RESUMEN
We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94 Delta rtx::Omega 1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94 Delta rtx::Omega 1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bradyrhizobium/metabolismo , Genes Bacterianos , Complejos Multienzimáticos/genética , Oxidorreductasas/genética , Propanolaminas/metabolismo , Transaminasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Bradyrhizobium/genética , Bradyrhizobium/fisiología , Cromatografía Liquida , Medios de Cultivo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Prueba de Complementación Genética , Espectrometría de Masas , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Mutagénesis , Fijación del Nitrógeno/efectos de los fármacos , Sistemas de Lectura Abierta , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Propanolaminas/farmacología , Análisis de Secuencia de ADN , Transaminasas/química , Transaminasas/metabolismoRESUMEN
Macroptilium atropurpureum is a model legume with a broad symbiont range for nodulation. We have achieved the first in vitro plant regeneration of this species using cv. Siratro. Hypocotyl explants excised from dark-grown seedlings generated slimy, friable calli after three weeks' culture on B5 medium containing 1-2 mg/l kinetin and 0.05 mg/l alpha-naphthaleneacetic acid. This was followed by the generation of green organogenic callus with shoot buds by subculturing the explants to hormone-free B5 medium 20 days after the start of culture. The green organogenic calli with shoot buds were maintained as organogenic callus by subculturing on the same medium, and shoots were elongated on hormone-free B5 medium. Elongated shoots were rooted on half-strength B5 medium. Most regenerated plants were morphologically normal, diploid and fertile, although tetraploid plants appeared at a low frequency (8%).
RESUMEN
Inhibitors of ethylene synthesis or its physiological function enhanced nodulation in Lotus japonicus and Macroptilium atropurpureum. In contrast, the application of 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, reduced the nodule number in these legumes. These results suggest that an ethylene-mediated signaling pathway is involved in the nodulation process even in the determinate nodulators.
Asunto(s)
Etilenos/metabolismo , Fabaceae/metabolismo , Fijación del Nitrógeno , Plantas Medicinales , Rosales/metabolismo , Bradyrhizobium/fisiología , Etilenos/antagonistas & inhibidores , Fabaceae/crecimiento & desarrollo , Fabaceae/fisiología , Rosales/crecimiento & desarrollo , Rosales/fisiologíaRESUMEN
Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.
Asunto(s)
Aminoácidos Cíclicos , Bradyrhizobium/metabolismo , Fabaceae/microbiología , Fijación del Nitrógeno/efectos de los fármacos , Plantas Medicinales , Propanolaminas/metabolismo , Aminoácidos/farmacología , Bradyrhizobium/genética , Bradyrhizobium/fisiología , Etilenos/biosíntesis , Etilenos/farmacología , Plásmidos/genética , Propanolaminas/farmacologíaRESUMEN
We isolated two muskmelon (Cucumis melo) cDNA homologs of the Arabidopsis ethylene receptor genes ETR1 and ERS1 and designated them Cm-ETR1 (C. melo ETR1; accession no. AF054806) and Cm-ERS1 (C. melo ERS1; accession no. AF037368), respectively. Northern analysis revealed that the level of Cm-ERS1 mRNA in the pericarp increased in parallel with the increase in fruit size and then markedly decreased at the end of enlargement. In fully enlarged fruit the level of Cm-ERS1 mRNA was low in all tissues, whereas that of Cm-ETR1 mRNA was very high in the seeds and placenta. During ripening Cm-ERS1 mRNA increased slightly in the pericarp of fruit before the marked increase of Cm-ETR1 mRNA paralleled climacteric ethylene production. These results indicate that both Cm-ETR1 and Cm-ERS1 play specific roles not only in ripening but also in the early development of melon fruit and that they have distinct roles in particular fruit tissues at particular developmental stages.