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Ben-Hong Wu, Shao-Hua Li, Marta Nosarzewski, and Douglas D. Archbold

). Metabolism of sorbitol by sink tissues in apple is primarily via the action of NAD + -dependent sorbitol dehydrogenase (NAD + -SDH, EC 1.1.1.14), which catalyzes the oxidation of sorbitol to fructose ( Loescher, 1987 ; Yamaguchi et al., 1994 ; Yamaki and

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Douglas D. Archbold

Following June drop, apple fruit growth depends on sorbitol import as the primary source of carbon. Sorbitol dehydrogenase plays a key role in carbohydrate metabolism by conversion of sorbitol to fructose, which then enters the general carbohydrate pool. Blocking the pathway and eliminating the source of sorbitol to the fruit by girdling the stem and defoliation after June drop resulted in a decline and eventual cessation of fruit growth. The fruit did not abscise however. Fruit sorbitol and starch levels declined while the fructose, glucose, and sucrose pools did not change. SDH activity declined to low levels and was not detectable in many fruit. The decline in SDH activity was evident 1 week after applying the treatments. A few fruit that resumed growth, presumably after the vascular connection was re-established across the girdle, exhibited normal SDH activity. Feeding sorbitol to whole fruit in vitro via the cut stem raised SDH activity in some fruit, although it was still below control levels.

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Valeria Sigal-Escalada and Douglas D. Archbold

Sorbitol dehydrogenase (SDH) is a key enzyme in apple fruit converting sorbitol into fructose. SDH activity in `Fuji' apple was reported to increase close to harvest, perhaps as part of the ripening process. Aminoethoxyvinylglycine (AVG) is used to delay fruit ripening and prevent fruit drop, though its effect on sorbitol metabolism is not known. To determine if the late season increase in SDH activity is common among apple cultivars and if AVG use affects SDH expression and activity, AVG was applied to `Lodi', `Redchief Delicious', and `Red Fuji' trees 4 weeks before harvest. Control and AVG-treated fruit were collected 1 week prior to, at, and 1 week after the normal harvest date for assessment of ethylene production over time after harvest and SDH presence and activity at harvest. Ethylene production in control fruit increased after harvest and AVG reduced it in all cultivars. `Redchief Delicious' fruit had the highest ethylene production of the treated samples. The levels of SDH activity in controls were similar across dates for `Redchief Delicious' and showed no consistent pattern in the other cultivars. `Redchief Delicious' and `Red Fuji' showed the highest and lowest levels of SDH activity, respectively. AVG did not affect SDH activity in `Redchief Delicious', and substantially increased SDH activity in `Red Fuji' on each of the three harvest dates, and, in `Lodi', only 1 week after normal harvest. SDH presence was confirmed through immunoblotting for all cultivars and harvest dates. Overall, fruit with the greatest reduction in ethylene production in response to AVG also showed changes in SDH activity.

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Hideaki Yamaguchi, Yoshinori Kanayama, Junichi Soejima, and Shohei Yamaki

Seasonal changes in the amounts of the NAD-dependent sorbitol dehydrogenase (NAD-SDH) (enzyme code, 1.1.1.14) protein in developing apple (Malus pumila Mill var. domestica Schneid) fruit were determined by immunoblotting analysis. The amounts of the enzyme protein were very low in young fruit and rose as fruit matured. The weak correlation between enzyme protein and NAD-SDH activity and also the changes in NAD-SDH specific activity suggested that there could be posttranslational modification to the pre-existing enzyme or isoenzyme(s) of NAD-SDH. The changes in the amounts of NAD-SDH protein did not show the same pattern as those in relative growth rate, which is used to express sink activity, especially in young fruit. The role of NAD-SDH on sink activity in apple fruit, therefore, could not be explained simply by the amount and activity of the enzyme. In young fruit, it seems that enzymes other than NAD-SDH would be more directly related with fruit growth.

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Motoko Iida, Nancy A. Bantog, Kunio Yamada, Katsuhiro Shiratake, and Shohei Yamaki

The regulation of NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14) by sugar was investigated by using sliced tissues of japanese pear (Pyrus serotina Nakai cv. Kousui) fruit in order to determine its role in the mechanism of sugar accumulation in fruit tissue. The results of the activities and steady-state levels of the protein and mRNA indicate that NAD-SDH in japanese pear fruit is among the sugar-inducible genes. By preincubating the sliced tissues for 16 hours in a medium without sugar, NAD-SDH activity declined and reached a stable level that was maintained for up to 40 hours. The washing procedure also reduced the sugar concentration in the apoplast and cytosol of the sliced tissues to low concentrations and enabled them to be manipulated by exogenous applications of carbohydrate solutions. Incubation of tissues in 50 or 100 mm sorbitol for 8 hours led to enhanced expression of the NAD-SDH gene as determined by increased mRNA and protein levels and enhanced enzyme activity. The presence of 100 mm glucose, sucrose, or mannitol also gave significant stimulation on the levels of activity, protein, and mRNA of NAD-SDH compared with those of control tissues bathed in media in which the osmotic potential had been adjusted to that of the sugar solutions by adding polyethylene glycol. However, fructose was ineffective in stimulating NAD-SDH activities and the level of the protein was not enhanced but the level of mRNA was increased. Therefore, it is suggested that NAD-SDH gene transcription is enhanced by each sugar investigated, and fructose appears to be unique as it also influences NAD-SDH at a post-transcriptional level.

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Riccardo Lo Bianco, Mark Rieger, and She-Jean S. Sung

Sorbitol is the major photosynthetic product in peach [Prunus persica (L.) Batsch.]. In sink tissues, sorbitol is converted to fructose via NAD+-dependent SDH. A new procedure is described that allows rapid, simple quantification of SDH activity in growing tissues. The procedure uses only 0.01 to 5 g of fresh tissue per sample, such that a single shoot tip, a single root tip, or ≈5 g of fruit flesh can be assayed for SDH activity. Storage of samples at 4 or -20 °C overnight resulted in significant loss of enzyme activity. Thus, freshly harvested tissues were ground with sand in buffer at 2 °C in a mortar and pestle, and the homogenate was centrifuged at 3000 g n to remove particulate matter and sand. The supernatant was desalted on a Sephadex G-25 column, and the eluent was assayed for SDH activity immediately. Activity was determined by measuring the production of NADH per minute in the assay mixture using a spectrophotometer (340 nm). Tris buffer at pH 9.0 was the best for extraction of peach SDH. Activity of SDH was strongly inhibited by dithiothreitol (DTT) in the extraction mixture and by DTT, L-cysteine, or SDI-158 in the assay mixture, similar to results reported for SDH from mammalian tissues. Peach SDH has a Km of 37.7 mm for sorbitol and a pH optimum of 9.5, similar to those reported for apple (Malus × domestica Borkh.) SDH. Unlike older protocols for SDH activity in plant tissues, the new procedure features reduced sample size (1/10 to 1/100 of that which was previously used), smaller volumes of buffer, fewer buffer ingredients, greatly reduced time for sample preparation, yet comparable or higher values of SDH specific activity. Following the same procedure, SDH activity was also measured in Prunus fremontii Wats., Prunus ilicifolia (Nutt.) Walp., and Marianna 2624 plum (P. cerasifera Ehrh. × P. munsoniana Wight & Hedr.).

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Riccardo Lo Bianco and Mark Rieger

The peach [Prunus persica (L.) Batsch (Peach Group)] fruit is a sink organ comprised of different types of tissue, which undergoes three distinct developmental stages during the growth season. The objective of this study was to characterize the activity and partitioning of sorbitol and sucrose catabolism within `Encore' peach fruit to determine whether the two forms of translocated carbon play different roles in the various fruit tissues and/or stages of development. Sorbitol catabolic activity was defined as the sum of NAD-dependent sorbitol dehydrogenase (SDH) and sorbitol oxidase (SOX) activities, whereas sucrose catabolic activity was defined as the sum of sucrose synthase (SS), soluble acid invertase (AI), and neutral invertase (NI) activities. Partitioning of sorbitol and sucrose catabolism in each tissue was calculated as percentage of total sorbitol or sucrose catabolic activity in the entire fruit. At cell division, sorbitol catabolic activity was similar in the endocarp and mesocarp, but lower in the seed. However, sorbitol catabolism was mostly partitioned into the mesocarp, due to its large size compared to that of other tissues. SDH was more active in the mesocarp, while SOX was more active in the endocarp. Sucrose catabolism was most active and partitioned mainly into the endocarp. At endocarp hardening, both sorbitol and sucrose catabolic activities were highest in the seed, but despite this, sucrose catabolism was partitioned mostly in the mesocarp. At cell expansion, sorbitol and sucrose catabolic activities were still higher in the seed only when expressed on a weight basis and similar in mesocarp and seed when expressed on a protein basis. Both sorbitol and sucrose catabolism were partitioned mostly into the mesocarp. Sorbitol and sucrose contents were generally higher in the tissues that exhibited lower catabolic activities. All carbohydrates were always partitioned mostly into the mesocarp. Our results show that, at the cell division and endocarp hardening stages, sorbitol and sucrose catabolism are partitioned differently in the fruit and that SDH activity may play an important role in mesocarp cell division and final fruit size determination.

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Riccardo Lo Bianco and Mark Rieger

In peach [Prunus persica (L.) Batsch (Peach Group)], both sorbitol and sucrose are used for source to sink carbon (C) transport, yet their specific functions in fruit growth and development remain unclear. Growth rate (GR), respiration rate (R), carbohydrate content, and the activities of sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX), sucrose synthase (SS), acid invertase (AI), and neutral invertase (NI) were determined in `Encore' peaches to study the specific functions of sorbitol and sucrose in each phase of fruit development (an early period of rapid cell division, a relatively inactive intermediate stage where endocarp (pit) hardening occurs, and a final swelling due to cell expansion). Fruit growth and respiration rates (mol C/fruit per day) were always positively correlated, but the growth coefficient (gc) relating them was significantly higher at cell division, when maintenance respiration (Rm) was nearly absent. Sorbitol and sucrose appeared to participate equally in growth and maintenance respiration. Contents of sorbitol and sucrose both correlated positively to GR, and their rates of accumulation increased from early to late growth stages in similar fashion. SDH activity was always positively correlated with sink strength and GR, but with R only at endocarp hardening (r = 0.632). SOX activity was also correlated with sink strength and GR in the early (r = 0.514 and 0.553) and late (r = 0.503 and 0.495) growth phases, but not at endocarp hardening, and was correlated with R in two of three growth phases. Among sucrose cleavage enzymes, AI activity was positively correlated with sink strength, GR, and R more strongly than the others (r = 0.51 to 0.80), but only in the cell division and cell expansion periods. SS activity was correlated with sink strength and R only at endocarp hardening, and NI activity was generally not correlated to sink strength, GR, or R. We conclude that sorbitol and sucrose play similar roles in fruit development, and the enzymes associated with their metabolism work in concert to produce the observed changes in growth and respiration.

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Yong Zhang, Chunxia Fu, Yujing Yan, Xiaodan Fan, Yan’an Wang, and Ming Li

al., 1982 ). Sorbitol dehydrogenase (SDH) and sorbitol oxidase (SOX) catalyze the metabolism of sorbitol to fructose and glucose, respectively, in apple fruit ( Park et al., 2002 ; Yamada et al., 1998 ). Various researchers have examined changes in

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Dongfeng Liu, Junbei Ni, Ruiyuan Wu, and Yuanwen Teng

., 1999 ; Yamaki, 1986 ). Activity of NAD + -dependent sorbitol dehydrogenase (NAD + -SDH) is positively correlated with sink strength throughout peach ( Prunus persica L.) fruit development, whereas sorbitol oxidase (SOX) activity is not correlated with