Genome-wide Identification, Structural Features and Single-Cell Expression Atlas of the Carbonic Anhydrase Gene Family in Maize (Zea mays L.)

Maize is a cornerstone C₄ crop underpinning food, feed and bioenergy. Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the interconversion of CO₂ and HCO₃⁻, underpinning photosynthesis, pH homeostasis and stress acclimation. Despite extensive work on plant CA families, a maize-focused synthesis integrating genome-wide cataloging, structural divergence, regulatory logic, and cell-type specificity is still lacking. Here we present a genome-wide census and a single-cell-resolved expression atlas of the maize CA family. We identified 18 CA genes partitioning into α (8), β (5) and γ (5) subfamilies. Proteins span 206–452 aa, pI 6.09–10.58 and 22.67–49.36 kDa, with predominantly hydrophilic profiles. Phylogeny resolved three robust clades (UFboot >90%) and indicated closer affinity to rice than to Arabidopsis; synteny recovered 15 maize–rice and 3 maize–Arabidopsis orthologs. Promoters are enriched for ABRE, TCA-element and ARE, suggesting ABA, SA and redox regulation. Bulk RNA-seq across 21 tissues revealed strong tissue specificity (τ >0.7 for 12 genes), with β-CAs elevated in leaves and α-CAs in roots. Single-cell analyses further showed ZmβCA3 enriched in mesophyll cells and ZmαCA2 enriched in vascular/root-cap cells. Together, these data position β-CAs as chloroplast-centered hubs for C4 photosynthesis, while α/γ-CAs support ion/pH buffering and mitochondrial metabolism. We nominate cell-type-specific CA members as candidates for genetic improvement toward stress resilience in maize.