RESUMO
BACKGROUND AND AIMS: The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. METHODS: The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. KEY RESULTS: Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. CONCLUSIONS: The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Nicotiana/fisiologia , Tubo Polínico/crescimento & desenvolvimento , Ribonucleases/metabolismo , Autoincompatibilidade em Angiospermas/fisiologia , Microscopia Confocal , Microscopia de Fluorescência , Proteínas de Plantas/metabolismo , Polinização , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/ultraestrutura , Vacúolos/enzimologiaRESUMO
Microdomains, or lipid rafts, are transient membrane regions enriched in sphingolipids and sterols that have only recently, but intensively, been studied in plants. In this work, we report a detailed, easy-to-follow, and fast procedure to isolate detergent-resistant membranes (DRMs) from purified plasma membranes (PMs) that was used to obtain DRMs from Phaseolus vulgaris and Nicotiana tabacum leaves and germinating Zea mays embryos. Characterized according to yield, ultrastructure, and sterol composition, these DRM preparations showed similarities to analogous preparations from other eukaryotic cells. Isolation of DRMs from germinating maize embryos reveals the presence of microdomains at very early developmental stages of plants.
Assuntos
Microdomínios da Membrana/química , Nicotiana/química , Phaseolus/química , Fotossíntese , Zea mays/química , Detergentes/química , Microdomínios da Membrana/ultraestrutura , Phaseolus/ultraestrutura , Sementes/química , Sementes/ultraestrutura , Esteróis/análise , Esteróis/química , Nicotiana/ultraestrutura , Zea mays/ultraestruturaRESUMO
Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants.
Assuntos
Proteínas de Helminto/genética , Nicotiana/genética , Nicotiana/parasitologia , Doenças das Plantas/parasitologia , Tylenchoidea/fisiologia , Animais , DNA Complementar/genética , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Contagem de Ovos de Parasitas , Fenótipo , Doenças das Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/parasitologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas/parasitologia , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Nicotiana/ultraestrutura , Tylenchoidea/genética , Tylenchoidea/crescimento & desenvolvimento , Tylenchoidea/patogenicidadeRESUMO
Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.
Assuntos
Ferredoxinas/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Anabaena/genética , Anabaena/metabolismo , Sequência de Bases , DNA de Plantas/genética , Ferredoxinas/deficiência , Ferredoxinas/genética , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Microscopia Eletrônica de Transmissão , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Antissenso/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Nicotiana/ultraestruturaRESUMO
Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.
Assuntos
Ferredoxina-NADP Redutase/genética , Nicotiana/enzimologia , Nicotiana/genética , RNA Antissenso/genética , RNA de Plantas/genética , Cloroplastos/enzimologia , Expressão Gênica , Genes de Plantas , Fenótipo , Fotobiologia , Plantas Geneticamente Modificadas , Ribulose-Bifosfato Carboxilase/metabolismo , Oxigênio Singlete/metabolismo , Nicotiana/efeitos da radiação , Nicotiana/ultraestruturaRESUMO
Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.