Lupinus species differ in their requirements for iron

The effect of iron supply on the growth and nodulation ofLupinus angustifolius L. (Gungurru),Lupinus luteus L. (R-1171) andLupinus pilosus Murr. (P20957) was studied in acid solutions. Plants of the three species were grown together in the same solution and inoculated withBradyrhizobium (Lupinus) WU 425. Plants were then grown with or without applied NH₄NO₃. The lupin species differed greatly in their sensitivity to low iron concentrations in solution withL. pilosus being most tolerant andL. luteus most sensitive.L. pilosus had the highest iron concentration in tissues and had a higher ratio of iron concentration in the youngest fully expanded leaf blades (YEB) to that in roots than the other two species.L. luteus had higher iron concentrations in roots but lower iron concentration in YEB and shoots than didL. angustifolius. The requirements of internal iron for the maximal chlorophyll synthesis in YEB were 65 μg g^-1 forL. angustifolius andL. luteus, and 52 μg g^-1 forL. pilosus. In contrast to effects on growth, the three species had similar external iron requirements for nodule formation in roots and for maximal nitrogen concentrations in shoots. The results indicate that iron tolerant lupin species require lower internal and external iron supply and have a greater ability than sensitive species to translocate iron from roots to shoots.     

Physical Mapping of Recombinant Plasmids Containing Broad Bean Chloroplast Ribosomal RNA Genes

Two BamHl fragments containing broad bean chloroplast rRNA genes were cloned using the bacterial plasmid pBR322 as a vector and Escherichia coli HB101 as host bacterial. Physical maps of the two cloned ct DNA BamHI fragments containing rRNA genes were constructed by cleavage with several restriction endonucleases and Southern blot hybridization with E. coli 16S-23S rRNAs. Recombinant plasmids pVFBI6 and pVFB32 contain a 16S rRNA sequence on the 4.70 kb BamHl fragment, a 23S rRNA sequence and 4.5S/5S rRNA sequences on the 5.65 kb BamHl fragment, respectively.     

Chromosomal Mapping and Molecular Characterization of Ribosomal RNA Genes in Lebias fasciata (Teleostei, Cyprinodontidae)

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.     

Variability of globulin composition in cultivars and individually tested seeds of yellow lupin (Lupinus luteus L.)

A study on globulins, major storage proteins in yellow lupin seeds, called conglutins, was conducted using SDS polyacrylamide gel electrophoresis. In this paper, an extensive and not yet published list of yellow lupin conglutins is presented. The patterns of subunits of major conglutins in seeds of three yellow lupin cultivars were similar to each other, varying only in the level of some polypeptides. Investigations of seeds of cultivar Parys showed considerable quantitative differences in major subunits. Some minor subunits occurred only in some seeds and were absent in the others. Great differences were shown between single individuals in the amount of subunits of conglutin which is of the most nutritional value due to high content of methionine.     

Ribosomal RNA genes inQuercus spp. (Fagaceae)

The taxonomy of the genusQuercus is still unclear. In order to elucidate the taxonomy of Mediterranean oaks we have analyzed ribosomal RNA genes ofQuercus cerris, Q. coccifera, Q. trojana, Q. ilex, Q. suber, andQ. macrolepis by means of Southern blot hybridization. Oak nuclear DNA was extracted from root tips of 300 acorns and from catkins of single plants. EcoRI and BamHI restriction endonucleases were used. DNA electrophoresis and rRNA/DNA hybridization were performed usingVicia faba rRNA 18 S and 25 S as probes. The rRNA genes of all the species studied have an identical restriction mapping in the 18 S and 25 S regions, while differences in length are present in the intergenic regions.Q. cerris possesses at least four types of genes of 12.1, 11.5, 8.5, and 8.3 kb;Q. coccifera at least three types of 12.4, 10.4, and 10.1 kb;Q. trojana possesses the same rRNA genes asQ. cerris plus another gene type 12.0 kb long, with EcoRI and BamHI restriction sites in the intergenic spacer;Q. ilex at least three types of 12.4, 10.85, and 9.5 kb;Q. suber at least five types of 11.5, 11.0, 8.6, 8.5, and 8.3 kb;Q. macrolepis, finally, at least seven types of 11.5, 11.0, 10.2, 8.6, 8.5, 8.3, and 8.15 kb.Q. coccifera andQ. ilex rDNA appears quite different respect to other species examined, while high similarity seems to exist betweenQ. cerris, Q. trojana, Q. suber, andQ. macrolepis. These results are in agreement with the taxonomic model proposed bySchwarz for the genusQuercus.     

Species-specific EcoRI repetitive elements of at least 16 kb in length are present in Lupinus luteus

Summary A genomic DNA library of Lupinus luteus cv. Ventus was constructed in the phage vector EMBL3 using Mb oI-digested DNA. Screening with a 1070 bp labelled repetitive unit from L. luteus yielded several DNA clones. The repetitive family is composed of elements whose length is at least 16 kb. The average copy number of the cloned fragments is 5.0 × 10⁴ per haploid genome and constitutes approximately 3% of the total L. luteus genome. The homologous repeats were found in all ten cultivars of L. luteus tested but were not detected in two cultivars each of the closely related species Lupinus albus and Lupinus angustifolius. The EcoRI family fragments could thus be considered as species-specific DNA elements. These fragments may be useful as molecular markers in the genetic manipulation of L. luteus.     

Ribosomal RNA genes in biotypes ofScilla peruviana (Liliaceae)

Scilla peruviana biotypes have different chromosome numbers due to changes in the nucleolar chromosomes and polyploidy. We have examined two diploid (2n = 15 and 2n = 16) and two tetraploid biotypes (2n = 28 and 2n = 32). From the results of rRNA/DNA filter hybridizations it appears that rDNA percentages of the diploid biotypes are, approximately, 2.2-fold higher than those of the tetraploid biotypes. To examine the rRNA gene structure we have utilizedSouthern blot hybridization after DNA digestions with three restriction enzymes: Eco RI, Hind III and Bam HI. From the band analysis of both single and double digestions it has been possible to reveal the presence, in the diploid biotypes, of three gene types, heterogeneous both for length and for nucleotide sequences in the external spacer. The three rRNA genes are 12 600, 12 700, and 12 800 base pairs long and they have a different position of the Hind III sites in the external spacer. On the other hand, a single gene type of 12 600 base pairs, identical to the first type of the diploid biotypes, surprisingly exists in the tetraploid biotypes. Considerations on the rRNA gene regulation and evolution are made.     

Sugars as a metabolic regulator of storage protein mobilization in germinating seeds of yellow lupine (Lupinus luteus L.)

The intensity of protein reserve activation in yellow lupine (Lupinus luteus L.) organs cultured in vitro in the presence of saccharose and without sugar in the medium was studied. Isolated embryo axes, excised cotyledons and seeds deprived of their testae were grown on Heller medium: a) with 60 mM saccharose (+S), b) without sugar (−S) and c) for 72 hours without saccharose + for 24 hours in the presence of saccharose (−S→+S). Using nitroanilide substrates, exo- and endopeptidase proteolytic activity was investigated in enzymatic extracts. Proteolytic activity was examined in organs isolated from swollen seeds and also in organs cultured for 24, 48, 72 and 96 hours. The proteolytic activity was higher in organs cultured on medium without saccharose. Stimulation of the proteolytic activity on the first day of culture was not significant, but intensified in the successive days of culture. The organ subcultured onto a medium with saccharose (−S→+S) caused a significant limitation of proteolytic activity, even to a markedly lower level in comparison to that activity level in the material continuously supplemented with saccharose. Observations of ultrastructure in Transmission Electron Microscopy revealed increased protein body degradation in the absence of saccharose in the medium and an increased degree of cell vacuolization, which may be indicative of intensifying autophagic processes under conditions of carbohydrate deficit.     

pH above 6.0 reduces nodulation in Lupinus species

Lupinus angustifolius and Lupinus pilosus differ substantially in root growth in response to high solution pH with L. angustifolius showing much greater sensitivity to pH above 6. This study examined the effect of pH above 6 on nodulation of these two species in buffered solution. Shoot weight and root weight and length in L. pilosus was not significantly affected by pH, whereas the growth of shoots and roots of L. angustifolius was markedly impaired by increasing pH. Total root length, the number of lateral roots, and the length of individual lateral roots were greatly decreased, resulting in decreased uptake of iron and phosphorus. In addition, L. angustifolius had a higher internal requirement for iron than L. pilosus. A solution pH above 6 decreased the number of nodule initials and nodules similarly in both species but decreased nodule mass much more in L. angustifolius. The effect of high pH on nodule formation occurred prior to that on host shoot growth. High pH also decreased nitrogen concentration and content in both species but to a greater extent for L. angustifolius. The results suggested that pH above 6 has a specific effect in the impairment of nodulation in lupins.     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI propidium iodide - SLO streptolysin O     

苜蓿核糖体基因物理定位及染色体荧光分带

利用核糖体基因为探针对,二倍体和四倍体苜蓿(Medicago sativa)进行原位杂交,结果表明,45s在四倍体、二倍体种中总是以单位点位于核仁组织区,5s则有2～3个位点;以二倍体种的基因组DNA为探针的原位杂交表明,蓝花苜蓿(M.coerulea)和黄花苜蓿(M.falcata)均能与四倍体染色体进行杂交,仅杂交信号强弱的染色体数目有差别;荧光染料DAPI使苜蓿的染色体显示带纹,蓝花苜蓿的DAPI带与C-带基本一致.文章对四倍体苜蓿的可能来源进行了讨论.     

Cluster-root production and organic anion exudation in a group of old-world lupins and a new-world lupin

We examined the capacity of several Old-World lupin species (Lupinus luteus L., L. hispanicus Boiss. et Reuter and L. angustifolius L.) and one species of a New-World lupin (L. mutabilis Sweet) to form cluster roots under a range of conditions in solution culture. The effect of the synthetic auxin, IBA (indole-3-butyric acid), on cluster-root development in L. luteus and L. albus L. provided with an adequate phosphorus (P) supply was also investigated. In addition, the effect of a high nitrate-N (NO₃-N) supply on the efflux of citrate and malate from roots of L. angustifolius was examined to determine if specific regions of the root system exuded these organic anions. When P-deficient, L. hispanicus, L. luteus and L. mutabilis formed cluster roots that secreted organic anions. Citrate was generally the dominant organic anion exuded, although succinate was also exuded in large quantities from L. luteus. Citrate efflux by L. hispanicus and L. luteus was at least comparable to that reported for P-deficient L. albus[up to 1.092 nmol g^–1 fresh weight (FW) s^–1], but was over an order of magnitude lower in L. mutabilis (0.036 nmol g^–1 FW s^–1). Citrate and malate were not detected in significant amounts from either the lateral roots or the root tips of any species grown under P-sufficient or -deficient conditions. Citrate efflux from cluster roots of L. luteus showed a diurnal pattern, similar to that reported for L. albus, with maximum efflux during the day, and declining to a minimum before dawn. IBA added to the nutrient solution induced cluster-root formation on both L. albus and L. luteus at concentrations of P that would normally suppress the production of these roots. However, the IBA-induced cluster roots did not exude significant amounts of citrate. Although L. angustifolius did not produce cluster roots when P-deficient, it produced cluster-like root structures that exuded citrate (0.053 nmol g^–1 FW s^–1) when grown at a high nitrate-N (NO₃-N) supply. L. angustifolius did not exude significant citrate or malate from lateral roots or root tips when grown at either high or low NO₃-N supply. Our findings for L. hispanicus and L. luteus are the first reports of cluster-root formation in response to P deficiency for these Old-World species, and for L. mutabilis, it is the first report of cluster roots for a New-World lupin species. These reports indicate that evolutionary and biogeographical aspects of cluster-root formation in the genus Lupinus need to be revised. Furthermore, investigation is warranted to determine the capacity of species of the large group of New-World lupins to form cluster roots in soils of their native habitats.     

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.     

Ribosomal RNA integrity and rate of seed germination

The integrity of ribosomal RNA (the percentage of complete, un-nicked molecules) in seeds was studied by electrophoresis under denaturing conditions. Two batches of carrot seed, harvested at different stages of maturity, and four batches ofNicotiana seed stored for various times were used. Within each species, there was a correlation between the integrity of the rRNA of the dry seed and the rate of germination of that seed. In carrot seed, there was extensive degradation of existing rRNA in both the embryo and endosperm during the first two days of imbibition.     

The role of copper in nitrogen fixation in Lupinus luteus L.

The effect of copper nutrition on symbiotic N₂ fixation in Lupinus luteus L. was studied. Copper nutrition increased the yield, total nitrogen content and dry weight of nodules. The control plants did not produce pods. Copper deficiency limited iron uptake and its translocation to the nodules. Nodules of copper-deficient plants contained less than half the leghaemoglobin concentration of copper-adequate plants and about one third the polyphenol oxidase activity, tested with catechol as a substrate.     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

The Physical Gene Hsp70 Map on Polytene Chromosomes of Anopheles darlingi from the Brazilian Amazon

In situ hybridization was used to determine the physical location of the Hsp70 genes in salivary polytene chromosomes of Anopheles darlingi from Manaus and Macapá, Brazil, and to assess the usefulness of the Hsp70 locus as a genetic marker in A. darlingi populations. In both populations, the double markings corresponding to the Hsp70-12A and Hsp70-14A genes were located on the right arm of chromosome 2. The Hsp70 locus was considered to be an excellent marker for studying chromosomal evolution and relationships among A. darlingi populations.     

Mapping QTL for Grain Yield and Plant Traits in a Tropical Maize Population

The vast majority of reported QTL mapping for maize (Zea mays L.) traits are from temperate germplasm and, also, QTL by environment interaction (QTL × E) has not been thoroughly evaluated and analyzed in most of these papers. The maize growing areas in tropical regions are more prone to environmental variability than in temperate areas, and, therefore, genotype by environment interaction is of great concern for maize breeders. The objectives of this study were to map QTL and to test their interaction with environments for several traits in a tropical maize population. Two-hundred and fifty-six F_2:3 families evaluated in five environments, a genetic map with 139 microsatellites markers, and the multiple-environment joint analysis (mCIM) were used to map QTL and to test QTL × E interaction. Sixteen, eight, six, six, nine, and two QTL were mapped for grain yield, ears per plant, plant lodging, plant height, ear height, and number of leaves, respectively. Most of these QTL interacted significantly with environments, most of them displayed overdominance for all traits, and genetic correlated traits had a low number of QTL mapped in the same genomic regions. Few of the QTL mapped had already been reported in both temperate and tropical germplasm. The low number of stable QTL across environments imposes additional challenges to design marker-assisted selection in tropical areas, unless the breeding programs could be directed towards specific target areas.     

Agrobacterium-mediated transformation of Lupinus mutabilis L. using shoot apical explants

A procedure for regenerating plants of Lupinus mutabilis from shoot apices, from which the leaf primordia and initial cell layer(s) of the apical meristem were removed, has been used to generate transgenic plants following Agrobacterium tumefaciens-mediated gene delivery. Transformation competent cells, from which buds developed, were located at the periphery of the apical meristem. Kanamycin resistant plants were obtained which expressed β-glucuronidase activity. Integration of the neomycin phosphotransferase II and β-glucuronidase genes into the genomes of transgenic plants was confirmed by non-radioactive DNA-DNA hybridisation. This is the first report of the generation of transgenic plants in L. mutabilis.