Mobilization and Utilization of Cyanogenic Glycosides - NCBI

Plant Physiol. (1988) 86, 711-716

0032-0889/88/86/0711/06/$01 .00/0

Mobilization and Utilization of Cyanogenic Glycosides THE LINUSTATIN PATHWAY Received for publication June 1, 1987 and in revised form August 24, 1987

DIRK SELMAR*, REINHARD LIEBEREI, AND BOLE BIEHL Botanisches Institut der Technischen Universitat Braunschweig Mendelssohnstr. 4, Postfach 3329, D-3300

Braunschweig, Federal Republic of Germany In addition, a linustatin-splitting diglucosidase in Hevea is described. From its activity in different developmental stages and in different tissues it is suggested that this enzyme is involved in the metabolism of cyanogenic glycosides. It is deduced that linustatin is a metabolite in the pathway by which linamarin is metabolized and utilized.

ABSTRACT In the seeds of Hevea brasiliensis, the cyanogenic monoglucoside linamarin (2-b3-D-glucopyranosyloxy-2-methylpropionitrile) is accumulated in the endosperm. After onset of germination, the cyanogenic diglucoside linustatin (2-[6-f8-D-glucosyl-,f-D-glucopyranosyloxyJ-2-methylpropionitrile) is formed and exuded from the endosperm of Hevea seedlings. At the same time the content of cyanogenic monoglucosides decreases. The linustatin-splitting diglucosidase and the .3-cyanoalanine synthase that assimilates HCN, exhibit their highest activities in the young seedling at this time. Based on these observations the folowing pathway for the in vivo mobilization and metabolism of cyanogenic glucosides is proposed: storage of monoglucosides (in the endosperm)-gucosylation-transport of the diglucoside (out of the endosperm into the seedling)-cleavage by diglucosidase-reassimilation of HCN to noncyanogenic compounds. The presence of this pathway demonstrates that cyanogenic glucosides, typical secondary plant products serve in the metabolism of developing plants as N-storage compounds and do not exclusively exhibit protective functions due to their repellent effect.

MATERIALS AND METHODS Seed Drainage. The seed drainage technique is described elsewhere (17). Additionally, some of these seed drainage experiments were run in a gastight system in which a stream of moistened air was used to exchange the atmosphere in the experimental system continuously. By bubbling this air through 5 ml of 1 M NaOH any HCN liberated from the seedling during the experiment was trapped for quantitative determinations. HCN Determination. The HCN was estimated with the Merck Spectroquant kit for cyanide (data sheet 130 259 8 Do dt/5, Fa. Merck). This assay is based on the method of Aldridge (1).

Linustatin (2-[6-l3-D-glucosyl-,3-D-glucopyranosyloxy]-2-meth-

ylpropionitrile) is a relatively rare cyanogenic diglucoside found in seeds of Linum usitatissimum (18) and in green tissues of different species of Passiflora (19). Quite recently, linustatin has also been observed in seeds of Hevea brasiliensis (17) together with the related cyanogenic monoglucoside linamarin (2-13-Dglucopyranosyloxy-2-methylpropionitrile). It has been shown that during the development of H. brasiliensis seedlings linamarin is metabolized to noncyanogenic compounds without any liberation of HCN (10). Furthermore, the amount of linamarin stored in the endosperm (11) and the occurrence of the HCN metabolizing enzyme f-cyanoalanine synthase in the growing seedling (9) indicate, that for consumption linamarin has to be transported from the endosperm into the young seedling (15). As a highly active linamarase is present in the apoplastic space between endosperm storage tissue and cotyledons (15), the linamarin transported out of the endosperm would be split as soon as it enters this extracellular space. In order to be protected against this cleavage by linamarase, linamarin has to be transported in a modified form, which cannot be split by the linamarin-cleaving Hevea ,3-glycosidase (linamarase). This enzyme, which occurs in all Hevea tissues does not split linustatin (16). For this reason it was assumed that linustatin functions as a protected transport form of linamarin (17). This paper demonstrates that linustatin occurs in Hevea seedlings only at that developmental stage when the content of cyanogenic monoglucoside linamarin decreases. 711

Alkaline samples were neutralized with HCl before the test was carried out. The determination of the HCN-potential is described by Lieberei (12). Enzyme Preparation. To prepare protein solutions for the enzyme tests, plant material was frozen in liquid N2 and crushed. Then the powdered material was homogenized in a blender (3 x 10 s) with 20 mm phosphate buffer (pH 6.5) (3 ml buffer per g fresh weight). The homogenate was squeezed through four layers of cheesecloth. The supernatant-was either used directly for enzyme tests or it was concentrated by precipitation with ammonia sulfate (15-85% saturation). A subsequent gel filtration was carried out with a G-150-gel (Spehadex, Pharmacia). Enzyme Testing. p3-Glucosidase was estimated according to Hosel and Nahrstedt (7) using 2 mM p-nitrophenyl-f3-glucoside in Mcllvaine buffer (pH 5.6). Linamarase activity was estimated by the determination of the HCN produced. Enzyme preparations were incubated with 10 mM linamarin (exact incubation conditions are described elsewhere (16). To obtain a complete decay of the hydroxynitrile produced in the course of the cleavage of linamarin and to stop the enzyme reaction the incubation mixture was made alkaline by adding 0.1 N NaOH. Diglucosidase activity was determined by estimating the HCN liberated from the acetone cyanohydrin or mandelonitrile, produced in the course of the cleavage of linustatin or amygdalin, respectively. An alkaline treatment guarantees the total breakdown of hydroxynitriles. Incubation conditions: Mcllvaine buffer (pH 4.5), T = 30°C, substrate concentration: 10 mm incubation time: 10 to 60 min, depending on the enzyme activity. The reaction was stopped by adding 1 ml of 0.5 N NaOH to the incubation mixture (2.5 ml). As the HCN-test was carried out in a final volume of 5 ml, the volume was adjusted by adding 0.5 ml H20 and 1 ml 0.5 N HCl (directly before the cyanide test was

Plant Physiol. Vol. 86, 1988

SELMAR ET AL.

712 carried out).

f3-Cyanoalanine synthase activity was measured according to Blumenthal et al. (2) as modified by Lieberei et al. (10). In this procedure H2S produced during the enzyme reaction is analyzed by determination of the absorbence of methylene blue, formed from H2S and N,N-dimethyl-p-phenylenediamine. Protein Determination. Protein was determined fluorometrically. Samples were incubated with Fluram (Roche) in 200 mM borate buffer (pH 9.3). The fluorescing solutions were measured in a spectral fluorometer, excitation: 390 nm, emission: 480 nm. A calibration curve was made with BSA. In those cases where the samples contained amino acids, the method of Bradford (3) was adopted. Gas Chromatography. Aliquots of methanolic extracts from freeze-dried plant-material were taken to dryness, dissolved in 20 ,ul pyridine, and silylated with 50 ,u1 N,N-bistrimethylsilyltrifluoroacetamide and 20 ,ul trimethylchlorosilane. Several ,u1 of the solution were injected into a capillary GLC system, using a DB-5-column (30 m x 0.32 mm), He (1 ml/min) as carrier gas, injector: 260°C, FID: 2700. Temperature program: 240 to 280°C, 1°C/min. TLC. The TLC was run with silica gel 60 F 254 aluminum-foil (Fa. Merck). Mobile phase: methanol/chloroform/15% NH4OH in water (2/2/1). Sugars and glucosides were detected with anisaldehyde/sulfuric acid according to Stahl (20). The dry TLC plates were sprayed with the reagent (anisaldehyde/glacial acetic acid/concentrate H2SO4, 1/100/2) and developed 30 min at 110°C.

RESULTS Linustatin Exudation. The amount of linustatin exuded from the endosperm during different developmental stages of Hevea seedlings was determined by using the seed drainage technique of Selmar (17). Figure 1 shows different characteristic patterns for the appearance of linustatin in the drainage liquid for three individual plants. In all cases there are two distinct time periods in which linustatin is detectable: one immediately after starting the drainage and one later at variable times. These variations in time until linustatin appears are due to the different develop-

mental stages when the drainage was started. In Figure 1 the developmental seedling stages are demonstrated by the extent of the young leaves. In all cases the appearance of lirustatin is correlated to the same developmental stage of the seedling. Exactly when the young leaves reach the leaf stage B (9) linustatin is found in the endosperm exudate. This occurrence of linustatin never lasts longer than 48 h. Linustatin is transported out of the endosperm via cotyledons into the young seedling only during the main growth phase of the leaves, corresponding to that developmental phase, when the content of cyanogenic glucosides decreases (Fig. 2). In addition to the occurrence of linustatin which is correlated to the developmental leaf stage, linustatin also appears in the drainage liquid immediately after the experiments were started. As several experiments were started at different developmental stages, this occurrence of linustatin cannot be correlated with a specific developmental seedling stage, but must be due to injuries of the endosperm, when the drainage was initiated. The occurrence of injuries is demonstrated by the fact that, in contrast to noninjured seedlings, the treated seedling liberated about 0.7 ,umol HCN during the entire drainage experiment. This amount corresponds to 0.4% of the total cyanogen content of the seedling. These injuries led to an artificial bleeding of cell compounds, including linamarin which is split by the Hevea,3glycosidase and gives rise to the HCN-liberation but also of linustatin. As the,B-glycosidase is not able to split this diglucoside, linustatin is appearing in the drainage liquids immediately after starting the experiment. Consequently, the diglucoside must be present already in the seeds, being synthesized at least partially in earlier seedling stages, but the transport out of the endosperm occurs only during the significant decrease of cyanogenic glycosides in seeds, endosperm, and seedlings. Linustatin Cleavage. The assumption that the utilization of cyanogenic glucosides during the seedling development takes place outside the endosperm and that linustatin is a transport form for linamarin implies that all linamarin metabolized has to be transformed to linustatin. Consequently, in Hevea seedlings an enzyme must be present which is able to hydrolyse this cyan-

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