Anthocyanin biosynthesis genes location and expression in wheat–rye hybrids

Studies into gene expression in a foreign background contribute toward understanding of how genes derived from different species or genera manages to co-exist in a common nucleus, on the one hand, and help to estimate possible effectiveness of wide hybridization for cultivated plant improvement, on the other hand. The aim of this study was to investigate conservation of wheat and rye expression networks, using the anthocyanin biosynthesis pathway (ABP) genes as a model system. We isolated and analyzed ABP genes encoding enzymes acting at different steps of the pathway: chalcone-flavanone isomerase (CHI), flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), and anthocyanidin-3-glucoside rhamnosyltransferase (3RT). The rye ABP genes locations we determined (Chi on chromosome 5RL, F3h on 2RL, Ans on 6RL, 3Rt on 5RL, the regulatory Rc—red coleoptile—gene on 4RL) were in agreement with the rearrangements established between rye and wheat chromosomes. Expression of the ABP structural genes was studied in wheat–rye chromosome addition and substitution lines. F3h activation by the Rc gene was found to be critical for the red coleoptile trait formation. It was shown that the rye regulatory Rc gene can activate the wheat target gene F3h and vice versa wheat Rc induces expression of rye F3h. However, lower level of expression of rye F3h in comparison with that of the two wheat orthologues in the wheat–rye chromosome substitution line 2R(2D) was observed. Thus, although work of the wheat and rye ABP gene systems following the formation of wheat–rye hybrids is finely coordinated, some divergence exists between rye and wheat ABP genes, affecting level of gene expression.     

α-isoamylases of rye seeds

Rye seeds contained 5 α-type amylases. Three behaved like typical α-amylases and were called Aα-amylases. Two showed chemical activity like α-amylase, but behaved differently physically and were called Bα-amylases. The latter were partly inactivated at pH 3·3 and at 70°. They were more resistant to EDTA than were the Aα-amylases. Barley and wheat seeds contained amylases behaving like Bα-amylases. Aleurone layers contained relatively large amounts of Aα-amylases. Relative amounts of Aα-and Bα-amylases depended on temperature during germination. Bα-amylases remained active for a longer time after germination than Aα-amylases.     

α-Amylase structure and activity

Two computerized methods of predicting protein secondary structure from amino acid sequences are evaluated by using them on the α-amylase ofAspergillus oryzae, for which the three-dimensional structure has been determined. The methods are then used, with amino acid alignments, to predict the structures of other α-amylases. It is found that all α-amylases of known amino acid sequence have the same basic structure, a barrel of eight parallel stretches of extended chain surrounded by eight helices. Strong similarities are found in those areas of the proteins believed to bind an essential calcium ion and at that part of the active site that catalyzes bond hydrolysis in the substrates. The active site, as a whole, is formed mainly of amino acids situated on loops joining extended chain to the adjacent helix. Variations in the length and amino acid sequence of these loops, from one α-amylase to another, provide the differences in binding the substrates believed to account for the known variations in action pattern of α-amylases of different biological origins.     

α-Amylase production in aqueous two-phase systems with Bacillus amyloliquefaciens

-Amylase production was studied in Bacillus amyloliquefaciens in aqueous two-phase systems composed of polyethyleneglycol (PEG)/dextran T500. Cells and enzyme were obtained in different phases when phase systems were applied to the growth media. Effects of different molecular weights and concentrations of polymers on differences of enzyme separation were established. The effect of PEG used in the system to the release of enzyme was investigated.     

α-Amylase Expression under Anoxia in Rice Seedlings: An Update

Rice grains can germinate under anoxia, while most other cereals fail to behave similarly. The ability of rice grains to degrade starch, thanks to the successful production of -amylase even in the absence of oxygen is likely one of the factors allowing rice to germinate anaerobically. In this paper we review the most recent results concerning the physiology and molecular biology related to the expression of -amylase genes under anoxia. The current view is that expression of sugar-starvation induces isoforms of -amylase playing a major role during germination under anoxia, while gibberellin induces -amylase predominating under aerobic conditions.     

α-Amylase production in batch and continuous cultures by Bacillus caldolyticus

Summary The concentration and productivity of -amylase increased remarkably, 15- and 11-fold respectively, in a continuous culture of Bacillus caldolyticus DSM 405 compared with batch culture, provided starch was used as the sugar source in a casitone medium. In the casitone medium with or without glucose hardly any improvement of enzyme production was observed in continuous culture. The addition of a small amount of starch to the glucose-casitone medium had a marked effect in stimulating amylase formation in continuous culture but no effect in batch culture.It was suggested that the higher production of -amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration.     

α-Amylase production by Bacillus amyloliquefaciens in a membrane recycle bioreactor

A strain of Bacillus amyloliquefaciens was cultivated in a membrane recycle bioreactor for the production of an extracellular -amylase. Continuous cultivations of B. amyloliquefaciens in the recycle fermentor led to higher cell mass and volumetric productivities than the ones obtained in batch or chemostat cultivations; the -amylase activities were lower than in the batch mode but significantly higher than in the chemostat mode. The operation of the membrane recycle bioreactor was sometimes disturbed by high broth viscosity leading to a stronger fouling of the membrane.     

Mapping of genes for male-fertility restoration in ’Pampa’ CMS winter rye (Secale cereale L.)

Hybrid rye breeding and seed production is based on the cytoplasmic male sterility (CMS)-inducing Pampa (P)-cytoplasm. For restoring male fertility in the hybrids, dominant, nuclear restorer genes are necessary. However, current pollinator lines are only partial restorers. Effective restorers were recently detected in the German inbred line L18 and in materials originating from the Argentinian rye cultivar Pico Gentario and an Iranian primitive rye accession called IRAN IX. F₂ populations were developed for each of these three restorer sources to map the responsible genes by means of RFLP (restriction fragment length polymorphism) markers. For this purpose, homo- and heterologous DNA probes were used leading to 101 polymorphic marker loci in total. For phenotypic evaluation, 100 to 134 randomly chosen plants from each of the populations were cloned and grown at two or three locations with two plants each. Segregation ratios of pollen fertility in the F₂ populations with L18 and IRAN IX were in accordance with a monogenic dominant inheritance. The segregation pattern for Pico Gentario indicated complementary gene action. Major dominant restorer genes were detected on chromosomes 1RS (L18) and 4RL (Pico Gentario, IRAN IX). The gene on 1RS explained 54% of the phenotypic variation and that on 4 RL 59% and 68% in the Pico Gentario and IRAN IX populations, respectively. Additionally, three minor genes from L18 were identified on chromosomes 3RL, 4RL and 5R. In the Pico Gentario population, a dominant modifier gene contributed by the female parent was found on chromosome 6R. This gene significantly enhanced the expression of the major restorer gene but on its own was not able to restore any degree of fertility. The map-distances between the major restorer loci and at least one flanking marker were small in all three F₂ populations (5–6 cM). In Pico Gentario an unfavorable linkage exists between the major restorer gene and a QTL for plant height. Since highly effective restorers are scarce in actual breeding populations, the major restorer genes detected on chromosomes 1 RS and 4RL should be introgressed into actual restorer lines. This is facilitated by using the closely linked molecular markers described. Received: 10 February 2000 / Accepted: 31 March 2000<@head-com-p1a.lf>Communicated by G. Wenzel     

Search for nonallelic structural genes for γ-chains of fetal hemoglobin in some primates

Peptide CB-3 is the indicator of the presence and activity of nonallelic structural genes for the human -chain. An equivalent peptide has been isolated from the HbF of the marmoset (Saguinus fuscicollis), the rhesus monkey (Macaca mulatta), the orangutan (Pongo pygmaeus), and the gorilla (Gorilla gorilla gorilla). The sequence of CB-3 from the marmoset and the rhesus monkey, although different from that of human CB-3, gives no evidence of heterogeneity of the HbF. However, the HbF of the rhesus monkey is itself heterogeneous, and there probably is a difference in another part of the -chain. The CB-3 peptide of the orangutan is definitely heterogeneous: the heterogeneity is in position 135, however, rather than in position 136 as in human CB-3. The gorilla has the same type of heterogeneity at position 136 as does the human, but the proportions of the two types of chains might differ in the two species.The work was supported in part by Research Grants HL-05168 and HL-02558 from the National Institutes of Health, U.S. Public Health Service, and by NIH-Division of Research Resources Grant FR-00165.     

α-Amylase production with Bacillus subtilis in the presence of PEG and surfactants

Summary -Amylase production with Bacillus subtilis was studied in the presence of PEG 600 and PEG 3350 as well as different surfactants: Trixon X-100, Tween 80, CTAB (cetylammonium-bromide) and SDS (sodiumdodecylsulphate) at concentrations resulting in comparable decreases in surface tension. Only PEG 600, at a concentration of 200 g/kg, was found to increase the enzyme production. Cell growth estimated as optical density at 620 nm and viable counts were not influenced by either PEG or surfactants. The results are discussed in relation to -amylase production in aqueous two-phase systems.     

α-Amylase and glucoamylase production by Schwanniomyces castellii

A chromogenic substrate (Cibachron blue-amylose), and soluble starch and maltose were used to characterize the amylolytic system from Schwanniomyces castellii 3754. The strain was able to produce inducible -amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) when grown on different C sources. The effect of the C source was slightly different for -amylase and glucoamylase production. Melezitose, maltose and soluble starch enhanced both -amylase and glucoamylase synthesis to nearly the same extent; amylose, trehalose and cellobiose particularly induced -amylase synthesis. The optimal pH for the release of both amylases was 5.5–7.0; maximal -amylase synthesis, on the other hand, was observed in the medium buffered at pH 6.0. The optimal pH for -amylase and glucoamylase activity was in the range of 4.5–7.2 and 4.2–5.5, respectively. Temperatures allowing maximal activity were 45°C for -amylase and 45–52°C for glucoamylase; a rapid decline of both activities was observed just above these temperatures.The species Schwanniomyces castellii (together with Schw. alluvius) is now considered to be synonymous with Schw. occidentalis var. occidentalis (Kreger-Van Rij 1984).     

Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat

Background

Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed.

Results

The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome.

Conclusion

Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.     

α-Amylase,Limit Dextrinase,and α-Glucosidase Enzymes in Barley and Malt

Abstract

A parallel discussion is presented of recent developments in fermentation monitoring and control in both research institutes and in industry. The discussion is based around the areas of measurement (on-line and off-line), data conditioning and analysis, modeling, fault analysis, and control. Recent progress in industrial fermentation monitoring and control is used as a guide to identify potential areas of research that might have a most rapid and direct impact on industrial fermentation operation.     

α-Amylase production by Bacillus subtilis and B. amyloliquefaciens in different PEG solutions

-Amylase production by Bacillus subtilis and Bacillus amyloliquefaciens was investigated in polyethyleneglycol (PEG)-containing growth medium. Five different molecular weight PEGs (600, 3000, 4000, 8000 and 20,000) were used. Enzyme production with B. subtilis increased 21% in medium containing 5% PEG 3000, but enzyme production with B. amyloliquefaciens increased 31% in medium containing 5% PEG 600 and 21% in medium containing 2% PEG 8000.     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Cell Bound α-Amylase in Aspergillus oryzae

It was previously reported that α-amylase accumulation is caused within the mycelium grown in a phosphate deficient medium and the concentration of anions or pH in a surrounding medium is responsible for its liberation. As it was subsequently found that α-amylase liberation from the mycelium of Aspergillus oryzae is stimulated by peptone, an attempt was made on purification of effective substances from it. The present paper describes on purification and properties of phosphopeptides found as an effective substance for α-amylase liberation, and discusses on the stimulation effect, comparing with the effects on pH and concentration of anions which were previously observed.     

α-Amylase production by immobilized whole cells of Bacillus subtilis

Summary Whole cells of Bacillus subtilis were immobilized in polyacrylamide gel prepared from 5% total acrylamide (85% acrylamide and 15% N,N-methylenebisacrylamide). Production of -amylase by the immobilized whole cells was attempted in a batch system. -Amylase produced by the immobilized whole cells was about three times larger than that produced by washed cells at optimum conditions. The reusability of the immobilized whole cells and washed cells was examined. The activity of -amylase production by washed cells decreased with increasing use cycles. On the other hand, the activity of the immobilized cells increased gradually, and it reched a steady state after seven cycles. -Amylase was produced from a simple reaction medium containing 1% meat extract and 0.05% yeast extract by the immobilized whole cells. The rate of -amylase production by the immobilized whole cells was the same as in submerged cultivation using starch bouillon medium. Growth of B. subtilis in polyacrylamide gel was observed by electron microscopy.     

Microsatellite mapping of genes that determine supernumerary spikelets in wheat (T. aestivum) and rye (S. cereale)

The wheat and rye spike normally bears one spikelet per rachis node, and the appearance of supernumerary spikelets is rare. The loci responsible for the ‘multirow spike’ or MRS trait in wheat, and the ‘monstrosum spike’ trait in rye were mapped by genotyping F₂ populations with microsatellite markers. Both MRS and the ‘monstrosum’ trait are under the control of a recessive allele at a single locus. The Mrs1 locus is located on chromosome 2DS, co-segregating with the microsatellite locus Xwmc453. The placement of flanking microsatellite loci into chromosome deletion bin 2DS-5 (FL 0.47–1.0) delimited the physical location of Mrs1 to the distal half of chromosome arm 2DS, within the gene rich region 2S0.8. The Mo1 locus maps about 10 cM from the centromere on chromosome arm 2RS. The similar effect on phenotype of mo1 and mrs1, together with their presence in regions of conserved synteny, suggest that they may well be members of an orthologous set of Triticeae genes governing spike branching. The practical importance of the MRS spike is that it produces more spikelets per spike, and thereby enhances the sink capacity of wheat, which is believed to limit the yield potential of the crop.     

Sulfhydryl Groups in Crystalline Sweet Potato β-Amylase

By means of amperometric mercurimetric titration, the –SH groups of native and oxidized sweet potato ²-amylase have been determined.

Although eight titratable –SH groups were found in the native enzyme molecule, no distinction between essential and non-essential –SH groups was observed. A partially active enzyme after treatment with o-iodosobenzoate was crystallized by salting out with ammonium sulfate, and only an extensively oxidized one from water upon dialysis. The oxidized enzyme did not restore its activity upon treatment with sodium thioglycolate. Oxidation by iodine was also carried out from which it was presumed that the oxidation of the enzyme either by o-iodosobenzoate or by iodine does not proceed through the “ all or none ” mechanism, and also that the essentiality of –SH groups would rather be indirect.