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The acclimation of plants to water deficit is the result of many different physiological and biochemical mechanisms. To gain a better understanding of drought stress acclimation and tolerance mechanisms in Populus cathayana Rehder, we carried out an integrated physiological and comparative proteomic analysis on the drought stress responses of two contrasting populations originating from wet and dry regions in western China. The plantlets were subjected to continuous drought stress by withholding soil water content at 25% of field capacity (FC) for 45 days, while the control treatments were kept at 100% FC. Drought stress significantly inhibited plant growth, decreased net photosynthetic rate and stomatal conductance of leaves, increased the relative electrolyte leakage and malondialdehyde (MDA) content, and, at the same time, accumulated soluble sugars and free proline in both populations tested. The population from the dry climate region exhibited stronger tolerance to drought stress compared with the wet climate population. The proteomic analyses resulted in the identification of 40 drought-responsive proteins. The functional categories of these proteins include the regulation of transcription and translation, photosynthesis, cytoskeleton, secondary metabolism, HSPs/chaperones, redox homeostasis and defense response. The results suggest that poplars’ tolerance to drought stress relates to the control of reactive oxygen species (ROS) and to osmoprotective capacity. The differential regulation of some drought-responsive proteins, such as HSPs and the enzymes related to redox homeostasis and regulation of secondary metabolism, plays an important role in poplars’ tolerance and acclimation to drought stress. In conclusion, acclimation to water deficit involves changes in cellular metabolism and the regulation of gene networks. The present study not only provides new insights into the mechanisms of acclimation and tolerance to drought stress in different poplar populations but also provides clues for improving poplars’ drought tolerance through breeding or genetic engineering. [ABSTRACT FROM AUTHOR]