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Figure 3. Frequency distribution of galactomannan yields (percentage of the dry weight or the seed) (A-C) and mannosergalactose ratio (D-F) in species of the subfamilies of the Leguminosae. Data obtained from the compilation shown in Table 1. (A and D) -Caesalpinioideae; (B and E) - Mimosoideae; (C and F) - Faboideae.

The deposition of galactomannan during seed development was also studied ultrastructurally in fenugreek [113]. These authors observed that newly synthesised galactomannan was associated with the intracisternal space of the rough endoplasmic reticulum and then secreted to the cell walls.

Although Sioufi et al. [114] detected the presence of UDP-galactose and GDP-mannose in seeds of T. foenum-graecum, it was only in 1982 that Campbell and Reid [115] showed that particulate fractions obtained from endosperms of fenugreek, which behaved as endoplasmatic reticulum under ultracentrifugation, were capable to transfer [l4C]-mannose from GDP-[U-14C] -mannose to a water soluble polysaccharide that could not be distinguished from galactomannan.

Edwards et al. [116] reported that particulate enzyme preparations isolated from developing endosperm of fenugreek and guar were highly effective in the formation of polysaccharide from either GDP-mannose (supplemented with divalent ions such as Mg2+, Mn2+or Ca2+) or a mixture of GDP-mannose and UDP-galactose. However, no galactosyl transfer was observed without the presence of GDP-mannose, indicating that the galactosyltransferase activity is dependent on the formation of the mannan chain. The degree of galactose substitution could be manipulated in vitro by varying the concentrations of GDP-mannose at constant and saturating UDP-galactose concentrations. In both cases (fenugreek and guar) lowering the concentration of GDP-mannose greatly increased the percentage of galactose substitution from 20 to 93% and 21 to 70% for fenugreek and guar respectively. On the basis of experiments where enzyme preparations "primed" or "unprimed" with GDP-[U-I4C]-mannose were followed by incubation with UDP-galactose alone or UDP-galactose and GDP-mannose, the authors concluded that transfer of galactose from UDP-galactose to pre-formed mannan is restricted to the residues that remain closely associated with the transferase activity.

In order to probe how the galactomannan biosynthetic process during seed development can result in galactomannans with different mannose:galactose ratios, Edwards et al. [117] performed a comparative study using developing seeds of fenugreek (M/G=l.l), guar (M/G=1.6) and Senna occidentalis (M/G=3.3). They followed galactomannan content and mannosyl- and galactosyl- transfer during maturation of seeds of the three species and found that both enzymes peak during the period when speed of galactomannan formation is higher. Whereas the M/G ratios for fenugreek and guar were constant during the whole developmental period, in seeds of Senna the M/G ratio increased from about 2.3 to 3.3. This increase in M/G ratio was associated with a concomitant increase in activity of a-galactosidase in the endosperm, leading to the suggestion that these two phenomena might be related. On the basis of these results, the authors concluded that in fenugreek and guar, the genetic control of the M/G ratio in galactomanan appears to be based only on the biosynthetic process, whereas in Senna the galactomannan production is probably controlled by the biosynthetic process in the beginning of maturation and later on by debranching, galactose being taken out by a-galactosidase present during late galactomannan deposition. Recently, Aq├╝ila [68] reported a drastic increase in M/G ratio in developing seeds of Senna macranthera var. nervosa, which corroborates these results.

4.4.2. Galactomannan mobilisation: control and metabolism

Although a large number of plant species have been reported to possess galactomannan in their seeds [2, 118, 56, 77] only a few have their post-germinative metabolism studied in detail. Among legumes, the most studied species are guar (Cyamopsis tetragonolobus), fenugreek (Trigonella foenum-graecum) and carob (Ceratonia siliqua). In these species, galactomannan has been shown to be broken down by three hydrolases: a-galactosidase, endo-p-mannanase and p-mannosidase.

The mobilisation of galactomannan in some other legumes has also been studied [119, 120, 121, 122], confirming the presence of the three enzymes mentioned above and also confirming that galactomannan mobilisation is performed through hydrolysis. In all cases studied, the polysaccharide is disassembled to its monosaccharide constituents (free mannose and galactose) at the same time as sucrose is produced in the endosperm (Figure 4). Apparently, sucrose is the sugar of transport that will take the products of storage mobilisation (carbon and energy) to the growing embryo. Starch is transiently produced in the cotyledons [108, 122] and recently Dirk et al. [123] proposed that storage cell wall degradation and starch synthesis might be biochemically related.

McCleary and co-workers [120, 124] performed careful studies on the fate of the products of guar galactomannan after mobilisation. They detected the activities of phosphomannoisomerase and phosphoglucoisomerase in the endosperm and suggested that these enzymes are probably responsible for the epimerisation of mannose into glucose, which will probably be used for sucrose synthesis in the endosperm. These authors fed growing embryos with radioactive mannose and galactose and demonstrated that the products of galactomannan mobilisation in guar are used for several biochemical processes in the growing plantlet [124].

McCleary and Matheson [125] detected multiple forms of a-galactosidases in guar seeds, but only one of them was associated to the endosperm. It has been experimentally demonstrated that this purified a-galactosidase is capable of hydrolysing galactose branches from the polymer. It is a single polypeptide (MW 40.5 KDa) and its gene has been cloned and sequenced [126].

Guar endo-P-mannanase has been purified by McCleary [124] and demonstrated to act on galactomannan polymer, producing a mixture of oligosaccharides. Reid and Edwards [3] highlighted the fact that as the endo-P-mannanase from legume seeds act as true mannanase, its hydrolytic action on galactomannans being hindered by galactose substitution. This underlines the importance of the prior debranching of the polymer and probably explains the very high levels of a-galactosidase activity found during galactomannan mobilisation in vivo in species containing highly substituted polymers.

Exo-P-mannanase (or P-mannosidase or P-D-mannoside mannohydrolase) from guar seeds has been purified to homogeneity by McCleary and Matheson in 1982 [127]. This enzyme has shown specificity on manno-oligosaccharides and no activity on the polymer [124], On the basis of these results it can be suggested that this enzyme has the function of hydrolysing manno oligosaccharides produced by the concerted action of a-galactosidase and endo-P-mannanase.

In all legume species studied, galactomannan mobilisation starts after germination (i.e. radicle protrusion). It has been demonstrated that in endosperms of seeds of Trigonella foenum-graecum and Ceratonia siliqua, the enzymes a-galactosidase and endo p-mannanase in the former and a-galactosidase in the latter are synthesised de novo [128, 129],

In 1985, Spyropoulus & Reid [130] demonstrated that isolated endosperms of seeds of T. foenum-graecum are not capable to mobilise galactomannan completely. However, they do

Figure 4. Biochemical pathways involved in the catabolism of galactomannan and further metabolism of its products by the embryo. When aleurone layer is not present, it is likely that the hydrolases are produced within each endosperm cell and secreted outwards to the cell wall where the same reactions occur (man, mannose, gal, galactose; SPS, sucrose phosphate synthase; Man-6-P, mannose-6-phosphate; Fru-6-P, fructose-6-phosphate; UDP-gal, uridine diphosphate galactose; Suc-6-P, sucrose-6-phosphate). Redrawn from [4]

Figure 4. Biochemical pathways involved in the catabolism of galactomannan and further metabolism of its products by the embryo. When aleurone layer is not present, it is likely that the hydrolases are produced within each endosperm cell and secreted outwards to the cell wall where the same reactions occur (man, mannose, gal, galactose; SPS, sucrose phosphate synthase; Man-6-P, mannose-6-phosphate; Fru-6-P, fructose-6-phosphate; UDP-gal, uridine diphosphate galactose; Suc-6-P, sucrose-6-phosphate). Redrawn from [4]

so either when incubated in a great volume of water or after they have been thoroughly washed with water. On this basis, the authors also proposed the existence of a water-soluble inhibitor and the observation that a-galactosidase activity was significantly inhibited in non water-washed endosperms, led them to suggest that the inhibitor might be acting by preventing enzyme production in the aleurone layer.

Although inhibition of galactomannan breakdown in endosperms of T. foenum-graecum increased with increasing water stress, the activity of the hydrolases was not affected [131]. Based on these results, the authors concluded that water stress does not inhibit galactomannan degradation by preventing enzyme production, but probably by preventing the sink of the degradation products (mannose and galactose), which would inhibit enzyme activity. They still reported that in stressed seeds, reducing sugar levels were fourfold higher than in unstressed controls and that stressed isolated endosperms had very high reducing sugar levels (nine times higher than in the endosperms of unstressed seeds).

On the other hand, those same authors observed that if stress was applied just before the beginning of galactomannan breakdown, there was observable inhibition of production of endo-[3-mannanase and (3-mannosidase, but not of a-galactosidase. The conclusion was that water stress can affect production of endosperm hydrolases if the stress is applied before the start of galactomannan breakdown and that the lack of effect on a-galactosidase might be related to the timing of the synthesis of these enzymes.

The few attempts to induce polysaccharide degradation using gibberellic acid failed, suggesting that legume systems have a different metabolic control when compared with mannan and starch containing seeds. On the other hand, jasmonic acid and its precursor linolenic acid have recently been shown to inhibit galactomannan degradation in carob and fenugreek [132],

Abscisic acid has been shown to act as a potent inhibitor of galactomannan degradation in fenugreek [128], carob [129] and Sesbania marginata (unpublished). In fact, our results clearly indicate that germination is retarded but not inhibited completely. Thus, it is possible to speculate that ABA has a general role (in legumes and non-legumes) as a modulator (rather than inhibitor) of the biochemical and physiological interactions between the endosperm and the embryo following germination and early growth. Its presence inhibits galactomannan degradation and only when it is leaked out or metabolised, its seed water relations function resumes and the transference of carbon and energy between both organs, i.e. storage function, starts.

In all legume seeds studied, as galactomannan is broken down, a correspondent increase in dry weight of the cotyledons is observed. Bewley et al. [133] observed quantitatively and by electron transmission microscopy, a transient accumulation of starch in the cotyledons and axis of fenugreek, during galactomannan mobilisation from the endosperm. Starch was shown to be synthesised and quickly mobilised from cotyledons with the concomitant increase in a-amylase activity. Similar results were observed for carob by Seiler [129] and for Sesbania marginata by Buckeridge and Dietrich [122])\. Leung et al. [134] reported that plantlets of T. foenum-graecum, grown without the endosperm, showed a 30% decrease in relation to control intact seeds, thus characterising the galactomannan as reserve.

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