RESUMEN
delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower.
Asunto(s)
Arabidopsis/genética , Ciclopentanos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/genética , Secuencia de Aminoácidos , ADN Bacteriano/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutación , Oxidorreductasas/química , Oxilipinas , Fenotipo , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
In addition to OPR1 and OPR2, two isoenzymes of 12-oxophytodienoate reductase, a third isoform (OPR3) has recently been identified in Arabidopsis thaliana (L.) Heynh. The expression of the OPR3 gene is induced not only by a variety of stimuli, such as touch, wind, wounding, UV-light and application of detergent, but also by brassinosteroids. The three enzymes were expressed in a functional form in Escherichia coli, and OPR2 was additionally expressed in insect cell cultures and overexpressed in A. thaliana. Substrate conversion was analyzed using a stereospecific assay. The results show that OPR3 effectively converts the natural (9S,13S)-12-oxophytodienoic acid [Km = 35 microM, Vmax 53.7 nkat (mg protein)-1] to the corresponding 3-2(2'(Z)-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) stereoisomer while OPR1 and OPR2 convert (9S,13S)-12-oxophytodienoic acid with greatly reduced efficiency compared to OPR3. Thus, OPR3 is the isoenzyme relevant for jasmonate biosynthesis.
Asunto(s)
Arabidopsis/enzimología , Ciclopentanos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/metabolismo , Reguladores del Crecimiento de las Plantas/biosíntesis , Arabidopsis/metabolismo , Immunoblotting , Isoenzimas/metabolismo , Oxilipinas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Plants respond to situations requiring the initiation of inducible defence reactions with a complex array of signalling events that ultimately result in the activation of sets of defence genes. Among the chemical signals involved in the induction of defence reactions are cyclic oxylipins derived from C18- or C16-unsaturated fatty acids, the octadecanoids and the hexadecanoids. Key to understanding octadecanoid biology are the C18-metabolite 12-oxophytodienoic acid (OPDA) and the C12-compound jasmonic acid which is biosynthetically derived from 12-oxophytodienoic acid. Different octadecanoids likely have different biological functions. The bouquet of signalling compounds, rather than any single compound, is probably decisive for the biological response that results. This means that the processes regulating the pool sizes of different octadecanoids and their distribution within the plant are key to understanding octadecanoid biology. Recent results, including the cloning of several enzymes of the octadecanoid biosynthetic pathway, have provided first insights into these processes and into how the octadecanoid system is linked to other defence-related signalling pathways of the plant cell.
Asunto(s)
Ácidos Grasos Insaturados/fisiología , Enfermedades de las Plantas , Transducción de Señal , Ciclopentanos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Modelos Químicos , OxilipinasRESUMEN
Monoclonal antibodies were raised and selected against recombinant Plantago major PmSUC2 sucrose carrier protein. Epitopes of two monoclonal antibodies (PS2-1A2 and PS2-4D4) were mapped using N-terminally truncated PmSUC2 proteins and a lambda library displaying random PmSUC2 peptides. PS2-1A2 recognizes an octapeptide close to the N-terminus of PmSUC2, PS2-4D4 binds to a decapeptide at the very C-terminus. Analyses of antibody binding to yeast protoplasts with functionally active, tagged PmSUC2 protein revealed that both epitopes are located in cytoplasmic domains of PmSUC2. These results support a model for plant sucrose transporters containing 12 transmembrane helices with the N-terminus and the C-terminus on the cytoplasmic side of the plasma membrane.
Asunto(s)
Proteínas Portadoras/ultraestructura , Proteínas de Transporte de Membrana , Proteínas de Plantas/ultraestructura , Sacarosa/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Bacteriófago lambda , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Membrana Celular/química , Polaridad Celular , Citoplasma/química , Mapeo Epitopo , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Plantago , Plantas Medicinales , Conformación Proteica , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
The genes of two closely related 12-oxophytodienoic acid reductases (EC 1.3.1.42), OPR1 and OPR2, were identified on a 7079-bp-long genomic fragment from Arabidopsis thaliana (L.) Heynh. The organization of these two genes was determined and putative cis elements were identified. Promoter-beta-glucuronidase (GUS) fusions expressed in transgenic Arabidopsis thaliana and Nicotiana tabacum L. plants revealed differences in OPR-promoter-driven GUS expression in flowers. While the OPR1 promoter directed GUS expression in young seeds, the OPR2 promoter directed pollen-specific expression. Both OPR1 and OPR2, were predominantly expressed in roots. Stress treatments, like local and systemic wounding, UV-C illumination and coldness, resulted in transient changes in steady-state OPR mRNA levels, but no concurrent changes in polypeptide level or enzyme activity were detected.