Translocation of Photosynthates into Vacuoles in Spinach Leaf Protoplasts

Abstract

A method was developed for the isolation of vacuoles from the mesophyll protoplasts of spinach leaf, employing the discontinuous Ficoll density gradient centrifugation technique. Isolated vacuole preparations were judged to be free from other organellar fractions based on the assays of marker enzyme activities of individual organelles.Using this isolation method, a time-dependent translocation of '4C-labeled photosynthates into vacuoles was determined. In contrast to a significant transport of 4C organic acids such as malate and citrate within 10 to 15 minutes '4C neutral sugars and amino acids were barely transported into vacuoles during 40 minutes incubation, in spite of the fact that a relatively large amount of these compounds are found in the vacuoles. It was also found that a majority of I"Cqsucrose remains in the cytosoL apparently not actively moving into the vacuoles. Overall results appear to suggest that vacuoles are not actively engaged in photosynthetic carbon metabolism in spinach leaf protoplasts.The isolation of central vacuoles from various higher plant tissues has been reported by numerous investigators who have shown that several solutes, e.g. salts, neutral sugars, amino acids, organic anions, and secondary metabolites, accumulate in vacuoles, and the mechanism(s) of transport of these substances across tonoplasts have been discussed (15). It has also been established that some lytic enzymes such as proteinase, DNase, phosphodiesterase, and a-mannosidase are localized in vacuoles (19) and that these enzymes are possibly involved in the hydrolytic breakdown of storage substances as well as cellular components (27). Therefore, in some aspects, the function of plant vacuoles is similar to animal lysosomes. An example of the dynamic function of plant vacuoles is provided by the diurnal rhythmic fluctuation in the contents of vacuoles isolated from Sedum telephium and the role of these vacuoles in the organic acid metabolism of this CAM plant (10). Active transport of sugars into vacuoles has been demonstrated using sugarbeet (3, 5), sugarcane (22), and rubber tree (2). The mechanism of ion transport into vacuoles is largely unknown, but experimental evidence is available showing the involvement of ATPase in the ion movement associated with vacuoles (11,17,24,25).In MATERIALS AND METHODSProtoplasts. Leaf of spinach (Spinacia oleracea cv Kyoho), freshly harvested in the field, was used throughout this investigation. Protoplasts were prepared according to the two-step enzymic digestion method described by Nishimura and Akazawa (18) after a slight modification; in the second step, 2% Driselase was added to 2% Cellulase Onozuka. Also, to avoid contamination with epidermal protoplasts, the final preparation of protoplasts obtained by the centrifugation at 100g for 3 min was washed twice with 0.7 M mannitol containing 3% Percoll. Epidermal protoplasts were not sedimented in 0.7 M mannitol containing 3% Percoll. The precipitate was suspended with 0.7 M mannitol and washed twice with 0.7 M mannit...

Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflata

“…After digestion, released protoplasts were passed through a 150-μm nylon mesh and washed with 0.7 M mannitol for three times. Protoplasts were treated with DEAE-dextran solution for the lysis and released vacuoles were separated by the discontinuous Ficoll gradient centrifugation as previously described by Asami et al [ 74 ].…”

Fructose 2,6-Bisphosphate Hydrolyzing Enzymes in Higher Plants

“…Such a relative specificity for the i3-anomeric form of sugar phosphates has already been observed for an acid phosphatase of mung bean (7). The acid phosphatase of spinach leaf has been previously shown to be localized in the vacuole (1). Phosphatase 11 This enzyme specifically cleaves the osyl-phosphate linkage of several phosphate sugars.…”

A Simple Method for Estimating Intactness of Spinach Leaf Protoplasts by Glycolate Oxidase Assay

“…Studies on plant protoplasts have made a marked progress in recent years in various fields of plant biology (5), including (a) photosynthesis (4,11,18); (b) organelle isolation (2,(15)(16)(17)(18)(19)(20); (c) protein synthesis (12); (d) developmental studies of plant cells and cell fusion (3,23,25); and (e) metabolite transport (2,7). Due to the technological advancement of producing potentially powerful microbial hydrolases, preparation of protoplasts from various types of plant cells has been reported (5).…”