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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.

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

Sorbitol is the major photosynthetic product in peach. In sink tissues, sorbitol is converted to fructose via the NAD-dependent enzyme sorbitol dehydrogenase (SDH). A new assay is described that allows rapid, simple quantitation of SDH activity in growing shoot tips, root tips, and fruits. The activity was measured on the crude extract desalted with a Saphadex G-25 column to eliminate small molecules such as sugars and nucleotides. Optimum buffer type and pH for the enzyme as well as degradation by proteolytic enzymes and stability over time were determined in the present study. Inhibition by dithiothreitol (DTT) was detected at an inhibitor concentration as low as 2 mM, proving the similarity with mammalian SDH. Storage of samples at 4°C overnight resulted in significant loss of enzyme activity. Using this assay, we also correlated SDH activity with sink strength in peach.

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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.).