Cloning and characterization of NADP-mannitol dehydrogenase cDNA from the button mushroom, Agaricus bisporus, and its expression in response to NaCl stress.

Mannitol, a six-carbon sugar alcohol, is the main storage carbon in the button mushroom, Agaricus bisporus. Given the physiological importance of mannitol metabolism in growth, fruit body development, and salt tolerance of A. bisporus, the enzyme responsible for mannitol biosynthesis, NADP-dependent mannitol dehydrogenase (MtDH) (EC 1.1.1.138), was purified to homogeneity, and MtDH cDNA was cloned, sequenced, and characterized. To our knowledge, this represents the first report on the isolation of a cDNA encoding an NADP-dependent mannitol dehydrogenase. The MtDH cDNA contains an open reading frame of 789 bp encoding a protein of approximately 28 kDa. The N-terminal and internal amino acid sequences of the deduced protein exactly matched the ones determined from the purified MtDH subunit, whereas the amino acid composition of the deduced protein was nearly identical to that of the purified MtDH. The MtDH cDNA showed high homology with a plant-induced short-chain dehydrogenase from Uromyces fabae. Phylogenetic analysis based on amino acid sequences from mannitol(-1-phosphate) dehydrogenases indicated a close relationship between the substrate specificity of the enzymes and phylogenetic differentiation. Salt-stressed fruit bodies showed an overall increase in mannitol biosynthesis, as was evident from the increase in MtDH activity, MtDH abundance, and MtDH RNA accumulation. Furthermore, the MtDH transcript level seems to be under developmental control, as MtDH RNA accumulated during maturation of the fruit body.

Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3.

Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.

Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures.

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 mumol h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elution. Sodium dodecylsulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immuno-reactive 40-kD protein.

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.

Carbon gain and photosynthetic response of chrysanthemum to photosynthetic photon flux density cycles.

Most models of carbon gain as a function of photosynthetic irradiance assume an instantaneous response to increases and decreases in irradiance. High- and low-light-grown plants differ, however, in the time required to adjust to increases and decreases in irradiance. In this study the response to a series of increases and decreases in irradiance was observed in Chrysanthemum x morifolium Ramat. "Fiesta" and compared with calculated values assuming an instantaneous response. There were significant differences between high- and low-light-grown plants in their photosynthetic response to four sequential photosynthetic photon flux density (PPFD) cycles consisting of 5-minute exposures to 200 and 400 micromoles per square meter per second (mumol m(-2)s(-1)). The CO(2) assimilation rate of high-light-grown plants at the cycle peak increased throughout the PPFD sequence, but the rate of increase was similar to the increase in CO(2) assimilation rate observed under continuous high-light conditions. Low-light leaves showed more variability in their response to light cycles with no significant increase in CO(2) assimilation rate at the cycle peak during sequential cycles. Carbon gain and deviations from actual values (percentage carbon gain over- or underestimation) based on assumptions of instantaneous response were compared under continuous and cyclic light conditions. The percentage carbon gain overestimation depended on the PPFD step size and growth light level of the leaf. When leaves were exposed to a large PPFD increase, the carbon gain was overestimated by 16 to 26%. The photosynthetic response to 100 mumol m(-2) s(-1) PPFD increases and decreases was rapid, and the small overestimation of the predicted carbon gain, observed during photosynthetic induction, was almost entirely negated by the carbon gain underestimation observed after a decrease. If the PPFD cycle was 200 or 400 mumol m(-2) s(-1), high- and low-light leaves showed a carbon gain overestimation of 25% that was not negated by the underestimation observed after a light decrease. When leaves were exposed to sequential PPFD cycles (200-400 mumol m(-2) s(-1)), carbon gain did not differ from leaves exposed to a single PPFD cycle of identical irradiance integral that had the same step size (200-400-200 mumol m(-2) s(-1)) or mean irradiance (200-300-200 mumol m(-2) s(-1)).

Photosynthetic dynamics in chrysanthemum in response to single step increases and decreases in photon flux density.

The time-course of CO(2) assimilation rate and stomatal conductance to step changes in photosynthetic photon flux density (PPFD) was observed in Chrysanthemum x morifolium Ramat. ;Fiesta'. When PPFD was increased from 200 to 600 micromoles per square meter per second, the rate of photosynthetic CO(2) assimilation showed an initial rapid increase over the first minute followed by a slower increase over the next 12 to 38 minutes, with a faster response in low-light-grown plants. Leaves exposed to small step increases (100 micromoles per square meter per second) reached the new steady-state assimilation rate within a minute. Both stomatal and biochemical limitations played a role during photosynthetic induction, but carboxylation limitations seemed to predominate during the first 5 to 10 minutes. Stomatal control during the slow phase of induction was less important in low-light compared to high-light-grown plants. In response to step decreases in PPFD, photosynthetic rate decreased rapidly and a depression in CO(2) assimilation prior to steady-state was observed. This CO(2) assimilation ;dip' was considerably larger for the large step (400 micromoles per square meter per second) than for the small step. The rapid photosynthetic response seems to be controlled by biochemical processes. High- and low-light-grown plants did not differ in their photosynthetic response to PPFD step decreases.