PHYSIOLOGICAL STUDIES ON THE CYANOGENIC GLUCOSIDES IN LOTUS ARABICUS L.

YASH PAL ABROL

Division of I!lant Physiology and Phytotron, Indian A,£;ricultural Research Institute, Delhi-12

SuMMARY

Observations on the occurrence of cyanogenic glucosides, linamarin and lotaustralin, in Lotus arabicus L., are reported. During the vegetative stage, a major portion of the cyanogens is localised in the leaves. With the onset of flowering, cyanogen content on per plant basis shows a sharp decrease but flower buds and flowers have a high content. Experiments on feeding carbon-14 labelled L-valine and L-isoleucine to flower buds reveals that the compounds are synthesised in these parts. lYiature seeds do not contain any detectable amounts of the cyanogens.

I:-!TRODUCTION

Very little work relating to the physiological studies on cyanogenic glucosides in higher plants has been reported. This is very likely due to the predominant view held until recently that a very small number of plants are cyanophoric and that cyano­ genic glucosides are inert end products of metabolism (Robinson, 1930). Work of physiological nature is however of interest in view of the recent observations that cyanogenic glucosidcs arc widely distributed (Dilleman, 1958; Hegnaucr, 1964), HCN is metabolised by seedlings of cyanophoric as well as non-cyano­ phoric plants (Blumenthal-Goldschmidt, Butler and Conn, 1963 Tschiersch, 1964; Nigam and Ressler, 1964), cyanogenic gluco­ sides are metabolically active rather than inert end products (Abrol, Uribe and Conn, 1965; Abrol and Conn, 1966; Abrol, Conn and Stoker, 1966) and are very likely involved in the synthesis of various neurotoxic and neurolathvritic amino acids (Ressler, 1962 ; Tschiersch, 1964). · THE CYANOGENIC GLUCOSIDES IN LOTUS ARABICL'S L. 109

Two cyanogenic glucosides, linamarin (et-hydroxy-iso­ butyronitrile-p-D-glucose) and lotaustralin (a-hydroxy-a-methyl­ butyronitrile-p-D-glucose) occurring in Lotus arabicus L. have been shown to account fully for the total cyanide content released by grinding the plant tissue ( Abrol and Conn, 1965; Abrol and Conn, 1966). This paper records the changes in these cyanogenic compounds with age of the plant as well as their localisation in different parts.

MATERIALS AND METHODS

The seeds of L. arabicus L. were supplied by M. Villax, Rabat, Morocco. The plants were grown in 12 inch clay pots in the greenhouse of the University of California campus at Davis. Uniformly labelled L-isoleucine-I4C (sp. act. 14 · 3 pc/pm) and L-valine-'4C (200 pcjpm) were obtained from New England Nuclear Corporation. Procedures for feeding labelled precur­ sors, chromatographic isolation and identification of linamarin and lotaustralin have been outlined earlier (Butler and Conn, 1964). The following solvents were used in the present study : l. Methylethyl Ketone-acetone-water (30 : 10 : 6 v/v) 2. Butanol-Pyridine-water (6 : 4 : 3 vjv) 3. Isopropanol-acetic acid-water (70 : 5 : 25 v/v) All radioactivity measurements were made in Packard's scintilla­ tion counter. Plant extracts were made by freezing the plant material in liquid nitrogen, grinding in a mortar and transferring the resulting powder to boiling 80 per cent ethanol. The suspension was then filtered and the filtrate evaporated to dryness under reduced pressure using a flash evaporator. The residue left on evaporation was dissolved in a known volume of water for analysis. Hydrogen cyanide was liberated from the cyanogenic glucosides by incubation in aqueous solution with linamarase, for two hours in a closed system. (Linamarase was purified approximately twentyfold from linseed meal (Coop, 1940). Carbon dioxide free air was then passed through the system and the HCN carried over in the air stream into 10 m1 of 0 · 1 N NaOH. Aliquots of this trapping solution were assayed for cyanide by the co Iori metric method of Aldridge ( 1944). 110 YASH PAL ABROL

RESULTS AND DISCUSSION

Linamarin and lotaustralin arc present in approximate ratio of 7 : 3 until the plants start flowering. Thereafter, the shift is to roughly 1 : 1 (Table I). This is in conformity with the Butler's ( 1965) observation that these two cyanogenic glucosides invariably occur together and that linamarin is preferentially synthesised in species of several genera. This also suggests that very likely a similar biosynthetic route is involved in the con­ version of their amino acid precursors viz. valine and isoleucine to linamarin and lotaustralin respectively (Butler and Butler, 1960).

Table I. The linamarin and lotaustralin content in Lotus arabicus L.

Age, days Lin a marin Lotaustralin Total Content

/L moles/gram (fresh wt.) 3 4·31 (72·73) 1·()2 (27·27) 5·93 37 9·49 (69·60) 4·1') (30·40) 13·64 77 7·09 (71·40) 2·84 (28·60) 9·93 137 (flowering) 2·l'i C>6·90) 1·62 (43·10) 3· 77

(The figures in parenthesis indicate the percentage of the total)

Studies on changes in the amount of total cyanogenic glucosides with age reveals that when expressed on fresh weight basis, increase is observed up to about 25 days followed hy a gradual decrease thereafter which becomes somewhat marked when plants start flowering. On per plant basis, however, there is an increase till the onset of flowering, which is followed by a sharp decrease. Only the aerial portions were examined because roots do not contain any cyanogenic compounds (Figure 1). Resuli:s on cyanogenic glucosides content in different parts of the plant show that during the vegetative stage, the major portion is present in the leaflets, the stem part accounting for very little. Flower buds and flowers have a high content. Dry yellow leaflets shed in large numbers from the mature plants contain the cyanogenic glucosides. The mature seeds are devoid of these compounds (Table II). Sharp decrease in the cyanogen content with the onset of flowering contrasts markedly with the observation as to their localisation in different plant parts, because flower buds and THE CYANOGENIC GLUCOSIDES IN LOTUS ,JRABICUS L. 111

CYANIDE CONTENT (1-L moles) PER GRAM (fresh weight) N 0

0"' .,. r r 0 • "l) "l) fT1 I'T1 :u :u (;) "l) :u r- 0"' )> )> 3: ;z l> • --i G) IT1 - 0 CD ~ 0 • ::.-"' z E e. !!!, "0 0 ~ l> • "'~ 0 -< 0 • :> "' 0 • i\5 0 • ::;:; 0

a; )( • • 0 • •

N ~ ~ ~ m 0 0 0 0 0 CYANIDE CONTENT (JJ moles) PER PLANT (aerial portion) Fig. 1 Cyanide content, fully accounted for by the two cyanogenic glucosides, linamarin and lotaustralin in L. arabicus L. expressed on fresh water and per plant (aerial portion) basis.

Table II. Amount of total cyanogenic glucosides zn different parts of plants of Lotus arabicus L.

~-- ~ ·----· Yellow Flower Age, days Stem Leaflets Leaflets* buds Flower 1-L moles per gram (fresh wt.) 37 1·22 6·35 57 2·10 13·86 137 (flowering) 1·30 1·66 21·04 32·90 52·28 -·------~----·------~----- ~------*Shed from the plant. 112 Y ASH PAL ABROL flowers have high content. To ascertain as to whether this high content is due to translocation from vegetative parts or the synthesis is taking place in flowering parts, uniformly labelled L-valinc-'4C and L-isoleucine-'~c were fed to flower buds. It was observed that of carbon-14 is incorporated into linamarin and lotaustralin, thus suggesting the likelihood of synthesis taking place in the flower buds itself. One cannot draw conclusions about the physiological significance of the cyanogenic glucosides from the observations reported in this communication. The task becomes difficult as meagre data are available on other cyanophoric plants. A few plants which have been investigated reveal a rather incongruous picture. For example, Ludke (1953) reported that mature seeds of flax are a rich source of cyanogenic glucosides while the dry yellow leaflets are completely devoid of these compounds. In Sorghum, however, it was observed in our laboratory that seeds as well as dry leaves do not contain any detectable amounts of cyanogens. Detailed studies on a number of cyanophoric plants may help to reveal the physiological role of cyanogenic glycosides.

AcKNOWLEDGEMENT

This work was done while the author was working as a Postdoctoral Fellow in the Department of Biochemistry and Biophysics, University of California, Davis, California. He is thankful to Professor Eric E. Conn for the facilities.

REFERENCES

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