Subcellular Localization of 2-(β-d-Glucosyloxy)-Cinnamic Acids and the Related β-glucosidase in Leaves of Melilotus alba Desr.

Abstract

The distribution of the glucosides of trans-and cis-2-hydroxy cinnamic acid and of the /8 glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts. (12,13). One is the glucoside of 2-hydroxy-trans-cinnamic acid (o-coumaric acid) or o-coumaryl glucoside. The other is the glucoside of 2-hydroxy-cis-cinnamic acid (coumarinic acid) or coumarinyl glucoside, also known as 'bound coumarin' (12). A ,B-glucosidase which also occurs in sweet clover leaves hydrolyzes the coumarinyl glucoside (bound coumarin) but is inactive towards the other glucoside (12, 13). When clover leaves are disrupted, the glucosidase liberates coumarinic acid, which spontaneously lactonizes and forms coumarin.Recent studies have shown that dhurrin, another plant glucoside, and the enzymes catalyzing its decomposition are located in different cellular compartments (11). In this paper, we examine the localization of these two glucosides (referred to as the coumarin-glucosides) and the related ,B-glucosidase in leaves of M. alba using epidermal and mesophyll tissues, mesophyll protoplasts, and vacuoles isolated from mesophyll protoplasts. Assay of o-Coumaric Acid and Two Related Glucosides. oCoumaric acid and the two isomeric coumarin-glucosides were determined by the fluorometric method of Haskins and Gorz (5). Tissue samples (lower epidermis or mesophyll with upper epidermis attached) were ground in 95% ethanol in a mortar with a pestle and filtered through a weighed glass filter (Whatman GF/ C). The filtrates from lower epidermis and mesophyll plus upper epidermis were made to 5 and 25 ml, respectively, with 95% ethanol, and their A at 649 nm and 665 nm was measured imnmediately to determine their Chl content (22). Five ml of each alcoholic filtrate was dried in vacuo, and 5.0 ml water were added to dissolve the residue. A 1-ml aliquot of the well-mixed aqueous extract was added to 9 ml 2.5 N NaOH in a Pyrex test tube. After mixing, two 1.0-ml aliquots of the alkaline solution were withdrawn, added to 9 ml water in a test tube, and saved for fluorometric analysis to determine any o-coumaric acid present. The alkaline solution remaining (8.0 ml) was autoclaved for 45 min at 120°C to hydrolyze the coumarin glucosides present in the original sample (12). The autoclaved solution was cooled, and 1-ml samples were diluted to 10 ml with water. These and the earlier nonautoclaved diluted samples were exposed to UV light (peak near 360 nm) for 15 min at a distance of approximately 15 cm from the filter to obtain an equilibrium mixture of the cis-and trans-isomer...

Conformational Changes of Apigenin 7‐O‐(6‐O‐malonylglucoside), a Vacuolar Pigment from Parsley, with Solvent Composition and Proton Concentration

“…Metabolism and storage of other phenolic compounds, such as cyanogenic glycosides [ 171, 2-coumaric acid glucoside [18], coumaryl glucosides [I91 and anthocyanins [20], have been studied recently in plants. These phenol glucosides, but not the respective aglycons, were shown to be deposited in vacuoles.…”

Tissue Level Compartmentation of (R)-Amygdalin and Amygdalin Hydrolase Prevents Large-Scale Cyanogenesis in Undamaged Prunus Seeds

“…Although tissue level compartmentation was also observed in light-grown sorghum leaves (Kojima et al, 1979), it remains to be seen whether this strategy is common to all cyanogenic species. Current evidence with other cyanophoric systems (Kojima et al, 1983;Kakes, 1985;Frehner and Conn, 1987;Mkpong et al, 1990) has been interpreted largely in terms of subcellular compartmentation similar to that shown by coumarinyl glucoside and its respective /3-glucosidase in sweet clover (Oba et al, 1981). However, further studies will be necessary in these cases to unequivocally exclude tissue level compartmentalization.…”

“…glucosinolates [Bones et al, 1991;Poulton and Mprller, 19931 and o-coumaric acid glucosides [Oba et al, 1981]), the large-scale degradation of cyanogenic glycosides to HCN occurs only upon tissue disruption (Conn, 1981). It is generally assumed that premature cyanogenesis is avoided in undamaged plants by compartmentalization of the glycoside and its catabolic enzymes at the tissue or subcellular levels.…”

Intracellular Localization of Lunularic Acid and Prelunularic Acid in Suspension Cultured Cells of Marchantia polymorpha

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