Seasonal changes in the amounts of the NAD-dependent sorbitol dehydrogenase (NAD-SDH) (enzyme code, 184.108.40.206) 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.
Hideaki Yamaguchi, Yoshinori Kanayama, Junichi Soejima, and Shohei Yamaki
Motoko Iida, Nancy A. Bantog, Kunio Yamada, Katsuhiro Shiratake, and Shohei Yamaki
The regulation of NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 220.127.116.11) 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.
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.).
Riccardo Lo Bianco, Mark Rieger, and She-Jean S. Sung
Terminal portions of `Flordaguard' peach roots [Prunus persica (L.) Batsch] were divided into six segments and the activities of NAD+-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX), sucrose synthase (SS), soluble acid invertase (AI), and soluble neutral invertase (NI) were measured in each segment 10, 15, and 20 days after seed germination. The same type of experiment was conducted with terminal portions of `Flordaguard' and `Nemaguard' peach shoots except that one of the six segments consisted of the leaflets surrounding the apex. Independent of the age of individual roots, activities of SDH and AI were consistently highest in the meristematic portion and decreased with tissue maturation. In shoots, AI was the most active enzyme in the elongating portion subtending the apex, whereas SDH was primarily associated with meristematic tissues. A positive correlation between SDH and AI activities was found in various developmental zones of roots (r = 0.96) and shoots (r = 0.90). Sorbitol and sucrose contents were low in roots regardless of distance from tip, while sucrose showed a decreasing trend with distance and sorbitol, fructose, and glucose increased with distance from the meristem in shoots. Activity of SDH in internodes, but not apices, correlated with shoot elongation rate of both cultivars, whereas activities of other enzymes did not correlate with shoot elongation rate. We conclude that AI and SDH are the predominant enzymes of carbohydrate catabolism and the best indicators of sink growth and development in vegetative sinks of peach.
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
Takaya Moriguchi, Kazuyuki Abe, Tetsuro Sanada, and Shohei Yamaki
Abbreviations: DTT, dithiothreitol; NAD+-SDH, NAD+-dependent sorbitol dehydrogenase; SPS, sucrose-phosphate synthase; SS, sucrose synthase. 1 Present address: Laboratory of Horticulture, School of Agriculture, Nagoya Univ., Chikusa, Nagoya, 464
Douglas D. Archbold* and Marta Nosarszewski
Acquiring sufficient carbohydrate is essential for successful apple fruit set. Sorbitol may be the dominant carbohydrate imported by growing fruit, and the rate of sorbitol accumulation may be a function of NAD-dependent sorbitol dehydrogenase (SDH; EC 18.104.22.168) activity. Prior work indicated that SDH activity from whole fruit (seeds plus cortex) increased for 2 or 3 weeks after initiation of fruit growth and then declined through 5 weeks. Using SDH activity assays, an SDH-specific antibody, and SDH-specific probes in Northern analyses, it is evident that SDH is expressed and is active in both apple seed and cortex tissue during the first few weeks of fruit growth. On a per unit protein basis, SDH activity in seeds increased by the pattern described above while that in fruit was generally lower and constant. During this same period of time, the sorbitol content of the expressed sap of apple shoots was analyzed. The sorbitol concentration was 50- to 100-fold higher than the sucrose concentration. The concentrations of both carbohydrates changed in parallel to the change in SDH activity of whole fruit and seeds. The lowest SDH activity and sap sorbitol levels preceded and/or coincided with the beginning of the natural fruit drop (or June drop) period.
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.
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 22.214.171.124), which catalyzes the oxidation of sorbitol to fructose ( Loescher, 1987 ; Yamaguchi et al., 1994 ; Yamaki and
Yong Zhang, Chunxia Fu, Yujing Yan, Xiaodan Fan, Yan’an Wang, and Ming Li
, and zinc fertilization Plant Soil 306 37 48 Yamada, K. Oura, Y. Mori, H. Yamaki, S. 1998 Cloning of NAD-dependent sorbitol dehydrogenase from apple fruit and gene expression Plant Cell Physiol. 39 1375 1379 Yamaguchi, H. Kanayama, Y. Soejima, J. Yamaki