When exposed to drought stress many plants reprogram their gene expression to activate adaptive biochemical and physiological responses for survival. However, most of the well-studied adaptive responses are common between drought-sensitive and drought-tolerant species, making it difficult to identify the key mechanisms underpinning successful drought tolerance in crops. We developed a sorghum experimental system that compares between drought-sensitive (ICSB338) and enhanced drought-tolerant (SA1441) varieties. We show that sorghum activates a swift and robust stomatal shutdown to preserve leaf water content when water stress has been sensed. Water uptake is enhanced via increasing root cell water potential through the rapid biosynthesis of predominantly glycine betaine and an increased root-to-shoot ratio to explore more soil volume for water. in addition to stomatal responses, there is a prompt accumulation of proline in leaves and effective protection of chlorophyll during periods of water limitation. Root and stomatal functions rapidly recover from water limitation (within 24 h of re-watering) in the drought-tolerant variety, but recovery is impaired in the drought-sensitive sorghum variety. Analysis of the root proteome revealed complex protein networks that possibly underpin sorghum responses to water limitation. common and unique protein changes between the two sorghum varieties provide new targets for future use in investigating sorghum drought tolerance. Drought stress is a major barrier to agricultural productivity 1 , primarily causing damaging water deficits in plant cells. Drought-induced osmotic stress triggers loss of cell turgor, which affects the rate of cell expansion and overall cell size, ultimately restricting plant growth and development 2. In sub-Saharan Africa, drought is often associated with very high temperatures, the combination of which severely limits crop productivity 1 , particularly in rain-fed agricultural systems. Several climatic models predict increases in surface temperatures and drought episodes by the year 2050 3 , further threatening global food security 4. Therefore, it is important to understand plant adaptive responses to drought in order to improve breeding for drought tolerant crops 5. Plants respond to the effects of drought stress through a complex network of mechanisms aimed at sustaining cellular metabolism under the prevailing conditions. The mechanisms of drought avoidance, escape and tolerance in a variety of plant species have been extensively reviewed 2,6-8. While drought escape is important in desert ephemerals that rapidly complete their life cycles before the onset of water deficits 2 , drought avoidance and tolerance are significant response mechanisms in many other plant species. For instance, plants can avoid drought by conserving water through various anatomical features. These include an extensive root system, a thick waxy cuticle, and reduced leaf surface area attained through leaf rolling, folding or shedding. In addition, changes in stomatal number,...