Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Occurrence of alpha-amylase in the axis of germinating peas

α-Amylase was found in the axis portion of ungerminated pea seeds (Pisum sativum var. Alaska). The occurrence of this enzyme was demonstrated with crude homogenates (also containing β-amylase) using three different methods: the hydrolysis of β-limit dextrin, the change in absorption spectra for the iodine-starch complex, and the increase in reducing materials relative to the decrease in starch. The first method was used to quantitate the changes in α-amylase activity during germination. The increase in total amylase activity (primarily β-amylase) paralleled germination; the accumulation of α-amylase activity was not initiated for an additional day. The increased α-amylase activity was related to epicotyl growth. Approximately half of this activity was found in the etiolated stem, the distribution being higher in growing than in nongrowing portions.     

Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1-->3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase

Polyclonal antibodies raised against barley (1→3,1→4)-β-d-glucanase, α-amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley. In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding α-amylase or (1→3,1→4)-β-d-glucanase, while in the aleurone α-amylase and (1→3,1→4)-β-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1→3,1→4)-β-d-glucanase, α-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation products were identical for each enzyme.     

Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain

α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl₂, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl₂. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

     

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

Background and Aims

α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone.

Methods

α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties.

Key Results

The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population.

Conclusions

The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.     

Starch metabolism in the leaf sheaths and culm of rice

The levels of starch and dextrin, free sugars, soluble protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and ADP-glucose starch synthetases—were assayed in the leaf sheaths and culm of the rice plant (Oryza sativa L., variety IR8) during growth.     

Amylase synthesis and stability in crested wheatgrass seeds at low water potentials

Drying of seeds of Agropyron desertorum (Fisch. ex Link) Schult. did not result in breakdown of α-amylase nor impair the ability of seeds to resume its synthesis when moistened again. β-Amylase activity did not change during 5 days of germination at a water potential of 0 atmosphere nor during 40 days of incubation at −40 atmospheres. Seeds synthesized α-amylase at 0, −20, and −40 atmospheres, but not at −60 atmospheres. At 0 and −20 atmospheres, the log of α-amylase activity was linearly related to hastening of germination. But at −40 atmospheres, seeds synthesized α-amylase during a time when there was little hastening of germination. Thus, it appears that other biochemical reactions are less drought-tolerant than synthesis of α-amylase. It is concluded that inhibition of α-amylase synthesis is not a controlling factor in the germination of these seeds at low water potentials.     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Enzymes of starch metabolism in the developing rice grain

The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and starch synthetase —were assayed in dehulled developing rice grains (Oryzasativa L., variety IR8). Phosphorylase, Q-enzyme, and R-enzyme had peak activities 10 days after flowering, whereas α- and β-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.     

In Vivo Synthesis and Turnover of alpha-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds

α-Amylase activity increased in attached cotyledons of germinated Vigna mungo seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double immunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. α-Amylase contents, determined by single radial immunodiffusion method, changed in parallel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of α-amylase in V. mungo cotyledons.

The rate of incorporation of the label from [³H]leucine into α-amylase and the ratios of dpm in α-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of α-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons α-amylase was synthesized and accumulated, because of low degrading activity during incubation.

     

beta-Amylases in Cereals : A Study of the Maize beta-Amylase System

β-Amylase of maize (Zea mays L.) caryopses was studied during development and germination by means of enzymic, electrophoretic, and immunochemical techniques. β-Amylase activity increased during caryopsis development to a maximum value at the beginning of the water content plateau (at this stage the enzyme was located primarily within the pericarp) and then decreased. Almost no β-amylase (activity or antigen) was found in either free or bound forms in the mature maize caryopsis. The activity increased again during seedling growth and reached much higher values. Both the aleurone layer (to a major extent) and the scutellum produced and secreted β-amylase during germination, the secretion being stimulated by Ca²⁺. No posttranslational modification of the enzyme was detected during germination. The molecular specific activity of the enzyme remained unchanged during the observed periods, indicating that the regulation of the activity is based essentially on protein turnover. The enzyme from developing and germinating caryopses was found to be identical in terms of antigenicity, isoelectric point, and molecular mass to the β-amylases extracted from the roots and the leaves of the maize seedling. The maize β-amylase resembles in all respects the ubiquitous β-amylase described for rye and wheat, whereas the major β-amylase of those cereals appears to be lacking in the maize caryopsis.     

Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems

Most of the activity of an α-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic α-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the α-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this α-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the β-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.     

Control of alpha-Amylase Development in Cotyledons during and following Germination of Mung Bean Seeds

Developmental patterns of α-amylase in Vigna radiata cotyledons during and following germination were quite different depending on the differences in the treatments of cotyledons during the imbibitional stage. When axis-detached cotyledons were imbibed in water with seed-coats attached, α-amylase activity did not increase and remained low. On the other hand, when the cotyledons were imbibed in water after seed-coat removal, the enzyme activity increased markedly. If the axis was attached to the cotyledons, α-amylase showed a marked development even under the former imbibition conditions. These changes in the enzyme activity were in parallel with those in the enzyme content, and the content, in turn, was dependent upon the availability of mRNA for α-amylase. We propose that the regulation of the development of α-amylase in cotyledons may involve some factor(s) inhibitory to accumulation of α-amylase mRNA, which is present in dry cotyledons and can be removed from cotyledons by leakage or by the presence of the axis.     

Alpha-amylase synthesis in wheat kernels as influenced by the seed coat

The effect of seed coat removal on the synthesis of α-amylase isoenzymes in wheat was investigated. The immature wheat endosperm-aleurone (seed coat and embryo detached) produced considerably less α-amylase activity than immature whole or de-embryonated wheat kernels, when incubated under identical conditions of 18.5 C and 99% humidity, in the presence or absence of gibberellic acid (GA₃). The incubated endosperm-aleurone also exhibited unique α-amylase isoenzyme composition when compared to the isoenzyme compositions of incubated whole and de-embryonated immature and mature wheat kernels both in the presence or absence of GA₃. Subsequent studies indicated that the seed coat may contain factor(s) required for normal α-amylase isoenzyme synthesis.     

Effects of 6-methoxy-2-benzoxazolinone on the germination and α-amylase activity in lettuce seeds

Germination of lettuce seeds was inhibited by 6-methoxy-2-benzoxazolinone (MBOA) at concentrations greater than 0.03 mmol/L. MBOA also inhibited the induction of α-amylase activity in the lettuce seeds at concentrations greater than 0.03 mmol/L. These two concentration–response curves for the germination and α-amylase indicate that the percentage of the germination was positively correlated with the activity of α-amylase in the seeds. Lettuce seeds germinated around 18 h after incubation and inhibition of α-amylase by MBOA occurred within 6 h after seed incubation. These results show that MBOA may inhibit the germination of lettuce seeds by inhibiting the induction of α-amylase activity.     

Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds

α-Amylase levels in intact seeds of barley (Hordeum vulgare L. cv. Himalaya) reach a maximum at 3 to 4 days of germination while gibberellin levels continue to increase beyond 6 days of germination. In contrast to its effect on half seeds, gibberellic acid does not increase the total amount of α-amylase produced in germinating seeds. The inability of gibberellic acid to stimulate α-amylase production is not related to its availability; rather, evidence suggests that a factor(s) in whole seeds prevents further enhancement of α-amylase formation and accumulation. Hydrolysis products accumulate in the subaleurone space of the endosperm of germinating seeds up to concentrations of 570 milliosmolar. Chromatography of these hydrolysis products indicate the presence of maltose and glucose. Calculations based on reducing sugar determinations show that glucose accounts for as much as 57% of the solutes present in the endosperm fluid. Both maltose and glucose in the range of 0.2 to 0.4 M effectively inhibit the production of α-amylase by isolated barley aleurone layers. This inhibition is quantitatively similar to that brought about by solutions of polyethylene glycol and mannitol. On the basis of these data we propose that hydrolysis products which accumulate in the starchy endosperm of germinating seeds function to regulate the production of hydrolytic enzymes by the aleurone layer.     

Independent Regulatory Aspects and Posttranslational Modifications of Two beta-Amylases of Rye : Use of a Mutant Inbred Line

We have examined the occurrence/disappearance, tissue location, and posttranslational modification of β-amylase proteins in rye (Secale cereale L.) kernels at three physiological stages (development, maturity, germination) with a normal inbred line and a mutant line exhibiting a high but incomplete β-amylase deficiency. This deficiency corresponds to a lack of accumulation of β-amylase activity in the endosperm and does not affect the level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct β-amylases (I and II) were detected in the normal line (II being the major constituent) and only one (I) in the mutant line. I and II display very similar electrophoretic polymorphism. In both lines, I appears to be ubiquitous, although it disappears from the outer pericarp during ripening. Antigen II was present only in the normal line and appears to be specific for the endosperm and perhaps for the maternal green tissues of the seed. Posttranslational modifications occurring during germination, which are mimicked by the action of papain, affect II but not I. The two groups of β-amylases are discussed in relation to recent reports indicating the presence of two types of β-amylase with different functions and gene loci in barley and wheat.     

Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.     

Specific Determination of alpha-Amylase Activity in Crude Plant Extracts Containing beta-Amylase

The specific measurement of α-amylase activity in crude plant extracts is difficult because of the presence of β-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70°C for 20 min, HgCl₂ treatment, and the use of the α-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.), and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While β-amylase can liberate no color alone, over 10 International units per milliliter β-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at β-amylase activities over 1,000 International units per milliliter. Two starch azure procedures were developed to eliminate β-amylase interference: (a) the dilution procedure, the serial dilution of samples until β-amylase levels are below levels that interfere; (b) the β-amylase saturation procedure, addition of exogenous β-amylase to increase endogenous β-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These two procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in β-amylase activity or inhibitory effects of the commercial β-amylase. The β-amylase saturation procedure was found to be preferable with most species. The heat treatment was satisfactory only for malted barley, as α-amylases in alfalfa and soybeans are heat labile. Whereas HgCl₂ proved to be a potent inhibitor of β-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited α-amylase in barley malt. The reported α-amylase activities in crude enzyme extracts from a number of plant species are apparently the first specific measurements reported for any plant tissues other than germinating cereals.