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The accumulation of high amounts of specific metabolites in transgenic plants increases tolerance to salt and water stress . This effect has been observed in tobacco after the transfer of genes or cDNAs encoding enzymes that lead to the biosynthesis of mannitol, Gly betaine, Pro, trehalose, or fructan (Tarczynski et al., 1993;Kishor et al., 1995;Nomura et al., 1995;Pilon-Smits et al., 1995;Holmstrom et al., 1996). Because many naturally salt-or drought-tolerant plants accumulate such compounds when stressed, these metabolites are considered to be compatible solutes that act by providing osmotic adjustment and by lowering the osmotic potential, i.e. increasing the capacity to retain water (LeRudelier and Boullard, 1983). Other mechanisms by which these metabolites might enhance resistance to stress are, however, still not well understood. Some of these metabolites could act, for example, as scavengers of hydroxyl radicals-extremely toxic, short-lived active oxygen species that have no known enzymatic systems for breakdown (Smirnoff and Cumbes, 1989;Smirnoff, 1993;Asada, 1994;Shen et al., 1997)--or their protective function might be in maintaining the hydration sphere of proteins under water-stress conditions (Galinski, 1993;Papageorgiou and Murata, 1995).We have previously shown that tobacco, transformed to accumulate mannitol, survives high salinity better when stressed at the beginning of the exponential growth phase (Tarczynski et al., 1993). However, the amount of mannitol accumulated was usually not greater than approximately 6 to 8 pmol g-' fresh weight, possibly because of the fact that the Escherickia coli mtlD gene product catalyzed an equilibrium reaction that could also result in a conversion of mannitol-6-P to Fru-6-P, owing to the equal affinity of MtlD for either mannitol-6-P or Fru-6-P (Teschner et al., 1990). The direction of the reaction is determined by a pH-dependent change in affinity for NAD/NADH. W e decided to generate tobacco plants with a capacity for higher polyol production by exploiting a pathway that is induced in the halophyte ice plant (Mesembryantkemum crystallinum) following salt stress (Vernon and Bohnert, 1992). This pathway shunts carbon from Glu-6-P to myoinositol and then to methylated inositols. Methylated inositols are formed by a specific O-methyltransferase, IMT1, which seems to be absent in tobacco. IMTl methylates myo-inositol to form D( +)-ononitol (1D-1-O-methyl-myoinositol). Our expectation was that D-ononitol, which is dependent on the concentration of myo-inositol, might accumulate and provide stress tolerance, but it also seemed possible that the activity of the methyltransferase would generate myo-inositol deficiency.Tobacco (Nicotiana tabacum L., cv SRl) plants, transformed with the IMTl cDNA from M. crystallinum producing the enzyme D-myo-inositol methyltransferase (Vernon et al., 1993), were phenotypically normal. We compare here the physiology of the transformed line, I5A, with untransformed tobacco cv SR1. When the 15A plants were salt stressed or water...

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 mol g ؊1 fresh weight were found. Plants that accumulated up to 2 to 3 mol g ؊1 fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 mol g ؊1 fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 mol g ؊1 fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.

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