Introduction

Since the beginning of civilisation, mankind has made use of plant polysaccharides as key to survival and to adapt to Earth's environmental conditions as well as to find new technologies and explore and conquer the world. Either as food (starch in cereals for example) or shelter (cellulose in wood) or fast locomotion on land or water (wood in carriages and boats), the physico-chemical properties of these very versatile substances permitted multiple uses. As civilisation evolved, increasing development of new technologies occurred, the polysaccharides being among the essential factors leading to their improvement. Sailing would not be possible without wood, writing would not be possible without paper (cellulose) and large populations feeding would not be possible without starch.

The refinement of the technologies led to the discovery and the control of new materials (plastics for ex.) and building of new equipment which nowadays are not strictly based on polysaccharides anymore such as computers, metal boats etc. However, it is not wrong to say that polysaccharides properties could not be substituted (yet?) by most of the recent

"■Corresponding author: [email protected] or [email protected] Supported by FAPESP/BIOTA n5: 98/05124-8

technologies. For instance, most of our clothing are still made of polysaccharide (cellulose) and so is our food (starch and some cell wall polysaccharides).

Less abundant polysaccharides have similar importance. Certain polymers that occur in legume seeds are known, since ancient times, to form very viscous solutions and this property was used for example in the refined techniques of mummification by the Egyptians [1, 2]. In recent times, this same property has been used for several technological purposes. The galactomannan gum is nowadays used in many different types of industrialised foods (icecream and soups for example) as well as in a number of other industrial sectors (pharmaceutical, paper, explosives and others).

These mannose containing substances (the galactomannans) are present mainly in seeds of plants belonging to the family Leguminosae, but as research progresses, mannans are being found in several other families and, due to differences in chemical structure which lead to distinct properties, it has been found that they may serve other purposes, either in the plant and/or for technological applications.

Even today, very few species are used as a source of mannan for industrial applications and as a consequence they are the most studied species on this respect. Nevertheless, galactomannans are present in seeds of many species of legumes. It is now established for a few species that these polymers are storage polysaccharides and are degraded by specific enzymes following germination. After isolation, some of these enzymes became tools to probe the fine structure of galactomannans and, through this knowledge, are leading to a much better understanding of their properties. Once the enzymes are isolated, the way is paved to start the search for the genes that encode them and this will enable an even better control of the structure and consequently the use of these polymers [3, 4],

Therefore, it can be said that the properties of galactomannans have just started to be discovered and their status of "new material" can be retaken whenever we find a new application or when we find another function in the biological system where it occurs, i.e. the seed. In this context, it is very important to continue the screening for new sources as well as searching for the biological functions played by this group of polysaccharides in the plant tissues where they occur.

Together with an intelligent form of search for new structures provided by the chemotaxonomic studies, the cloning of the genes that encode for the synthases of galactomannans is one of the most difficult tasks to achieve. But it is probably worth trying, since the power of controlling polysaccharide structure by synthesising them in vitro or modifying their assembly in vivo will certainly be among the most important and valuable technologies of the 21 st century.

This is one of the scenarios found as this chapter is being written. In the review that follows, we shall focus on some details in the lines of thinking described above, with the aim to show some of the main approaches adopted by scientists to study this important group of polysaccharides in recent years. The focuses of the chapter will be on the occurrence of galactomannan in legumes and how chemotaxonomical information can be used for searching of galactomannan with desired structures. Also, the importance of galactomannan for the physiology of the plant tissue where it occurs, as well as some aspects of control of metabolism will be discussed in an attempt to shed some light on how galactomannan appeared and was modelled during evolution. This approach is important in the context that understanding the relationship between structure-biological function-evolutionary meaning will help to understand the importance of galactomannan for the adaptation of key species to the tropical environments, which are one of the most important reservoirs of biological diversity on Earth. Understanding how they grow and adapt to their own environment will provide means to exploit these species in a sustainable fashion. Also, we will try to correlate all this information with the purpose of finding even finer tools to control production and quality of galactomannans for our own use.

2. MANNANS AMONG THE CELL WALL STORAGE POLYSACCHARIDES

The cell wall storage polysaccharides (CWSP) have been usually classified into three groups: mannans, xyloglucans and galactans [5, 3, 6]. Such a classification is based on structure and composition, the mannans being also divided into pure mannans, glucomannans and galactomannans [4].

A recent model has been proposed for the plant cell wall [7, 8] in which three matrices (cellulose-hemicellulose, pectin, protein) are thought to exist. The CWSP might be considered as magnifications of these carbohydrate matrices [6]. Mannans are polymers that, when present as CWSP, form a matrix that apparently lack cellulose and pectin in same proportions that occur in primary cell walls.

In many cases the CWSP appear to be special depositions of a single polysaccharide below a "normal" primary cell wall. It is therefore reasonable to suppose that during its deposition, either cellulose biosynthesis is suppressed or synthesis of hemicelluloses is enhanced. Although it is not understood how can a stable cell wall be assembled without (or with minimal amounts of ) cellulose, it is possible that this might be related with the facilitation of the mobilisation processes, which could be considerably more complex if cellulose were present.

Although the focus of the present chapter will be on the galactomannans present in legumes, in order to understand the role and origin of these polysaccharides it is worthwhile to compare it with similar polymers that occur in other plant families. That is why some information on mannans present in seeds of non-legumes was included.

3. THE MANNAN GROUP

Species from different plant families of mono and dicots store mannose-containing polysaccharides in their seeds. All are based on variations of a P-mannan backbone, which may be interrupted with D-glucose, and/or branched with a-(l-»6) linked galactose. Depending on their structure and branching degree, these CWSP may play distinct roles, from hardness in Palmae to water relations control in the Leguminosae.

The pure mannans are artificially defined as polymers having 90% or more mannose with a linear chain of 13-( 1 —»4)-linked manopyranosyl residues with up to 10% of these residues substituted by single units of a-(l—>6)-linked galactoses. Mannans are structurally related to the galactomannans, but having fewer galactosyl branch-points. For this reason they are (unlike the galactomannans) insoluble in water and self-interactive, being to some extent crystalline in the cell wall [9]. Mannans are found in monocotyledons [e.g. Phoenyx dactylifera and Phytelephas macrocarpa (ivory nut mannan)] and in dicotyledons [5].

Phoenix dactylifera seed germination was studied by Sachs [10] and extended by Keusch [11]. In these seeds, a small conical embryo develops slowly and its cotyledon is transformed in a haustorium, which absorbs the products of degradation of the reserves following germination. In those seeds, the hydrolytic enzymes endo-P-mannanase and (3-mannosidase were detected in the dissolution zone near the haustorium. Besides, it has been proposed that the haustorium might not be responsible for the production of the enzymes [11], Instead, enzymes would be activated in the endospermic cells, which are living and metabolically active, by an unknown signal coming from the haustorium. The authors, however, did not rule out the hypothesis that inactive enzymes could have been secreted by the haustorium and activated in the endosperm.

Mannans possibly have other functions apart from being reserve substances. The great hardness of the date seed has been attributed to the presence of this polymer in endosperm cell walls. In fact, mannan-containing seeds are, in general, very hard and resistant to mechanical damage.

Species like pepper [12], celery [13], tomato [14], lettuce [15], coffee [16] and datura [17] have been reported to possess mannan or have an endosperm capable of producing endo P-mannanase. In most of these cases, endosperm degradation is induced by gibberellic acid and in some cases inhibited by abscisic acid (ABA).

Recently, Bewley et al. [18] cloned a cDNA encoding an endo P-mannanase from tomato. They found a low homology with fungal mannanases (28-30%) and also found that it is expressed in the endosperm but not in other parts of the plant. This will possibly be a valuable tool for studying the control of one of the most important enzymes involved in mannan mobilisation, especially concerning the mechanism of action of the hormones (gibberellin and ABA) in mannan containing seeds.

It is believed that in tomato and lettuce, mannan plays its function by increasing the hardness of the endosperm. The mannan present in the endosperm surrounding the root cap of tomato has been shown to be an important constraint to radicle protrusion [19, 14], It was found that the weakening of the endosperm, which is induced by gibberellin, facilitates germination. Groot and Karssen [20] presented results showing that the effect on galactomannan degradation induced by gibberellin can be reversed by ABA. Some authors affirm that as mannans present in the endosperm are also mobilised after germination (for ex. in tomato [21] and coffee [22], [23]) they can, to a certain extent, be considered as storage compounds.

The observations cited above, on palms and other non-legume mannan containing seeds, suggest that this polymer may be considered as a bifunctional molecule. This polysaccharide might play a role as a constrictor of radicle protrusion during germination, probably setting the time when radicle should start growing, and as a storage polysaccharide after germination, the latter mainly in palms and coffee seeds where mannan yield is higher.

Of the CWSP of the mannan group, the glucomannans are the least studied. They can be extracted with alkali from seeds of some species of Liliaceae (Asparagus officinalis [24] and Edymion mutans [25], Scilla nonscripta [26]), and Iridaceae (Iris ochroleuca and 1. sibirica [27]). The structures of glucomannans have been determined by methylation analysis. They have a linear P-(l->4)-linked backbone containing almost equal numbers of p-glucopyranosyl and P-mannopyranosyl residues. Some branching (3-6%), with single (l-»6) (probably a-linked) galactopyranosyl residues occur.

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