Journal of Experimental Botany, Vol. 54, No. 384, pp. 901-911,
March 1, 2003
© 2003 Oxford University Press
The role of two isoenzymes of 
-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination
Received 4 April 2002; Accepted 22 October 2002
1 Facultad de Ciencias, Universidad de Chile, Departamento de Biología, Casilla 653, Santiago, Chile
2 Facultad de Ciencias Básicas, Universidad Metropolitana de Ciencias de la Educación, Departamento de Biología, Casilla 147, Santiago, Chile
3 To whom correspondence should be addressed. Fax: +56 2 271 7580. E-mail: lcardemi{at}uchile.cl
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Abstract |
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Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by
-amylase. There are several -amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T0) to a different one observed 90 h after imbibition (T90). The objective of this research was to study the roles of two purified -amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T0 digested starch granules mainly from quiescent seeds, while the isoenzyme T90 digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T0 digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T90 digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T0 and T90 digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T90 digested starch granules previously digested by the isoenzyme T0. This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.
Key words: -Amylase, amyloplasts, Araucaria araucana, isoenzymes, starch granules.
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Introduction |
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In storage organs, as well in other non-photosynthetic tissues of higher plants, starch is the only carbohydrate reserve synthesized and stored in the amyloplasts of these tissues (Liu and Shannon, 1981; Shannon, 1989). In seeds of the conifer tree Araucaria araucana (Mol.) Koch, 61% of the dry weight is starch (Cardemil and Reinero, 1982). Starch is 31% of the dry weight of the embryo and 63% of the dry weight of the megagametophyte. The starch is digested during germination mainly by
-amylase in the embryo and by -amylase and starch phosphorylase in the megagametophyte (Cardemil and Varner, 1984). Seeds germinate after 40 h of imbibition using sucrose that is synthesized from glucose, the product of starch hydrolysis (Cardemil and Reinero, 1982). The sucrose supplied by the megagametophyte is absorbed by the embryo cotyledons, acting as haustorial organs, and then transported into the embryonic tissues for seedling growth and development (Cardemil et al., 1990; Lozada and Cardemil, 1990).
In barley, -amylases are coded by two gene families. One codes for the high isoelectric point (pI) -amylases and the other for the low pI -amylases (Jacobsen and Higgins, 1982; Rogers et al., 1992). Both gene families are induced by gibberellic acid (GA3) but with different sensitivity to this hormone (Jacobsen and Higgins, 1982; Rogers et al., 1992, 1994). Both gene families are negatively regulated by abscisic acid (ABA), which suppresses the GA3 induction (Gómez-Cadenas et al., 1999).
In A. araucana, there are also multiple forms of -amylases (Salas and Cardemil, 1986). The -amylases from the embryo and megagametophyte show a complex pattern that changes during germination, with differences in their relative mobilities (Rm) and pIs (Salas and Cardemil, 1986). These isoforms can be defined as isoenzymes since they have differences in amino acid composition and susceptibility of peptide cleavage by cyanogen bromide (Acevedo and Cardemil, 1994).
Seven -amylase isoenzymes have been detected at the onset of imbibition, with isoelectric points that are slightly different from those found after 90 h of imbibition (Salas and Cardemil, 1986). The molecular masses of these isoenzymes vary between 47 and 56 kDa (Acevedo and Cardemil, 1994). Accumulation of these -amylases seems to be regulated by GA3 and by ABA (Acevedo and Cardemil, 1997).
Starch granules are also complex. Their size and shape depends on the botanical source (Banks and Muir, 1980; Shannon, 1989), gene-line variation (Bertoft et al., 2000), stage of development and starch hydrolysis (Sargeant, 1979; Steup, 1988; Lauro et al., 1999).
In cereals, starch granule size also affects the mode of isoenzyme attack, since the isoenzymes secreted by the aleurone cells have to be adsorbed and absorbed into the starch granule (MacGregor, 1979, 1980; MacGregor and Ballance, 1980; Kruger and Marchylo, 1985). In maize, starch granules from different genetic lines can be more resistant or be more susceptible to digestion by -amylase from B. amyloliquefaciens, depending on the structure of amylopectin and dextrin (Bertoft et al., 2000).
The question arises as to what is the functional base underlying this complex pattern of -amylase isoenzymes? The complex pattern of -amylase isoenzymes may be related to the available substrate and, therefore, related to the degree of amyloplast development and starch digestion.
In spite of the work that has been done on cereals, the authors wanted to address this question with a seed of a plant not often studied, a conifer, which has starch granules not only in the reserve tissue, but also in the embryo. In the present work, the in vitro digestion of A. araucana starch granules of different sizes, taken at two times before germination and during early seedling growth, exactly before imbibition (T0) and 90 h (T90) after the beginning of the seed imbibition was studied. The starch granules were incubated with the two major purified embryonic -amylase isoenzymes, one isoform extracted from the embryos before seed imbibition (T0) and the other extracted from embryos after 90 h (T90) of seed imbibition. The major focus of this work was on the -amylase isoenzymes and the starch granules from the embryos of the seeds although work was also done on a mixture of partially purified isoenzymes and starch granules from the megagametophytes.
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Materials and methods |
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Plant material
Seeds of Araucaria araucana (Mol.) Koch were collected in the Chilean forest of Malalcahuello, latitude 37° 5' S. After collection, seeds were stored at 5 °C until used.
Germination conditions
Seeds without coats were washed with 0.25% (w/v) of commercial sodium hypochlorite and germinated in plastic trays containing wet vermiculite as has been described by Cardemil and Reinero (1982).
-Amylase extraction and partial enzyme purification
Embryos or megagametophytes were sterilized for 15 min in 0.25% (w/v) of commercial sodium hypochlorite and rinsed several times with distilled water. Tissues were homogenized using 2 ml g–1 fresh weight of extraction buffer. This extraction buffer contained 0.05 M sodium acetate, pH 4.8, 0.02 M CaCl2, 0.06 M NaCl. To inactivate ß-amylases, starch phosphorylases, and other heat susceptible proteins, supernatants were heated for 15 min at 70 °C (Loyter and Schramm, 1962). After heating, samples were cooled down on ice and centrifuged for 5 min at 5000 g. The pellet was discarded. -Amylase was precipitated by the method of Loyter and Schramm (1962) using 4% (w/v) oyster glycogen (Acevedo and Cardemil, 1994).
Further enzyme purification
The two major embryonic isoenzymes used in the in vitro experiments of starch hydrolysis were preparatively purified from the partially purified extract obtained from isolated embryos (without megagametophytes) as described above. These were the isoenzymes of 50.2 kDa, relative mobility (Rm) 0.49 and pI 4.8 from the embryos of quiescent seeds (isoenzyme T0) and the isoenzyme of Rm 0.51 and pI 5.4 from embryos 90 h after the start of imbibition (isoenzyme T90) (Salas and Cardemil, 1986; Acevedo and Cardemil, 1994). The isoenzymes were preparatively purified by SDS-polyacrylamide-gel electrophoresis performed at 10 °C with a loading of 20 µg of protein per well with the partially purified -amylase extract. After electrophoresis the gel was divided lengthwise with one half used to detect the isoenzymes by staining with Coomassie Blue. In the other half the isoenzymes were detected by enzyme activity (zymogram) as described by Acevedo and Cardemil (1994). -Amylase activity was recovered from each band by exchanging the SDS with Triton X-100 for 60 min at room temperature (Rosenthal and Lacks, 1977; Jameel et al., 1984). The gel half was then incubated with soluble starch solution, and activity of the protein bands detected by staining with 2% KI–I2 (w/v) (Juliano and Varner, 1969). After staining, the protein band of the isoenzymes were cut and subjected to electroelution according to the method described by Sreekrishna et al. (1980). For this, the gel strip containing the protein band was placed inside a dialysis bag with 3 ml of 0.0125 M Tris-HCl buffer containing 0.096 M glycine, pH 8.3. The electroelution was done for 12 h at 60–80 mA. The SDS was eliminated from the bag by dialysing against a mix containing acetone, triethylamine, acetic acid, and water at a ratio 85:5:5:5 (by vol.) for 45 min.
In the case of the megagametophytes, specific isoenzymes were not purified due to the low levels of -amylases in this tissue.
Enzyme activity and protein assays
The -amylase activity was assayed using the starch–I2–KI method (Jones and Varner, 1967; Acevedo and Cardemil, 1994). One enzyme unit was defined as 1 mg of degraded starch min–1 g–1 dry weight at 37 °C. The protein concentration was determined according to Bradford (1976) using bovine serum albumin as standard.
Starch granules isolation
Quiescent seeds and seedlings after 90 h of seed imbibition were selected to separate the embryos from megagametophytes. Each tissue was sterilized with 0.5% (w/v) commercial sodium hypochlorite for 15 min, followed by three rinses with distilled water. Each tissue was homogenized at 4 °C with the starch granule extraction buffer containing 0.05 M HEPES pH 7.5, 0.175 M sucrose; 0.001 M MgCl2, 0.01 M KCl, 1 mg ml–1 BSA, 0.001 mg ml–1 sodium azide, and 0.001 M EDTA. The extracts were filtered through four layers of cheesecloth and the supernatants were centrifuged for 10 min at 9980 g. The supernatants were eliminated and the pellets were resuspended in 1 ml of starch granule extraction buffer. The starch granules were lyophilized in a freeze-drier for 48 h (MacGregor, 1979).
Starch granule purification
Embryo and megagametophyte starch granule extracts (3.3x109 granules ml–1) of each germination time (starch granules isolated at T0 and starch granules isolated at T90) were resuspended in 200 µl of a 20% (w/v) sucrose solution and layered on top of a discontinuous sucrose gradient made up of 2 ml of 85% (w/v), 4 ml of 80% (w/v), 3 ml of 65% (w/v), and 2 ml of 50% (w/v). The gradient was centrifuged in a Chriss UJ1 centrifuge for 16 min at 200 g. Each step in the gradient was sampled (500 µl) with a Pipetman. The samples were further centrifuged for 10 min in an Eppendorf centrifuge at 9980 g. After centrifugation, the supernatant was used to calculate the refraction index of the sucrose solution in order to determine the sucrose concentration of the fraction at which starch granules of a particular size were sedimentated. The refraction index of the supernatant of each fraction was determined using a Bausch and Lomb Refractometer. Sucrose solutions of different concentrations were used to calibrate the instrument. For this, an aliquot of 20 µl of each supernatant was deposited on its plate and the refractive index was determined. The specific gravity of each fraction was also determined with the use of iscotables which correlates refractive indexes with specific gravities and with sucrose concentrations.
The pellets were washed several times with the starch granule extraction buffer. Samples (µl) were stained with KI–I2 solution and observed under the light microscope to evaluate the degree of purification and the starch granule size.
Determination of starch granules sizes
The diameters of the starch granules of each gradient were determined using an eyepiece micrometer calibrated with a stage micrometer. The diameters were corroborated afterwards by measurements performed under a scanning electron microscope.
In vitro starch granule digestion
Starch granule solutions containing 106 starch granules from the embryonic and megagametophytic tissues at T0 and at T90 were gently shaken with a buffer containing 0.05 M sodium acetate, pH 4.8, 0.02 M CaCl2, 0.06 M NaCl, 1 µg ml–1 sodium azide (to avoid bacterial and fungal contamination). The starch granules were incubated for 90 h with 0.02 units of -amylase (approximately 12 µg of protein) at 37 °C. Starch granules of three sizes were obtained from the sucrose gradients and digested in two different ways: (1) with the purified isoenzyme from the same tissue and from the same time of germination, and (2) with the purified isoenzyme from the same tissue but extracted at a different time of germination.
Other experiments were performed with starch granules extracted and purified from the embryos and from the megagametophytes and incubated for 90 h at 37 °C with 0.02 units of a mixture of partially purified isoenzymes extracted from the embryonic and megagametophytic tissues at two times during seed imbibition (a mixture of partially purified -amylase isoenzymes T0 and a mixture of partially purified -amylase isoenzymes T90). Embryo and megagametophyte starch granules isolated at T0 and isolated at T90 were incubated: (1) with the isoenzyme mixture of its own age and from its own tissue and (2) from a different age and from a different tissue. Controls without enzyme were also run. The starch granules were kept suspended by constant gentle shaking during incubation with the partially purified isoenzyme mixture. After incubation, each sample was centrifuged at 9980 g for 10 min. The supernatants were saved for the determination of the total amount of reducing sugars released during starch granule digestion. The starch granule pellets were washed with the incubation buffer, without calcium and containing 0.001 M EDTA, and centrifuged at 9980 g for 10 min. The washes were repeated twice to stop the enzymatic activity. Finally the starch granules were lyophilized and stored at 4 °C until observation using light microscopy and scanning electron microscopy.
In another set of experiments, starch granules isolated from embryos at T0 were incubated with 0.02 units of the purified isoenzyme T0 extracted from the same embryonic tissue at 37 °C. After 90 h of incubation with the enzyme, the starch granules were centrifuged, and the supernatant saved for quantification of reducing sugars. The starch granule pellet was washed twice with incubation buffer without enzyme. After the second wash, the starch granules were incubated for another 90 h at 37 °C with 0.02 units of the isoenzyme T90, pI 5.4. The amount of reducing sugars released after the second incubation was quantified after 135 h and 180 h of incubation. The same type of experiment was also performed with embryonic starch granules isolated at T90, incubated first with 0.02 units of the purified isoenzyme T90 for 90 h at 37 °C and then incubated with 0.02 units of the purified isoenzyme T0 for another 90 h.
The time of 90 h of incubation was chosen in all these experiments because the isoenzyme T0 is present in the embryos during this period of time and disappears after 90 h of seed imbibition; at this time the isoenzyme T0 is replaced by other isoenzymes, among them, the isoenzyme T90.
Reducing sugar determinations
The release of reducing sugars as products of the starch granule digestion by -amylase was determined in the supernatants obtained by centrifugation of the reaction mix after digestion. The amounts of reducing sugar residues were determined by the 3,5-dinitrosalicylic acid method (Henson and Stone, 1988). For this, 100 µl of each supernatant was incubated with 500 µl of the reagent and heated in a water bath at 100 °C for 5 min. The reaction was stopped by placing the tubes on ice. Each sample was diluted with 5 ml of distilled water and the absorption was read at 540 nm. The reducing sugar concentration was expressed in µmol of glucose equivalents.
Scanning electron microscopy
After digestion, the starch granules were fixed in 3% (v/v) of glyceraldehyde for 150 min and washed after fixation with 0.1 M phosphate buffer pH 7.4. After the wash, the starch granule suspensions were centrifuged at 9980 g for 10 min. The pellets were dehydrated with aqueous solutions of increasing acetone concentration: 30, 50, 70, and 90% (v/v), followed by two 10 min rinses with 100% acetone. Dried starch granules were attached to aluminium plates with polyglycine and covered with gold 20 nm deep in a Polaron E-5000 sputter at 12 mA for 3 min. The samples were observed with a Phillips EM 300 G scanning electron microscope.
Tissue printing
Tissue printing was performed to provide a different approach to determining the -amylase activities at T0 and T90 of embryonic and megagametophytic tissues. Hand-cut cross-sections of the embryos, megagametophytes without embryos, and complete seed sections (with embryo and megagametophyte surrounding tissue) taken at these two times of imbibition were printed on agarose films. The films were prepared using a solution of 1% (w/v) soluble starch and 1.25% (w/v) agarose in 0.05 M sodium acetate, pH 4.8, 0.02 M CaCl2, 0.06 M NaCl. The mix was spread on a petri dish and dried to form a thin layer of starch-containing agarose gel. The sections were deposited on the film and the petri dish was incubated at 25 °C for 20 min. After incubation, the sections were removed and the films were stained with 2% KI-I2 (w/v). Areas having amylase activity appeared white on the gel with a dark background.
Statistical analysis
The significant differences were evaluated by Tukey HSD test.
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Results |
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In vivo
-amylase activity detection by tissue printingTissue printing was performed to test if
-amylases were active in the embryonic and megagametophytic tissues at two times of imbibition (T0 and T90). The major -amylase activity was in the embryonic tissue at T0. Therefore, the main enzyme responsible for starch hydrolysis in the A. araucana seeds during the first hours of imbibition is the embryo -amylase. Megagametophytes at this time showed a very slight activity (Fig. 1A) However, -amylase activity increased in the megagametophytic tissue at T90, compared to T0 (Fig. 1B) (Salas and Cardemil, 1986). At this developmental stage, there was higher -amylase activity in the megagametophytic cells located on the internal border next to the embryo cavity, in some other scattered areas of the parenchyma tissue, and in the external cortex (Fig. 1A, B).
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Starch granule sizes
Light microscopy and scanning electron microscopy of the purified starch granule fractions collected from the sucrose gradient showed that all the starch granules of the megagametophyte have similar sizes with an average diameter of 13.8±3.2 µm (Table 1). In the embryo, however, there were different sizes of starch granules at all times of imbibition. In the light of this, the embryonic starch granules were separated into three groups by the sucrose gradient denoted small, medium, and large, (Table 1; Fig. 2A–D). The fractionation results also show that each fraction contained only starch granules of a particular size (Fig. 2B–D), while the starch granules isolated from the embryonic tissue were of all sizes (Fig. 2A).
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Pattern of embryonic isoenzymes and purification of the two major embryonic
-amylases of T0 and T90The major
-amylase isoenzymes from T0 and T90 were purified for in vitro starch granule digestion. The results of the preparative purification of the enzyme T0 is shown in Fig. 3A. Seven isoenzymes can be detected in the embryos of quiescent seeds (Fig. 3B) and seven isoenzymes can be detected in the seedlings after 90 h of seed imbibition (Fig. 3C). The isoenzyme T0 was previously characterized with an Rm 0.49 and a pI 4.8. Additionally, it has a molecular mass of 50.2 kDa (Acevedo and Cardemil, 1994). The major isoenzyme T90 has an Rm 0.51 and a pI 5.4 (Salas and Cardemil 1986). This enzyme was purified as well as the isoenzyme T0 (data not shown).
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In vitro starch granules digestion by
-amylaseIn vitro starch granule digestion by
-amylase was monitored by (a) scanning electron microscopy and (b) quantification of the reducing sugars released from starch digestion.
(a) Scanning electron microscopy
Embryonic starch granules isolated at T0 and incubated with isoenzyme T0 showed digested regions on the surface (Fig. 4A) as compared to control starch granules incubated in buffer without the enzyme (Fig. 4B). No digestion was observed when starch granules isolated at T0 and taken from embryonic tissue were incubated with the isoenzyme T90 (Fig. 4C). In the case of starch granules isolated at T90, these showed a further digestion when they were incubated with isoenzyme T90 (Fig. 4D) as compared to the control starch granules isolated at T90 (incubated without the enzyme) (Fig. 4E). The starch granules isolated at T90 were not digested with isoenzyme T0 (Fig. 4F).
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(b) Quantification of reducing sugars released
Reducing sugars released from the in vitro digestion of embryonic starch granules with the purified isoenzymes T0 and T90 agreed with the scanning electron microscopic observations (Fig. 4A, B). Large starch granules extracted from embryonic tissue at T0 and incubated with purified isoenzyme T0 (Fig. 3A) released more glucose equivalents than the medium and small-sized starch granules (P <0.05, data not shown). There were small amounts of glucose released when small, medium and large starch granules isolated at T90 were incubated with isoenzyme T0 (Fig. 5A).
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Small and medium starch granules isolated at T90 incubated with purified isoenzyme T90 released more glucose equivalents than the large-sized starch granules (P <0.05, data not shown). Small, medium and large starch granules isolated at T0 released very few glucose equivalents when they were incubated with the purified
-amylase isoenzyme T90 (Fig. 5B).
Other experiments were performed with starch granules extracted and purified from the embryos and from the megagametophytes and incubated with a mixture of partially purified isoenzymes extracted from the embryonic and megagametophytic tissues at two times of imbibition, as described in the Materials and methods. As with purified individual -amylase isoenzymes, a mixture of partially purified isoenzymes extracted from embryos at T0 and T90 digested embryonic starch granules of their own imbibition stage better: 9.6 versus 3.6 glucose equivalents for 90 h (P <0.05) (Table 2). The partially purified megagametophytic isoenzyme mixtures of T0 digested the starch granules of its own time slightly better: 5.2 versus 4.7 glucose equivalents for 90 h (P <0.05) while T90 digested the embryonic starch granules of both times equally well: 7.7 versus 7.5 glucose equivalents for 90 h (P=0.09) (Table 2).
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The mixture of partially purified embryonic isoenzymes extracted at T0 digested the megagametophytic starch granules of both germination stages equally well (P=0.072) (Table 3), but less than the embryonic starch granules when these were of the same germination stage as the mixture of isoenzymes (Table 2). The mixture of partially purified embryonic isoenzymes T90 digested the megagametophytic starch granules of its own stage significantly better (P=0.02). The mixture of partially purified megagametophytic isoenzymes extracted at both times of imbibition digested the megagametophytic starch granules of both imbibition stages equally well (P=0.089 and P=0.067) (Table 3).
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In vitro sequential digestion of embryonic starch granules by the two purified
-amylase isoenzymes T0 and T90To determine if the isoenzymes T0 and T90 may function sequentially to digest starch granules, embryonic starch granules isolated at T0 were first incubated with the isoenzyme T0, followed by digestion with the isoenzyme T90. A control experiment was performed in which starch granules incubated with the isoenzyme T0 for 90 h were left with the same isoenzyme for another 90 h (Fig. 6A). Similar experiments were performed with starch granules isolated at T90 incubated first with isoenzyme T90, and then digested with isoenzyme T0. A control experiment was run with starch granules isolated at T90 incubated for another 90 h with isoenzyme T90 (Fig. 6B). The quantification of released reducing sugars showed that the starch granules isolated at T0 can be further digested by isoenzyme T90 following initial digestion with isoenzyme T0. However, these starch granules were not digested further by isoenzyme T0 (Fig. 6A). By contrast, the starch granules isolated at T90 could not be digested by isoenzyme T0, but could be further digested by isoenzyme T90 (Fig. 6B).
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Tissue printing and seed distribution of the enzyme activity
The tissue printing results showed that
-amylase was active in vivo in both megagametophyte and embryo tissues, both in quiescent seeds and 90 h after imbibition. The results are in agreement with those reported by Cardemil and Reinero (1982) showing that, in quiescent seeds, the enzyme is more active in the embryo than in the megagametophyte and that the activity increases in the megagametophytes at 90 h of imbibition (Salas and Cardemil, 1986). Therefore, in quiescent seeds and before 90 h of seed imbibition, the -amylase isoenzymes from the embryo appear to be responsible for the greatest degree of starch digestion. No cross-contamination of the -amylases from embryo and megagametophyte occurs in A. araucana since the -amylase isoenzymes are not secreted out from the cells. Indeed, the megagametophytic cavity where the embryo is located does not show any -amylase activity (Cardemil and Reinero, 1982; Salas and Cardemil, 1986).
Furthermore, tissue printing allowed details of the enzyme activity in different parts of the megagametophyte to be seen. In megagametophytic tissue, the distribution of the enzyme after 90 h of imbibition was uneven. The external cortical parenchyma and the parenchyma cells surrounding the embryo showed higher -amylase activity at this time.
The distribution of starch granules and -amylase in seeds of A. araucana is different from cereal grains. In A. araucana, starch is stored in amyloplasts in the embryos and megagametophytes and the enzyme is produced in both tissues (Cardemil and Reinero, 1982; Reinero et al., 1983). In cereals, starch is stored in the endosperm and the -amylase is produced in the aleurone cells, but not in the embryos (Yomo and Varner, 1971). Therefore, in cereals, the enzyme is mobilized from the aleurone to the endosperm to digest the starch (Yomo and Varner, 1971), but in A. araucana, -amylase is not mobilized in seeds. The higher activity of -amylase found in the cortical parenchyma of the megagametophytes 90 h after imbibition probably indicates the tissue where the enzyme is synthesized and accumulated (Cardemil and Reinero, 1982). The higher amylolytic activity found in the parenchyma cells surrounding the cavity where the embryo is lying may facilitate soluble sugar transport from the megagametophyte to the embryonic cotyledons, which function as haustorial organs (Lozada and Cardemil, 1990).
Starch granules are digested on the basis of the developmental stage of seed, the stage of imbibition/germination, and on the basis of the tissue from which they originate
The digestions of starch granules isolated at T0 and T90 by the isoenzymes T0 and T90 were detected using scanning electron microscopy as concavities formed in the surface of the starch granules and by the amount of glucose released from them. These results indicated that embryonic isoenzymes T0 and T90 digested starch granules of their own imbibition time better (i.e. isoenzyme T0 digested starch granules isolated at T0 better than those isolated at T90). Therefore, the results from these experiments with purified isoenzymes indicate that the T0 and T90 isoenzymes discriminate on the basis of the developmental stage of the seed and the stage of imbibition/germination. In the living cells, the developmental stage of starch granules of reserve tissues may reflect the starch structure (Banks and Muir, 1980: Kaimuna, 1988; Steup, 1988), i.e. the length of the polyglucan chain and the ratio between amylose and amylopectin. As a consequence, the starch in the starch granule will change as it is digested by different enzymes. The A. araucana starch granules isolated at T0 are undigested while the starch granules isolated at T90 are partially digested by isoenzyme T0 and therefore, have a different starch structure. It is possible then that the isoenzymes T0 and T90 may discriminate the stage of starch granules digestion because the starch structure is different at these two times of imbibition (Kruger and Marchylo, 1985; Lauro et al., 1999).
The mixture of partially purified embryonic isoenzymes showed the same tendency as the purified embryonic isoenzymes with respect to the germination stage of the embryonic starch granules. It digested the starch granules of their own imbibition stage better. The mixture of partially purified embryonic isoenzymes also digested the starch granules extracted from the same tissue better, digesting less well starch granules extracted from the megagametophyte. Probably, the large size of megagametophytic starch granules makes a difference for the adsorption and absorption of the embryonic -amylase isoenzymes. The mixture of partially purified megagametophytic isoenzymes differentiated less the digestion of embryonic and megagametophytic starch granules isolated from a different age. The megagametophyte mixture of isoenzymes digested the embryonic starch granules as well as their own starch granules.
Isoenzymes may differentiate the starch granules sizes
The embryonic starch granules of A. araucana are more variable in size and smaller than those of the megagametophyte (as described by Cardemil and Reinero, 1982). Isoenzyme T0 seems to digest large embryonic starch granules better, while isoenzyme T90 seems to digest smaller starch granules better. MacGregor (1979, 1980) found two barley -amylases isoenzymes which differed significantly in their effectiveness in degrading large starch granules. Kruger and Marchylo (1985) similarly found that the two major groups of wheat -amylases, GI and GIII, differ in their capacity of adsorption and absorption by starch granules of different sizes. Sargeant (1979) and Lowy et al. (1981) found that the change of isoenzyme patterns during wheat grain development correlated with maturation of the endosperm amyloplasts. However, from their work it is not clear if the isoenzymes discriminate between the stage of development/germination or the starch granule size or both. Size, stage of development and degree of starch granule digestion appear to be related to the specific function of a particular isoenzyme. Therefore, activity of different -amylase isoenzymes may depend upon starch granule size, stage of development, degree of digestion, and a combination of them.
As in the case of cereals (MacGregor, 1979, 1980; MacGregor and Ballance, 1980; Kruger and Marchylo, 1985), A. araucana -amylases need to be taken into the amyloplast since these isoenzymes are cytoplasmically localized (Cardemil and Reinero, 1982). Fannon et al. (1992), and Huber and Bemiller (1997) have given evidence that corn and sorghum starch granules have channels that connect a central cavity with the external environment. These channels open to the exterior through pores. It is possible that starch granules of different sizes could differ in the number and diameter of these pores making a difference in the adsorption and absorption of -amylase isoenzymes.
Scanning electron microscopy revealed that the purified isoenzymes T0 and T90 digested the starch granules from the surface. The granules were not broken in fragments nor did they appeared to have multiple holes as occurs with the reserve starch granules of legume cotyledons and of barley endosperm (Steup, 1988; Lauro et al., 1999).
The two isoenzymes digested the starch granules sequentially
That the two purified -amylases can specifically digest starch granules of a specific stage of development was corroborated by the experiments involving sequential digestion of starch granules. These experiments may have simulated in vitro what may happen in vivo. It may be assumed that isoenzyme T90 only hydrolyses the starch of the starch granules which have been first hydrolysed by isoenzyme T0. Probably, isoenzyme T0 can digest intact starch granules with a smooth surface, and therefore, this isoenzyme cannot digest starch granules for more than 90 h of incubation. The starch of starch granules digested by isoenzyme T0 seems to be a good substrate for isoenzyme T90. This isoenzyme can digest the same starch granule beyond 90 h of incubation.
However, it is important to keep in mind that what happens with in vitro digestion of A. araucana starch granules may be different from that in vivo, since in vivo the starch granules will be attacked by several -amylase isoenzymes and starch will be hydrolysed by other enzymes such as the starch phosphorylases, debranching enzymes and glucosidases (Cardemil and Varner, 1984). Therefore, in vivo starch digestion will release sugars in amount and speed compatible with the embryo and seedling requirements for growth and development.
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Acknowledgements |
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The authors wish to thank the Departamento Técnico de Investigación of the Universidad de Chile for funding provided to Liliana Cardemil, to the Dirección de Investigación of the Universidad Metropolitana de Ciencias de la Educación for financial support (grant GAF DL 92-4) provided to Elba Acevedo. The technical assistance of Angélica Vega is acknowledged. This research was part of Juana J Waghorn’s dissertation to fulfil the requirements for the title of Profesor de Estado en Biología y Ciencias Naturales at the Universidad Metropolitana de Ciencias de la Educación.
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