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Regenerating oak seedlings by different methods impacts taproot architecture, root-to-shoot allometry and, potentially, soil water use and plant growth under water limitations. In the present study, Quercus robur (L.) seedlings regenerated by four different methods – sowing (acorn-sown seedlings), sowing followed by shoot clipping (coppiced), sowing followed by root-pruning (root-pruned), and container planting with the root plug intact (containerized) – were subjected to four water-shortage regimes – full watering and 75, 50, and 25% of full watering. These treatments were used to test the hypotheses that root morphology, anatomy and biochemistry varied among regeneration methods, and, thus, differentially affected plant responses to water-shortages. For most traits, the response to water shortage of acorn-sown and containerized seedlings was similar, and opposite to that of coppiced and root-pruned seedlings. Water shortage did not change tree allocation to total root biomass among regeneration methods but altered the partitioning between absorptive and structural roots within the root system. Acorn-sown and containerized seedlings produced more pioneer roots, and coppiced and root–pruned seedlings more highly-branched fine roots with greater specific root length and specific root surface area under greater water shortage. Thus, acorn-sown and containerized oaks appeared to be primed for water foraging, and coppiced and root-pruned oaks for water absorption efficiency in response to water shortage. Water shortage caused a reduction in nonstructural carbohydrate concentrations in fine roots of acorn-sown and containerized seedlings and in the taproot of coppiced and root-pruned seedlings. Results generally indicated that seedling response to water limitation depended on the regeneration method, highlighting the importance of root–to–shoot allometry, taproot development, and carbon reserve mobilization for plant functioning under water shortage. Acorn-sown and containerized oaks seedlings might be more suitable for forest regeneration in sites characterized by severe water shortage periods. The reduction of taproot starch reserves under strong water shortage may render root-pruned oak trees more susceptible to topsoil water deficit than acorn-sown trees. This work contributes to understand drought acclimation and to identify the most adequate forestry regeneration practices to adapt forest ecosystems to ongoing climatic changes.