A bacterial gene encoding mannitol-lphosphate dehydrogenase, mtlD, was engineered for expression in higher plants. Gene constructions were stably incorporated into tobacco plants. The mtlD gene was expressed and translated into a functional enzyme in tobacco, resulting in the synthesis and accumulation of mannitol, which was identified by NMR and mass spectroscopy. Mannitol concentrations exceeded 6 jumol/g (fresh weight) in the leaves and in the roots of some transformants, whereas this sugar alcohol was not detected in these organs of wild-type tobacco plants or of untransformed tobacco plants that underwent the same regeneration scheme. These experiments demonstrate that branchpoints in plant carbohydrate metabolism can be generated by which novel gene products can utilize endogenous substrates to divert metabolic energy into novel compounds. Additionally, the system described here allows for physiological studies in which the responses of wild-type and transgenic tobacco to various environmental stimuli can be compared directly. Such studies will facilitate our understanding of the roles of sugar alcohols (e.g., in stress tolerance) in higher plants.Metabolic engineering (1) was examined as an approach to gain insight into the roles of sugar alcohols or polyols in higher plants. These simple carbohydrates are widely distributed among organisms including bacteria, algae, fungi, higher plants, insects, and mammals (2-5). In plants, the importance of sugar alcohols is reflected by the estimation that metabolism of these compounds, rather than that of sugars, contributes to about 30% of the annual global primary carbon production (4). Some sugar alcohols, notably mannitol and sorbitol, are major photosynthetic products of, and can accumulate to high levels in, various higher plant species (3,4,6). These sugar alcohols are relatively widespread in higher plants, with mannitol, for example, having been detected in over 50 families (3). Mannitol and sorbitol are also translocated by some higher plant species (e.g., ref. 7), indicating a role as a storage compound. Other sugar alcohols, such as dulcitol and ribitol, are considerably less prevalent in higher plants and are apparently characteristic of species representing only a few families (3, 4).The functions of sugar alcohols in higher plants (and organisms in general) are not clear. A commonly held belief is that these compounds may confer beneficial traits on those species where they are found, rather than being simply intermediates of carbohydrate metabolism. Suggested physiological roles for sugar alcohols include osmoregulation (3,8) and service as compatible solutes (5, 9), storage of reduced carbon and energy (3), regulation of coenzymes (3, 10), and neutralization of hydroxyl radicals (11). Evidence supporting these roles for sugar alcohols has been obtained primarily with fungi and animals; only limited studies exist regarding the functions of these compounds in higher plants (12)(13)(14)(15)(16)(17).It seemed possible, by means of gene engineeri...