Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses. We report the cloning of a full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5'-RACE technique. The cDNA encodes a soluble protein containing 318 amino acid residues. The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from other plants. A 5 x His tag was added to the N-terminus of the BNSEH1 and the construct was over-expressed in the yeast Pichia pastoris. The recombinant protein was recovered at high levels after Ni-agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana. BNSEH1 was shown to be a monomer by gel filtration analysis. The activity was low towards cis-stilbene oxide but much higher using trans-stilbene oxide as substrate with Vmax of 0.47 micro mol.min.mg-1, Km of 11 micro m and kcat of 0.3 s-1. The optimum temperature of the recombinant enzyme was 55 degrees C and the optimum pH 6-7 for trans-stilbene oxide hydrolysis. The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop. (+info)
Regulation of the pollen-specific actin-depolymerizing factor LlADF1.
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Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by approximately 60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy. (+info)
Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution.
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Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids. (+info)
Isocitrate lyase is essential for pathogenicity of the fungus Leptosphaeria maculans to canola (Brassica napus).
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A pathogenicity gene has been identified in Leptosphaeria maculans, the ascomycetous fungus that causes blackleg disease of canola (Brassica napus). This gene encodes isocitrate lyase, a component of the glyoxylate cycle, and is essential for the successful colonization of B. napus. It was identified by a reverse genetics approach whereby a plasmid conferring hygromycin resistance was inserted randomly into the L. maculans genome. Twelve of 516 transformants tested had reduced pathogenicity on cotyledons of B. juncea and B. napus, and 1 of these 12 had a deletion of the isocitrate lyase gene, as well as an insertion of the hygromycin resistance gene. This mutant was unable to grow on fatty acids, including monolaurate, and the isocitrate lyase transcript was not detected. When the wild-type gene was reintroduced into the mutant, growth on monolaurate was restored and pathogenicity was partially restored. L. maculans isocitrate lyase is produced during infection of B. napus cotyledons, while the plant homologue is not. When 2.5% glucose was added to the inoculum of the isocitrate lyase mutant, lesions of sizes similar to those caused by wild-type isolate M1 developed on B. napus cotyledons. These findings suggest that the glyoxylate pathway is essential for disease development by this plant-pathogenic fungus, as has been shown recently for a fungal and bacterial pathogen of animals and a bacterial pathogen of plants. Involvement of the glyoxylate pathway in pathogenesis in animals and plants presents potential drug targets for control of diseases. (+info)
Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves.
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Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature. (+info)
Cauliflower mosaic virus is preferentially acquired from the phloem by its aphid vectors.
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Cauliflower mosaic virus (CaMV) is transmitted in a non-circulative manner by aphids following the helper strategy. Helper proteins P2 and P3 act as a bridge between virions and the aphid cuticle. Electronic monitoring of aphid stylet activities (EPG technique), transmission tests and electron microscopy showed that CaMV is preferentially acquired from the phloem by its most common aphid vectors, Brevycorine brassicae and Myzus persicae. We also found that CaMV is semipersistently transmitted and that the rate of acquisition does not follow a typical bimodal curve. Instead, the virus could be acquired from non-phloem tissues at a low and fairly constant rate after one or more intracellular punctures within a few minutes, but the probability of acquisition rose significantly when aphids reached the phase of committed ingestion from the phloem. The acquisition rate of CaMV did not increase with increasing number of intracellular punctures, but the total duration of intracellular puncture was one of the variables selected by the stepwise logistic regression model used to fit the data that best explained acquisition of CaMV. Furthermore, aphids reaching the phloem faster had a higher probability of acquiring the virus. Our results support the hypothesis that multiple intracellular punctures of epidermal and mesophyll cells result in loading aphids with the CaMV-encoded aphid transmission factor (P2), and that aphids, in most cases, subsequently acquire CaMV particles during phloem sap ingestion. Consistently, immunoelectron microscopy showed that P3-virions are frequently found in the sieve element lumen, whereas P2 could not be detected. (+info)
Stability in vitro of the 69K movement protein of Turnip yellow mosaic virus is regulated by the ubiquitin-mediated proteasome pathway.
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Plant viruses move to adjacent cells with the use of virus-encoded cell-to-cell movement proteins. Using proteins produced by in vitro translation, we present evidence that the '69K' movement protein of Turnip yellow mosaic virus (TYMV) is recognized as a substrate for the attachment of polyubiquitin chains and for subsequent rapid and selective proteolysis by the proteasome, the ATP-dependent proteolytic system present in reticulocyte lysate. Truncation of the 69K protein suggests the existence of two degradation signals within its sequence. We propose that selective degradation of virus movement proteins may contribute to the previously reported transient nature of their accumulation during infection. (+info)
The coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step.
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Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of Turnip crinkle virus (TCV) strongly suppresses PTGS. The Agrobacterium infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and double-stranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants. (+info)