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Shifting patterns of precipitation and rising temperatures have highlighted forest vulnerability to heat- and drought-induced stress. For systems that face water-limitation, either from short-term, seasonal dry periods or longer-term droughts, plasticity of root system function establishes the ability of individuals to meet atmospheric demand and maintain physiological function. This functional plasticity is determined by an individual’s intrinsic properties and their interactions within the community and environment. Given limitations to in situ measurement, improved model representation of below-ground structural and functional complexity has provided means for exploring these ecophysiological feedbacks between drying soil and trees across biomes. This research addresses individual, ecosystem, and basin scale responses to water limitation by examining (i) the role of below-ground structure and ecophysiological controls on water uptake across functional gradients (i.e., low diversity vs. high diversity ecosystems); (ii) identifying and expanding the utility of novel proxies of hydraulic function; and (iii) exploring the feasibility of monitoring drought response at large scales using a parsimonious model of surface energy partitioning. Modeled root water uptake from both temperate and tropical systems highlight that independent of functional strategy, root lateral interactions at the tree scale directly impact the depth distribution of water uptake and plant hydraulic status. A newly developed index of root system interaction provides an amenable axis with which to explore the tradeoffs between structural investment and resource acquisition. Laboratory and field analysis show that conventional technologies used to measure sap flow velocity may contain hidden information regarding a tree’s hydraulic state. This low frequency signal may also serve well as a proxy for below-ground response to the drying soil, providing valuable validation for future modeling efforts. Finally, the feasibility of hourly, basin scale estimates of the land-surface energy budget partition are tested. The Maximum Entropy Production model is successfully applied to the Amazon River Basin, a highly complex region prone to strong seasonal droughts, elucidating avenues of future research needed to more fully link ecosystem and hydrologic processes. The methodologies developed and expanded in this work provide new avenues for assessing tree-scale water fluxes and hydraulic state, providing a means for observing and testing hypotheses related to ecophysiological response across spatiotemporal scales.