COLLOQUIUM The Dual Role of Mannitol as Osmoprotectant and Photoassimilate in Celery D.M. Pharr1, J.M.H. Stoop2, J.D. Williamson3, M.E. Studer Feusi2, M.O. Massel4, and M.A. Conkling5 North Carolina State University, Box 7609, Raleigh, NC 27695-7609

Mannitol, a six carbon noncyclic sugar alcohol, is the most abun- that the native enzyme was probably a dimer. Polyclonal antibodies dant polyol in nature, occurring in bacteria, fungi, algae, lichens, and raised against the enzyme were used in immunocytochemical studies in at least 70 species of vascular plants (Bieleski, 1982; Lewis, 1984). demonstrating that M-6-PR was located in the cytosol of photosyn- Various roles have been postulated for mannitol in these organisms, thetic mesophyll cells, as depicted in the model (Fig. 1) (Everard et al., including carbon storage, free radical scavenging, osmoregulation, 1993a). This result confirmed an earlier conclusion concerning the and service as a compatible solute (Bieleski, 1982; Loescher, 1987). location of this enzyme based on cell fractionation studies (Rumpho et Some of these roles are of obvious importance in stress tolerance, al., 1983) and clearly implies the need for a massive quantity of particularly in tolerance to salt stress, which invariably causes NADPH in the cytosol of the mesophyll cells during mannitol biosyn- osmostress of varying durations in plants. Mannitol is also a phloem- thesis. This requirement for cytosolic reductant in mannitol-forming translocated photoassimilate in some vascular plants (Bieleski, 1982; plants poses an interesting problem in that photochemically produced Loescher, 1987). This article focuses on the enzymatic synthesis and NADPH is generated exclusively within the chloroplast, and NADPH degradation of mannitol in celery (Apium graveolens L. var. dulce) cannot pass directly through the chloroplast membranes into the plants and the role of these pathways in controlling mannitol accumu- cytosol. NADPH is supplied indirectly by shuttling another metabolite lation in stressed plants. generated within the chloroplast to the cytosol (Fig. 1). Subsequent to the photosynthetic reduction of 3-PGA to triose phosphate within the BIOCHEMISTRY OF MANNITOL SYNTHESIS AND chloroplast, triose phosphate is exported to the cytosol in exchange for CATABOLISM 3-PGA. The triose-phosphate is oxidized in the cytosol to 3-PGA by a nonreversible triose-phosphate dehydrogenase, generating NADPH Mannitol synthesis from NADP. The resulting NADPH then can be used for the reductive step in mannitol synthesis. Nonreversible cytosolic triose-phosphate In celery plants, mannitol is a major product of leaf photosynthesis dehydrogenase activity is present at as much as five times higher and constitutes as much as 50% of the phloem-translocated activity in extracts of celery leaves than in extracts from leaves of photoassimilate (Loescher et al., 1992). The remainder of the translo- plants that exclusively form sucrose as the photosynthetic export cated photoassimilate is largely sucrose. The partitioning of photosyn- product (Rumpho et al., 1983). The overall result of this metabolite thetically fixed CO2 between mannitol and sucrose (Fig. 1) is a shuttle, providing for NADP reduction in the cytosol, is a transfer of function of leaf age, with mannitol favored in more mature leaves reducing equivalents to the cytosol from the chloroplasts with no net (Davis et al., 1988). The biosynthetic pathway of mannitol from gain or loss of C or phosphate within the cytosol or chloroplasts. photosynthetic intermediates was first described in celery in the early 1980s (Rumpho et al., 1983). More recently, the pathway also was Mannitol catabolism demonstrated in privet (Ligustrum vulgare L.) (Loescher et al., 1992). Mannitol is formed in the cytosol of photosynthetic leaf cells by the Carbon derived from translocated products of photosynthesis is NADPH-dependent reduction of mannose-6-P to mannitol-1-P, con- used in nonphotosynthetic (sink) tissues as a storage reserve for later stituting a reduction of carbon-1. The subsequent removal of Pi from use or in the immediate production of energy and carbon skeletons for mannitol-1-P by a phosphatase produces mannitol. Sucrose and man- synthetic reactions to build cellular components (Fig. 2). This scheme nitol biosynthesis share common intermediates originating from triose is a critical aspect of growth in all plants, as assimilation of translo- phosphates exported from chloroplasts and leading to the production cated photosynthates is the only means of C gain in nonphotosynthetic of fructose-6-P (Fig. 1). In lower organisms, such as bacteria, yeast, tissues. In celery, radioactively labeled sucrose supplied to excised and algae, mannitol is formed by the reduction of carbon-2 in fructose tissues is readily converted to CO2, whereas the conversion of mannitol or fructose-6-P to form mannitol or mannitol-1-P, respectively. Thus, to CO2 is largely restricted to actively growing sinks (Fellman and vascular plants synthesize mannitol by a different set of enzymatic Loescher, 1987). Petioles of celery contain a substantial storage pool reactions than other organisms. of mannitol, with ≈80% of the mannitol being in vacuoles of the petiole The enzyme catalyzing the reduction of mannose-6-P to mannitol- storage parenchyma cells and the remainder in the cytosol (Keller and 1-P, mannose-6-P reductase (M-6-PR), was purified to homogeneity Matile, 1989). Activity of the mannitol synthetic enzyme, M-6-PR, is from celery (Loescher et al., 1992). The molecular mass of the native extremely low in petiole parenchyma tissue, supporting the hypothesis enzyme was estimated, by physical methods, to be about 58 kD. The that mannitol is accumulated from the phloem translocation stream molecular mass of the monomer, as determined by sodium dodecyl (Keller and Matile, 1989). sulfate (SDS) gel electrophoresis, was 35 kD, leading to the conclusion The initial enzymatic step by which mannitol enters the central metabolism in sink tissues of vascular plants was only recently discovered. A mannitol-oxidizing enzyme, NAD-dependent Received for publication 17 Mar. 1995. Accepted for publication 8 Apr. 1995. mannitol:mannose 1-oxidoreductase, was identified in celeriac [Apium This work was supported in part by the North Carolina Agricultural Research graveolens var. rapaceum (Mill.) Gandich.] plants and subsequently Service Raleigh, NC 27695; U.S. Dept. of Agriculture Competitive Grant no. in celery (Stoop and Pharr, 1992). Unlike M-6-PR, mannitol dehydro- 9302250; the Belgian American Educational Foundation (J.M.H. Stoop); and genase (MDH) is not active in photosynthetic leaves. MDH occurs the Swiss National Foundation (M.E. Studer Feusi). Use of trade names does only in actively growing sink tissues of celery and celeriac plants, and not imply endorsement of the products named nor criticism of similar ones not the enzyme is absent or barely detectable in photosynthetic leaves and named. The cost of publishing this paper was defrayed in part by the payment mature petioles that store mannitol. Distribution of MDH activity of page charges. Under postal regulations, this paper therefore must be hereby throughout celery plants (Stoop and Pharr, 1992) mirrors the previ- marked advertisement solely to indicate this fact. 1Professor, Dept. of Horticultural Science. ously reported capacity of the tissues to convert mannitol to CO2 2Postdoctoral Research Associate, Dept. of Horticultural Science. (Fellman and Loescher, 1987). Additionally, the activity of the en- 3Visiting Assistant Professor, Dept. of Horticultural Science. zyme in various tissues of the plant is inversely correlated with tissue 4Laboratory Research Technician, Dept. of Horticultural Science. mannitol concentration (Stoop and Pharr, 1992). Collectively, these 5Associate Professor, Dept. of Genetics. observations strongly suggest a role for MDH in mannitol catabolism.

1182 HORTSCIENCE, VOL. 30(6), OCTOBER 1995 Fig. 1. Simplified scheme showing the biosynthesis of mannitol from photosynthetically fixed CO2.

The celery MDH is unlike mannitol dehydrogenases found in lower organisms. The latter enzymes are all 2-oxidoreductases that catalyze oxidation of mannitol or mannitol-1-P at carbon-2 to form fructose or fructose-6-P as a product. The enzyme from celery and celeriac is a 1-oxidoreductase that catalyzes the oxidation of mannitol at carbon-1 to form mannose. NAD functions as the electron acceptor while NADP does not. A somewhat analogous mannitol-oxidizing enzyme that attacks carbon-1 was isolated from mitochondria of the land snail (Helix aspersa Miller); however, the snail enzyme does not depend on pyridine nucleotide as a cosubstrate (Vorhaben et al., 1980). Aside from the single report in celery and celeriac, there is apparently no precedent for the existence of a NAD-dependent mannitol 1- oxidoreductase. It will be of interest to learn if the occurrence of this particular type of mannitol dehydrogenase is restricted to vascular plants. Recently, our laboratory has isolated highly purified MDH from mannitol-grown celery suspension culture. Unlike the biosyn- thetic enzyme, MDH is apparently a monomer. Molecular size of the native enzyme as estimated by gel filtration chromatography was ≈52 kD. The denatured protein runs as a single 46-kD band on SDS gels (unpublished). Polyclonal antibodies raised against the SDS-denatured peptide are cross-reactive with the enzyme from celery plants. In celery, mannose derived from the oxidation of mannitol subse- quently is phosphorylated at carbon-6 by hexokinase to form man- nose-6-P, which in turn can be isomerized by phosphomannose isomerase (PMI) to fructose-6-P (Stoop and Pharr, 1992, 1993, 1994a). Fructose-6-P is a central metabolite that can be used in the formation of many important compounds for growth and respiratory energy production (Fig. 2). Extracts of celery contain relatively high levels of PMI (Stoop and Pharr, 1992, 1993, 1994a). This enzyme is critical for the use of mannose as a C source. Certain plants are incapable of rapidly metabolizing exogenously supplied mannose, probably due to their lack of sufficiently high levels of PMI (Herold and Lewis, 1977). In these species, mannose is toxic, apparently due to the accumulation of mannose-6-P with the resulting sequestration of cellular phosphate Fig. 2. Simplified scheme depicting the assimilation of mannitol in (Herald and Lewis, 1977). Cell suspension cultures of celery grow nonphotosynthetic sink tissues, starting with its oxidation to mannose.

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