Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post‐photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post‐photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth‐and‐differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02–0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions. Summary statement: The proportion of oxygen in leaf cellulose which is derived from leaf sucrose in Lolium perenne is much more constant and predictable than was hitherto assumed, encouraging reconstructions of photosynthetic information [ABSTRACT FROM AUTHOR]