β-Carbonic anhydrase SlβCA4 is required for normal growth, carbon assimilation and stomatal plasticity in tomato

Abstract

β-Carbonic anhydrases (βCAs) catalyze the reversible interconversion of CO2 and bicarbonate and contribute to plant carbon metabolism and environmental responses. However, the physiological functions of individual βCA isoforms in crops remain poorly understood. Here, we investigated two chloroplast-localized βCAs, SlβCA1 and SlβCA4, in tomato (Solanum lycopersicum). We combined subcellular localization, CRISPR/Cas9 mutagenesis, grafting, leaf gas-exchange measurements, and controlled environmental treatments to examine their roles in growth, photosynthesis, and stomatal developmental plasticity. Both proteins localized to chloroplasts but their disruption resulted in markedly different phenotypes. While SlβCA1 mutants showed largely normal growth and photosynthesis, SlβCA4 mutants exhibited severe root and shoot growth defects. Grafting mutant shoots onto wild-type rootstocks partially restored vegetative growth, indicating that impaired root function contributes to the growth defects. SlβCA4 mutants also showed reduced CO2 assimilation and stomatal conductance, while electron transport remained largely unaffected. Elevated CO2 partially improved leaflet growth but did not restore plant height or root elongation. Furthermore, unlike wild-type plants, SlβCA4 mutants failed to increase stomatal index in response to elevated temperature. These findings reveal distinct physiological roles for chloroplast-localized βCAs in tomato and identify SlβCA4 as an important contributor to plant growth, photosynthesis, and temperature-dependent stomatal developmental plasticity.