Differential expression of rice α-amylase genes during seedling development under anoxia

The unique capability of rice (Oryza sativa L.) seedlings to grow under anoxic conditions may result in part from their ability to express -amylase and maintain the supply of sugar needed for energy metabolism. Previous studies have demonstrated that under aerobic conditions the Amy1 and Amy2 subfamily genes are regulated primarily by phytohormones while the Amy3 subfamily genes are induced during sugar starvation. The expression patterns for these -amylase genes were considerably different in anoxic vs. aerobic rice seedlings. The level of total -amylase mRNA under anoxic conditions was decreased in aleurone layers while it increased in the embryo. Anoxic conditions greatly diminished the expression of the Amy1A gene in aleurone. Conversely, expression of many Amy3 subfamily genes was up-regulated and prolonged in embryo tissues under anoxic conditions.     

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.     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

Phage lambda pL promoter controlled α-amylase expression inEscherichia coli during fermentation

Summary Phage lambda p_L promoter controlled expression of theBacillus stearothermophilus gene coding for a thermostable -amylase inE. coli was studied in shake flask cultures and in a laboratory fermenter. At an inducible temperature (40 °C) the final cell density was lower, but the total enzyme activity produced ca. 80% higher than at a non-inducible temperature (30 °C). Moreover, 17% of the total enzyme activity was found in the culture medium. The -amylase yield, production rate and proportion secreted were further increased by shifting the fermentation temperature after certain period of bacterial growth rather than at the beginning of fermentation.     

Direct screening for high-level expression of an introduced α-amylase gene in plants

A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature -amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for -amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlated with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.     

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     

Calcium sensitivity of α-actinin is required for equatorial actin assembly during cytokinesis

The actin cross-linking protein, α-actinin, plays a crucial role in mediating furrow ingression during cytokinesis. However, the mechanism by which its dynamics are regulated during this process is poorly understood. Here we have investigated the role of calcium sensitivity of α-actinin in the regulation of its dynamics by generating a functional calcium-insensitive mutant (EFM). GFP-tagged EFM (EFM-GFP) localized to the equatorial regions during cell division. However, the maximal equatorial accumulation of EFM-GFP was significantly smaller in comparison to α-actinin-GFP when it was expressed in normal cells and cells depleted of endogenous α-actinin. No apparent defects in cytokinesis were observed in these cells. However, F-actin levels at the equator were significantly reduced in cells expressing EFM-GFP as compared with α-actinin-GFP at furrow initiation but were recovered during furrow ingression. These results suggest that calcium sensitivity of α-actinin is required for its equatorial accumulation that is crucial for the initial equatorial actin assembly but is dispensable for cytokinesis. Equatorial RhoA localization was not affected by EFM-GFP overexpression, suggesting that equatorial actin assembly is predominantly driven by the RhoA-dependent mechanism. Our observations shed new light on the role and regulation of the accumulation of pre-existing actin filaments in equatorial actin assembly during cytokinesis.     

Hyper expression ofBacillus stearothermophilus α-amylase gene inBacillus subtilis

Summary Utilizing plasmids pUB110, pBR322 and pRG71, we have constructed a number of vectors which could be utilized for expression of cloned genes both in gram positive and gram negative bacteria. This report discusses the hyperexpression of -amylase gene ofB. stearothermophilus BR135 using these vectors.     

Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the -amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice -amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

Isoenzymes of α-amylase during pod development of field beans

The pod mesophyll of field beans accumulates large amounts of starch during stage 1 of embryogenesis, which is later utilized during stage 2. The activity of starch degradation in the pod is under metabolic control of the enclosed seeds. Changes in the isoenzyme pattern of α-amylase and not starch phosphorylase coincide with the beginning of the starch degradation period in the pods. Mesophyll cells of the pods contain the same α-amylase isoenzymes as the endocarp but exhibit a higher α-amylase activity that parallels the much higher starch content of this tissue in comparison to the endocarp. Regulation of starch breakdown may be mediated at least in part by the formation of a special α-amylase isoenzyme.     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

The role of endogenous gibberellin A₁ (GA₁) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA₁ and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA₁ and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA₁. These results indicate that the endogenous GA₁ biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R _t retention time - GC-SIM gas chromatography-selected ion monitoring     

Action pattern of human pancreatic α-amylase on maltoheptaose,a substrate for determining α-amylase in serum

An enzymatic assay for the determination of α-amylase in serum was developed which employed a soluble substrate, maltoheptaose, and a coupled enzymatic indicator reaction consisting of α-glucosidase and the hexokinase—glucose-6-phosphate dehydrogenase system. We used high-performance liquid chromatography (HPLC) to establish the action pattern of maltoheptaose under the test conditions: (A) the action pattern of α-amylase alone, (B) that of the combined action of α-amylase and α-glucosidase. Conducive to this effort was: the availability of pure maltoheptaose and human pancreatic α-amylase; the development of an adequate procedure for sample pretreatment (partition chromatography on a mixed-bed ion exchanger) and of an HPLC system for separation of substrate and reaction products without interference from by-products of the assay (partition chromatography on a cation-exchange column with acetonitrile—water); and the use of a new, very sensitive refractometric detector revealing sugar amounts as low as 40 ng.We derived the following stoichiometric equations:

The standard deviation of the rate coefficients is about 5%.     

Is 1,25-dihydroxyvitamin D required for reproduction?

The role of 1,25-dihydroxyvitamin D (1,25-(OH)2D) in avian and mammalian reproduction is examined. 1,25-Dihydroxyvitamin D is required, in both the avian and mammalian species, for maintenance of normocalcemia, adequate intestinal calcium absorption, bone turnover, and mineral homeostasis throughout the reproductive cycle--just as it is required in the nonlaying bird or nonpregnant, nonlactating mammal. In the avian species, 1,25-(OH)2D is required for ovulation and shell formation, transfer of calcium from the egg shell across the chorioallantoic membrane to the fetal circulation, and maintenance of fetal serum calcium, bone metabolism, and mineral homeostasis. In the mammalian species, 1,25-(OH)2D is required for normal ovulation, normal fetal and neonatal bone metabolism, milk production, and maintenance of normocalcemia and mineral homeostasis in the neonate. In the absence of 1,25-(OH)2D, however, embryogenesis (rat and chick) and neonatal development (rat) can proceed in such a way as to produce viable, normal appearing offspring. The classical effects of 1,25-(OH)2D deficiency (hypocacemia, inadequate intestinal calcium absorption, and bone mineralization) become increasingly apparent with advancing age but there are no other apparent major developmental abnormalities.     

The α-amylase genes in Oryza sativa: Characterization of cDNA clones and mRNA expression during seed germination

Summary Two cDNA clones, pOS103 and pOS137, were isolated which code for distinct -amylase isozymes in germinating rice seeds. Sequence analysis indicated that the clones encode polypeptides of approximately 48 kDa, both of which possess a signal peptide involved in directing secretion of the protein. Comparison of the two rice -amylase amino acid sequence showed that they are 76% similar to each other, while showing 85% to 90% similarity with other cereal -amylases. A comparison of eleven cereal -amylases also revealed three new conserved regions (I, II, and IV) not previously identified in the animal, bacterial, and fungal -amylases. Regions I and IV are sites for intron splicing while region II' is probably involved in calcium binding. One of the rice a-amylase cDNAs, pOS103, encodes a protein that has two potential N-glycosylation sites, one in the signal peptide and the other in the mature portion of the protein. The cDNA clone, pOS137, encodes an -amylase with a single glycosylation site in the signal peptide, suggesting that the mature OS137 isozyme is not glycosylated. Analysis of the expression of these genes in germinating rice seeds indicated that mRNA corresponding to pOS103 and pOS137 could be detected throughout a 48 h period of seed imbibition. RNA levels, however, were dramatically stimulated by treatment of embryoless half-seeds with exogenous GA₃. Our results demonstrate that at least two forms of -amylase are expressed in germinating rice seeds and that the expression of these genes is regulated by the phytohormone GA₃.Abbreviations GA gibberellic acid - GA₃ gibberellic acid₃; poly(A)^–, polyadenylated - PPA porcine pancreatic -amylase - SSC 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0 - TAA Taka-amylase