Mechanism and regulation of galactomannan biosynthesis in developing leguminous seeds. J. S. Grant Reid,* Mary E. Edwards,* Michael J. Gidleyt and Allan H.
Biochemistry of Plant Polysaccharides
Mechanism and regulation of galactomannan biosynthesis in developing leguminous seeds J. S. Grant Reid,* Mary E. Edwards,* Michael J. Gidleyt and Allan H. Clarkt *Department of Biological and Molecular Sciences, School of Natural Sciences, University of Stirling, Stirling FK94LA, U.K.and tUnilever Research Laboratory, Colworth House, Sharnbrook, Bedford MK44 I LQ, U.K. The galactomannans [ l ] are a group of watersoluble polysaccharides which are found in the endosperms of leguminous seeds. (Not all legume seeds are endospermic, but if an endosperm is present it will contain galactomannan). Structurally, legume-seed galactomannans comprise a linear ‘backbone’ of ( 1 4)-@-linked u-mannopyranosyl residues, which is laterally substituted by u-galactopyranosyl residues linked a, 1 6 to backbone mannose: +
-.
Gal 1 Gal 1 Gal 1 la la la h B B h B B 6 B - -Man 1 4Manl- 4Manl- 4Manl-4Manl- 4Man - 6 h 6 la la ta Gal 1 Gal 1 Gal 1
-
In the above partial structure all the positions of possible galactose substitutions on the backbone have been shown occupied. In nature this is not the case, the galactomannans from different leguminous species having different degrees of galactose substitution, ranging from about 2.5% (low substitution) to about 9.5% (high substitution). The degree of substitution reflects the taxonomic division of the Leguminosae [2, 31. Species from the more primitive largely tropical Leguminosae-Caesalpinioideae have galactomannans with low substitution, whereas medium to high substitution is typical of the more advanced Leguminosae-Faboideae. In the seed endosperm the galactomannans are localized in the cell walls. They are deposited there massively during seed development and are mobilized following seed germination. Functionally, therefore, they belong to the seed cell-wall storage polysaccharides [4], although they can have a biological relevance extending beyond that of a substrate reserve. It has been shown, for example, that the galactomannan in the endosperm of the fenugreek seed plays a role in the water relations of germination and seedling development. The hydrophilic properties of the galactomannan allow the imbibition by the seed endosperm of copious amounts of water, which is localized between the germinating embryo and the external environment. Since the galactomannan can lose most of its imbibed water with little change in the water-potential of the system, it then ‘buffers’ the
germinating embryo against desiccation during periods of drought following imbibition [51. Legume-seed galactomannans are used in industry [l]. For example in the food industry the galactomannans from guar (Cyamopsis tetragonoloba) and locust bean (Ceratonia siliqua) seed are used extensively as thickeners (viscosifiers) and stabilizers. The (low galactose) galactomannan of Ceratonziz (locust bean gum) is, in addition, used for special rheologies, for example the gels which are formed by interaction with xanthan gum [6]. The superior interactive properties of locust bean gum over guar gum have been attributed to its lower degree of galactose substitution, low galactose contents (consistent with water solubility) favouring interaction [71. We are studying galactomannan biosynthesis in the developing seed endosperms of three leguminous species which form galactomannans in vivo with high-, medium- and low-galactose substitution. These are fenugreek ( Trigonella foenum-graecum, galactomannan 96%-substituted), guar (Cyamopni tetragonoloba, galactomannan 6.5%-substituted) and Senna occzifentalk (galactomannan 30%-substituted). The work is aimed at identifying the enzyme systems which are responsible for the polymerization of galactomannans, and at the elucidation of the molecular mechanism underlying the genetic control of the degree of galactose substitution. Fenugreek, guar and senna plants are being grown successfully to flowering and fruiting under controlled-environment conditions. In the case of fenugreek and guar full analyses of developing seeds for galactomannan content and galactomannan composition (degree of substitution) have been carried out, thus identifying the period of development relevant to the synthetic process. These analyses demonstrated also that there was no change in the degree of galactose substitution of the galactomannan product post-deposition, indicating that the regulation in vivo of the degree of galactose substitution must be at the level of the biosynthetic machinery (J. S. G. Reid & M. Edwards, unpublished work). In fenugreek and guar particulate (membrane-bound) enzyme activities were present in extracts prepared from endosperms during galactomannan deposition. These catalysed the efi-
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cient transfer of galactosyl and mannosyl residues from UDP-[ U-14C]-u-galactoseand GDP-[ U-I4C]n-mannose to a polymeric product which was shown by enzymic analysis to be galactomannan [8]. In the absence of UDP-galactose, and in the presence of M 8 + or MnZ+ ions, the GDP-mannose-linked mannosyltransferase formed a linear (1 -.4)-/3-linked D-mannan. In the presence of UDP-galactose (plus M$+ and/or Mn2+) only a trace of polymeric product was formed, which was shown to be a glucan. In the presence of GDP-mannose and UDP-galactose, plus Mn2+,the enzyme system catalysed the formation of a galactomannan of the legume-seed structural type [8]. It has been demonstrated experimentally, by the use of the enzyme system to preform mannan at the site of synthesis, that galactose can be transferred from UDP-galactose only to the most recently transferred (i.e. the non-reducing terminal) mannose residue of the growing (ga1acto)mannan chain [8]. Using saturating substrate levels (80 pM-GDP-mannose, 800 pM-UDP-galactose) the galactomannan products obtained in vitro, using enzyme systems from fenugreek or guar, were of the low-galactose type. Higher levels of substitution, approaching but not exceeding those characteristic of the parent plant in vivo, were obtained by maintaining saturating UDP-galactose and reducing the concentration of the GDP-mannose substrate well below saturating levels. Mannosyl and galactosyl transfer are therefore not independent, but are not tightly coupled. Our current experimental model [81 for legumeseed galactomannan biosynthesis is therefore the co-operative interaction of two closely associated, membrane-bound glycosyltransferases: a GDPmannose-linked ( 1 4)-/3-1,-mannosyltransferase, and a specific, MnZ+-requiring a-i~galactosyltransferase. In fenugreek, guar and senna the levels in endosperm extracts of the galactomannan-forming mannosyl- and galactosyltransferaseactivities parallel closely the time-course of galactomannan deposition in vivo, confirming their involvement in the biosynthetic process in vivo 0. S. G. Reid & M. Edwards, unpublished work).
-.
Man1 -4Man
-
-
-
A
H
C
- - _ ~Man1 - 4 M a n l - 4 M a n l - 4 M a n l - 4 M a n
I
)
Enzyme
Galactomannans are hydrolysed also, but the galactosyl substitution of the mannan backbone influences the degree and pattern of hydrolysis [lo]. Galactosyl substitution at B or D would prevent hydrolysis of the mannosyl linkage between residues C and D, whereas substitution at A, C or E would not [ 101. This action pattern clearly dictates that the higher the degree of galactosyl substitution in a galactomannan, the lower the degree of hydrolysis achievable using the A. niger mannanase. It also limits the permitted structures of the galactomanno-oligosaccharide products of total hydrolysis. 'Permitted' hydrolysis products with degrees of polymerization up to seven are shown below [lo].
mannotriose
a specific galactosylmannobiose
(M,G)
a specific galactosylmannotriose
(M,G)
Gal 1 +
----
1)
1
4Man
H
I -4Man I -4Man I
Gal
Man 1 4Man 1 4Man
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+
mannobiose
Man 1 4Man 1 4Man
Man1
The molecular fine structures of the galactomannans formed in vitro by the enzyme systems have been investigated by a high-resolution enzymic fingerprinting technique. This involves the exhaustive hydrolysis of labelled galactomannans formed in vitro from 14C-labelled GDP-mannose and unlabelled UDP-galactose (mannose-labelled galactomannans), from ''C-labelled UDP-galactose and unlabelled GDP-mannose (galactose-labelled galactomannans), or from GDP-[ 14C]mannoseand UDP-[ ''C]galactose at the same specific radioactivity (galactose- and mannose-labelled galactomannans). Fragmentation was effected using the pure d o - ( 1 -.4)-/3-u-mannanase from Aspergzlllls niger [9], and followed by the quantitative determination of the labelled (ga1acto)mannooligosaccharide products, aftei t.1.c. separation, by digital autoradiography. The molecular mode of action of the A. nker mannanase has been well characterized [lo]. Five mannose residues of a (1 4)p-u-mannan chain are recognized by the enzyme, with cleaving occurring as indicated:
Biochemistry of Plant Polysaccharides
Man1
-
Gal 1
4Manl- 4Manl- 4Man 1 + 4Manl- 4Man t Gal
a specific digalactosylmannopentaose
(M,G,)
25
We are able to separate the above, and higher permitted oligosaccharides, by t.1.c. Labelled in vitro products are therefore hydrolysed completely using the A. niger endo-p-mannanase and the amounts of radioactivity associated with each galactomannan oligosaccharide product are determined quantitatively by digital autoradiography of t.1.c. plates. Data of this type are being used to determine the molecular tine-structure of galactomannans biosynthesized in vitro and, in conjunction with our experimental model of the biosynthetic mechanism, to define quantitatively the transfer specificities of the biosynthetic enzyme systems. The experimental data are handled via a computer program [ l l ] which simulates both the biosynthesis of galactomannans by the experimentally defined model (galactosyltransfer only to the newly transferred mannosyl residue) and the action of the A. niger /I-mannanase. The model currently in use incorporates a second-order Markov-chain assumption for the biosynthetic process, namely that the probability of galactosyl substitution at the newly transferred mannosyl residue (italicized below) is influenced by the state of substitution of its nearest-neighbour and its second-nearestneighbour mannosyl residue only. This involves four independent probabilities: Munf -4Man1-4Man1 - - - -
probability 0,O (Po,,,)
Gal 1
---
probability 1,0 (PI,,,)
Man1 4Man 1 4Man 1 - - - -
probability 0,1 (P(,, I)
Munl-4Man1-4Manl-
-
-.
1 Gal
Gal 1 Manl-4Man1-4Manlt Gal
---
probability 1 , l ( P , , , )
Thus for a given degree of galactosyl substitution (obtainable from the experimental data) the program will model the biosynthetic process, according different values of Po,,,PI,,, Po,land It will then model the endo-p-mannanase hydrolysis of the computer-biosynthesized galactomannans, predicting the relative proportions of the permitted galactomannan oligosaccharides released. For each experimental data-set (the endo-mannanase hydrolysis of a single, I4C-labelled biosynthetic product
formed in vitro), the computer-modelling process will therefore deliver a set of best-fit values of the four probabilities Po,, to Pl,l.The results of this exercise obtained to date are summarized in Table 1. The absolute values of Po,, to obtained experimentally are naturally dependent upon the degree of galactosyl substitution of the in vitro galactomannan. However, there is a remarkable consistency within the data for a given species (fenugreek, guar or senna) of the relative values of the four probabilities ('scaled probabilities' in Table 1). The values of the four scaled probabilities for the three species are moreover distinctly different. In Table 1, the zero-zero probability (Po,,)has been set at unity. If the figures for the fenugreek system are considered in isolation it is evident that the are considerably greater than values of Po,land unity while PI,,is at least equal to unity. The practical consequence of such biosynthetic 'rules' in a real system would be that any pattern of substitution at the two 'nearest-neighbour' mannosyl residues would positively enhance the probability of substitution of the newly transferred mannose residue ('pushing' towards high substitution). By contrast the data for guar and senna contain probabilities which are less than unity. In senna, for example, the value of is approximately 0.3, indicating that the biosynthetic enzyme system discriminates against further substitution following two contiguous galactosyl-substituted mannosyl residues. A further modelling exercise has been carried out using the scale probabilities from Table 1. In reality, the value of a probability cannot be greater than unity. The highest probability value (Po,",PI,", or PI,Jwithin the scaled probabilities for each species was therefore accorded the value 1.0 and the computer program was used to predict the degree of galactose substitution of a galactomannan biosynthesized under these limiting conditions. To our surprise (and satisfaction!) the predicted limiting galactose contents for the fenugreek and guar systems were in almost perfect agreement with the values for the native polysaccharides. In the case of senna the agreement was less close, and this system is currently being examined further. There was, of course, no reason to expect the biosynthetic systems to operate in vivo under limiting conditions.
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Table I
Calculated probabilities of galactose substitution at newly transferred (terminal, non-reducing) mannosyl residues with different substitution patterns at the nearest-neighbour and t h e second-nearest-neighbour mannose residues
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P , ”, Substitution at the nearest neighbour and no substitution at the second-nearest neighbour. etc.
Projected limiting degree of substitution
Scaled probabiIities Biosynthetic system Fenugreek Guar Senna
P0.o
PI.0
Po. I
1.0 1.0 1.0
I.18fO.2 1.58f0.2 1.39f0.2
1.89f0.5 1.04fO.I 0.43f0.3
The self-consistency of our data for fenugreek, for guar and for senna gives us no reason to reject the second-order Markov chain assumption built into our model. The probability of galactose substitution at a newly transferred mannosyl residue appears to be influenced only by the state of substitution of the nearest-neighbour and the secondnearest-neighbour mannosyl residue. Our results demonstrate clearly that the biosynthetic systems of fenugreek, guar and senna follow different biosynthetic rules, which we have defined quantitatively via the four probabilities PO.,)to In themselves these probabilities set an upper limit to the degree of galactosyl substitution achievable by a given biosynthetic system. In fenugreek and in guar the biosynthetic enzyme systems produce, in vivo, galactomannans with this limiting structure, indicating that in these systems it is the specificity of the glycosyltransferase systems alone which controls the degree of substitution. In senna, the biosynthetic ‘rules’ are followed but the native polysaccharide does not have the theoretical ‘limiting’ composition. The observed differences in transfer specificity between the galactomannan-synthesizing enzyme systems from the three species indicate differences in their molecular architecture. This is currently under investigation.
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PI,,
(%)
1.85f0.8
0.76f0.5
87f8 68 f 5
0.31f0.2
54f3
We are grateful to the Agricultural and Food Research Council, U.K. for financial support and encouragement.
1. Reid. J. S. G. (1985) in Hiochemistry of Storage Carbohydrates in Green Plants (Lky. 1’. M. & Dixon, R. A., eds.), pp. 265-2x8, Academic Press. London 2. Keid, J. S. G. & Meier, H. (1970) Z.I’flanzenphysiol. 62,89-92 3. Bailey, R. W. (1971) in Chemotaxonomy of the Leguminosae (Harborne, J. B., Houlter, L). & Turner, B. L., eds.). pp. 503-541, Academic Press, New York 4. Reid, J. S.G. (1985) Adv. Hot. Hes. 11, 125-155 5. Reid, J. S. G. & Bewley, J. 1). (1979) I’lanta 147, 145-150 6. Dea. I. C. M. & Morrison, A. (1985) Adv. Carbohydr. Chem. Hiochem. 31.241-312 7. Dea. I. C. M., Morris, E. R., Kees, L). A., Welsh. E. J., Barnes. H. A. & Price, J. (1977) Carbohydr. Res. 57, 249-272 8. Edwards, M., Hulpin. P. V.. Dea, 1. C. M. & Reid, J. S. G. (1989) Planta 178.4 1-5 1 9. McCleary, B. V. (1979) Phytochem. 18,757-763 10. McCleary, R. V. & Matheson, N. K. (1983) Carbohydr. Res. 119,191-219 11. McCleary, €3. V., Clark, A. H., Dea, 1. C. M. & Hees, D. A. (1985) Carbohydr. Res. 139,237-260
Received 10 September 1991
