D. Mason Pharr

Salicylic acid stimulates secretion of the normally symplastic enzyme mannitol dehydrogenase: a possible defense against mannitol-secreting fungal pathogens

The sugar alcohol mannitol is an important carbohydrate with well-documented roles in both metabolism and osmoprotection in many plants and fungi. In addition to these traditionally recognized roles, mannitol is reported to be an antioxidant and as such may play a role in host–pathogen interactions. Current research suggests that pathogenic fungi can secrete mannitol into the apoplast to suppress reactive oxygen-mediated host defenses. Immunoelectron microscopy, immunoblot, and biochemical data reported here show that the normally symplastic plant enzyme, mannitol dehydrogenase (MTD), is secreted into the apoplast after treatment with the endogenous inducer of plant defense responses salicylic acid (SA). In contrast, a cytoplasmic marker protein, hexokinase, remained cytoplasmic after SA-treatment. Secreted MTD retained activity after export to the apoplast. Given that MTD converts mannitol to the sugar mannose, MTD secretion may be an important component of plant defense against mannitol-secreting fungal pathogens such as Alternaria. After SA treatment, MTD was not detected in the Golgi apparatus, and its SA-induced secretion was resistant to brefeldin A, an inhibitor of Golgi-mediated protein transport. Together with the absence of a known extracellular targeting sequence on the MTD protein, these data suggest that a plant’s response to pathogen challenge may include secretion of selected defensive proteins by as yet uncharacterized, non-Golgi mechanisms.

Partial purification and characterization of mannitol: mannose 1-oxidoreductase from celeriac (Apium graveolens var. rapaceum) roots

A mannitol:mannose 1-oxidoreductase was isolated from celeriac (Apium graveolens var. rapaceum) root tips by fractionation with (NH4)2SO4, followed by chromatography on a Fractogel DEAE column and then concentration with (NH4)2SO4. This newly discovered mannitol dehydrogenase catalyzes the NAD-dependent oxidation of mannitol to mannose, not mannitol to fructose. The sugar product of the enzyme reaction was identified by three independent HPLC systems and by an enzymatically linked system as being mannose and not fructose or glucose. Normal Michaelis--Menten kinetics were exhibited for both mannitol and NAD with Km values of 72 and 0.26 mM, respectively, at pH 9.0. The Vmax was 40.14 mumol/h/mg protein for mannitol synthesis and 0.8 mumol/h/mg protein for mannose synthesis at pH 9.0. In the polyol oxidizing reaction, the enzyme was very specific for mannitol with a low rate of oxidation of sorbitol. In the reverse reaction, the enzyme was specific for mannose. The enzyme was strongly inhibited by NADH and sensitive to alterations of NAD/NADH ratio. The enzyme is of physiological importance in that it is mainly localized in root tips (sink tissue) where it functions to convert mannitol into hexoses which are utilized to support root growth. Product determination and kinetic characterization were carried out on an enzyme preparation with a specific activity (SA) of 30.44 mumol/h/mg protein. Subsequently, the enzyme was further purified to a SA of 201 mumol/h/mg protein using an NAD affinity column. This paper apparently represents the first evidence of the existence of a mannitol:mannose 1-oxidoreductase and also the first evidence of the presence of a mannitol dehydrogenase in vascular plants.

Developmental changes in carbohydrate concentration and activaties of sucrose metabolizing enzymes in fruits of two Capsicum annuum L. genotypes

Abstract Recently it was reported that hexose sugars accumulate in pepper ( Capsicum annuum ) fruit during ripening as a result of increased acid invertase (EC 3.2.1.26) activity. In this study, fruit of two pepper genotypes which differ in size, shape and carbohydrate concentration were evaluated during growth and development. Carbohydrate concentrations and sucrose metabolizing enzyme activities were measured. Starch and sucrose concentration increased midway through development and declined during fruit ripening. The increase in sucrose concentration was seemingly unrelated to activity of sucrose-phosphate synthase (SPS) (EC 2.4.1.14). As sucrose and starch concentrations declined during ripening, hexose sugar concentration increased. This change in carbohydrate composition was associated with increased acid and neutral invertase activity. Overall trends in carbohydrate pools and enzyme activities were similar between the two genotypes. However, the smaller jalapeno-shaped pepper had much higher carbohydrate concentrations and enzyme activities than the bell pepper. The most striking differences between the two genotypes were a very high concentration of hexose sugars (> 90 mg/g fresh wt.) and the extent to which activity of acid and neutral invertases increased during the final stages of ripening in the jalapeno-shaped pepper compared to the bell-shaped pepper. Thus, there may be an association between increased invertase activity and the accumulation of hexose sugars in pepper fruit during ripening.

Characterization of NAD-dependent mannitol dehydrogenase from celery as affected by ions, chelators, reducing agents and metabolites

Abstract NAD-dependent mannitol dehydrogenase (MTD) from celery (Apium graveolens L. var. dulce (Mill.) Pers.) provides the initial step by which mannitol is committed to central metabolism and plays a critical role in regulating mannitol concentration in the plant. The pH optimum for mannitol oxidation occurs at pH 9.5 whereas the optimum for mannose reduction occurs at pH 6.5. Michaelis–Menten kinetics were exhibited for mannitol and NAD with Km values of 64 and 0.14 mM, respectively at pH 9.5. The Km for mannose and NADH were 745 mM and 1.27 μM, respectively at pH 6.5. The high Km for mannose is consistent with a reaction in situ favoring mannitol oxidation rather than mannose reduction. The observed down-regulation of MTD in salt stressed celery is not due to a direct inhibition by NaCl or macronutrients. Inhibition by the chelator 1,10-phenanthroline suggests that zinc is required for MTD activity. Reducing agents DTT, DTE and β-mercaptoethanol inactivated MTD reversibly. At pH 7.0, ADP and to a lesser extend AMP and ATP were competitive inhibitors, with respect to NAD, having apparent Ki’s of 0.24, 0.64 and 1.10 mM, respectively.

A Nicotiana tabacum cell culture selected for accelerated growth on mannose has increased expression of phosphomannose isomerase

Abstract Phosphomannose isomerase (PMI), a key enzyme in mannose (Man) metabolism, is expressed at very low levels in many plant species. For example, measured PMI activity in Nicotiana tabacum (NT1) suspension cells is relatively low, resulting in slow metabolism of Man. Not surprisingly then, NT1 cultures were observed to grow six times faster on glucose (Glc) than on Man as sole carbon source. We report here the selection of a mutant NT1 cell line that grows four times faster on Man than the parental culture from which it was derived. This cell line had fivefold greater PMI activity than the parental culture, which likely contributes to the increased growth rate on Man. The selected line continued to express elevated PMI activity after transfer to Glc, suggesting a stable genetic change. However, the selected line grew more slowly than the wild type on Glc. This was likely due to a more than 50% reduction in hexokinase (HK) activity, an enzyme that is required for the phosphorylation and subsequent metabolism of Glc. Unlike HK, fructokinase activity was essentially unchanged in the mutant cell lines. However, activities of the carbohydrate metabolic enzymes phosphoglucose isomerase and 6-phosphogluconate dehydrogenase were also higher in mutant cells.