, or Rubisco activity or gene expression. The findings further emphasise the need for future work exploring the influence of stomatal sensitivity and mesophyll conductance on δ¹³C in maize.