Physiological and metabolomic analysis of a knockout mutant suggests a critical role of MtP5CS3 gene in osmotic stress tolerance of Medicago truncatula

In the model legume Medicago truncatula, Δ1-pyrroline-5-carboxylate synthetase (P5CS), the rate-limiting enzyme of proline biosynthesis, is encoded by three closely related genes, MtP5CS1, MtP5CS2, and MtP5CS3. While MtP5CS1 is constitutively expressed, MtP5CS2 and MtP5CS3 are induced by adverse environmental conditions, of which MtP5CS3 is prevalently expressed during drought and salinity stresses. Mtp5cs3, a transposon (Tnt1) insertion mutant of MtP5CS3 that cannot synthesize a mature protein, showed decreased proline accumulation and increased sensitivity to salinity, drought, and low water potential stresses, as evidenced by decreased seedling growth and chlorophyll content and increased hydrogen peroxide content. These defective phenotypes were complemented by externally supplied proline or ectopically expressed cDNA to the wild-type gene (MtP5CS3). Gas chromatography–mass spectrometry-based analysis of soluble metabolites revealed that some major metabolites contributing to osmotolerance, including certain amino acids, sugars, and polyols, accumulated more abundantly in the Mtp5cs3 roots than in the wild type, whereas a few other amino acids accumulated less during drought and salinity stresses. While such metabolic reconfiguration apparently fell short of compensating for proline deficiency in Mtp5cs3, overexpression of MtP5CS3 significantly increased tolerance of M. truncatula to salinity and low water potential stress. Thus, MtP5CS3 plays a crucial role in proline accumulation and osmotic stress tolerance of M. truncatula. Manipulation of this predominant proline biosynthetic gene will facilitate the development of environmentally stable legume crops.