Chapter 10
Downloaded via UNIV OF MASSACHUSETTS AMHERST on September 26, 2018 at 04:30:26 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Function and Variation of the ß-Glucosidase Linamarase in Natural Populations of Trifolium repens P. Kakes Free University, de Boelelaan 1087, 1081 H V Amsterdam, Netherlands
Cyanogenesis is the production of H C N by plants, and in some cases, by animals. Cyanogenesis is only apparent after damage of the cyanogenic organs or tissues of the plant. The mechanism responsable for cyanogenesis is hydrolysis of one or more cyanogenic substrates by a ß-glucosidase. The function of cyanogenesis is best studied in species that are polymorphic for cyanogenesis. Trifolium repens (white clover) is polymorphic for both the cyanogenic substrates and the ß -glucosidase linamarase. This situation makes it possible to study the function of linamarase in plants with and without the natural substrates. The most obvious function of cyanogenesis is defense against herbivores. However I was able to show that linamarase has no function in the deterrence of slugs and snails by the cyanogenic glucosides present in white clover. It is possible that linamarase has a function in the deterrence of other herbivores, but the evidence for this is weak. There is much variation in linamarase content in natural populations of white clover. The significance of this variation is discussed within the framework of the cost-benefit hypothesis. In white clover (Trifolium repens) cyanogenesis, i.e. the production of H C N is caused by the action of the cyanogenic p-glucosidase linamarase on two cyanogenic substrates: linamarin and lotaustralin (7) (See Figure 1) The substrates occur in varying proportions in leaf cells, presumably in the vacuole. Linamarase is an apoplastic enzyme, occurring mainly in the walls of the epidermal cells of the leaves (2). Linamarase activity in other organs of T.repens is low or undetectable (3), (Kakes, unpublished). The compartmentalisation of enzyme and substrate (Figure 2) insure that no detectable amount of H C N is produced in intact plants. As in other plants, the cyanogenic system in white clover is generally looked upon as one of the mechanisms of defense against herbivores. There is ample evidence that cyanogenesis protects plants against generalized herbivores (but not specialized ones) but of course this does not exclude other functions of the cyanogenic system or of its components.
0097-6156/93/0533-0145$06.00/0 © 1993 American Chemical Society
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
/?-GLUCOSIDASES: BIOCHEMISTRY AND MOLECULAR BIOLOGY
146 Ac
p -glucosidase
- c—
C
-glc + H 0 2
OH
+ glucose
C=N
C=N
Spontaneously or lyase
Rj
C =
0 + HCN
Figure 1. General scheme of the hydrolysis of cyanogenic substrates. The effects of the genes Ac and L i in T. repens are indicated
Figure 2. The localisation of linamarase and the presumed localisation of the cyanogenic substrates. (Reproduced with permission from Vakblad voor Biologen 1986, 66, 13. Copyright 1986.)
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
10. KAKES
P-Glucosidase Linamarase in Natural Populations
147
T. repens is is a species that is well suited for an investigation of the cyanogenic system as most populations contain cyanogenic and acyanogenic plants. In population studies only long established fields or natural stands should be used, to prevent contamination with cultivars. Fortunately gene flow is very limited in T.repens (4).The polymorphism is caused by variation in two genes: Li regulates the presence/absence of linamarase and Ac the presence/absence of the two cyanogenic substrates. The variation in A c and Li produce four so called cyanotypes that can be simply distinguished (Figure 3). Both genes have a dose effect: in plants with two active alleles twice as much product is produced, compared to plants with one active allele (5, 6). Genetical tests show that Ac and Li are unlinked. In this paper I will discuss the variation in linamarase and the possible functions of this enzyme in natural populations of white clover and some related species. Properties of Linamarase Linamarase in T.repens is homodimer with a subunit molecular mass of 62.000 Dalton. It is a glycoprotein and can represent up to 5% of the soluble proteins in young leaves (7). As mentioned before it is localized in the cell wall. In plants homozygous for the li allele no linamarase activity is found and also no anti-linamarase cross reactive material can be detected (8, 9). In genotypes and organs without linamarase activity a low but distinct p-glucosidase activity can be found with artificial substrates. This activity may be due to a p-glucosidase (or p-glucosidases) not under the control of the Li-gene (10). Kinetic studies show that the white clover linamarase has a broad substrate specificity, depending on the aglycone and on the type of glucosidic linkage (77). The two known substrates in T.repens are both split by linamarase. Distribution of the Li-alleles i n European Populations There are at least three alleles of L i differing in linamarase activity (6) but only plants homozygous recessive for the null allele (lili) can be simply distinguished from the other genotypes. It has long been known that in Europe the frequency of the null allele is low in populations around the Mediterranean Sea and gradually increases to the North (72). In Scandinavia and northern Russia, where the species has its northern limit, very few plants with linamarase have been found. A similar pattern is found when we study mountain populations: the higher the altitude, the lower the frequency of linamarase containing plants. The decrease is however not gradual: up to 600m high frequencies of the dominant allele prevail. Over 600m the range of frequencies broadens, with a definite dowward trend. As this pattern is observed both in the Cevennes in South-eastern France (13) (Figure 4) and in the Western Pyrenees (Kakes, unpublished) it is not likely to be a chance event. The corresponding latitudinal and altitudinal clines suggest that low temperatures, especially low winter temperatures, in some way determine the large scale distribution of Li. It should be noted that the frequency of Ac follows a similar pattern. Although in local populations almost every frequency of the four cyanotypes may be observed the large scale distributions of Li and Ac are correlated. Local populations often show linkage disequilibrium, i.e.the phenotype having both enzyme and substrate is found more often than expected on the basis of independent assortment of the alleles. (74), (15) Studies of the Function of the Cyanogenic System, i n Particular of Linamarase The occurrence in one population of plants with and without linamarin or linamarase poses a problem: If we assume that cyanogenesis has a function, why is it that most
American Chemical Society Library 1155 16th St.. N.W.
Esen; ß-Glucosidases Washington. DC.Society: 20036 ACS Symposium Series; American Chemical Washington, DC, 1993.
148
/9-GLUCOSIDASES: BIOCHEMISTRY AND MOLECULAR BIOLOGY
Added: Genotype:
Water
Substrate
Enzyme
Ac-LiAc-lili acacLiacaclili
+
+
+
-
-
+
-
+
-
Figure 3. The four cyanotypes that can be distinguished with semiquantitative tests.
o : Dommee-Anthony 1978 X : Kakes
1979
X : Kakes 1985
1.0 * 0.9-
1 0.8-
X
I
f 0.7 ^ 0.6 ° 0.5 S" OA 0.3-
x x
A: Till 1981
vX
V : Till 1982
o
0.2-
0.1500
1000
1500
Alt. in m
Figure 4. The distribution of the L i alleles in South-Eastern France. Results of different investigators and years have been combined. A l l data are from Till et al
U3) (Reproduced with permission Gauthier-Villars.)
from reference 13.
Copyright
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
1988
10. KAKES
(3-Glucosidase Linamarase in Natural Populations
149
populations contain individuals that do not have this system, or have an incomplete one? The variation in chemical defense in general has given rise to a considerable body of hypotheses and I will discuss only briefly one of them: The cost-benefit hypothesis (see also 76). It is postulated that the production and maintenance of a defensive system has a cost for the plant. Natural selection will favour such a system only so long as the benefits are greater than the costs. To study cost and benefit of cyanogenesis I produced a repeated backcross generation segregating for the genes Ac and Li. The different cyanotypes were followed from the germination of the seeds until seedset of the adult plants in the second year. The results (76) showed that for Ac the cost-benefit hypothesis works well: Plants that possess linamarin/lotaustralin (Acac) are less attacked by slugs and snails than acac plants, both in the seedling stage and as adult plants. Table I shows the result of the seedling experiment: the difference with the non grazed control (not shown) is significant for Ac but not for Li. Figure 5 demonstrates the effect of grazing on adult plants. Clearly the protection of seedlings by the presence of cyanogenic glucosides has a selective advantage, as we know that under natural conditions slugs and snails destroy a large proportion of the seedlings. The cost of defense appears when the plants reproduce, as shown in figure 6. Acac plants produce only half of the flowers and seeds compared to plants without linamarin/lotaustralin (acac). For linamarase the situation is different: Our results clearly show that linamarase is not necessary for the deterrence of slugs and snails. This result, although not obtained earlier, is not surprising, as the enzyme mixture in the gut of snails has a high linamarase activity (77), (Kakes, unpublished). It is possible that linamarase is necessary for the defense against other herbivores lacking linamarase activity in their guts, but the evidence for selective eating by organisms other than molluscs is weak. In a recent review Hughes (3) lists 10 papers in which selective eating of acyanogenic T.repens is reported. These 10 papers give 14 species or species groups. Ten of these are molluscs (slugs and snails), the remaining 4 are insects. However the two insect reports are far from convincing. In the first report (18) different reproduction of the aphids on cyanogenic and acyanogenic plants could well explain the data given by the authors. In the case of the other two insect species (79) not cyanotypes but commercial varieties differing in frequency of cyanogenic plants were compared. In none of the studies except that of Kakes (76) were the effects of Ac and Li studied separately. Several other differences between plants with and without linamarase have been reported in the literature: linamarase containing plants have bigger leaves and better survival as young plants (20), shorter stolons (27) and higher rootgrowth (22) compared to plants without the enzyme. These characteristics cannot be easily related to the known properties of the enzyme and possibly constitute the effects of other genes linked to Li. The frost resistance of lili plants, once thought of as the reason of the geographical clines, has not been confirmed by Kakes: of 80 plants comprising the four cyanotypes in equal frequencies four individuals died in the comparatively severe winter of 1985/1986. Two of the survivors were AcacLili, and the other two were acaclili. (See 76 for full experimental details) So we are here in a rather paradoxal situation: Although biochemical evidence suggests that linamarin and linamarase form a coordinated system, we lack direct experimental evidence that they serve a coordinated ecological function. There are several ways out of this dilemma: the first is that we may be looking in the wrong direction: linamarase could have a function that is related to the metabolism of the cyanogenic glucosides, but not to chemical defense. The second is that linamarase has indeed a function in the defensive system, but that we have as yet failed to study the right herbivores under the right conditions. The third possibility is that although linamarase has served some function in the past, it does no longer do so, at least not in the majority of extant white clover populations. This may not seem obvious, but we
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
150
/9-GLUCOSIDASES: BIOCHEMISTRY AND MOLECULAR BIOLOGY
Table I. Seedling Experiment Results Cyanotypes: Lili //// Acac 30 20
Total 50
acac
5
5
10
Total
35
25
60
706050-
I ^os
30-
5 io-
TJ
-10
I I
-20 -30
-40 -50 -60H -70 -80-90-
Acac Lili
Acac lili
acac Lili
acac lili
cyanotypes
Figure 5. Grazing index of the cyanotypes of T.repen&The difference in leaf area before and after a week of grazing by the snail Helix aspersa is expressed on the Y-axis as percentage of the difference in leaf area of a non grazed control.As the latter difference was positive, a negative value of the grazing index means that more leaf was removed by grazing than added by growth. The effect of Ac is significant but that of Li is not. 20
15-
Acac Lili
Acac lili
acac Lili
acac . lili cyanotypes
Figure 6. Mean dry weight of the ripe flower heads of the cyanotypes of T. repens. The effect of Ac is significant but that of Li i s not. Cyanotypes of the survivors of 178 seedlings of T.repens grazed for 18 days by Helix aspersa and Cepaea nemoralis. The proportion of Acac - acac plants differs significantly from a non-grazed control, but that of Lili -lili not. See also (16). Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
10.
KAKES
/3-Glucosidase Linamarase in Natural Populations
151
must realize that of the two species ancestral to T.repens one, T.nigrescens, with a southerly distribution is monomorphic for cyanogenesis, i.e. all individuals contain linamarase, whereas in the other species, T.occidentale, no plants with linamarase have been found to date, although the majority of plants contain linamarin/lotaustralin (23, Kakes, unpublished). So far I have discussed genetic variation in linamarase content. However there is also non-genetic variation. The expression of the L/-gene is dependent on the environment (24, 25). Especially at high temperatures the expression of certain genotypes is strongly depressed so that they are difficult to distinguish from lili plants. We have developed a method for the quantitative determination of linamarase in single leaflets of white clover. We are planning to use this method to study the effects of environmental factors on the expression of Li under natural conditions. A pilot experiment, to study the validity of the method, has unexpectedly shown that the leaflets of one leaf may show a two to threefold difference in linamarase content. It is difficult to believe that these differences are caused by the environment. It rather looks like an endogenous mechanism, although we will have to do more experiments to reach a firm conclusion. I mention these results for the following reason: in order to understand the function and variation of linamarase we must look upon the system from the point of view of the plant on one hand and from the point of view of the herbivores on the other. From the point of view of the plant it is irrelevant if some part of it is eaten by herbivores so long as survival and reproduction are not impaired. The herbivores, especially the smaller ones, see the world as a mosaic of more and less palatable plant parts. White clover adds to the complexity of their world both by genetic means (cyanogenic and acyanogenic clones growing intermingled) as by epigenetic means (environmental and endogenous variation). It is this complexity that prevents a herbivore from adapting to one specific type of plant. In a recent paper TillBottraud and Gouyon (26) show that under certain conditions natural selection will favour a mixture of cyanogenic and acyanogenic plants or leaves. It may well be that variation is an essential part of the natural function of linamarase. Conclusions We have seen that from a biochemical point of view linamarase has a well defined function: it is the only plant enzyme that effectively hydrolyzes the cyanogenic substrates. No other natural substrates have been discovered as yet, at least not in white clover. The evidence for its ecological function is less clear. Certainly it is not an essential enzyme: as we saw before it is absent from many plants and some populations and there is even one species closely related to T.repens in wich the substrates are present and the enzyme is presumably lacking. On the other hand there is circumstantial evidence of its function: That evidence is of a statistical nature: the correlated distributions of Ac and Li, both on a local and on a geographical scale. There is no direct experimental evidence that linamarase is necessary for the protection against herbivores. However, we have isolated observations of selective eating of lili plants by waterfowl. Although such observations do not carry much weight in themselves, they strenghten the conclusion from statistical data that there is indeed a function for linamarase. In any case, the coocurrence of plants with and without linamarase in most populations suggests that variation in linamarase content may be the evolutionary optimum for white clover in most populations. Literature cited 1. 2. 3.
Melville, J. ; Doak, B.W. N.Z.J. Sci. Techn. 1940, 22B, 67-71. Kakes, P. Planta 1985, 166, 156-160. Hughes, M . A . Heredity 1991, 66, 105-115.
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
152 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
/9-GLUCOSIDASES: B I O C H E M I S T R Y A N D M O L E C U L A R B I O L O G Y
Gliddon, C., Saleem, M. In: Genetic Differentiation and Dispersal in Plants P. Jacquard et al Eds.; Springer: Berlin, 1985, 293-308. Hughes, M . A . ; Stirling, J.D. Euphytica 1982, 3, 477-483. Maher, E.P.; Hughes, M . A . Biochem. Gen. 1973, 8, 13-15. Hughes, M.A.; Dunn, M . A . Plant Mol. Biol. 1982, 1, 169-181. Hughes, M.A.; Dunn, M . A ; Pearson, J.R. Heredity 1985, 55, 387-391. Kakes, P.; Eeltink, H . Z. Naturforsch. 1985, 40c, 509-513. Boersma, P.; Kakes, P.; Schram,.A.W. Acta Bot. Need. 1983.32, 39-47. Pócsi, I., Kiss, L.; Hughes, M.A.; Nánási, P. Arch. Biochem. Biophys. 1989, 272, 496-506. Daday, J. Heredity 1954, 8, 61-78. Till, I.; Kakes, P.; Dommée, B . Oecol. Plant 1988, 9, 393-404. Ennos, R.A. Genet. Res. 1982, 40, 65-73. Kakes, P. Acta Bot. Neerl. 1987, 36, 59-69. Kakes, P. Theor. Appl. Genet. 1989, 77, 111-118. Dirzo, R. ; Harper, J.L. J. Ecol. 1982, 70, 101-117. Dritschilo, W ; Krummel, J.; Pimentel, D. Heredity 1979, 42, 49-56. Mowat, D.J.; Shakeel, M . A . Grass Forage Sci. 1988, 43, 371-375. Ennos, R.A. Heredity 1981, 46, 127-132. Kakes, P. 1990. Euphytica 48, 25-43. Dommée, B.; Brakefield, P.M.; Macnair, M.R. Oecol. Plant 1980, 1, 367-370. Gibson, P.B.; Barnett, O.W; Gillingham, Y.T. Crop Sci. 1972, 12, 708-709. Waal, R. de. Thesis Landbouwhogeschool, Wageningen, The Netherlands
1942. 25. Till, I. Heredity 1987, 59, 265-271. 26. Till-Bottraud, I.; Gouyon, P.H. Am. Nat. 1992, 139, 509-520. R E C E I V E D January 12,
1993
Esen; ß-Glucosidases ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
