Barley (Hordeum vulgare L.) is an important cereal crop and suffers substantial yield loss under salinity. Little is understood of salinity perception and responses in roots, which involve complex changes at the physiological, metabolic, molecular, transcriptional, and genetic level. We develop new tools to unravel how plants respond to the perception of salinity. Evidence is accumulating that lipid signalling is an integral part of complex regulatory networks upon salinity by modifications of membrane lipids, which through the activity of phospholipases, lipid kinases and phosphatases that produce different classes of lipid and lipid-derived messengers. These provide spatial and temporal regulatory functions crucial for cell survival, growth and for an appropriate response of the plant to environmental stimuli. Initial analyses indicate that different tissue types within the root respond differently to salt stress in tolerant and sensitive cultivars. Here we study the root responses to salinity using a combination of RNA-sequencing and targeted metabolite and lipid analyses of three key sections of barley roots. In addition, we are using MALDI-FT-MS based imaging technologies to monitor spatial distributions of metabolites and lipids across root sections of salt-treated tolerant and sensitive barley genotypes. Transcriptomics results are now being integrated with spatial biochemical data, enhancing our understanding of system-wide and tissue-specific responses of roots to salinity stress. Given the lack of fundamental knowledge of the genes and proteins involved in signalling and lipid metabolism under salinity stress, and the enormous potential for biotechnological application in this area, our results provide insight into novel mechanisms responsible for salt tolerance of barley.