Differential expression of α-amylase genes in germinating rice and barley seeds

Steady-state levels of mRNA from individual -amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the -amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, -amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenver two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley -amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI -amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI -amylases always preceded those encoding low-pI -amylases. Two distinct differences in -amylase gene expression were observed between the two barley varieties. levels of high-pI -amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI -amylase mRNA and nearly four times as much low-pI -amylase mRNA than the slower-germinating Himalaya variety.     

Regulation of α-amylase activity in Amaranthus caudatus seeds by methyl jasmonate, gibberellin A3, benzyladenine and ethylene

Methyl jasmonate (JA-Me) at 10^–3 Minhibited Amaranthus caudatus seed germination anddecreased -amylase activity. Exogenous gibberellin A₃(GA₃) and ethylene, but not benzyladenine (BA), increased activity ofthe enzyme in the presence of JA-Me, with ethylene being the most effective. Theinhibitor of ethylene action, 2,5-norbornadiene (NBD) inhibited seed germinationand decreased -amylase activity. The inhibitory action of JA-Me onAmaranthus caudatus seed germination is associated with theinhibition of -amylase activity. Exogenous GA₃ and ethylenecontrol both -amylase activity and seed germination in the presence of JA-Me.     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

Red-light- and gibberellic-acid-enhanced α-galactosidase activity in germinating lettuce seeds,cv. Grand Rapids

Dry lettuce (Lactuca sativa L.) seeds (achenes) contain -galactosidase (EC 3.2.122) at a level which is maintained in the imbibed dormant state in darkness. Both red light (R) and gibberellic acid promote an increase in enzyme activity several hours prior to the completion of germination. Germination and enzyme activity are not essentially linked, however, for the latter can increase while the former is inhibited. -Galactosidase activity increases within the cotyledons and the endosperm following R stimulation, but the axis is essential to perceive the stimulus and to promote and maintain the increase in enzyme activity. A diffusible factor (or factors) is produced by and-or released from irradiated axes, and it migrates to the cotyledons (and possibly endosperm) to promote the increase in -galactosidase activity. Gibberellic acid, particularly in the presence of benzyladenine, can replace the requirement for irradiated axes.Abbreviations GA₃ gibberellic acid - R red light     

Sugar sensing and α-amylase gene repression in rice embryos

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) α-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the α-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the α-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization. Received: 22 April 1997 / Accepted: 9 September 1997     

A highly efficient and thermostable α-amylase from soya bean seeds

The α-amylase from soya bean seeds was purified by affinity precipitation, resulting in approx. 20-fold purification with approx. 84% recovery. The purified α-amylase had an optimum pH of 5.5, optimum temperature of 75?°C, Arrhenius energy of activation of 6.03?kcal/mol (1?kcal≈4.184?kJ) and a Km of 2.427?mg/ml (starch substrate). The enzyme had maximum substrate specificity for starch. Among the various metal ions tested, Co2+ and Mn2+ were found to be strong activators. The effect of thiol group modifying agents showed that the thiols of soya bean α-amylase are not directly involved in catalysis. The thermostability of the enzyme makes it suitable for starch liquefaction and the detergent industry respectively.     

Where do gibberellin biosynthesis and gibberellin signaling occur in rice plants?

To identify where gibberellin (GA) biosynthesis and signaling occur, we analyzed the expression of four genes involved in GA biosynthesis, GA 20-oxidase1 and GA 20-oxidase2 (OsGA20ox1 and OsGA20ox2), and GA 3-oxidase1 and GA 3-oxidase2 (OsGA3ox1 and OsGA3ox2), and two genes involved in GA signaling, namely, the gene encoding the alpha-subunit of the heterotrimeric GTP-binding protein (Galpha), and SLENDER RICE1 (SLR1), which encodes a repressor of GA signaling. At the vegetative stage, the expression of OsGA20ox2, OsGA3ox2, Galpha, and SLR1 was observed in rapidly elongating or dividing organs and tissues, whereas the expression of OsGA20ox1 or OsGA3ox1 could not be detected. At the inflorescence or floral stage, the expression of OsGA20ox2, OsGA3ox2, Galpha, and SLR1 was also observed in the shoot meristems and stamen primordia. The overlapping expression of genes for GA biosynthesis and signaling indicates that in these tissues and organs, active GA biosynthesis occurs at the same site as does GA signaling. In contrast, no GA-biosynthesis genes were expressed in the aleurone cells of the endosperm; however, the two GA-signaling genes were actively expressed, indicating that the aleurone does not produce bioactive GAs, but can perceive GAs. The expression of OsGA20ox1 and OsGA3ox1 was observed only in the epithelium of the embryo and the tapetum of the anther. Based on the specific expression pattern of OsGA20ox1 and OsGA3ox1 in these tissues, we discuss the unique nature of the epithelium and the tapetum in terms of GA biosynthesis. The epithelium and the tapetum are considered to be an important source of bioactive GAs for aleurone and other organs of the flower, respectively.     

β-galactosidase activity in the germinating seeds of Vigna sinensis

The β-galactosidase activity in cotyledons of Vigna sinensis increases during seed germination and is inhibited by cycloheximide. The increasing activity may be due to the de novo synthesis of enzyme protein. The enzyme has been partially purified by gel filtration and characterized with respect to some biochemical parameters. The optimum pH and optimum temperature are 4.5 and 55°, respectively and the enzymes follows typical Michaelis kinetics with K_m and V_max of 4.5 x 10^?4 M and 2.0 x 10^?5 mol/hr respectively. K_i for galactose and lactose are 4.5 and 220 mM, respectively. The energy of activation of the enzyme for p-nitrophenyl β-D-galactoside is 9.83 kcal/mol. The apparent relative MW of the enzyme as determined by gel filtration was 56000.     

Geographie variation of α-amylase, β-amylase, β-glucanase,pullulanase and chitinase activity in germinating Hordeum spontaneum barley from Israel and Jordan

The enzyme activities of -amylase, -amylase, -glucanase, pullulanase and chitinase were determined in extracts of wild barley (Hordeum vulgare ssp. spontaneum) germinated for five days under axenic standard conditions. The material comprises 257 accessions, for 242 of which the botanical territory of origin in Israel or Jordan is known. The enzyme activities based on soluble protein in the extracts showed significant differences (P<0.001) among the eleven territories. The territorial moisture parameters mostly correlate with the enzyme activities. As determined by one gene or oligogenes, the significant territorial differences and the correlation with moisture are thought to reflect natural selection of genes responsible for favourable activity, or of genes linked to the enzyme coding loci, or in a coadaptive manner, of physiologically allied genes. Genes for high -glucanase activity at germination are probably coadaptive with genes for high -glucan content of the grain. The generally low starch content of wild barley grains probably makes any high -amylase activity unnecessary at the germination stage. An inverse relationship appears between -glucanase and chitinase activity; these two enzymes are also pathogenesis related proteins.     

Regulation of Medicago sativa L. somatic embryos regeneration by gibberellin A3 and abscisic acid in relation to starch content and α-amylase activity

Somatic embryo quality is an important factor decisive for the efficiency of somatic embryogenesis. Addition gibberellic acid (GA₃) at a concentration of 1 μM to germination medium improved the regeneration of alfalfa somatic embryos. Inhibitory effect of ancymidol, an inhibitor of gibberellin biosynthesis, on germination and conversion may indicate that those processes require endogenous GAs. Since fluridone, an ABA biosynthetic inhibitor, at a concentration of 1 μM, had a slight stimulatory effect on germination of somatic embryos, it may be presumed that embryos contain a too high level of residual ABA after maturation phase (20 μM ABA is used at that phase). The observed improvement of regeneration of somatic embryos by GA₃ was correlated with acceleration of starch hydrolysis through α-amylase activity enhancement by GA₃. In contrast, the inhibitory effect of ABA on the above processes was probably related to inhibition of α-amylase activity and, in consequence, to delayed starch hydrolysis. It is suggested that α-amylase activity can be considered a good marker of the quality of Medicago sativa L. somatic embryos.     

Spatial distribution of β-amylase in germinating barley seeds based on the avidin-biotin-peroxidase complex method

Summary In comparison with two wild type barley cultivars, Sundance and Bomi, biochemical data show that the high-lysine mutant Hiproly contains abundant amounts of lysine-rich -amylase, whereas mutant Risø 1508, also a high-lysine mutant, contains negligible amounts of this enzyme. Immunocytochemical studies of germinating barley seeds, using both mono- and polyclonal antibodies to -amylase, support the biochemical findings of enzyme abundance in developing seeds. Three immunostaining methods for localization of -amylase were tested; of these, the avidin-biotin-peroxidase complex method, a relatively new procedure for study of plant tissues, is by far the most sensitive. -Amylase occurs predominantly in cytoplasm of the endosperm, with a minor, but previously unknown localization in the aleurone of the mid-region of the seeds. A spatial distribution of -amylase is seen. Endosperm in the upper and lower regions has the greatest amount of -amylase, with the amount decreasing toward the mid-region. In the mid-region, a limited aleurone localization of -amylase is found in all four barley strains. The function of this aleurone localization is unclear. In the endosperm, the abundance of -amylase appears to be inversely correlated with number of starch grains per unit area in Hiproly but not in Risø 1508, yet the rate of germination of the two mutants is essentially identical. Whether -amylase has a role in starch metabolism in these germinating barley seeds is unclear.Abbreviations Ab antibodies - ABC avidin-biotin-peroxidase complex - a -Amylase - BSA bovine serum albumin - DAB diaminobenzidine tetrahydrochloride dihydrate - FAA formalin: glacial acetic acid: ethyl alcohol - FITC fluorescein isothiocyanate - mAb mouse monoclonal antibodies - pAb rabbit polyclonal antibodies - PAP peroxidase-anti-peroxidase - PBS phosphate buffer saline - R-1508 Risø 1508     

Classification and characterization of the rice α-amylase multigene family

To establish the size and organization of the rice -amylase multigene family, we have isolated 30 -amylase clones from three independent genomic libraries. Partial characterization of these clones indicates that they fall into 5 hybridization groups containing a total of 10 genes. Two clones belonging to the Group 3 hybridization class have more than one gene per cloned fragment. The nucleotide sequence of one clone from Group 1, OSg2, was determined and compared to other known cereal -amylase sequences revealing that OSg2 is the genomic analog of the rice cDNA clone, pOS103. The rice -amylase genes in Group 1 are analogous to the -Amy1 genes in barley and wheat. OSg2 contains sequence motifs common to most actively transcribed genes in plants. Two consensus sequences, TAACA _G ^A A and TATCCAT, were found in the 5 flanking regions of -amylase genes of rice, barley and wheat. The former sequence may be specific to -amylase gene while the latter sequence may be related to a CATC box found in many plant genes. Another sequence called the pyrimidine box ( _T ^C CTTTT _T ^C ) was found in the -amylase genes as well as other genes regulated by gibberellic acid (GA). Comparisons based on amino acid sequence alignment revealed that the multigene families in rice, barley and wheat shared a common ancestor which contained three introns. Some of the descendants of the progenitor -amylase gene appear to have lost the middle intron while others maintain all three introns.     

Endotoxin-neutralizing activity and mechanism of action of a cationic α-helical antimicrobial octadecapeptide derived from α-amylase of rice

We have previously reported that AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice, is a novel cationic α-helical peptide that exhibited antimicrobial activity against human pathogens, including Porphyromonas gingivalis, Pseudomonas aeruginosa, Propionibacterium acnes, Streptococcus mutans, and Candida albicans. In this study, to further investigate the potential functions of AmyI-1-18, we examined its inhibitory ability against the endotoxic activities of lipopolysaccharides (LPSs, smooth and Rc types) and lipid A from Escherichia coli. AmyI-1-18 inhibited the production of endotoxin-induced nitric oxide (NO), an inflammatory mediator, in mouse macrophages (RAW264) in a concentration-dependent manner. The results of a chromogenic Limulus amebocyte lysate assay illustrated that the ability [50% effective concentration (EC₅₀): 0.17 μM] of AmyI-1-18 to neutralize lipid A was similar to its ability (EC₅₀: 0.26 μM) to neutralize LPS, suggesting that AmyI-1-18 specifically binds to the lipid A moiety of LPS. Surface plasmon resonance analysis of the interaction between AmyI-1-18 and LPS or lipid A also suggested that AmyI-1-18 directly binds to the lipid A moiety of LPS because the dissociation constant (K_D) of AmyI-1-18 with lipid A is 5.6 × 10⁻¹⁰ M, which is similar to that (4.3 × 10⁻¹⁰ M) of AmyI-1-18 with LPS. In addition, AmyI-1-18 could block the binding of LPS-binding protein to LPS, although its ability was less than that of polymyxin B. These results suggest that AmyI-1-18 expressing antimicrobial and endotoxin-neutralizing activities is useful as a safe and potent host defense peptide against pathogenic Gram-negative bacteria in many fields of healthcare.     

Regulatory interplay of the Sub1A and CIPK15 pathways in the regulation of α-amylase production in flooded rice plants

Rice (Oryza sativa L.) can successfully germinate and grow even when flooded. Rice varieties possessing the submergence 1A (Sub1A) gene display a distinct flooding-tolerant phenotype, associated with lower carbohydrate consumption and restriction of the fast-elongation phenotype typical of flooding-intolerant rice varieties. Calcineurin B-like interacting protein kinase 15 (CIPK15) was recently indicated as a key regulator of α-amylases under oxygen deprivation, linked to both rice germination and flooding tolerance in adult plants. It is still unknown whether the Sub1A- and CIPK15-mediated pathways act as complementary processes for rice survival under O(2) deprivation. In adult plants Sub1A and CIPK15 may perhaps play an antagonistic role in terms of carbohydrate consumption, with Sub1A acting as a starch degradation repressor and CIPK15 as an activator. In this study, we analysed sugar metabolism in the stem of rice plants under water submergence by selecting cultivars with different traits associated with flooding survival. The relation between the Sub1A and the CIPK15 pathways was investigated. The results show that under O(2) deprivation, the CIPK15 pathway is repressed in the tolerant, Sub1A-containing, FR13A variety. CIPK15 is likely to play a role in the up-regulation of Ramy3D in flooding-intolerant rice varieties that display fast elongation under flooding and that do not possess Sub1A.     

Regulatory polymorphism in midgut α-amylase activity and developmental rate of Drosophila subobscura

The correlation between the types of midgut -amylase activity and rates of preadult stages of development was studied in D. subobscura. It was found that flies having more midgut regions where activity of -amylase could be determined, develop slower than those with the activity of this enzyme expressed in only one or two out of five such regions. This relationship, however, is quite specific in relation to embryonal, larval, and pupal development, and characteristic of each particular phenotype. The adaptive meaning of such correlations is only presumed, but comparisons between the experimental and the original natural population suggest that specific types of balancing selection could affect the frequency of genes controlling the regulatory system of enzyme studied, and the frequency of genes controlling the variation of important fitness traits, such as the rate of preadult development.     

α-d-Galactosidase activity and galactomannan and galactosylsucrose oligosaccharide depletion in germinating legume seeds

Germinating seeds of lucerne, guar, carob and soybean initially depleted raffinose series oligosaccharides and then galactomannan. This depletion was accompanied by a rapid increase and then a decrease in α-galactosidase levels. Lucerne and guar contained two α-galactosidase activities, carob three and soybean four. One of these in each plant, from its location in the endosperm, time of appearance and kinetic behaviour, appeared to be primarily involved in galactomannan hydrolysis. This enzyme in lucerne had MW of 23 000 and could not be separated from β-mannanase by (NH₄)₂SO₄ fractionation, DEAE, CM or SE-cellulose chromatography or gel filtration, but only by polyacrylamide gel electrophoresis. In guar, carob and soybean, it could be separated by ion-exchange chromatography and gel filtration. In lucerne, carob and guar most of the total increase in activity was due to this enzyme. The other α-galactosidases had MWs of about 35 000 and could be separated from β-mannanase by dissection, ion exchange cellulose chromatography and gel filtration. They were located in the cotyledon-embryo and appeared to be primarily involved in galactosylsucrose oligosaccharide hydrolysis.