Biologically active secondary metabolites of barley. II ... - Springer Link

Journal of Chemical Ecology, Vol. 19, No. 10, 1993

BIOLOGICALLY ACTIVE SECONDARY METABOLITES OF B A R L E Y . II. P H Y T O T O X I C I T Y OF B A R L E Y ALLELOCHEMICALS

D . L . L I U 1 and J . V . L O V E T T *

Department of Agronomy and Soil Science University of New England Armidale, N.S.W., 2351, Australia Abstract--The release of alkaloids by barley was quantified by HPLC. Hordenine was released from the roots of barley in a hydroponic system for up to 60 days. The amount reached a maximum, 2/,g/plant/day, at 36 days, then declined. Effects on white mustard by hordenine and gramine included reduction of radicle length and apparent reduction in health and vigor of radicle tips. Transmission electron microscopic examination of white mustard radicle tips exposed to hordenine and gramine showed damage to cell walls, increase in both size and number of vacuoles, autophagy, and disorganization of organelles. The evidence of the morphological and primary effects of barley allelochemicals at the levels released by living plants indicates that the biologically active secondary metabolites of barley may lead to a significant role in selfdefense by the crop. Key Words--Allelopathy, allelochemicals, phytotoxin, gramine, hordenine, HPLC, TEM, micrograph, autophagy, barley, Hordeum .vulgare, Sinapis alba.

INTRODUCTION A few w e l l - d o c u m e n t e d e x a m p l e s o f primary effects o f alkaloids on associated plants are known. W a l l e r and B u r s t r o m (1969) reported that diterpenoid alkaloids o f Delphinium ajacis L. (larkspur) inhibited internode growth in Pisum sativum L. (pea), possibly through interference with the synthesis o f gibberellins. Olney (1968) s h o w e d that v e r a t r u m (Liliaceae) alkaloids inhibited the growth o f Avena sativa L. (oats) and Secale cereale L. (rye), apparently through *To whom correspondence should be addressed. ~Present address: Bureau of Sugar Experiment Stations, P.O. Box 651, Bundaberg, Queensland 4670, Australia. 2231 0098 0331/93/1000-2231507.00/0 9 1993 Plenum Publishing Corporation

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a specific effect on DNA stability. Tropane alkaloids, scopolamine and hyoscyamine, are present in the leachates of seeds and foliage ofDatura stramonium L. (Lovett et al., 1981) and soil of D. stramonium-infested fields (Levitt and Lovett, 1984). These compounds inhibited the growth of Helianthus annuus L. seedlings, possibly by interference with starch hydrolysis (Levitt et al., 1984). Lovett and Potts (1987) tested the hypothesis that the inhibition of early seedling growth of some crop plants by scopolamine was due to interference with gibberellin-stimulated food reserve metabolism. However, in germinating barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), they found no evidence of interference with gibberellin activity, although seedling growth of both cereals was inhibited by the alkaloids at the levels tested (Lovett and Potts, 1987). Therefore, no conclusions as to the mechanism of seedling growth of inhibition by scopolamine and hyoscamine could be drawn. Two alkaloids, gramine and hordenine, have been suspected of contributing to allelopathy by barley (Overland, 1966). Gramine is a constituent of barley leaves and is present in several barley cultivars, reaching concentrations up to 8 mg/g dry weight (Hanson et al., 1981) at day 12 after germination, then slowly declining (Schneider and Wightman, 1974). Gramine was reported as a factor in the resistance of several barley cultivars to the aphids Schizaphis graminum and Rhopalosiphumpadi (see, for example, Zuniga and Corcuera, 1986). Gramine is not present in barley seeds and roots (Schneider and Wightman, 1974). Hordenine is absent from barley seeds but appears in the roots from the first day of seed germination (Mann et al., 1963). Overland (1966) reported that both gramine and hordenine inhibited the growth of various plants including Stellaria media (L.) Cyr., Capsella bursa-pastoris (L.) Medic., and Nicotiana tabacum L. under controlled conditions. Allelopathic activity of barley has been confirmed in the present work (Liu and Lovett, 1993). Liu and Lovett (1990) reported that barley seedlings released gramine and hordenine into a bioassay system, and peak concentrations of 22 ppm gramine and 48 ppm hordenine were quantified by GC-MS from washings of filter paper after barley seed germination for four days. However, the recovered concentrations declined following a peak under the bioassay system where no nutrients were supplied. Thus, it is unknown if such release could be continuous by living plants growing with adequate nutrient and other resources. The objective of the present experiments was to identify and quantify gramine and hordenine during the growth of barley seedlings and to monitor the effects of these compounds on white mustard (Sinapis alba L.). METHODS AND MATERIALS Chemicals. All organic solvents used for high-performance liquid chromatography (HPLC) were HPLC grade. Chloroform used in the concentration procedure was purified for reuse by redistillation. Gramine and hordenine

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hemisulfate were purchased from Sigma Chemical Co. Pure hordenine was obtained by dissolving the product in 5 : 1 0.1 N sulfuric acid-ammonia solution (pH 11), partitioning with chloroform, and removing the chloroform in a stream of dry nitrogen.

Quantification of Gramine and Hordenine Released by Barley at Different Stages of Growth and Development. Three 5-day-old barley (H. vulgare cv. Triumph) plants were transplanted to a hydroponic system as described by Liu and Lovett (1993) for up to 60 days. Samples were collected at 5- to 10-day intervals. On the sampling day, total volume of solution in each container was measured and passed through a 0.2-/zm super membrane filter (Gelman Sciences). Samples (100 ml) were concentrated using the methods described by Lovett and Potts (1987). About 85 ml of the chloroform fraction was collected and evaporated to 5 ml in a water bath at 45~ The condensed organic phase was further evaporated in a stream of dry nitrogen to dryness. The dry residue was diluted by 0.5 ml HPLC mobile phase (see later paragraph) before analysis. HPLC was performed with a Waters Associates Liquid Chromatograph equipped with a model 680 automated gradient controller, Waters LC M-45 Solvent Delivery system, a Lambda-Maxmodel 481 LC spectrophotometer set at 219 nm, and a Waters 745 data module set at 1 mm/min. A/zBondapak C18 reversed-phase column was used throughout the study. Ten microliter solutions of reference compounds and samples were injected into the column using a SGE injector. The solvent was mixed by the dual pumping system in the proportion of 60 % 0.05 M KH2PO 4 pulsing triethylamine (TEA) and 40% CH3CN at pH 7.65. Solvent flow rate was 2 ml/min. After each sample analysis, mobile phase was injected one or two times to clean up the injector.

Effect on Root Elongation of White Mustard by Gramine and Hordenine. Standard solutions of gramine and hordenine were prepared. Concentrations of each alkaloid were set at three levels, namely, 0 ppm, 15 ppm (8.61 x 10 -2 mM for gramine and 9.08 x 10 -2 mM for hordenine), and 50 ppm (2.87 x 10 -1 mM for gramine and 3.03 x 10 -1 mM for hordenine). The experimental design was a 3 x 3 factorial, where the 0 ppm gramine + 0 ppm hordenine were treated as controls. Seeds of white mustard were surface-sterilized with 1.25 % (w/v) sodium hypochlorite solution for 2 rain before sterile distilled water rinsing; 10 seeds of white mustard were placed on filter paper in 9-cm-diameter sterile Petri dishes with one Whatman No. 1 filter paper to which were added 3 ml of the appropriate solution. Seeds were incubated in the dark at 25~ for three days after which the length of the radicles was measured (Liu and Lovett, 1990). The data were converted to percentage of reduction from the control and the synergistic or antagonistic responses of the two alkaloids calculated by the method introduced by Colby (1967):

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ijh E=

+

-

10--6

where Ig is the percent inhibition of growth by gramine at concentration Cg and Ih is the percent inhibition of growth by hordenine at concentration Ch. E is the expected percent inhibition of growth by gramine + hordenine at the concentration of Cg + Ch. When the observed response is greater than expected, the combination is synergistic; when less than expected, it is antagonistic.

Effect of Barley Alkaloids on Root Tip Ultrastructure of White Mustard. The experiment was conducted under axenic conditions. Ten surface-sterilized white mustard seeds were sown in 9-cm glass Petri dishes on filter paper moistened with 3 ml of solutions at concentrations of 0 ppm (sterilized distilled water for control), 22 ppm gramine, 48 ppm hordenine, 100 ppm gramine and 100 ppm hordenine, with three replications. A further 1 ml appropriate solution was added to each dish on day 3. The seeds were germinated in an incubator for four days at 25~ in the dark. The techniques for processing material for transmission electron microscopy (TEM) were as outlined by Liu and Lovett (1990). More than 20 sections from each root tip were examined. The ultrastructure of the third or fourth layers of the cells of root tips from both control and treatments were recorded on micrographs. RESULTS

Alkaloids Released by Barley in a Hydroponic System. Results of a preliminary experiment showed the presence of gramine and hordenine in samples from the hydroponic system. However, gramine was too low to be quantified. Hordenine was released into the hydroponic system up to 60 days (Figure 1). The amount reached a maximum at 36 days, then decreased. The highest amount of hordenine recovered was 2/~g/day from one barley plant. Gross Morphological Effects of Barley Alkaloids. Gramine and hordenine inhibited radicle elongation of white mustard at concentrations of 50 ppm or more. Combinations of gramine and hordenine showed a synergistic action (Table 1), according to equation 1 defined by Colby (1967). The values of synergism were higher when the two alkaloids were combined in equal concentrations. Effect of Barley Alkaloids on Root Tip Ultrastrueture of White Mustard. Cells of white mustard root tips from sterile distilled water showed distinct nuclei with intact nuclear membranes; intact organelles, including mitochondria; small vacuoles; and uniform cell walls (Figure 2). Cells from the treatment with 22 ppm gramine also showed distinct nuclei and normal cell walls (Figure 3). However, more vacuoles appeared in these cells, compared to control. It was noted that some of the vacuoles were tending

2235

METABOLITES OF BARLEY: PHYTOTOXICITY ~5

2.0 -'-" i

'~ ~o

t.5 7 1.0

e~ e 5 o ~

~-

la0

0.5

0

0.0

EO 30 40 50 Days a f t e r t r a n s p l a n t i n g

i0

60

2Q

FIG. 1. Amount of hordenine released by barley in a hydroponic system. Histogram for hordenine per plant in each sample interval and line for hordenine released by one barley plant per day. TABLE 1. EFFECT OF COMBINATIONS OF GRAMINE AND HORDENINE ON RADICLE LENGTH OF WHITE MUSTARD

Gramine (ppm)

Hordenine (ppm)

0 0 0 15 50 15 15 50 50

0 15 50 0 0 15 50 15 50

Radicle length (mm _+ SE) ~ 38.0 37.6 35.5 38.6 35.2 34.7 34.4 33.8 29.6

• 1.4 • 0.9 _+ 1.6 + 0.8 _+ 1.3 _ 2.1 + 2.1 + 1.7 _+ 0.7

a ab bc a bc c c c d

Reduction observed (%) 0 1.2 6.6 -1.6 c 7.3 8.8 9.5 11.1 22,0

Reduction expected (%)

Differenceb

-0.4 0.5 8.4 13.4

+9.2 +4.4 +2.7 +8.6

aMeans sharing a common letter are not significantly different at 5% level, by LSR. bDifferences (the % reduction observed minus % reduction expected) with a plus sign indicate synergistic effect. CA minus sign indicates stimulation compared to the distilled water control.

to join together and might become giant vacuoles (typically, see Figure 3a and c). Cells from the treatment with 48 ppm hordenine showed similar results for nuclei, which appeared to be intact, but had more and larger vacuoles (Figure 4), compared to the control. There was evidence also of damaged cell walls (Figure 4a and b) and giant vacuoles, which contained some membrane fragments from other organelles (Figure 4c). In the treatments with both 100 ppm gramine and hordenine, a further increase in vacuole size occurred, with evidence of distinct autophagy (typically,

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Fro. 2. T r a n s m i s s i o n electron m i c r o g r a p h o f the cells o f white m u s t a r d root tips four d a y s after c o m m e n c e m e n t o f germination; sterile distilled water. Scale bar = 1 # m . C W = cell wall; V = vacuole; M = m i t o c h o n d r i o n ; N = nucleus; N U = nucleolus; N m = nuclear m e m b r a n e .

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FIG. 3. Transmission electron micrographs o f the cells o f white mustard root tips four days after c o m m e n c e m e n t o f germination; 22 p p m gramine. Scale bar = 1 /zm. C W = cell wall; V or v = vacuole; Gv = giant vacuole; L = lipid; M = mitochondrion; N = nucleus; N U = nucleolus; N m = nuclear membrane; arrows indicate development o f giant vacuoles.

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Fro. 4. Transmission electron micrographs o f the cells o f white mustard root tips four days after c o m m e n c e m e n t o f germination; 48 p p m hordenine. Scale bar = 1 /~m. C W = cell wall; V or v = vacuole; Gv = giant vacuole; L = lipid; M or m = mitochondrion; N = nucleus; N U = nucleolus; N m = nuclear membrane; arrows indicate the development o f giant vacuoles and autophagy.

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FIG. 5. Transmission electron micrographs o f the cells o f white mustard root tips four days after c o m m e n c e m e n t o f germination; 100 p p m gramine. Scale bar = 1 ~m. C W = cell wall; V = vacuole; Gv = giant vacuole; L = lipid; M = mitochondrion; N = nucleus; N U = nucleolus; N m = nuclear membrane; arrows indicate autophagy.

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Fro. 6. Transmission electron microgmphs o f the cells o f white mustard root tips four days after c o m m e n c e m e n t o f germination; 100 p p m hordenine. Scale bar = 1 /zm. C W = cell wall; V or v = vacuole; Gv = giant vacuole; Cv = circular vacuole; L = l i p i d ; M = mitochondrion; N = nucleus; N U = nucleolus; N m = nuclear membrane; arrows indicate the development o f giant vacuoles and autophagy.

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see Figures 5d and 6d). At this concentration there were some circular vacuoles (Figures 5a, 6a and c), which might be created from individual small vacuoles (Figure 6b). In addition, some cell walls appeared to be damaged and some mitochondria showed evidence of disorganization (Figures 5 and 6). The nucleolus appeared normal, although there was evidence of less distinct nuclei and damaged nuclear membranes (Figure 6d). It was noted that many lipids appeared in the treatments (Figures 3 and 4), as compared with the control (Figure 2). In general, at the concentrations of alkaloids used in these studies, there was evidence of disorganization but not of gross levels of damage.

DISCUSSION

In the bioassay of the combined effect of gramine and hordenine, a synergistic interaction occurred. This agrees with the work of Einhellig and coauthors (Rasmussen and Einhellig, 1979; Einhellig et al., 1982), who tested a number of phenolic allelochemicals and demonstrated their synergistic effects.Rasmussen and Einhellig (1979) found that an equimolar combination of 3.3 mM with any two of ferulic, p-coumaric, and vanillic acids showed more inhibition of sorghum seed germination than the two allelochemicals combined at different concentrations. In addition, the equimolar combination also depressed seed germination more than a combination at different concentrations. In the combination of gramine and hordenine, the synergistic effects of equal concentrations were higher than unequal concentrations (Table 1). The evidence from electron microscopy showed the effect of allelochemicals on the ultrastructure of root tip cells of white mustard, including increases in both size and number of vacuoles (Figures 3-6). Matile (1978) reviewed the work in which the vacuoles of higher plants play fundamental roles in: (1) storage pools of intermediates such as enzymes, inorganic ions, primary and secondary metabolites; (2) regulation of turgot; and (3) detoxification. Matile (1976) discussed the detoxification of potentially toxic plant metabolites and concluded that cells protect themselves from compounds such as poisonous alkaloids by sequestering them into the vacuoles. In our experiments, cells treated with high concentrations of alkaloids (Figures 5d and 6d) showed an autophagic phenomenon (Matile, 1975, 1976, 1978; Matile and Wiemken, 1976), which is similar to phagocytosis in animal cells. This phenomenon has been considered as either representing the autophagic elimination of damaged organelles (Villiers, 1971) or the beginning of senescence of the cells (Matile, 1975, 1976; Matile and Wiemken, 1976). The latter cannot explain the results in our experiments since cells of the same age and location were compared between the control and alkaloid treatments. In the

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process of autophagy, portions of cytoplasm, including damaged mitochondria, are encircled by a number of small vacuoles that ultimately fuse together and form a giant vacuole (Figures 5a, b, d, and 6). This process may represent an active and energy-dependent response to damage (Matile and Wiemken, 1976). It has been noted that plants take up alkaloids from soil by processes similar to those in nutrient uptake (Winter, 1961). Investigations have shown that alkaloids can be freely taken up by higher plants, including alkaloid-free plants (Winter, 1961) or lower plants (Schneider and Stermitz, 1990). Lovett et al., (1981) suggested that allelopathy and responses to allelochemicals are part of a network of plant chemical communication. Plants may produce alkaloids as defensive chemicals to microorganisms and other plants; the alkaloids in the cells Of a producer can be stored, released, or detoxified. Plant vacuoles are a principal place for storage or detoxification. There are at least two advantages for alkaloids entering vacuoles. First, the alkaloids can be isolated to avoid contact with other organelles that may be damaged, as observed in the cells of root tips of white mustard treated with gramine (Figures 3 and 5) and hordenine (Figures 4 and 6). Second, alkaloids stored in vacuoles can be used as selfdefense chemicals to insects or other predators. Recent results on alkaloid uptake in isolated vacuoles contradict the classic alkaloid accumulation model (Deus-Neumannand Zenk, 1986). Vacuoles of alkaloid-containing plants take up only those alkaloids that are specific to this particular plant, while vacuoles of alkaloid-free plants do not take up alkaloids (Deus and Zenk, 1982; Deus-Neumann and Zenk, 1984). Thus it would be expected that toxic alkaloids may damage the organelles in cells of such plants and may, for example, cause dysfunction of enzyme systems (Benoit and Starkey, 1968, as cited in Rice, 1984). The appearance of many lipids after the treatment of root tips with gramine and hordenine in this experiment may be evidence of disruption of food metabolism (Figures 3 and 4), similar to the effects of benzylamine on root tip cells of Linum usitatissimum L. (Lovett, 1982). The effects of the tropane alkaloids, scopolamine and hyoscamine, on root tips of Helianthus annuus were the accumulation of lipid droplets and the abundance of amyloplasts (Levitt et al., 1984), indicating a general slowing down of the metabolism of food reserves. During the course of germination, the reserve foods, such as starch and lipids, are enzymatically degraded to low-molecular-weight carbohydrates for further metabolic utilization (Bonner and Varner, 1976). Therefore, retarded food metabolism may result from the dysfunction of enzymes by allelochemicals, as previously discussed. Hence, although the allelochemicals present are insufficient to cause the death of the seedlings, their metabolism is adversely affected (Levitt et al., 1984), consequently, a slow germination (Liu and Lovett, 1993) and short root length (Liu and Lovett, 1990) could be observed. For studies of primary effects of allelochemicals, transmission electron

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microscopy can be used to show direct evidence of such effects. When seedlings of white mustard were treated with 22 ppm gramine, there was a substantial response in the cells, typically, increases in the number and size of vacuoles (Figure 3). The same amount of alkaloids in bioassay could be expected to produce nonsignificantly different radicle lengths of white mustard (Liu, 1991), compared to cells from control. Therefore, when measuring a secondary effect, the noneffect (0% reduction) following stimulation cannot be interpreted as a true noneffect, but may only be expressed as "no difference" in a secondary indicator. In our work, the release of the two alkaloids, gramine and hordenine, has been established in the early seedling stage (Liu and Lovett, 1990) and in the later stage from a barley growing environment (Figure 1). The effects of these alkaloids at the levels quantified have been demonstrated at both gross morphological and ultrastructural levels. The results suggest that biologically active secondary metabolites of barley may play a significant role in self-defense by the crop. REFERENCES BONNER, J., and VARNER, J.E. (eds.) 1976. Plant biochemistry. Academic Press. New York, 925 pP. COLBY, S.R. 1967. Calculating synergistic and antagonistic responses of herbicide combinations. Weeds 15:20-22. DEUS, B., and ZENK, M.H. 1982. Exploitation of plant cells for the production of natural compounds.Biotechnol. Bioeng. 24:1965-1974. DEus-NEUMANN, B., and ZENK, M.H. 1984. A highly selective alkaloid uptake system in vacuoles of higher plants. Planta 162:250-260. DEus-NEUMANN, B., and ZENK, M.H. 1986. Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism. Planta 167:44-53. EINHELLIG, F.A., SCHON, M., and RASMUSSEN,J.A. 1982. Synergistic effects of four cinnamic acid compounds on grain sorghum. J. Plant Growth Regul. 1:251-258. HANSON, A.D., TRAYNOR,P.L., DTTZ,K.M., and REICOSKY, D.A. 1981. Gramine in barley forageeffects of genotype and environment. Crop Sci. 21:726-730. LEVITT, J., and LOVETT, J.V. 1984. Activity of allelochemicals of Datura stramonium L. (thornapple) in contrasting soil types. Plant Soil 79:181-189. LEVITT, J., LOVETT,J.V., and GARLICK,P.R. 1984. Datura stramonium allelochemicals: Longevity in soil, and ultrastmcture effects on root tip cells of Helianthus annuus L. New Phytol. 97:213218. LIU, D.L. 1991. Modelling plant interference and assessing the contribution of allelopathy to interference by barley. PhD thesis. University of New England, Armidale, N.S.W., 231 pp. LEu, D.L., and LOVETT, J.V. 1990. Allelopathy in barley: potential for biological suppression of weeds, pp. 85-92, in C. Bassett, L.J. Whitehouse, and J.A. Zabkiewicz (eds.). Alternatives to the Chemical Control of Weeds. Proceedings of an International Conference Rotorua, New Zealand, July 1989. Ministry of Forestry, FRI Bulletin 155. LIu, D.L., and LOVETT, J.V. 1993. Biologically active secondary metabolites of barley. I. Developing techniques and assessing allelopathy in barley. J. Chem. Ecol. 19:2217-2230.

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