Nocturnal sucrose does not reflect the hydrogen isotope composition of transitory starch in leaves as expected

The hydrogen isotope composition (δ²H) of cellulose is inherently linked to that of sucrose synthesized in leaves. Daytime sucrose is synthesized from triose phosphates produced by the Calvin-Benson-Bassham cycle, while nighttime sucrose is synthesized from remobilized transitory starch in leaves. Photosynthetic metabolism causes starch to be naturally ²H-depleted relative to triose phosphates. Thus, sucrose δ²H values should vary diurnally. However, this has not been tested. We made diel measurements of sucrose and starch δ²H in three species differing in their sucrose/starch dynamics (bean, radish and sunflower) under climate controlled and steady-state isotopic conditions. Leaf starch was degraded at night and ²H-depleted by around 90‰ compared with daytime sucrose. However, in all tested species we surprisingly observed no effect on nighttime sucrose δ²H and, instead, only species-specific differences. Consequently, the apparent isotope fractionation associated with sucrose biosynthesis (ε_sucrose) was indistinguishable between day and night and within -140‰ to -180‰ across the three species. The lack of day/night variation in ε_sucrose could originate from cytosolic sugar metabolism and ²H-enrichment at H-atom positions counteracting the ²H-depletion in starch. Using a simplified steady-state isotopic model of metabolism, we show that differences in day/night fluxes can reduce the expected differences between day/night ε_sucrose. From a practical perspective, this suggests that: (i) estimating ε_sucrose at a single time point might be sufficient to capture δ²H variation under steady-state conditions; and (ii) to extract more than one metabolically sensitive isotope signals from a given compound, position-specific isotope analysis will be required.