Phosphatidylinositol transfer protein (PITP) and phosphatidylinositol 4-kinase (PI4K) are very upstream regulatory elements of the phospholipid signaling pathway in the signal transduction network. Unlike in animal systems, their role in stress signaling is poorly understood in plants. To study this area, PITP- and PI4K-overexpressing transgenic barley lines were developed. Morphological and developmental abnormalities were surveyed and characterized. It was revealed that the overexpression of the upstream signalling genes led to more phenotypic abnormalities than in other transgenic studies working with effector genes or even transcription factors. We hypothesize that this high level of abnormalities is the consequence of the modulation of the very upstream signal transduction pathway elements. On the other hand, we also revealed that overexpression of the PITP and PI4K genes increased stress tolerance during hypoxic cold stress, but not during salinity stress. Differences were also found in the level of frost tolerance between the transgenic overexpression plants and the recipient Golden Promise line. Molecular analysis showed that this improvement was not related to the most important cold responsive transcription factors, the CBF genes. We conclude that the transgenic method may be useful to prove the role of an upstream signaling element; however, due to the many developmental consequences that occur as side effects, it is a less advisable approach to achieve improved stress tolerance.