Nitrogen fertilization and δ 18 O of CO 2 have no effect on 18 O‐enrichment of leaf water and cellulose in Cleistogenes squarrosa (C 4 ) – is VPD the sole control?

The oxygen isotope composition of cellulose (δ 18 O Cel ) archives hydrological and physiological information. Here, we assess previously unexplored direct and interactive effects of the δ 18 O of CO 2 (δ 18 O CO2 ), nitrogen (N) fertilizer supply and vapour pressure deficit (VPD) on δ 18 O Cel , 18 O‐enrichment of leaf water (Δ 18 O LW ) and cellulose (Δ 18 O Cel ) relative to source water, and p ex p x , the proportion of oxygen in cellulose that exchanged with unenriched water at the site of cellulose synthesis, in a C 4 grass ( Cleistogenes squarrosa ). δ 18 O CO2 and N supply, and their interactions with VPD, had no effect on δ 18 O Cel , Δ 18 O LW , Δ 18 O Cel and p ex p x . Δ 18 O Cel and Δ 18 O LW increased with VPD, while p ex p x decreased. That VPD‐effect on p ex p x was supported by sensitivity tests to variation of Δ 18 O LW and the equilibrium fractionation factor between carbonyl oxygen and water. N supply altered growth and morphological features, but not 18 O relations; conversely, VPD had no effect on growth or morphology, but controlled 18 O relations. The work implies that reconstructions of VPD from Δ 18 O Cel would overestimate amplitudes of VPD variation, at least in this species, if the VPD‐effect on p ex p x is ignored. Progress in understanding the relationship between Δ 18 O LW and Δ 18 O Cel will require separate investigations of p ex and p x and of their responses to environmental conditions.