Hydrogen isotope fractionation in plants with C₃, C₄, and CAM CO₂ fixation

Measurements of stable isotope ratios in organic compounds are widely used tools for plant ecophysiological studies. However, the complexity of the processes involved in shaping hydrogen isotope values (δ²H) in plant carbohydrates has limited its broader application. To investigate the underlying biochemical processes responsible for ²H fractionation among water, sugars, and cellulose in leaves, we studied the three main CO₂ fixation pathways (C₃, C₄, and CAM) and their response to changes in temperature and vapor pressure deficit (VPD). We show significant differences in autotrophic ²H fractionation (ε_A) from water to sugar among the pathways and their response to changes in air temperature and VPD. The strong ²H depleting ε_A in C₃ plants is likely driven by the photosynthetic H⁺ production within the thylakoids, a reaction that is spatially separated in C₄ and strongly reduced in CAM plants, leading to the absence of ²H depletion in the latter two types. By contrast, we found that the heterotrophic ²H-fractionation (ε_H) from sugar to cellulose was very similar among the three pathways and is likely driven by the plant's metabolism, rather than by isotopic exchange with leaf water. Our study offers new insights into the biochemical drivers of ²H fractionation in plant carbohydrates.