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 ABSTRACT In addition, a linustatin-splitting diglucosidase in Hevea is de- scribed. From its activity in different developmental stages and In the seeds of Hevea brasiliensis, the cyanogenic monoglucoside lina- in different tissues it is suggested that this enzyme is involved in marin (2-b3-D-glucopyranosyloxy-2-methylpropionitrile) is accumulated in the metabolism of cyanogenic glycosides. It is deduced that lin- the endosperm. After onset of germination, the cyanogenic diglucoside ustatin is a metabolite in the pathway by which linamarin is linustatin (2-[6-f8-D-glucosyl-,f-D-glucopyranosyloxyJ-2-methylpropio- metabolized and utilized. nitrile) is formed and exuded from the endosperm of Hevea seedlings. At the same time the content of cyanogenic monoglucosides decreases. The MATERIALS AND METHODS linustatin-splitting diglucosidase and the .3-cyanoalanine synthase that assimilates HCN, exhibit their highest activities in the young seedling at Seed Drainage. The seed drainage technique is described else- this time. Based on these observations the folowing pathway for the in where (17). Additionally, some of these seed drainage experi- vivo mobilization and metabolism of cyanogenic glucosides is proposed: ments were run in a gastight system in which a stream of mois- storage of monoglucosides (in the endosperm)-gucosylation-transport tened air was used to exchange the atmosphere in the experimental of the diglucoside (out of the endosperm into the seedling)-cleavage by system continuously. By bubbling this air through 5 ml of 1 M diglucosidase-reassimilation of HCN to noncyanogenic compounds. The NaOH any HCN liberated from the seedling during the exper- presence of this pathway demonstrates that cyanogenic glucosides, typical iment was trapped for quantitative determinations. secondary plant products serve in the metabolism of developing plants as HCN Determination. The HCN was estimated with the Merck N-storage compounds and do not exclusively exhibit protective functions Spectroquant kit for cyanide (data sheet 130 259 8 Do dt/5, Fa. due to their repellent effect. Merck). This assay is based on the method of Aldridge (1). 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 en- zyme tests, plant material was frozen in liquid N2 and crushed. Then the powdered material was homogenized in a blender (3 Linustatin (2-[6-l3-D-glucosyl-,3-D-glucopyranosyloxy]-2-meth- x 10 s) with 20 mm phosphate buffer (pH 6.5) (3 ml buffer per ylpropionitrile) is a relatively rare cyanogenic diglucoside found g fresh weight). The homogenate was squeezed through four in seeds of Linum usitatissimum (18) and in green tissues of layers of cheesecloth. The supernatant-was either used directly different species of Passiflora (19). Quite recently, linustatin has for enzyme tests or it was concentrated by precipitation with also been observed in seeds of Hevea brasiliensis (17) together ammonia sulfate (15-85% saturation). A subsequent gel filtra- with the related cyanogenic monoglucoside linamarin (2-13-D- tion was carried out with a G-150-gel (Spehadex, Pharmacia). glucopyranosyloxy-2-methylpropionitrile). It has been shown that Enzyme Testing. p3-Glucosidase was estimated according to during the development of H. brasiliensis seedlings linamarin is Hosel and Nahrstedt (7) using 2 mM p-nitrophenyl-f3-glucoside metabolized to noncyanogenic compounds without any liberation in Mcllvaine buffer (pH 5.6). of HCN (10). Furthermore, the amount of linamarin stored in Linamarase activity was estimated by the determination of the the endosperm (11) and the occurrence of the HCN metabolizing HCN produced. Enzyme preparations were incubated with 10 enzyme f-cyanoalanine synthase in the growing seedling (9) in- mM linamarin (exact incubation conditions are described else- dicate, that for consumption linamarin has to be transported from where (16). To obtain a complete decay of the hydroxynitrile the endosperm into the young seedling (15). As a highly active produced in the course of the cleavage of linamarin and to stop linamarase is present in the apoplastic space between endosperm the enzyme reaction the incubation mixture was made alkaline storage tissue and cotyledons (15), the linamarin transported out by adding 0.1 N NaOH. of the endosperm would be split as soon as it enters this extra- Diglucosidase activity was determined by estimating the HCN cellular space. In order to be protected against this cleavage by liberated from the acetone cyanohydrin or mandelonitrile, pro- linamarase, linamarin has to be transported in a modified form, duced in the course of the cleavage of linustatin or amygdalin, which cannot be split by the linamarin-cleaving Hevea ,3-glyco- respectively. An alkaline treatment guarantees the total break- sidase (linamarase). This enzyme, which occurs in all Hevea tis- down of hydroxynitriles. Incubation conditions: Mcllvaine buffer sues does not split linustatin (16). For this reason it was assumed (pH 4.5), T = 30°C, substrate concentration: 10 mm incubation that linustatin functions as a protected transport form of lina- time: 10 to 60 min, depending on the enzyme activity. The re- marin (17). action was stopped by adding 1 ml of 0.5 N NaOH to the incu- This paper demonstrates that linustatin occurs in Hevea seed- bation mixture (2.5 ml). As the HCN-test was carried out in a lings only at that developmental stage when the content of cyan- final volume of 5 ml, the volume was adjusted by adding 0.5 ml ogenic monoglucoside linamarin decreases. H20 and 1 ml 0.5 N HCl (directly before the cyanide test was 711 712 SELMAR ET AL. Plant Physiol. Vol. 86, 1988 carried out). mental stages when the drainage was started. In Figure 1 the f3-Cyanoalanine synthase activity was measured according to developmental seedling stages are demonstrated by the extent Blumenthal et al. (2) as modified by Lieberei et al. (10). In this of the young leaves. In all cases the appearance of lirustatin is procedure H2S produced during the enzyme reaction is analyzed correlated to the same developmental stage of the seedling. Ex- by determination of the absorbence of methylene blue, formed actly when the young leaves reach the leaf stage B (9) linustatin from H2S and N,N-dimethyl-p-phenylenediamine. is found in the endosperm exudate. This occurrence of linustatin Protein Determination. Protein was determined fluorometri- never lasts longer than 48 h. cally. Samples were incubated with Fluram (Roche) in 200 mM Linustatin is transported out of the endosperm via cotyledons borate buffer (pH 9.3). The fluorescing solutions were measured into the young seedling only during the main growth phase of in a spectral fluorometer, excitation: 390 nm, emission: 480 nm. the leaves, corresponding to that developmental phase, when the A calibration curve was made with BSA. In those cases where content of cyanogenic glucosides decreases (Fig. 2). the samples contained amino acids, the method of Bradford (3) In addition to the occurrence of linustatin which is correlated was adopted. to the developmental leaf stage, linustatin also appears in the Gas Chromatography. Aliquots of methanolic extracts from drainage liquid immediately after the experiments were started. freeze-dried plant-material were taken to dryness, dissolved in As several experiments were started at different developmental 20 ,ul pyridine, and silylated with 50 ,u1 N,N-bistrimethylsilyltri- stages, this occurrence of linustatin cannot be correlated with a fluoroacetamide and 20 ,ul trimethylchlorosilane. Several ,u1 of specific developmental seedling stage, but must be due to injuries the solution were injected into a capillary GLC system, using a of the endosperm, when the drainage was initiated. The occur- DB-5-column (30 m x 0.32 mm), He (1 ml/min) as carrier gas, rence of injuries is demonstrated by the fact that, in contrast to injector: 260°C, FID: 2700. Temperature program: 240 to 280°C, noninjured seedlings, the treated seedling liberated about 0.7 1°C/min. ,umol HCN during the entire drainage experiment. This amount TLC. The TLC was run with silica gel 60 F 254 aluminum-foil corresponds to 0.4% of the total cyanogen content of the seed- (Fa. Merck). Mobile phase: methanol/chloroform/15% NH4OH ling. These injuries led to an artificial bleeding of cell com- in water (2/2/1). Sugars and glucosides were detected with an- pounds, including linamarin which is split by the Hevea,3- isaldehyde/sulfuric acid according to Stahl (20). The dry TLC glycosidase and gives rise to the HCN-liberation but also of lin- plates were sprayed with the reagent (anisaldehyde/glacial acetic ustatin. As the,B-glycosidase is not able to split this diglucoside, acid/concentrate H2SO4, 1/100/2) and developed 30 min at 110°C. linustatin is appearing in the drainage liquids immediately after starting the experiment. Consequently, the diglucoside must be RESULTS present already in the seeds, being synthesized at least partially Linustatin Exudation. The amount of linustatin exuded from in earlier seedling stages, but the transport out of the endosperm the endosperm during different developmental stages of Hevea occurs only during the significant decrease of cyanogenic gly- seedlings was determined by using the seed drainage technique cosides in seeds, endosperm, and seedlings. of Selmar (17). Figure 1 shows different characteristic patterns Linustatin Cleavage. The assumption that the utilization of for the appearance of linustatin in the drainage liquid for three cyanogenic glucosides during the seedling development takes individual plants.