Identifying sources of hydrogen isotope fractionation in plant carbohydrates and lipids under low carbohydrate supply

Appreciation for the influence of plant metabolism on the hydrogen isotope composition (δ²H) of plant compounds has increased in recent years, adding new opportunities to understand how plants respond to environmental change. In general, where carbon supply is limited (e.g. at the beginning of the growth season, in darkness, under low CO₂), the resulting δ²H of newly produced plant compounds tends to be ²H-enriched. The source of the ²H-enrichment has yet to be identified but hypotheses include: 1) a direct effect of reduced photosynthesis; 2) change in the partitioning of photoassimilates to sucrose/transitory starch; 3) use of longer-term starch reserves; and/or, 4) increased isotopic exchange with water suggesting increased metabolite cycling. To test these ideas, we utilised samples collected from a study which grew tree saplings under 100% and 6% of ambient sunlight in the field (Weber et al. 2018, New Phytologist, 222: 171-182).  In general, relative growth rate was attenuated by a species-specific amount that ensured homeostasis of non-structural carbohydrate (NSC) concentrations; however, not before an initial two-fold reduction in NSC levels – the time period the current study focussed on. Cellulose was purified and n-alkanes extracted from leaves of six deciduous species (Betula pendula, Carpinus betulus, Fagus sylvatica, Prunus avium, Quercus petraea and Tilia platyphyllos) in July 2016, around one year after the shade treatment began. The isotopic difference of cellulose between shaded and full sunlit grown leaves (ε_shade-sun) was significantly different from zero, with species-specific offsets ranging between 20 to 70‰. In comparison, the treatment effect was minimal for the C-chain length concentration weighted δ²H values of n-alkanes, ranging between -7 to +10‰. To narrow down the source of ²H-enrichment in leaf cellulose, the δ²H of sucrose and starch are currently being analysed in samples collected from stem wood (without bark) before bud-break in March 2016, and in leaf and stem material sampled after leaf-out in July 2016. An increase in sucrose δ²H values from shade leaves relative to controls would support Hypothesis 1 and 2; ²H-enriched storage starch relative to leaf sucrose would support Hypothesis 3; and an increase in the δ²H of sucrose in stem wood relative to storage starch (before bud-break), would support Hypothesis 4. Our study has important implications for interpreting ²H-enrichment of plant compounds with respect to reduced C supply.