Alternative transcriptional initiation of OsβCA1 produces three distinct subcellular localization isoforms involved in stomatal response regulation and photosynthesis in rice.

Plants adjust the size of their stomatal openings to balance CO2 intake and water loss. Carbonic anhydrases (CAs) facilitate the conversion between CO2 and HCO3 -, and the OsβCA1 mutant in rice (Oryza sativa) shows similar traits in carbon fixation and stomatal response to CO2 as the dual βCA mutants in Arabidopsis thaliana. However, the exact role of OsβCA1 in these processes was unclear. We used gene editing, molecular biology, and plant physiology to study how OsβCA1 contributes to carbon fixation, stomatal opening, and CO2 responses. OsβCA1 produces three isoforms (OsβCA1A, OsβCA1B, and OsβCA1C) through alternative transcriptional initiation, which localize to the chloroplast, cell membrane, and cytosol, respectively. Protein measurements revealed that OsβCA1A/C and OsβCA1B contribute 97 and 3% to OsβCA1, respectively. By creating specific mutants for each isoform, our results found that the chloroplast and cell membrane isoforms independently participate in carbon fixation and regulation of stomatal aperture. Furthermore, the complete knockout of OsβCA1 caused a delayed response to low CO2. Our findings provide new insights into the generation and function of different OsβCA1 isoforms, clarifying their roles in CO2 diffusion, CO2 fixation and stomatal regulation in rice.