Purification and Some Properties of Plant Endonuclease from Scallion Bulbs

A plant endonuclease with 3′-nucleotidase activity was purified from scallion bulbs to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 38,000 by SDS-PAGE and 40,000 by Sephadex G-100 gel filtration. The enzyme rapidly hydrolyzed yeast RNA and denatured calf thymus DNA to acid-soluble substances, and hydrolyzed the plasmid pBR322 to yield small DNA fragments at low enzyme concentrations. These four activities were eliminated by treatments with EDTA and tetraethylenepentamine. The enzyme had maximum activity at pH 8.5-9.0 for 3′-AMP, 3′-GMP, and 3′-UMP, at pH 6.5 for 3′-CMP and yeast RNA, and at pH 6.0 for denatured calf thymus DNA and pBR322. During digestion of yeast RNA by the enzyme at pH 6.5, 5′-GMP was released most rapidly, followed by 5′-UMP, 5′-AMP, and 5′-CMP. These properties were different from those of endonucleases isolated from other sources such as mung bean sprouts and wheat seedlings.     

Pedigree assessment using RAPD-DGGE in cereal crop species

Summary The introduction of molecular biology methodologies to plant improvement programs offers an invaluable opportunity for extensive germplasm characterization. However, the detection of adequate DNA polymorphism in self-pollinating species remains on obstacle. We have optimized a denaturing-gradient-gel electrophoresis (DGGE) system which, when used in combination with random amplified polymorphic DNA (RAPD) analysis, greatly facilitates the detection of reproducible DNA polymorphism among closely related plant lines. We have used this approach to estimate pedigree relationships among a spectrum of plant materials in wheat, barley and oat. Based on analysis with one or two primers, we were able to distinguish soft from hard winter wheat, and 2-rowed from 6-rowed barley. Further analysis with additional primers allowed resolution of polymorpisms even among closely related lines in highly selected populations. We placed 17 cultivars of oat into two distinct clusters that differed significantly from previous oat pedigree assessments. We believe that DGGE-RAPD is a superior method for detecting DNA polymorphism when compared to RFLP, agarose-RAPD, or polyacrylamide-RAPD methods.     

The involvement of wheat and common bean lectins in the control of cell division in the root apical meristems of various plant species

The effects of wheat germ agglutinin (WGA) and phytohemagglutinin (PHA) at the concentration of 1 mg/l on the rate of cell division in the root apical meristem of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), rice (Oryza sativa L.), and common bean (Phaseolus vulgaris L.) seedlings were compared. WGA enhanced cell division in the roots of barley and rice approximately similarly as in wheat roots but did not affect division of meristematic cells in the roots of common bean seedlings. In contrast PGA enhanced mitotic activity in the root apical meristem of common bean seedlings but did not affect division in the wheat and barley roots. Seedling treatment with lectins shifted the hormonal balance in them toward accumulation of growth activators (IAA and cytokinins). The relationship between lectin and hormonal systems in the control of cell division is discussed.     

Inheritance of nitrite reductase and regulation of nitrate reductase, nitrite reductase, and glutamine synthetase isozymes

Banding patterns of nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS) from leaves of diploid barley (Hordeum vulgare), tetraploid wheat (Triticum durum), hexaploid wheat (Triticum aestivum), and tetraploid wild oats (Avena barbata) were compared following starch gel electrophoresis. Two NR isozymes, which appeared to be under different regulatory control, were observed in each of the three species. The activity of the more slowly migrating nitrate reductase isozyme (NR1) was induced by NO3- in green seedlings and cycloheximide inhibited induction. However, the activity of the faster NR isozyme (NR2) was unaffected by addition of KNO3, and it was not affected by treatments of cycloheximide or chloramphenicol. Only a single isozyme of nitrite reductase was detected in surveys of three tetraploid and 18 hexaploid wheat, and 48 barley accessions; however, three isozymes associated with different ecotypes were detected in the wild oats. Inheritance patterns showed that two of the wild oat isozymes were governed by a single Mendelian locus with two codominant alleles; however, no variation was detected for the third isozyme. Treatment of excised barely and wild oat seedlings with cycloheximide and chloramphenicol showed that induction of NiR activity was greatly inhibited by cycloheximide, but only slightly by chloramphenicol. Only a single GS isozyme was detected in extracts of green leaves of wheat, barley, and wild oat seedlings. No electrophoretic variation was observed within or among any of these three species. Thus, this enzyme appears to be the most structurally conserved of the three enzymes.     

Excretion of 5′-Nucleotides from Bacterial Cells

Incubating the dried cells of Brevibacterium sojae No. 425-40 in alkaline buffer, the excretion of 5′-nucleotides accompanying with the decrease of intracellular RNA was observed. Then the determination of the optimum condition of the excretion and the investigation on the enzyme responsible for the degradation of endogenous RNA were carried out.

In the experiments using sonicate and disrupted cells, it appeared that orthophos-phate and Mg⁺⁺ might be accelerative or essential for the degradation of endogenous RNA and, in addition to four 5′-nucleotides (AMP, GMP, UMP and CMP), each nucleoside 5′-diphosphate was also contained in its degraded products. Nucleoside 2′- or 3′-monophos-phates were not detected. Although it was not clear whether phosphodiesterase concerned with the degradation of intracellular RNA or not, it was suggested that polynucleotide phos-phorylase acted mainly on the degradation.

The maximal excretion of 5′-nucleotides from dried cells was obtained by suspending 1 to 2% of dried cells in 0.05 M carbonate-bicarbonate buffer (pH 10) and incubating it at 60°C for two to three hours. Orthophosphate and Mg⁺⁺ were not required for the excretion.     

Isolation and characterization of multiple forms of malt deoxyribonuclease

A deoxyribonuclease (DNase), similar to bovine pancreatic DNase, has been isolated from germinating barley. Commerically available malt was used as source of the enzyme. The purification procedure involves (a) ammonium sulfate fractionation (45–65% saturation), (b) CM-cellulose chromatography at pH 4.7 and (c) DEAE-cellulose chromatography at pH 8. DEAE-cellulose separates the enzyme into 4 distinct forms, designed as DNases A, B, C, and D. DNase A and B may be rechromatographed on DEAE-cellulose employing a CaCl₂ instead of Tris-HCl gradient. Both forms appear homogeneous on regular and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In addition, both forms have a sp. act. of ca 700 units per A unit at 280 nm, similar to the potency of the pancreatic enzyme. DNase C and D, which are present in relatively small quantities in malt, were not characterized. The MWs of DNases A and B, as estimated by the SDS gel electrophoresis techniques, are near 32 000, slightly larger than that of the pancreatic enzyme. In the presence of either Mn²⁺ or Mg²⁺, the pH-activity profile of the barley enzyme is similar to that obtained with the pancreatic enzyme. Like the pancreatic enzyme, barley DNase is protected by Ca²⁺ from inactivation. The amino acid compositions of the A and B forms are about the same; a comparison of the malt and pancreatic enzymes shows many similarities but major differences in the amounts of glutamic acid, proline and glycine. The hydrolysis products of DNA by malt DNase are indistinguishable from those obtained with pancreatic DNase. Further hydrolysis of these products by snake venom phosphodiesterase shows malt DNase to be a 5′-phosphate producer. Deoxythymidine 3′,5′-di-p-nitrophenyl phosphate, one of the synthetic substrates of pancreatic DNase, is also hydrolysed by malt DNase.     

Shoot apical meristem: A sustainable explant for genetic transformation of cereal crops

Summary Immature zygotic embryo has been the widely used explant source to develop embryogenic callus lines, cell suspensions and protoplasts for transformation of cereal crops including maize, wheat, rice, oat, barley, sorghum, and millet. However, the lack of competence of immature embryos in certain elite lines is still a barrier to rontine production of transgenic cereal crops in certain commercial cultivars. In addition, a great deal of effort is required to produce immature embryos, manipulate cultures, of immature embryos or their cell suspensions, and cryoperserve cultures for further use. In addition, undifferentiated cells may have reduced regenerability after a few months, of in vitro culture. Alternative explants and regeneration systems for efficient transformation of cereal crops are needed to avoid or reduce the above limitations. During the past decade, scientists have successfully manipulated the shoot apical meristerms from seedlings of maize oat, sorghum, millet, wheat, and barley in an effort to develop a less genetype-dependent and efficient cereal regneration system that can be maintained in vitro for long pertiods of time without the need for cryopreservation. Furthermore, apical mesistem regeneration systems were used to stably transform maize, wheat, rice, oat, barley, sorghum, and millet.     

Failure to Detect Cyclic 3', 5'-Adenosine Monophosphate in Healthy and Crown Gall Tumorous Tissues of Vicia faba

Attempts were made to provide proof for the occurrence of cyclic 3′,5′-adenosine monophosphate in healthy and crown gall tissues of Vicia faba. Although our purified extracts gave positive readings in the Gilman binding assay for cyclic AMP, they were not digested by a specific cyclic 3′,5′-adenosine monophosphate phosphodiesterase from beef heart. The extracts were digested, however, by a partially purified cyclic nucleotide phosphodiesterase from carrot tissue, which attacks both cyclic 2′,3′- and 3′,5′-nucleotides. The data indicate that the substances detected in the V. faba extracts are perhaps cyclic 2′,3′-nucleotides, a possible RNA degradation product.     

Rice wine brewing with sprouting rice and barley malt

Though alcoholic beverages are widely made with barley malt in Western countries, as well as in Asiatic countries today, alcoholic beverages are rarely made with sprouting rice. Rice wines were obtained from cooked nonglutinous rice using sprouting rice and barley malt as saccharifying agents with compressed baker's yeast and Kyokai no. 9 yeast, and a comparative study was conducted of the resulting rice wines. The saccharifying activity of barley malt was higher than that of sprouting rice. The amounts of ethyl alcohol, volatile aromatic components, and reducing sugars in the rice wine made with barley malt were higher than those in the wine made with sprouting rice. The rice wine made with barley malt was faintly brownish in color and had heavy, complicated and vulgar characteristics. By contrast, sprouting rice wine was colorless and had light, simple and refined characteristics in terms of both aroma and taste. Sprouting rice wine made with Kyokai no. 9 yeast contained about 8% ethanol with an acidity of around 4.1. Sprouting rice was found to be applicable as a saccharifying agent for ethanol fermentation, as is barley malt. The quality of the sprouting rice wine was further improved through the use of Kyokai no. 9 yeast.     

Osservazioni Preliminari sui Sistemi Degradanti dell'RNA nei semi di Ricino

Abstract

Preliminary observations on the enzymatic degradation of RNA in castor bean seeds. — Cocucci, Maggio, Monroy and Marrè have shown the decrease of RNA content during ripening in castor bean seeds, and its increase during germination. Furthermore, these Authors have demonstrated that in the dry ripe seeds the ribosomes are undetectable, and that they increase rapidly during germination. Two peaks of ribosomes are easily detected upon ultracentrifugal analysis in germinating seeds (Cocucci and Sturani). These observations were the basis for our investigations of the enzymes of RNA metabolism in castor bean seeds. This paper deals with our preliminary observations on RNA degrading enzymes in these tissues. We have been able to measure RNase activity, phosphodiesterase, 3′-,5′- and 2′-nucleotidases in castor bean seeds at different stages of development. RNase activity (measured in crude extracts) changes little during the ripening process, its rate corresponding to 40–50 μMoles of nucleotides liberated from RNA per hour and per gram of fresh weight. In the dry seeds, RNase activity is 30–40 μMoles of nucleotides/h.g.f.w., and it increases to about 60–70 μMoles/h/g.f.w. after 72 hours of germination.

Phosphodiesterase activity is about 4–5 μMoles/h.g.f.w.

The following rates have been found in seeds almost completely ripe seeds for 3′-, 5′- and 2′-nucleotidase activities, respectively 45–50 μMoles/h.g.f.w.; 6–7 μMoles/h.g.f.w.; 8 μMoles/h.g.f.w.; ATP-ase activity was of about 80–100 μMoles of phosphate liberated /h.g.f.w. - The high activity of 3′-nucleotidase, of the same order of that of RNase, suggests that these two enzymes are responsible for degradation of RNA to nucleosides and inorganic phosphate. Further investigations are being carried on to define the biochemical properties of castor bean RN-ase.     

Characterization of the cultivable microbiota of sprouts and their potential for application as protective cultures

The microbiota of ten seeds and ready-to-eat sprouts produced thereof was characterized by bacteriological culture and denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments of the 16S rRNA gene. The predominant bacterial biota of hydroponically grown sprouts mainly consisted of enterobacteria, pseudomonades and lactic acid bacteria (LAB). For adzuki, alfalfa, mung bean, radish, sesame and wheat, the ratio of these bacterial groups changed strongly in the course of germination, whereas for broccoli, red cabbage, rye and green pea the ratio remained unchanged. Within the pseudomonades, Pseudomonas gesardii and Pseudomonas putida have been isolated and strains of the potentially pathogenic species Enterobacter cancerogenes and Pantoea agglomerans were found as part of the main microbiota on hydroponically grown sprouts. In addition to the microbiota of the whole seedlings, the microbiota of root, hypocotyl and seed leafs were examined for alfalfa, radish and mung bean sprouts. The highest and lowest total counts for aerobic bacteria were found on seed leafs and hypocotyls, respectively. On the other hand, the highest numbers for LAB on sprouts were found on the hypocotyl. When sprouting occurred under the agricultural conditions, e.g. in soil, the dominating microbiota changed from enterobacteria to pseudomonades for mung beans and alfalfa sprouts. No pathogenic enterobacteria have been isolated from these sprout types. Within the pseudomonades group, Pseudomonas jessenii and Pseudomonas brassicacearum were found as dominating species on all seedling parts from soil samples. In practical experiments, a strain of P. jessenii was found to exhibit a potential for use as protective culture, as it suppresses the growth of pathogenic enterobacteria on ready-to-eat sprouts.     

Degradation of Nucleic Acids and Related Compounds by Microbial Enzymes

The culture filtrate of a strain of Bacillus subtilis decomposed ribonucleic acid into 5′-nucleotides and into other intermediates which released orthophosphate by an arsenate-resistant phosphatase. Under the best conditions examined in these experiments, about 50 per cent of ribonucleic acid was converted into 5′-nucleotides.

The culture filtrate of a strain of Bacillus brevis showed slight activities of ribonuclease and/or phosphodiesterase which produced 5′-nucleotides from ribonucleic acid, but showed predominant activity of 5′-adenylic acid degrading phosphatase.     

Involvement of bacterial quorum-sensing signals in spoilage of bean sprouts

Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10(8) to 10(9) CFU/g. AHL-producing bacteria (Enterobacteriaceae and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of N-3-oxo-hexanoyl-l-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (I gene) from a spoilage Pectobacterium was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in Erwinia carotovora. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in Enterobacteriaceae. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensing-regulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.     

Specific 5S ribosomal RNA primers for plant species i dentification in admixtures 3

The DNA sequences of the spacers between the 5S ribosomal RNA genes were determined for the cereals maize, barley, soghum, rye, rice, oat, and wheat. Species-specific primers were designed from the spacer region. PCR with these primers and a common primer from the conserved 5S ribosomal RNA gene sequence was investigated as a method for detection of the seven cereal species. DNA from these species could be specifically detected in mixtures. This technique could find application in the determination of the composition of admixtures or processed cereal products. The protocol described has potential for general application in the identification of plant species.     

Occurrence and synthesis of lectin in coleoptiles of germinating wheat,rye and rice seedlings

Occurrence, synthesis and localization of lectins in coleoptiles of 3-day old seedlings of wheat, rye, barley and rice were studied by a combination of high resolution ion-exchange chromatography, in vivo labelling with ³⁵S-cysteine and immunocytochemistry. Whereas no lectin can be isolated or localized in barley coleoptile, 1.9 and 40 ng of lectin per coleoptile was obtained from wheat and rye respectively. Wheat germ agglutinin was localized in the outer layer of the wheat coleoptile and both inner and outer layers of rye coleoptile displayed a specific reaction. In rice, 250 ng of lectin is present in the coleoptile and is distributed throughout this organ. Wheat coleoptiles synthesize no lectin and rye coleoptiles synthesize minute amounts while those from developing rice seedlings incorporate reasonable amounts of ³⁵S-cysteine into lectin.Abbreviations FPLC Fast Protein Liquid Chromatography - GlcNAc N-acetylglucosamine - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.     

Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants overproducing an oat cell-wall-bound thionin

Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field. The thionins purified from seed and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens; however, we found that no endogenous rice thionin genes alone are enough for resistance to two major seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and B. glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria. Thus, we isolated thionin genes from oat, one of which was overexpressed in rice. When wild-type rice seed were germinated with these bacteria, all seedlings were wilted with severe blight. In the seedling infected with B. plantarii, bacterial staining was intensively marked around stomata and intercellular spaces. However, transgenic rice seedlings accumulating a high level of oat thionin in cell walls grew almost normally with bacterial staining only on the surface of stomata. These results indicate that the oat thionin effectively works in rice plants against bacterial attack.     

Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in growing bean sprouts

AIMS: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. METHODS AND RESULTS: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20,000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ beta-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. CONCLUSIONS: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.     

Studies on cyclic adenosine 3' ,5'-monophosphate levels, Adenylate cyclase and phosphodiesterase activities in the dimorphic fungus Mucor rouxii.

The germination of spores of Mucor rouxii into hyphae was inhibited by 2 mm dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 mm cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 mm) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air.Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.     

Host range,mating type and population structure of Magnaporthe sp. of a single barley field in São Paulo state,Brazil

Brazil is blast disease hot spot because severe epidemics have occurred among wheat, triticale, rye, barley and oat crops. Although the first outbreak of barley blast appeared in 1998, little information is available. Therefore, this study aimed to examine host range, mating type composition and population structure of Magnaporthe sp. from a single barley field in São Paulo, Brazil. To examine pathogenicity, 25 Magnaporthe isolates were inoculated on five, three, two and two cultivars of barley, wheat, oat and rice, respectively, and one cultivar each of rye, corn, sorghum, triticale and certain weeds (Cenchrus echinatus, Setaria geniculata, Brachiaria plantaginea and Eleusine indica). Mating type distribution of 33 isolates was investigated by molecular tools. The genotypic divergence of 41 barley and five wheat isolates was investigated by 15 random amplified polymorphic DNA primers and unweighted pair group method with arithmetic mean. The host range of the barley blast pathogen included wheat, oat, rye and triticale but not rice and weeds. Sexual reproduction appeared to not be involved in the high genotypic diversity because only a single isolate, MAT1‐2, was identified. The majority of barley isolates clustered together with wheat blast, except for four, suggesting a different origin.