Differential expression of β-conglycinin genes in nodulated and non-nodulated isolines of soybean

Nodulated (T202) and non-nodulated (T201) isolines of soybean (Glycine max [L.] Merr.) were cultivated in a rotated paddy field in Niigata, Japan. The pods, and seeds were harvested at 7-day intervals until maturity, and the subunit compositions of seed storage proteins were analyzed by SDS-PAGE. The β-subunit of β-conglycinin could scarcely be detected in the non-nodulated isoline, T201, at any period throughout seed development, although it was a major component in T202. The accumulation of α′- and α-subunits of β-conglycinin, together with the acidic and basic subunits of glycinin, appeared about one week later in seeds of T201 than in those of T202, perhaps due to a shortage of nitrogen and growth retardation. Northern hybridization could not detect the β-subunit mRNA in immature T201 seeds, while it was pronounced in T202. These results indicate that the suppression of the β-subunit in the non-nodulating isoline T201 is regulated at the level of mRNA accumulation. The α′(α)-subunit mRNAs were actively expressed in both isolines. Total nitrogen concentration was consistently lower in T201 than T202. No significant difference was observed in either the free amino acid or ureide concentrations in seeds, although the concentration of sucrose was considerably lower in T201 seeds and pods compared with T202. This result indicates the possibility that β-subunit accunmlation was regulated not only directly by total nitrogen concentration but also by carbohydrate concentrations. Nitrogen regulation of storage protein subunit levels of soybean seed were evaluated using T201 and T202. Greenhouse-grown plants were subjected to different levels and timing of nitrate treatments. The culture solution (2, 5 or 10 mM NO₃–was supplied from flowering, 42 days after planting (DAP), until maturation (137 DAP), or switched from 2 to 10 mM, or from 10 to 2 mM at 61 DAP. With a continuous 2 mM NO₃–treatment, seed dry weight and N concentration of the T201 plants were significantly lower than those in the T202 plants due to the lack of N₂ fixation by the non nodulated T201 plants. However, when adequate NO₃ was supplied, N concentration and dry weight were similar in T201 and T202 seeds. When 5 mM NO₃ was supplied, the subunit proportion of the seed storage protein was similar in non-nodulating and nodulating isolines. On the other hand, when plants received a low level of NO₃ (2 mM), the β-conglycinin proportion was lower in T201 than in T2O2. Furthermore, in the nodulating T202 plants treated with 10 mM NO₃–the proportion of β-conglycinin increased markedly. The results indicate that non-nodulated T201 has a normal, non-defective, β-subunit gene and that limited N availability decreases accumulation of β-conglycinin, whereas high N availability increases the proportion of β-conglycinin in soybean seeds, irrespective of whether N was derived from N₂ fixation or from NO₃ absorption.     

A new rapid method of glycolate test by diethyl ether extraction,which is applicable to a small amount of bacterial cells of less than one milligram

The glycolate test is a method to discriminate N-acyl groups of muramyl residue in peptidoglycan of bacterial cell walls by color reaction without purification of the cell walls. The glycolyl residue presents red purple color by heating with 0.02% 2,7-dihydroxynaphthalene (DON) dissolved in concentrated H(2)SO(4). Instead of the previous column methods for quantitative analysis, a qualitative method by solvent works was developed to simplify and to miniaturize the analysis. In this method, solvents played two roles, removal of interfering materials and extraction of glycolic acid from the cell hydrolysates. Of several solvent systems tested, diethyl ether was studied in detail on such properties as the efficiency of glycolic acid extraction under several conditions, the ability of removing various interfering compounds, and the advantage on evaporation procedure of the solvent from extracts. DON reaction of the second diethyl ether extract from cell hydrolysate of "Micromonospora nigra" JCM 3328 showed a clear red purple color of a strong absorbance at 530 nm, which is the same as that of authentic glycolic acid. The solvent method was applied to 20 strains of typical actinomycete species whose acyl types have already been known (Uchida and Seino, 1997). All glycolate test positive strains showed the clear red purple color mentioned above, whereas acetyl type strains revealed no apparent color by the same procedures. Additional experiments indicated that the glycolate test could be determined with less than 1 mg of actinomycete cells by using a smaller amount of DON reagent and ordinary polypropylene tubes. The new method was discussed for advantages in the identification of actinomycetes and for possible applications to other fields.     

Development of adventive embryos in cauline leaves of Sargassum macrocarpum (Fucales, Phaeophyta)

Spontaneous formation and development of adventive embryos were observed in cauline leaves of Sargassum macrocarpum in laboratory culture. Semi-spherical swellings, which were 200–250 μm in diameter, arose from the surface of cauline leaves of thalli cultured for 4 months from zygotic embryos. Swellings became cylindrical protuberances and grew into ‘daughter’ thalli with one or two small cauline leaves. These thalli detached from ‘mother’ thalli and attached to the surface of culture vessels by rhizoids produced within 1 week after detachment. Each daughter thallus developed into an individual thallus exhibiting the same morphological processes as zygotic embryos.     

Yeast Artificial Chromosome Clones of Rice Chromosome 2 Ordered Using DNA Markers

Yeast artificial chromosome (YAC) clones were ordered for thephysical mapping of rice chromosome 2, the last of the 12 ricechromosomes to be assigned YACs by the Rice Genome ResearchProgram. A total of 128 restriction fragment length polymorphismmarkers and 4 sequence-tagged site (STS) markers located onour high-density genetic map were used for YAC clone landing.By colony/Southern hybridization and polymerase chain reactionscreening, a total of 239 individual YACs were selected fromour YAC library of 6934 clones covering six genome equivalents.The YACs located on the corresponding marker positions in thelinkage map formed 43 contigs and islands and were estimatedto encompass about 50% of the length of rice chromosome 2.     

Mapping of Sequence-Tagged Sites in Rice by Single Strand Conformation Polymorphism

The conditions for efficient single-strand conformation polymorphism(SSCP) detection were examined for its application to mappingof DNA regions in the rice genome. Temperature for electrophoresisand glycerol concentrations in gel affected SSCP patterns significantly.The optimal detection conditions for SSCP also depends on thenucleotide sequences of fragments analyzed. Fragments over 300bp show complicated patterns depending on their nucleotide sequencesand were not suitable for SSCP analysis. Seventy primer pairswere designed from the sequence data available to amplify DNAregions as sequence tagged sites (STSs), and 39 of these STSswere found to generate SSCP between japonica rice (Nipponbare)and indica rice (Kasalath) in at least one of the experimentalconditions. The maps of DNA fragments amplified from 186 F₂-plantDNAs with 17 primer pairs were successfully determined. Thisdirect mapping method of the amplified DNA fragments with PCRis simple and quite sensitive, and can be used to set markersin the gap regions of a genetic linkage map.     

Fluorescence spectrophotometric analysis of melanins in the house mouse

We extracted the yellow melanin (phaeomelanin), black melanin (eumelanin), and mixed type of melanin from dorsal hair of dominant yellow (A ^y /a), non-agouti (a/a), and agouti (A/A) mice, respectively. Spectrophotometric and fluorescence spectrophotometric analysis demonstrated that the yellow melanin was qualitatively distinct from the black melanin and that the agouti hair contained both types of pigment.This work was supported by Grant 244004 from the Ministry of Education. Part of this work was presented at the X International Pigment Cell Conference.     

The glycerophosphoryl diester phosphodiesterase-like proteins SHV3 and its homologs play important roles in cell wall organization

Despite the importance of extracellular events in cell wall organization and biogenesis, the mechanisms and related factors are largely unknown. We isolated an allele of the shaven3 (shv3) mutant of Arabidopsis thaliana, which exhibits ruptured root hair cells during tip growth. SHV3 encodes a novel protein with two tandemly repeated glycerophosphoryl diester phosphodiesterase-like domains and a glycosylphosphatidylinositol anchor, and several of its paralogs are found in Arabidopsis. Here, we report the detailed characterization of mutants of SHV3 and one of its paralogs, SVL1. The shv3 and svl1 double mutant exhibited additional defects, including swollen guard cells, aberrant expansion of the hypocotyl epidermis and ectopic lignin deposits, suggesting decreased rigidity of the cell wall. Fourier-transform infrared spectroscopy and measurement of the cell wall components indicated an altered cellulose content and pectin modification with cross-linking in the double mutant. Furthermore, we found that the ruptured root hair phenotype of shv3 was suppressed by increasing the amount of borate, which is supposed to be involved in pectic polysaccharide cross-linking, in the medium. These findings indicate that SHV3 and its paralogs are novel important factors involved in primary cell wall organization.     

The autoregulation of nodulation mechanism is related to leaf development

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.     

Taxonomic distribution of <Emphasis Type="Italic">Streptomyces</Emphasis> species capable of producing bioactive compounds among strains preserved at NITE/NBRC

The taxonomic distribution of Streptomyces species capable of producing bioactive compounds was investigated. Nine hundred and six strains were tested for the following four biological activities: antimicrobial, anti-tyrosinase, antioxidant, and hemolytic. Approximately 30% of strains tested showed antimicrobial activities, except for anti-Escherichia coli activity, which was present in only a few strains, while the rates of positivity for the anti-tyrosinase, antioxidant, and hemolytic activities were much lower. The distribution of Streptomyces strains capable of producing bioactive compounds was analyzed by the taxonomy based on 16S rRNA gene sequences. Moreover, the strains of Streptomyces hygroscopicus tested were divided into two clades in the phylogenetic tree, and all of the strains belonging to one clade showed antibacterial and antifungal activities. For detection of polyenes, the UV-visible spectra of metabolic extracts in the strains showing antifungal activities were measured. It was suggested that Streptomyces strains produce universal active compounds under different growth conditions. Further information on the relationship between the microbial taxonomy and the bioactive compounds produced would be useful for the utilization of industrial microorganisms.