DAF yields a cloned marker linked to the soybean (Glycine max) supernodulation nts-1 locus

We report a further characterization of the genomic region containing the soybean supernodulation gene NTS-1. We performed a search for new markers linked to NTS-1 by combining DNA amplification fingerprinting (DAF) and bulked segregant analysis (BSA). The search resulted in one cloned polymorphism (B44-456) linked in trans, 8.5cM from the locus. Southern hybridization showed duplication of the B44-456 sequence in the soybean genome. Additionally, a DNA database search revealed one Arabidopsis thaliana genomic clone from chromosome I possessing 62% homology to the B44-456 marker. A relatively low number of polymorphisms were identified by several PCR marker technologies for this soybean genomic region, providing an additional support for its highly conserved and/or duplicated organization.

The surface accessibility of the glycine receptor M2-M3 loop is increased in the channel open state

Mutations in the extracellular M2-M3 loop of the glycine receptor (GlyR) alpha1 subunit have been shown previously to affect channel gating. In this study, the substituted cysteine accessibility method was used to investigate whether a structural rearrangement of the M2-M3 loop accompanies GlyR activation. All residues from R271C to V277C were covalently modified by both positively charged methanethiosulfonate ethyltrimethylammonium (MTSET) and negatively charged methanethiosulfonate ethylsulfonate (MTSES), implying that these residues form an irregular surface loop. The MTSET modification rate of all residues from R271C to K276C was faster in the glycine-bound state than in the unliganded state. MTSES modification of A272C, L274C, and V277C was also faster in the glycine-bound state. These results demonstrate that the surface accessibility of the M2-M3 loop is increased as the channel transitions from the closed to the open state, implying that either the loop itself or an overlying domain moves during channel activation.

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha (1) homomeric and alpha (1)beta heteromeric glycine receptors (GlyRs), At low (0.03 muM) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (greater than or equal to0.03 muM) concentrations it irreversibly activated both alpha (1) homomeric and alpha (1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin, Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the beta-amino acid agonist, taurine

The beta -amino acid, taurine, is a full agonist of the human glycine receptor al subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta -amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta -amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta -amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alpha beta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alpha beta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alpha beta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alpha beta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin las an allosterically acting 'competitive' antagonist) binds to this residue.

Fast neutron mutagenesis of soybean (Glycine soja L.) produces a supernodulating mutant containing a large deletion in linkage group H

We describe for the first time the application of fast neutron mutagenesis to the genetic dissection of root nodulation in legumes. We demonstrate the utility of chromosomal deletion mutations through production of a soybean supernodulation mutant FN37 that lacks the internal autoregulation of nodulation mechanism. After inoculation with microsymbiont Bradyrhizobium japonicum, FN37 forms at least 10 times more nodules than the wild type G. soja parent and has a phenotype identical to that of chemically induced allelic mutants nts382 and nts1007 (NTS-1 locus). Reciprocal grafting of shoots and roots confirmed systemic shoot control of the FN37 nodulation phenotype. RFLP/PCR marker pUTG132a and AFLP marker UQC-IS1 which are tightly linked to NTS-1 allowed the isolation of BAC contigs delineating both ends of the deletion. The genetic/physical distance ratio in the NTS-1 region is 279 kb/cM. The deletion is estimated to be about 460 kb based on the absence of markers and bacterial artificial chromosomes (BAC) ends as well as genetic and physical mapping. Deletion break points were determined physically and placed within flanking BAC contigs.

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor ( GlyR) alpha(1) subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha(1) subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His(107) from one subunit and His(109) from an adjacent subunit. This was tested by co-expressing alpha(1) subunits containing the H107A mutation with alpha(1) subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha(1) homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha(1) subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha(1)beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha(1) homomers. The binding of zinc at the interface between adjacent alpha(1) subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.

Seleção de variedades de soja (Glycine Max (L.) Merril ): I. Analises Bromatológicas

O presente trabalho foi realizado com o fito de estabelecer uma seleção prévia entre 32 variedaeds de soja (Glycine max. (L.) Merril), com base nas análises bromatológicas das sementes. Foram determinados teores de umidade (U), cinzas (RM), proteína bruta (PB), gordura bruta ou extrato etéreo (EE), fibra bruta (F) e extrativo não nitrogenado (ENN). As análises da variância permitem tirar as seguintes conclusões: 1) Entre as variedades classificadas no grupo forrageiro e comestível, tem-se: Mandarin 8a (40,16% PB), Bicolor de Calai (39,64% PB) e Aliança (38,70% PB). 2) De acordo com a classificação estabelecida, para o grupo produtor de óleo, as variedades Lee, Hood, Lincoln (Blanco) Improved Pelican (2), Lincoln (Morado) e Improved Pelican (1), se destacam com 21,76, 21,65, 21,62, 21,35, 21,13 e 21,04% de EE respectivamente. 3) As variedades que apresentaram menor percentagem de fibra são: Hernónjn.0 107, Hood, Mogiana, Hill e Selection n.° 135, com 9,59; 9,62; 9,68; 10,02 e 10,12% F respectivamente. 4) Encontrou-se uma correlação positiva entre o teor de proteína bruta e o de cinzas.

Efeitos de doses crescentes de nitrogênio e da inoculação de bactérias fixadoras de nitrogênio atmosférico, na cultura da soja (Glycine max, Merril)

Para estudar os efeitos de doses crescentes de nitrogênio, na presença e ausencia de Rhizobium, na cultura da soja, foram instalados ensaios em dois solos arenosos pobres em mate ria orgânica dos municipios de Herculandia e de Regente Feijó. No ensaio de Herculandia constatou-se efeitos de Rhizobium, enquanto no de Regente Feijo houve efeitos linear e cúbico das doses de nitrogênio, nao sendo observado efeito de inoculação. Em ambos os casos notaram-se cloroses nos tratamentos sem nitrogênio, todavia no ensaio de Regente Feijo foi temporária enquanto no de Herculândia a clorose persistiu ate o fim do ciclo nas parcelas sem inoculaçao e sem nitrogênio.

Influência do tamanho sobre a conservação, germinação e vigor de sementes de soja (Glycine max (L.) Merr.)

O presente trabalho foi desenvolvido no Laboratório de Sementes do Departamento de Agricultura e Horticultura da E. S. A. «LUIZ DE QUEIROZ», constando de testes de germinação e de vigor (envelhecimento prococe) realizados em seis épocas bimestrais, com sementes de três cultivares de soja (Santa Rosa, IAC-2 e Viçoja), de três tamanhos (Peneira 17-grandes, Peneira 16-médias e Peneira 15 pequenas), conservadas em dois ambientes diferentes: em câmara seca e em ambiente não controlado. A análise dos dados obtidos permitiu conclusões como: a germinação variou entre cultivares e foi diretamente proporcional ao tamanho das sementes; houve decréscimo da germinação no decorrer do período considerado, sendo mais acentuado para sementes pequenas; o vigor foi maior para sementes pequenas; as sementes conservadas em câmara seca superaram as demais em todas as circunstâncias.

Efeitos do tratamento de sementes de algodão (Gossypium hirsutum L.), arroz (Oryza sativa L.) e soja [Glycine max (L.) Merril] com alguns fungicidas não mercuriais

Esta pesquisa foi conduzida com o objetivo de estudar o comportamento de sementes de arroz, algodão e soja, quando tratadas com íungicidas não mercuriais, através de testes de germinação e de vigor. Foram utilizados os seguintes produtos: Arasan (Thiram 75% i.a.), Panoctine (Guazatine 75% i.a.) e Terracoat (23,2% PCNB + 5,8% Terrazole). A dosagem do 1º produto foi de 100 g/100 kg de sementes enquanto que, para os outros dois, foram de 200, 400 e 600 cm³/100 kg. Nas condições do experimento, as três dosagens de Panoctine foram tóxicas para sementes de arroz e a dose mais alta prejudicou o vigor de soja e de algodão. Por outro lado Arasan e Terracoat não prejudicaram a germinação e vigor de arroz e de soja e apresentarm efeitos benéficos sobre o vigor de sementes de algodão.

Deficiências de macronutrientes na sota (Glycine max L., Merril, var. IAC-2)

Sintomas de deficiência de macronutrientes foram induzidos na soja, var. IAC-2. Foi verificado o efeito da omissão de N, R, K, Ca, Mg e S no crescimento, produção e composição mineral das folhas.

Destacadas de soja (Glycine max (L.) merril absorção de cálcio e fósforo por raízes var. IAC-2)

A absorção do cálcio e do fósforo por raízes destacadas da soja var. IAC-2 foi estudada com ajuda de traçadores. Foram verificados os efeitos da concentração iônica externa do tempo, do pH, da temperatura, da aeração e de venenos respiratórios. Os dados sugerem que a absorção do cálcio tenha se dado passivamente, sendo ativa a do fósforo. A absorção cresceu com o pH e a temperatura. Os valores das constantes de Michaelis encontrados concordam com os da literatura.

Nutrição mineral de leguminosas tropicais: I. Absorção dos macronutrientes pela centrosema (Centrosema pubescens Benth.), siratro (Macroptilium atropurpureum cv. 'Siratro') e soja perene (Glycine wightii Willd.) cultivadas em condições de campo

As leguminosas tropicais constituem opção como fonte de alimento para o gado, e como revestimento para proteger taludes e aterros contra a erosão. Com a finalidade de se conhecer o habito alimentar das leguminosas tropicais centrosema (Centrosema pubescens Benth.), siratro (Macroptilium atropurpureum cv. 'Siratro') e soja perene (Glycine wightii Willd.) instalou-se o presente ensaio no campo, em Piracicaba, SP, visando obter dados para estabelecer equações para o crescimento, para a concentração e extração dos macronutrientes (N, P, K, Ca, Mg e S), e para a matéria seca digestível das folhas e caules, "in vivo", no bovino, em função da idade da planta. Da apreciação geral dos resultados obtidos, verificou-se diferenças entre as três espécies quanto aos fatores estudados, tais como: a. A máxima produção de matéria seca é da soja perene, seguida pela centrosema e siratro, ocorrendo entre 134 e 140 dias. A maior velocidade no crescimento ocorre entre 83 e 91 dias, para as três espécies. b. O caule apresenta as maiores concentrações de potássio; as folhas, todos os outros macronutrientes. Existem diferenças entre as espécies quanto aos teores de nitrogênio, potássio, cálcio, magnesio e enxofre. O enxofre é absorvido e acumulado continuamente pela centrosema. c. A maior velocidade de acumulação de nitrogênio, fósforo, magnesio e enxofre pelas folhas ocorre entre 80 e 95 dias para as três espécies; de potássio aos 83 e 90 dias para a centrosema e soja perene, respectivamente; de cálcio, aos 116 e 135 dias para a soja perene e siratro. A quantidade maxima e a idade em que ocorre são: d. Existem diferenças na digestibilidade da matéria seca das partes entre as leguminosas. Essa digestibilidade aumenta com a idade da centrosema e do siratro; na soja perene, a digestibilidade mínima ocorre aos 74 dias (39,2%) para o caule e 88 dias (30,2%) para a folha. A matéria seca digestível das folhas de centrosema é 29,8% e das do siratro, 20%, aos 42 dias de idade; aos 147 dias, a da soja perene e 58,8%, seguida do siratro, 56,5%, e da centrosema, 44,0%.