Xyloglucan mobilisation in cotyledons of developing plantlets of
Hymenaea courbaril
L. (Leguminosae-Caesalpinoideae)
Marco Aure´lio Silva Tine´
a,b, Angelo Luiz Cortelazzo
b, Marcos Silveira Buckeridge
a,*
aSec¸a˜o de Fisiologia e Bioquı´mica de Plantas,Instituto de Botaˆnica,Caixa Postal4005,CEP01061-030,Sa˜o Paulo,SP,Brazil bDepartamento de Biologia Celular,UNICAMP,Campinas,SP,Brazil
Received 9 April 1999; received in revised form 16 November 1999; accepted 16 November 1999
Abstract
Many seeds contain storage compounds that are used by the embryo/plantlet as a source of nutrients after germination. In seeds ofHymenaea courbaril, a leguminous tree, the main reserve consists of a structurally unusual xyloglucan stored in thickened walls of the cotyledon cells. The present work aimed to studyH.courbarilxyloglucan metabolism during and after germination in order to compare its degrading system with the other known xyloglucan containing seeds. Polysaccharide degradation occurred after germination between 35 and 55 days after planting. The activities ofa-xylosidase,b-glucosidase,b-galactosidase and XET rose during the period of xyloglucan disassembling but a low level of endo-b-glucanase activity was detected, suggesting that this XET has high affinity for the oligosaccharides. The pH optimum ofb-galactosidase was different from thea-xylosidase,b-glucosidase and XET optima suggesting that the former may be important in the control of the mobilisation process. A tentative model for xyloglucan disassembling in vivo is proposed, whereb-galactosidase allows the free oligosaccharides to bypass a transglycosylation cycle and be disassembled by the other exo-enzymes. Some ecophysiological comparisons among H. courbaril and other xyloglucan storing seeds are discussed. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Xyloglucan;Hymenaea courbaril; Seed; Storage; Cell wall; XET
www.elsevier.com/locate/plantsci
1. Introduction
Many legume seeds are known to accumulate carbohydrates as storage compounds. Usually, the first reserves to be broken down during germina-tion are the raffinose family oligosaccharides, whereas cell wall polysaccharides such as galac-tomannan [1], galactan [2] and xyloglucan [3,4] are thought to be reserves for the growing plantlet, being degraded after germination [5]. Among legumes, some members of the subfamily Cae-salpinoideae are known to accumulate xyloglucan in the cotyledons [6 – 9]. These polymers are
present in storage cell walls of species such as tamarind (Tamarindus indica) [6],Copaifera langs
-dorffii and Hymenaea courbaril [4,8].
Seed storage xyloglucan have a cellulosic b -(1,4)-glucan backbone, branched at some points with a-(1,6)-xylopyranosyl (forming with the glu-cose of the backbone a disaccharide assigned X) or
b-(1,2)-D-galactopyranosyl-a(1,6)-D-xylopyranosyl
(forming a trisaccharide assigned L) [10]. Except for the absence of terminal fucosyl units a -(1,2)-linked to theb-D-galactopyranosyl groups, there is
a remarkable similarity between seed storage xy-loglucans and structural xyloglucan from primary walls of dicotyledons [11]. The basic polymer molecule is composed of heptasaccharide repeat-ing units of XXXG with variation in the substitu-tion with galactose generating oligosaccharides structures like XLXG, XXLG and XLLG.
Abbre6iations:b-gal,b-galactosidase; XET,
xyloglucan-endo-trans-glycosylase;a-xyl,a-xylosidase;b-glc,b-glucosidase.
* Corresponding author. Tel.: +55-11-5584-6300, ext. 287; fax:
+55-11-577-3687.
E-mail address: [email protected] (M.S. Bucke-ridge)
Although Leguminosae seems to be the princi-pal family to possess storage xyloglucan in seeds, a member of Tropaeolaceae (Tropaeolum majus) is the xyloglucan-containing system best studied to date. The work by Reid and collaborators has shown that the rate of xyloglucan mobilisation in this species coincided with the increase and later decrease in the levels of four hydrolytic enzymes: endo-b-glucanase, b-galactosidase (b-gal), a -xy-losidase (a-xyl) and b-glucosidase (b-glc) [12]. Since then, the hydrolases were purified to homo-geneity and some of their properties were studied. Endo-b-glucanase was purified by Edwards et al. [13], and further studies on its mode of action on xyloglucan were performed by Fanutti et al. [14], demonstrating that this enzyme is a xyloglucan-endo-transglycosylase (XET).
As far as we know, the only legume which had its hydrolases studied is Copaifera langsdorffii. Buckeridge et al. [4] inferred the presence of these enzymes through the detection of glucose, galac-tose and xylose released by crude enzymic extracts containing endogenous water soluble xyloglucan. They also detected the exo enzymes (b-gal, a-xyl and b-glc) using artificial substrates.
We now present data on the mobilisation of the xyloglucan of H. courbaril, a native tropical legume known to accumulate up to 40% of the seed dry weight as xyloglucan [8]. Recently a new oligosaccharide was isolated and identified in this seed [15]. The presence of this oligosaccharide makes this polysaccharide unique among others described in the literature, possibly with implica-tions in the metabolism of the cell wall.
2. Material and methods
2.1. Seed origin and germination conditions
Seeds of H. courbaril L. were provided by the Seed Department of the Institute of Botany at Sa˜o Paulo. The quiescent seeds had an average mass of 4.7491.18 g and only seeds between 3 and 6.5 g were used in the experiment to avoid variations caused by very different seed sizes. The seeds were scarified individually by abrading the seed coat with sand paper, weighed, soaked in commercial sodium hypochlorite solution for 5 min, washed with tap water for 10 min, soaked in distilled water for 12 h and planted in vermiculite. Trays
with the seeds regularly spaced were kept at 25°C under a 12 h photoperiod. The regular distribution of the seeds in the trays allowed the calculation of the relative dry weight (i.e. the weight of the embryo or the pair of cotyledons divided by the weight of the quiescent seed), because the initial weight of each seed was known. Every 5 days, 20 seeds were collected until the abscission of the cotyledons which occurred 75 days after planting. Of the 20 seeds collected, 15 were used for dry mass measurements, two were used for micro-scopic observations and three were subjected to carbohydrate extraction and analyses and mea-surement of enzyme activities. The seeds used for the studies of mass allocation were dissected and had their cotyledons and embryo dried for 24 h at 80°C and weighed for the determination of dry weight. The values were expressed in relative dry weight as a result of the difference in the size of the seeds used.
2.2. Light microscopy
The seeds collected for microscopic observations were fixed with p-formaldehyde (4%)/ glutaralde-hyde (2.5%) for 24 h at 5°C, dehydrated in a series of ethanol (70, 80, 95 and 100%, successively) at room temperature, included in Paraplast and 8mm sections were obtained [16]. For staining with iodine, the sections were covered with a solution of I2/KI (0.5/1%) and photographed immediately.
2.3. Biochemical analyses
All polymeric or oligomeric xyloglucan used in this work was obtained as described in [15]. The seeds collected for biochemical analyses were cut into pieces of approximately 5 mm and the pool was separated into two groups: one was dried as described before, ground and stored at −20°C until usage for carbohydrate analyses; the other was homogenised in 50 mM sodium acetate buffer (pH 5) with NaCl (200 mM), centrifuged (10 000×g, 10 min) and the supernatant was used as crude extract for determination of enzyme activities.
rang-ing from 2.6 to 7.6 (with increases of 0.2 units). Sodium acetate buffer (pH 5) was used in all assays in the rest of the work. In the viscometric assays, the fall in viscosity, characteristic of endo activity, was observed only when xyloglucan oligosaccharides were added to the assay medium (see below). Therefore, limit digest xyloglucan oligosaccharides were added to the assay and the enzyme named XET.
The choice of substrates was based on the knowledge gathered after the studies by Reid and co-workers [12 – 14,17]. For measuringa-xyl activ-ity, the release of pentose (measured according to [18]) from cellulase limit digest oligosaccharides (obtained as in [15]) was used. The enzymatic extract (20ml) was incubated with 25 ml of sodium acetate buffer (100 mM pH 5.0) and 20 ml of a 20 mg ml−1oligosaccharide solution for 24 h at 30°C
and the amount of pentose released was measured.
b-gal was assayed against pNP-b-D
-galactopyra-noside (Sigma) [10 ml of crude extract, 10 ml of sodium acetate (100 mM pH 5.0) and 10 ml of 50 mM substrate, incubated for 30 min at 40°C].
b-glc was assayed similar to b-gal, but using
pNP-b-D-glucopyranoside as substrate. XET was
as-sayed viscometrically using purified H. courbaril
xyloglucan as substrate (400 ml of 1.2%
xyloglu-can, 10 ml of a 20 mg ml−1oligosaccharide
solu-tion, 50 ml of enzymatic extract and 60 ml of 1 M sodium acetate pH 5, incubated at 30°C and the flow through a 0.2 ml pipette was measured every 5 min). The activity of endo-glucanase was mea-sured as described above but without addition of oligosaccharides. It was calculated as the slope of the line obtained from the logarithm of the flow time versus incubation time.
2.4. Xyloglucan extraction and analysis
For extraction of polysaccharides, 100 mg of dried seeds were extracted with ethanol (4×750ml of 80% ethanol, 80°C, 10 min) and then with water (4×1 ml, 80°C, 180 min). The insoluble material was extracted with 4 M KOH with 26 mM sodium borohydride (3×1 ml, 180 min, room tempera-ture). The alkali extracted polymer was neutralised with acetic acid and both water and alkali ex-tracted polysaccharides were dialysed against dis-tilled water and freeze dried. The polysaccharides were hydrolysed with sulphuric acid according to Ref. [19] or hydrolysed with cellulase (Megazyme-Australia) according to Ref. [15]. The monosac-charide products of acidic hydrolysis were analysed by HPAEC-PAD (Dionex) using iso-cratic elution (23 mM NaOH, 0.8 ml min−1) for
20 min with a PA-1 column (CARBOPAK). The proportions among monosaccharides were cor-rected according to detector sensitivity to each monosaccharide, calculated by using equimolar standards. The xyloglucan oligosaccharides were analysed in a gradient of sodium acetate from 75 to 116.5 mM in isocratic NaOH (150 mM) for 40 min (1 ml min−1) using the same column.
3. Results
3.1. Mass allocation and morphologic
characteristics
Fig. 1 shows the changes in the dry mass of the cotyledon and embryo. The first drop in the cotyledon dry weight occurred between 10 and 20 days and was as a result of the seed coat natural detachment. Germination of most of the seeds occurs 35 days after planting and during the next 20 days the embryo dry mass increases while the mass of the cotyledons decrease. The primary
Fig. 2. Sections of parenchymatic cells of the cotyledons of
Hymenaea courbarilstained with iodine. In quiescent seeds (a)
the storage xyloglucan (W) and protein bodies ( ) can be observed. During xyloglucan breakdown (b), starch grains ( ) appear in the cytoplasm. After xyloglucan mobilisation (c), the storage wall has been completely dissembled and only a few starch grains can be seen. Bars=10mm.
leaves expand to their full length by day 45. It is only after germination that xyloglucan degrada-tion takes place. Mobilisadegrada-tion starts after day 40 being complete by day 70, when only residual amounts of xyloglucan are left into severely shrunken cotyledons. Xyloglucan solubility in-creases from days 25 to 45, prior to degradation.
3.2. Light microscopy
Fig. 2 shows transversal cuts of the storage parenchyma of cotyledons of H. courbaril at dif-ferent stages of xyloglucan mobilisation. In quies-cent seeds, the appearance of the thick storage cell walls is smooth and protein bodies were observed into the cytoplasm (Fig. 2a). At the beginning of xyloglucan disassembling (day 40) the smooth ap-pearance of the cell wall had disappeared. The staining with iodine was altered and lamellae could be observed. At the same time, starch could be seen in the cytoplasm and the thickness of the wall increased two fold (Fig. 2b). After nearly complete mobilisation (day 60) few xyloglucan could be detected by iodine staining (Fig. 2c) and some starch remained in the cytoplasm. Walls were much thinner, accounting for only 10% of the initial thickness.
3.3. Biochemical analyses
For the optimisation of assay conditions, the pH optima of the hydrolases were determined (Fig. 3).b-glc,a-xyl and XET have maximal activ-ities around pH 4.5, although the latter was active in a broad range of pHs (from 4 to 7). Exception was made for b-gal, which presented a sharp peak of activity at pH 3.2 and less than 30% of its activity at pH 5.0, in which the other hydrolases are most active.
In the XET/endo-b-glucanase assay, the pres-ence of xyloglucan oligosaccharides was required for the detection of activity (Fig. 4). Although a mixture of oligosaccharides with different struc-tures and molecular weights was used, an appar-ently Michaelis-Menten kinetics was obtained, which led us to consider the oligosaccharides as substrates for the transglycosylation reactions.
used, high variability was observed on the enzyme activities during the time course. Fig. 5 shows that
b-glc and b-gal activities were already present in quiescent seeds (but rise during xyloglucan disas-sembling) whereas a-xyl and XET activities were
not detected at the beginning and rose during and after germination, respectively. The cellulase activ-ity raised together with transglycosylation activactiv-ity, indicating that theHymenaea XET has both activ-ities, like Nasturtium XET [14] or contamination of the enzyme extract with xyloglucan which, dur-ing the incubation, generates oligosaccharides.
3.4. Carbohydrate analysis
The changes in xyloglucan structure following germination and plantlet growth were probed by monosaccharide and limit digest oligosaccharides analyses by HPAE chromatography. Table 1 shows monosaccharide analysis of the xyloglucan extracted with hot water and alkali from the cotyledons of quiescent seeds and from 30, 45 and 75 days old cotyledons. The proportion of ara-binose in the water-extracted polymer increased after mobilisation to a proportion close to the one found in the alkali extracted polysaccharide. This arabinose is probably present in xyloglucan molecules, because acid hydrolysis of cellulase limit digest oligosaccharides, which hydrolysed nearly 70% of the xyloglucan from H. courbaril, yields arabinose in approximately the same pro-portions (data not shown). Galactose and xylose also showed changes in proportion. Their propor-tion (amount of monosaccharides for each of the four glucoses) dropped from 1.2 and 3.2, respec-tively, during imbibition, to 0.9 and 2.9 after 30 days and rose back to 1.1 – 1.2 and 3 – 3.3, respec-tively, following mobilisation. These data can be compared with the analyses of the limit digest oligosaccharides (Fig. 6), which show that the peak assigned to oligosaccharide XXLG dramati-cally decreases at the beginning of xyloglucan disassembling, when compared to the other peaks. Together with the change in solubility (see Fig. 1), these data suggest that xyloglucan was somehow processed before complete mobilisation to the embryo.
4. Discussion
4.1. Seedling growth and xyloglucan mobilisation
The seeds of the tropical legume H. courbaril
take approximately 30 days to germinate. They are large seeds and water imbibition takes about one
Fig. 3. Activities of xyloglucan-disassembling enzymes present in crude extracts of 40 days old cotyledons of Hymenaea courbarilat different pHs.A-activity of XET (, expressed in arbitrary units) and b-glucosidase (2, assayed upon pNP-b -glucopyranoside). B-activity of b-galactosidase ( , assayed upon pNP-b-galactopyranoside) anda-xylosidase (, assayed upon xyloglucan oligosaccharides).
Fig. 5. Enzymatic activities detected in crude extract ofHymenaea courbarilcotyledons following germination and early plantlet growth.A-a-xylosidase, assayed upon xyloglucan oligosaccharides;B-b-galactosidase, assayed upon pNP-b-D-galactopyranoside;
C-b-glucosidase, assayed upon pNP-b-D-glucopyranoside; and D-XET assayed by viscosimetry with () and without () xyloglucan oligosaccharides, in arbitrary units.
third of the germination period (10 days), the rest consisting of metabolic activities that culminate in radicle protrusion. Only after germination, the mobilisation of xyloglucan from the cotyledons starts. This could be concluded from the reduction of the cotyledon average dry mass with an increase in the dry mass of the embryo (Fig. 1), the reduc-tion of the cell wall thickness after this period, the appearance of starch in the cell (indicating a large input of carbohydrate into the cell — see Fig. 2) and also by the rise in the activities of the enzymes involved in xyloglucan disassembling (Fig. 5). De-spite the presence of other reserves in the cotyle-dons of quiescent seeds (sucrose, raffinose family oligosaccharides and protein bodies, data not shown), it is only during xyloglucan mobilisation that the dry mass of the embryo changes significantly.
The aspect of the cotyledon cell walls after xyloglucan mobilisation (Fig. 2C) indicates that some structural elements of the wall are not mo-bilised after germination. The presence of two populations of xyloglucan with different
ex-tractability can be suggested from the data in shown in Table 1. Most of the xyloglucan present in quiescent seeds can be extracted with hot water.
Table 1
Proportion of monosaccharides obtained by acidic hydrolysis of the polysaccharide extracted from the seed powder with water and KOH (4 M)a
Days after planting
0 30 45 75
1.1 0.3
0.2 Water Ara 0.2
1.2 0.9
Gal 1.1 1.2
Glc 4.0 4.0 4.0 4.0
3.0 3.3
2.9 3.2
Xyl
KOH Ara 0.7 0.7 1.1 1.4
Gal 1.2 0.9 1.0 1.1
Glc 4.0 4.0 4.0 4.0
3.2
Xyl 2.9 3.0 3.3
aThe area of the glucose peak was considered 4 and the
Fig. 6. Changes in the proportions of the limit digest xyloglu-can oligosaccharides before breakdown of the polysaccharide. The known peaks are (a) XXXG; (b) XXLG; (c) XLLG; and (d) XXXXG.
4.2. Biochemical aspects of xyloglucan
disassembling
Three facts suggests that a modification in the fine structure of the xyloglucan occurs prior to mobilisation: (1) the change in polymer solubility that made the polymer more easily extractable immediately before disassembling; (2) change in the appearance the of the cell wall; and (3) change in the polymer structure. The existence of enzymes with exo-activities, especially b-gal, which is the only one reported to hydrolyse the polymer [20], could explain the changes in the fine structure of the xyloglucan prior to mobilisation. Although a family of b-galactosidases occurs in seeds, only some of its members were purified and character-ised [21,22] and they show differences in specific-ity. The diversity of b-galactosidases and their modes of action suggest a high complexity in the process of degalactosylation of the xyloglucan. For example, the b-gal from C. langsdorffii [22] was shown to hydrolyse galactose from XLLG and XLXG, but not from XXLG. The
characteri-sation of this purified enzyme led to similar results
for Hymenaea concerning the pH optimum for
activity, i.e. a peak at pH 3.2.
As a pH optimum around 5 is expected for wall enzymes [23], the difference observed between the pH optima for b-galactosidase (pH 3.2) and the other enzymes (a-xyl, b-glc and XET) suggest that
b-gal activity may limit, under certain circum-stances, xyloglucan disassembling. This can be im-portant in the control of the process, because the disassembling cannot proceed without the retrieval of the galactose [24]. The acidic growth theory [25] and the model for the control of cell expansion based on the differences of pH optima of the enzymes involved in the process [26] are two previ-ous examples of regulation of cell wall metabolism based on pH.
The requirement of xyloglucan oligosaccharides for detection of viscosity decrease in the assay system for endo-b-glucanase suggests that in H.
courbaril, the enzyme responsible for the release of
xyloglucan fragments from the cell wall is a xy-loglucan endo-transglycosylase (XET). However, differently from the enzyme purified from T. ma
-jus, this XET has a very low activity against purified polymer in the absence of oligosac-charides.
4.3. An impro6ed model for storage xyloglucan
disassembling
In some aspects, our data appear to differ from what has been observed by Edwards et al. [12] in cotyledons of T. majus, in which the activities of the four enzymes overlap. On the basis ofT. majus
system, Buckeridge and Reid [5] proposed a model for xyloglucan degradation in which the polymer would enter a linear disassembling process which would lead from polymer to monomer directly, beginning with the action of one XET (with endo-glucanase activity at low concentrations of oligosaccharides). The fragments released would be hydrolysed by the sequential action of b-gal,
a-xyl and b-glc, producing free galactose, xylose and glucose.
It is possible that in species of the Leguminosae, the xyloglucan degrading system works differently from theT. majusone. Thus, some changes in this model can be suggested to explain the results presented here and by Buckeridge et al. [4], ob-tained with other seeds (from legumes) that store xyloglucan (H.courbaril,C.langsdorffii). This new tentative model (Fig. 7) proposes that seed xy-loglucan degradation occurs in at least three steps: (1) transglycosylation; (2) degalactosylation; and (3) oligosaccharide disassembling. Such a model is
not linear, but would have two different cycles (transglycosylation and disassembling) which would be linked by the action of b-gal. In a condition of low b-gal activity, the fragments gen-erated by the XET would not be disassembled and the rise in their concentration would lead to an increase in endo-transglycosylation activity, halt-ing hydrolysis and avoidhalt-ing an excessive rise in the input of carbohydrate into the cell. After the increase in b-gal activity, these fragments of xy-loglucan would be degalactosylated and subse-quently enter the second cycle of degradation, releasing free monosaccharides.
With only a few cell wall storage systems bio-chemically studied so far, it is difficult to suggest a general model that describes the disassembling of the storage xyloglucan in every seed. As different species are exposed to different environmental conditions, this is likely to reflect distinct strategies of adaptation, requiring diverse physiological and biochemical characteristics. As H. courbaril is a tropical tree with seeds much larger thanT.majus, the disassembling of xyloglucan at the same time in all the cells might lead to an imbalance of the source/sink relation, therefore resulting in an over-load of the transport system towards the embryo. Indeed, the accumulation of starch inside cotyle-don cells suggests that the input of carbohydrate into the cell is greater than the output. If the proposed model is correct, the presence of transg-lycosylation even at low oligosaccharide concen-tration (contrarily to T. majus) might serve to make the disassembling system of H. courbaril
partially reversible, unless b-gal is active. This would delay degradation and possibly synchronise it with a relatively slower pace of mobilisation (30 days from start to end of degradation). This con-trasts with T. majus which, being a faster grower, degrades xyloglucan within an interval of only 5 days [12].
4.4. Ecological meaning of storage xyloglucan
mobilisation strategy
Our microscopic observations suggest that the breakdown of xyloglucan starts in the cells near the vascular bundles and spreads towards the rest of the cotyledons later on (data not shown). Legume seeds have different patterns of storage mobilisation [27] and it possibly represents an optimisation of sugar transport. Under the
condi-Fig. 7. Tentative model proposed for the in vivo disassem-bling of the storage xyloglucan in seeds. The polysaccharide is first hydrolysed by the XET characterising a period ofTrans
tions used in this work to detect enzyme activi-ties, these positional differences in activities were probably lost and instead of a sharp peak of activity, a smooth rise and a plateau were ob-served.
Distinct ecological strategies could also explain some differences between these two plants. Since
T. majus is herbaceous and germinates in areas
with higher light intensity than H. courbaril, its plantlets would have to grow faster in order to achieve reproductive success in less than a year.
H. courbaril plantlets, on the other hand, grow
slower under the canopy and may stay under shadowed conditions for much longer periods. This requirement of an internal source of carbo-hydrate for longer periods could explain the larger seed found in tropical trees, the greater amount of xyloglucan and the longer period for its mobilisation as well as the biochemical differ-ences among the xyloglucan disassembling sys-tems.
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