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. In all cases there are two distinct time periods place outside the endosperm and that linustatin is a transport in which linustatin is detectable: one immediately after starting form for linamarin implies that all linamarin metabolized has to the drainage and one later at variable times. These variations in be transformed to linustatin. Consequently, in Hevea seedlings time until linustatin appears are due to the different develop- an enzyme must be present which is able to hydrolyse this cyan-
a
LEAF-STAGES OF THE SEEDLING LEAF-STAGES OF THE SEEDLING LEAF-STAGES OF THE SEEDLING
- -%2. a 5 2- I (A 0 P- I- z z z . 4 * 2.0 -a PLANT 1 ° T PLANT 2 -j PLANT 3 'U - 'U 1.5- I= 3 In C C 'U a x U 2 Ul 1.0 J 'U z - < z 4 O- 1- 0.5. 1~- I- CI- ; 0 z z z 0 c 0 24 48 72 96 120 144 0 24 48 72 96 120 144 0 24 48 72 96 120 144 0 DRAINAGE DURATION (h) DRAINAGE DURATION (h) DRAINAGE DURATION (h)
______.1-I
FIG. 1. Plants 1, 2, and 3. The amounts of linustatin are determined by gas chromatographic analysis. Amygdalin was used as internal standard. Linustatin as reference was isolated according to Smith et al. (18). Ten relative units correspond to a linustatin exudation of 1.2 /.g/h and seedling. MOBILIZATION AND UTILIZATION OF CYANOGENIC GLYCOSIDES 713
SEEDLING STAGES Relative activity Protein (% ) ( OD 280 ) 100 2
0- Lo Ko 0- 0.3.7 10 14 -19 - 0 O 3 7 ~~~~~~1014 19I 0 3 7 10 14 19 days after gurmimaties days after germination
12 o
E. 10 FIG. 4. Diglucosidase and ,3-cyanoalanine synthase activity were de- termined as described in "Materials and Methods" (n = 6). To calculate .o 8 the rate of consumption, the differences in the cyanogen content of the -8 total seedling (and the endosperm) within two seedling stages are divided by the number of days required for this diminution (n = 6). Contents of soluble protein per endosperm and of the green parts of the young a 6 0 seedling, respectively are given (C). A combination (multiplication) of data mentioned in (A) and (B) with those in (C) reveals the enzyme activities per total organ. As the protein content in the endosperm de- 4- creases whereas that of the young seedling increases, the activity curves out of (A) and (B) calculated on a total-organ-basis will not modify the quantitative activity curves but will lead to far steeper slopes. 2
0 days after germination
15% of the original content. In the course of this consumption, HCN produced, the hydrolytic cleavage of cyanogenic glucosides the cyanogenic glycosides are metabolized by hydrolytic en- must take place in the same seedling organ where the 3- zymes, but during this growth phase HCN liberation does not cyanoalanine synthase is sufficiently active. During the time occur (10). That means the HCN produced in the course of the that the utilization of cyanogens takes place, B-cyanoalanine enzymic cleavage of cyanogenic glycosides followed by the dis- synthase-activity is high only in the growing seedling itself but sociation of the corresponding cyanohydrins must be refixed and not in the endosperm. For being metabolized in the seedling, transformed into noncyanogenic compounds. IPor this transfor- linamarin has to be transported out of the endosperm storage mation of 'cyanogen nitrogen' into 'noncyanogen nitrogen' only tissue into the young plant via the apoplastic space between them. two enzymes are known to occur in higher plants: ,3-cyanoalanine As linamarase is present in this apoplastic zone (15), linamarin synthase and rhodanese (14). Although rhodanese has been re- must be protected against enzymic cleavage. Symplastic transport ported to occur in Hevea (5), its possible role in HCN fixation or transport by vesicles from endosperm to the genetically dif- was excluded by Lieberei (9) because of its very low activity in ferent young plant can be excluded. So, the protection of lina- Hevea. marin during its apoplastic passage has to take place by producing To guarantee an immediate and complete refixation of the a chemically modified transport-form which cannot be split by MOBILIZATION AND UTILIZATION OF CYANOGENIC GLYCOSIDES 715 the Hevea f-glycosidase (linamarase) (16). (19) are known to contain linustatin. Assuming the linustatin The results presented here suggest that the diglucoside linu- pathway to be more generally distributed in cyanogenic plants, statin functions as such a transport-form. These findings permit linustatin should be detectable in a lot of plant species. First the postulation of the following process for the mobilization and evidence for this assumption was recently given by positive linu- utilization of cyanogenic glycosides (Fig. 5): In the endosperm statin proofs in several plants (M Frehner, D Selmar, unpub- the stored linamarin is glucosylated to form linustatin. This proc- lished data). Probably linustatin occurs in all plants containing ess starts directly after dehiscence. During the germination phase linamarin, but only very low concentrations may be detectable in which the primary leaves expand, linustatin is transported out during certain developmental stages. of the endosperm via the apoplast and cotyledons into the grow- Apart from linustatin, some other cyanogenic diglucosides are ing parts of the young seedling, where a diglucosidase splits off known. Neolinustatin was found besides linustatin in flax seeds gentiobiose. The HCN produced by the dissociation of the re- (18). It may be regarded as 'lotaustralin-glucoside.' Plants which sulting acetone cyanohydrin is fixed immediately by,3-cyano- contain both linamarin and lotaustralin may be expected to reveal alanine synthase. The f-cyanoalanine produced may then be not only linustatin as a transport form of linamarin but in analogy hydrolyzed to produce asparagine (4). also neolinustatin as a glycosylated transport form of lotaustralin. This linustatin pathway illustrates clearly that stored secondary Just recently, several Hevea species which contain small amounts plant products are not terminal metabolites or waste products of lotaustralin (11) besides linamarin have been thoroughly an- but are involved in the plant's metabolism to provide reduced alyzed. Indeed, in these plants traces of neolinustatin are de- nitrogen in certain developmental stages. tectable besides linustatin (D. Selmar, unpublished data). Analogous Pathways in Other Plants. Until now this pathway The best known cyanogenic diglucoside is amygdalin, occur- has been demonstrated only in germinating Hevea seedlings, but ring in several Rosaceae. Like linustatin, its sugar moiety is gen- data about the occurrence of linustatin in the phloem sap of tiobiose. Amygdalin can be regarded as a prunasin-glucoside. It Hevea trees compel that this pathway is also used in adult Hevea is stored in bitter almonds, apricot kernels, or in fruits of wild plants (15). Furthermore, the high activity of 83-cyanoalanine cherries whereas high amounts of its corresponding monoglu- synthase and diglucosidase in ungerminated Hevea seeds may coside prunasin are observed in the leaves. At the first sight this indicate that linustatin might be transported into the endosperm storage of diglucosides in seeds is a contradiction to the 'diglucoside- during the seed-filling. transport-scheme.' However, it should be noted that the storage Besides Hevea, only flax seeds (18) and some Passiflora species organs in almonds and apricots are cotyledons, not endosperm. In plants the endosperm is the primary storage tissue, whereas the cotyledons are secondary derived storage organs. So, the occurrence of the diglucoside amygdalin in cotyledons that func- tion as storage organs is totally consistent with the existence of a modification process of cyanogenic monoglucosides in those plants which contain amygdalin and prunasin, respectively. Anal- ogous to the linustatin pathway found in Hevea in these plants the monoglucoside prunasin may be synthesized in the devel- oping endosperm. For its apoplastic transport into the cotyledons and its 'intermediate storage' prunasin may be transformed to amygdalin. Acknowledgments-The authors wish to thank Prof. Dr. E. E. Conn (University of California, Davis) for his helpful discussions and the critical reading of the manuscript. We express our gratitude to Prof. Dr. A. Nahrstedt (Westfalische- Wilhelms-Universitat, Munster, FRG) for his support for the gas chromatographic analysis and for the use of his GLC equipment. We thank Volker Schmidtmann for his helpful assistance with the GLC analysis.
LITERATURE CITED 1. ALDRIDGE WN 1944 A new method for the estimation of microquantities of cyanide and thiocyanide. Analyst 69: 262-265 2. BLUMENTHAL-GOLDSCHMIDT S, HR HENDRICKSON, YP ABROL. EE CONN 1968 Cyanide metabolism in higher plants. III. The biosynthesis of j3- cyanoalanine. J Biol Chem 243: 301-322 3. BRADFORD M 1976 A rapid and sensitive method for the quantitation of mi- crogram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 4. CASTRIC PA. KJ FARNDEN, EE CONN 1972 The formation of asparagine from ,3-cyanoalanine. Arch Biochem Biophys 152: 62-69 5. CHEW M-Y 1973 Rhodanese in higher plants. Phytochemistry 12: 2365-2367 6. HENDRICKSON HR, EE CONN 1969 Cyanide metabolism in higher plants. IV: Purification and properties of the /-cyanoalanine synthase of blue lupine. J Biol Biochem 244: 2632-2640 7. HOSEL W, A NAHRSTEDT 1975 Spezifische Glucosidasen fur das Cyanglucosid Triglochinin, Reinigung und Charakterisierung von f3-glucosidasen aus Alocasia macrorrhiza Schott. Z Physiol Chem 356: 1265-1275 FIG. 5. Schematical representation of the linustatin pathway for the 8. KUROKI G, PA LIZOTTE, JE POULTON 1984 Catabolism of (R)-amygdalin and mobilization and utilization of Before the (R) vicianin by partially purified,-glucosidases from Prunus serotina Ehrh. cyanogenic glucosides. nitrogen and Davallia trichomonoides. Z Naturforsch 39: 232-239 stored in linamarin can be utilized, the cyanogenic monoglucoside has 9. LIEBEREI R 1984 Cyanogenese und Resistenz; Habilitationsschrift. Natur to be glucosylated to linustatin. After this mobilization the cyanogenic wissenschaftliche Fakultat, TU Braunschweig, FRG diglucoside is transported out of the endosperm into the growing seed- 10. LIEBEREI R, D SELMAR, B BIEHL 1985 Metabolization of cyanogenic gluco- There linustatin is and the HCN sides in Hevea brasiliensis. Plant Syst Evol 150: 49-50 ling. split by diglucosidase corresponding 11. LIEBEREI R, A NAHRSTEDT, D SELMAR, L GASPAROTTO 1986 The occurrence to the related acetone cyanohydrin reacts with cysteine to 8-cyano- of Lotaustralin in the genus Hevea and changes of HCN-potential in devel- alanine, which can be hydrolyzed to asparagine. oping organs of Hevea brasiliensis. Phytochemistry 25: 1573-1578 716 SELMAR ETAL. Plant Physiol. Vol. 86, 1988
12. LIEBEREi R 1988 Relationship of cyanogenic capacity (HCN-C) of the rubbcr 16. SELMAR D, R LIEBEREI, B BIEHL, J VOIGT 1987 Linamarase in Hevea-a tree (Hevea spec.) to the susceptibility to Microcyclus ulei, the agent causing nonspecific, 3-glycosidase. Plant Physiol 83: 557-563 South American leaf blight. J Phytopathol In press 17. SELMAR D, R LIEBEREI, B BIEHL, A NAHRSTEDT, V SCHMIDTMANN, V WRAY 13. MEVENKAMP G 1986 Charakterisierung und intrazellul3re Lokalisierung der 1987 Occurrence of the cyanogen linustatin in Hevea brasiliensis. Phyto- 13-Cyanoalaninsynthase in Blattern von Hevea brasiliensis. Diplomarbeit, chemistry 26: 2400-2401 Naturwissenschaftliche Fakultat, TU Braunschweig, FRG 18. SMITH CR, D WEISLEDER, RW MILLER 1980 Linustatin and neolinustatin: 14. MILLAR, JM, EE CONN 1980 Metabolism of hydrogen cyanide by higher plants. cyanogenic glycosides of linseed meal that protect animals against sclenium Plant Physiol 65: 1199-1202 toxicity. J Org Chem 45: 507-510 15. SELMAR D Cyanogenese in Hevea-zwei Wege zur Metabolisierung cyano- 19. SPENCER KC, DS SEIGLER, A NAHRSTEDT 1986 Linamarin. lotaustralin and gener Glycoside. Ph.D. thesis, Naturwissenschaftliche Fakultat, TU Braun- neolinustatin from Passiflora species. Phytochemistry 25: 645-647 schweig, FRG 20. STAHL E 1967 Dunnschichtchromatographie, 2. Auflage, Springer-Verlag. Berlin