Chlorophyll fluorescence responses to CO₂ availability reveal crassulacean acid metabolism in epiphytic orchids

Crassulacean acid metabolism (CAM), a specialized mode of photosynthetic carbon assimilation characterized by nocturnal fixation of atmospheric CO₂ and vacuolar malic acid storage, is found in a wide variety of vascular plant species, mainly those inhabiting water-limited environments. Identifying and characterizing diverse CAM species enhances our understanding of the physiological, ecological, and evolutionary significance of CAM photosynthesis. In this study, we examined the effect of CO₂ elimination on chlorophyll fluorescence-based photosynthetic parameters in two constitutive CAM Kalanchoe species and six orchids. In CAM-performing Kalanchoe species, the effective quantum yield of photosystem II showed no change in response to CO₂ elimination during the daytime but decreased with CO₂ elimination at dusk. We applied this method to reveal the photosynthetic mode of epiphytic orchids and found that Gastrochilus japonicus, Oberonia japonica, and Bulbophyllum inconspicuum, but not B. drymoglossum, are constitutive CAM plants, which were also confirmed by malate determination. Our data propose a novel approach to identify and characterize CAM plants without labor-intensive experimental procedures. Although B. drymoglossum leaves had relatively high malate content, they did not depend on it to perform photosynthesis even under water-deficient or increased light conditions. Anatomical comparisons revealed a notable difference in leaf structure between B. drymoglossum and B. inconspicuum; B. drymoglossum leaves possess large water storage tissue internally, unlike B. inconspicuum leaves, which develop pseudobulbs. Our findings suggest different evolutionary adaptations to water deficit between closely related B. drymoglossum and B. inconspicuum.