Topsoil depth substantially influences the responses to drought of the foliar metabolomes of Mediterranean forests

• Topsoil depth, rather than moisture, was correlated with the vegetation structure.
• Shallow soils may not be able to sustain the structures of larger Quercus ilex trees.
• Drought effects on the metabolomes of Q. ilex trees are enhanced in shallower soils.

The upper soil provides support, water, and nutrients to terrestrial plants and is therefore crucial for forest dynamics. We hypothesised that a tree's metabolic activity (and therefore its metabolome; the total set of metabolites) would be affected by both the depth of upper soil layers and water availability. We sampled leaves for stoichiometric and metabolomic analyses once per season from differently sized Quercus ilex trees under natural and experimental drought conditions representing the likely conditions in the coming decades). Although the metabolomes varied according to tree size, smaller trees did not show higher concentrations of biomarker metabolites related to drought stress. However, the effect of the drought treatment on the metabolomes was greatest for small trees growing in shallow soils. Our results suggest that tree size is more dependent on the depth of the upper soil, which indirectly affects a tree's metabolome, rather than on the moisture content in the upper soil. Metabolomic profiling of Q. ilex supports our finding that water availability in the upper soil is not necessarily correlated with tree size. The higher impact of drought on trees growing in shallower soils nevertheless indicates that any increase in the frequency, intensity, and duration of drought − as has been projected for the Mediterranean Basin and other areas − would affect small trees most. Metabolomics has proved to be a useful means for investigating the links between plant metabolism and environmental conditions.