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The accumulation of sugar alcohols and other low molecular weight metabolites such as proline and glycine-betaine is a widespread response that may protect against environmental stress that occurs in a diverse range of organisms. Transgenic tobacco plants that synthesize and accumulate the sugar alcohol mannitol were engineered by introduction of a bacterial gene that encodes mannitol 1 -phosphate dehydrogenase. Growth of plants from control and mannitol-containing lines in the absence and presence of added sodium chloride was analyzed. Plants containing mannitol had an increased ability to tolerate high salinity.

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Hydroxyl radicals may be responsible for oxidative damage during drought or chilling stress. We have shown that the presence of mannitol in chloroplasts can protect plants against oxidative damage by hydroxyl radicals (B. Shen, R.C. Jensen, H.J. Bohnert [1997] Plant Physiol 11 3: 11 77-1 183). Here we identify one of the target enzymes that may be protected by mannitol. lsolated thylakoids in the presence of physiological concentrations of Fez+ generated hydroxyl radicals that were detected by the conversion of phenylalanine into tyrosine. The activity of phosphoribulokinase (PRK), a thiol-regulated enzyme of the Calvin cycle, was reduced by 65% in illuminated thylakoids producing hydroxyl radicals. Mannitol (1 25 mM) and sodium formate (15 mM), both hydroxyl radical scavengers, and catalase (3000 units mL-') prevented loss of PRK activity. In contrast, superoxide dismutase (300 units mL-') and glycine betaine (125 mM) were not effective in protecting PRK against oxidative inactivation. Ri bulose-l,5-bisphosphate carboxylase/oxygenase activity was not affected by hydroxyl radicals. We suggest that the stress-protective role of mannitol may be to shield susceptible thiol-regulated enzymes like PRK plus thioredoxin, ferredoxin, and glutathione from inactivation by hydroxyl radicals in plants.

The pathway from glucose 6-phosphate (G 6-P) to myoinositol 1-phosphate (Ins 1-P) and myo-inositol (Ins) is essential for the synthesis of various metabolites. In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo-inositol O-methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, D-ononitol and D-pinitol, which serve as osmoprotectants. This paper describes transcripts for the enzyme, Inps1, encoding myo-inositol 1-phosphate synthase (INPS1), from the ice plant. Two Inps-like sequences are present in the genome. The deduced amino acid sequences of the cloned transcript are 49.5% and 87-90%, respectively, identical to those of yeast and other higher plant sequences. Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10-fold during salinity stress. Inps1 induction is by transcription, similar to the induction of Imt1. In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt-stressed. The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection. The stress-induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences.

To investigate the potential role of a polyol, mannitol, in oxidative stress protection, a bacterial mannitol-1 -phosphate dehydrogenase gene was targeted to chloroplasts by the addition of an aminoterminal transit peptide. Transgenic tobacco (Nicofiana fabacum) lines accumulate mannitol at concentrations ranging from 2.5 to 7 pmol/g fresh weight. Lhe BSI-31 accumulated approximately 1 O0 mM mannitol in chloroplasts and was identical to the wild type in phenotype and photosynthetic performance. l h e presence of mannitol in chloroplasts resulted in an increased resistance to methyl viologen (MV)-induced oxidative stress, documented by the increased retention of chlorophyll in transgenic leaf tissue following M V treatment. In the presence of MV, isolated mesophyll cells of BS1-31 exhibited higher CO, fixation than the wild type. When the hydroxyl radical probe dimethyl sulfoxide was introduced into cells, the initial formation rate of methane sulfinic acid was significantly lower in cells containing mannitol in the chloroplast compartment than in wild-type cells, indicating an increased hydroxyl radicalscavenging capacity in BS1-31 tobacco. We suggest that the chloroplast location of mannitol can supplement endogenous radicalscavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals.