The hydrogen isotopic composition (delta H-2) of plant compounds is increasingly used as a hydroclimatic proxy; however, the interpretation of delta H-2 values is hampered by potential coeffecting biochemical and biophysical processes. Here, we studied delta H-2 values of water and carbohydrates in leaves and roots, and of leaf n-alkanes, in two distinct tobacco (Nicotiana sylvestris) experiments. Large differences in plant performance and biochemistry resulted from (a) soil fertilization with varying nitrogen (N) species ratios and (b) knockout-induced starch deficiency. We observed a strong H-2-enrichment in sugars and starch with a decreasing performance induced by increasing NO3-/NH4+ ratios and starch deficiency, as well as from leaves to roots. However, delta H-2 values of cellulose and n-alkanes were less affected. We show that relative concentrations of sugars and starch, interlinked with leaf gas exchange, shape delta H-2 values of carbohydrates. We thus provide insights into drivers of hydrogen isotopic composition of plant compounds and into the mechanistic modeling of plant cellulose delta H-2 values.