Plant Molecular Biology 47: 621–631, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 621 Analysis of celery (Apium graveolens) mannitol dehydrogenase (Mtd) promoter regulation in Arabidopsis suggests roles for MTD in key environmental and metabolic responses Eli Zamski 1 , Wei-Wen Guo 1 , Yuri T. Yamamoto 2 , D. Mason Pharr 1 and John D. Williamson 1,∗ Departments of 1 Horticultural Science and 2 Forestry, North Carolina State University, Raleigh, NC 27695-7609, USA ( ∗ author for correspondence; e-mail john - williamson@ncsu.edu) Received 11 September 2000; accepted in revised form 9 July 2001 Key words: carbohydrate translocation and regulation, gene regulation, mannitol metabolism, plant-pathogen interaction, salt and osmotic stress Abstract Of the growing list of promising genes for plant improvement, some of the most versatile appear to be those involved in sugar alcohol metabolism. Mannitol, one of the best characterized sugar alcohols, is a significant photosynthetic product in many higher plants. The roles of mannitol as both a metabolite and an osmoprotectant in celery (Apium graveolens) are well documented. However, there is growing evidence that ‘metabolites’ can also have key roles in other environmental and developmental responses in plants. For instance, in addition to its other properties, mannitol is an antioxidant and may have significant roles in plant-pathogen interactions. The mannitol catabolic enzyme mannitol dehydrogenase (MTD) is a prime modulator of mannitol accumulation in plants. Because the complex regulation of MTD is central to the balanced integration of mannitol metabolism in celery, its study is crucial in clarifying the physiological role(s) of mannitol metabolism in environmental and metabolic responses. In this study we used transformed Arabidopsis to analyze the multiple environmental and metabolic responses of the Mtd promoter. Our data show that all previously described changes in Mtd RNA accumulation in celery cells mirrored changes in Mtd transcription in Arabidopsis. These include up-regulation by salicylic acid, hexokinase-mediated sugar down-regulation, and down-regulation by salt, osmotic stress and ABA. In contrast, the massive up-regulation of Mtd expression in the vascular tissues of salt-stressed Arabidopsis roots suggests a possible role for MTD in mannitol translocation and unloading and its interrelation with sugar metabolism. Abbreviations: 3-OMG, 3-O-methylglucose; dGlc, 2-deoxyglucose; HK, hexokinase; MTD, mannitol dehydroge- nase protein/enzyme (also mannitol:mannose 1-oxidoreductase; EC pending); Mtd, mannitol dehydrogenase gene or transcript; PR, pathogenesis-related; SA, salicylic acid Introduction The sugar alcohol mannitol is an important osmolyte and compatible solute that confers salt tolerance in a number of higher plants (Stoop et al., 1996). In vascular plants, mannitol is oxidized directly to man- nose by the catalytic activity of an NAD-dependent mannitol dehydrogenase (MTD; EC pending), a 1- oxidoreductase (Stoop et al., 1996). This enzyme differs from microbial mannitol dehydrogenases (EC 1.1.1.67 and 1.1.1.138) which are 2-oxidoreductases (i.e. convert mannitol or mannitol-1-phosphate to fruc- tose or fructose-6-phosphate). MTD also catalyzes the initial reaction by which translocated mannitol is committed to central metabolism.