Calcium carbonate-induced CO2 generation via the β-class carbonic anhydrase CAN4 promotes morphological development in Hypsizygus marmoreus

Abstract

This study investigated the roles of the carbonic anhydrase gene (CAN4) and the hydrophobin genes ABH2 and ABH3 in the morphological development of Hypsizygus marmoreus under different inorganic supplementation conditions. Cultivation with 0.1% CaCO3 significantly induced CAN4 expression by day 7, correlating with the highest CO2 accumulation, enhanced hyphal branching, and active oidiogenesis. In contrast, Na2CO3 treatment resulted in minimal CAN4 expression and limited morphological development, highlighting the importance of direct CO2 release. CaCl2 supplementation led to a moderate increase in CAN4 expression, suggesting that Ca2+ may interact with carbonate ions derived from dissolved atmospheric CO2 to precipitate CaCO3, thereby contributing indirectly to CO2 availability. Furthermore, expression of ABH2 and ABH3 was also upregulated under CaCO3 supplementation conditions, linking CO2 sensing to the activation of developmental pathways. These findings support a model in which CaCO3 enhances CO2 generation, activates CAN4, and subsequently triggers cAMP / PKA signaling to promote hydrophobin expression and aerial hyphal development. Ultimately, CaCO3 functions as a key regulator of fungal differentiation through both metabolic and signaling pathways, providing valuable insights for optimizing mushroom cultivation.