Metabolic engineering of proline biosynthesis to enhance plant osmotolerance

Proline has been reported to accumulate during osmotic stress in many plant species and has been proposed to be directly involved in stress adaptation. However, whether or not a constitutive accumulation of proline can facilitate osmotic adjustment and adaptation has not been established. The enzyme catalyzing the rate-limiting reaction for proline biosynthesis, Δ-1-pyrroline-5-carboxylate synthetase (P5CS) has been cloned from several species and it has been shown to be allosterically feed-back inhibited by proline. Following what has been proposed in bacteria a few years ago, we rationalized that overexpression of a mutated P5CS gene lacking feed-back inhibition would be an appropriate strategy to increase the constitutive level of proline. We therefore cloned tomPRO2, the gene encoding the tomato P5CS, and we isolated 15 tomPRO2 mutations allowing the overaccumulation of high levels of proline in bacteria. Sequence analysis of three of these mutants revealed novel single amino acid substitutions in the γ-glutamyl kinase, the domain of tomPRO2 that is sensitive to feed-back inhibition. By overexpressing the mutated tomPRO2 in Arabidopsis plants, we were able to increase the constitutive level of proline 10 fold. In contrast with other reports, the transgenic plants did not show differences in cell osmotic potentials, fresh weights and dry weights, relative to their controls, when grown in presence or absence of NaCl. Overexpression of tomPRO2 and its mutated derivatives in the yeast Saccharomyces cerevisiae led to a 20 fold increase of the basal proline level. However, expression of the plant gene(s) in a yeast genetic background deficient in the structural gene for proline oxidase, led to an increase in the proline content of more than 100 fold. In the yeast strains expressing the plants genes, the cellular proline concentration was inversely proportional to their growth rate. This caused an apparent salt tolerance so that, at 250 mM NaCl, the growth of the wild type strain was inhibited 30% whereas the growth of proline overproducing strains was not inhibited. Such tolerance was noticeable only with NaCl-induced and LiCl-induced stress, but not with sorbitol-induced stress, suggesting that it was ion-specific and not osmo-specific