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71.
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Characterization of cis-elements required for vascular expression of the cinnamoyl CoA reductase gene and for protein-DNA complex formation 总被引:1,自引:0,他引:1
Lacombe E Van Doorsselaere J Boerjan W Boudet AM Grima-Pettenati J 《The Plant journal : for cell and molecular biology》2000,23(5):663-676
Cinnamoyl-CoA reductase (CCR) catalyses the first specific step in the biosynthesis of monolignols, the monomeric units of lignins. We examined the developmental regulation of the Eucalyptus gunnii EgCCR promoter by analysing the expression of EgCCR-GUS fusions in tobacco. EgCCR promoter activity was strongest in lignified organs (stems and roots) consistent with the EgCCR mRNA level in these organs. Histochemical analysis showed expression in vascular tissues (cambium, young differentiating xylem, ray cells, internal and external phloem) of stems and roots in agreement with in situ hybridization data. Promoter deletion analysis and gain-of-function experiments identified the sequences between positions -119 and -77 as necessary and sufficient for expression in vascular tissues of stems. Electrophoretic mobility-shift assays showed that this region is specifically recognized by nuclear proteins present in tobacco stems, giving rise to two retarded complexes, LMC1 and LMC2. Using overlapping EgCCR fragments and mutated oligonucleotides as competitors in gel-shift assays, two DNA-protein interaction sites were mapped. Finally, the role of protein-protein interactions in the formation of the LMC1 and LMC2 complexes was investigated using the detergent sodium deoxycholate, and protein fractionation onto a heparin Sepharose column. 相似文献
74.
M L Boerjan W M Baarends H J Ruven 《The journal of histochemistry and cytochemistry》1991,39(1):135-138
We describe a cytochemical staining procedure for succinate dehydrogenase (SDH) activity in pre-ovulatory mouse oocytes. The oocytes were embedded in low gelling temperature agarose and treated with caffeine before cytochemical staining in the presence of nitro blue tetrazolium (NBT), phenazinemethosulfate (PMS), and succinate. This resulted in intense staining of the oocytes by formazan precipitate. The level of aspecific formazan production in the absence of succinate was very low. We applied the procedure to oocytes matured in vitro and found that the location of the formazan precipitate as a result of SDH activity correlated well with the location of mitochondria. The chromatin of the cytochemically stained oocytes could subsequently be analyzed by means of the DNA-specific fluorochrome DAPI. In pre-ovulatory oocytes, we found a correlation between chromatin organization and the location of mitochondria: in oocytes with an intact germinal vesicle the mitochondria were uniformly distributed in the cytoplasm, as shown by fine grains of formazan precipitate. In oocytes with condensed chromatin the mitochondria apparently had clustered, because the formazan precipitate was more coarse in these cells. 相似文献
75.
A simple, rapid, and quantitative high-pressure liquid chromatography radio method is described for the determination of in vivo (14)C-labeled l-ascorbate, dehydro-l-ascorbate, and total l-ascorbate of Arabidopsis thaliana cell suspensions upon incubation of cultures with exogenous d-[(14)C]mannose. The same radio-HPLC conditions can be used to follow the products of in vitro enzymatic conversions of GDP-d-mannose by enzyme extracts of A. thaliana, namely GDP-l-galactose, GDP-4"-keto,6"-deoxy-d-mannose, and GDP-l-fucose. In particular, an accurate assay for GDP-d-mannose 3",5"-epimerase, a key enzyme of the plant vitamin C pathway, is presented. 相似文献
76.
77.
Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins 总被引:9,自引:0,他引:9
Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity
of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem,
mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence
analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized
proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The
identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in
the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic
acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function
of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping
enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one
α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels
in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our
data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing
may yield information complementary to that from EST sequencing strategies.
Received: 28 June 1999 / Accepted: 3 September 1999 相似文献
78.
Leplé JC Dauwe R Morreel K Storme V Lapierre C Pollet B Naumann A Kang KY Kim H Ruel K Lefèbvre A Joseleau JP Grima-Pettenati J De Rycke R Andersson-Gunnerås S Erban A Fehrle I Petit-Conil M Kopka J Polle A Messens E Sundberg B Mansfield SD Ralph J Pilate G Boerjan W 《The Plant cell》2007,19(11):3669-3691
79.
80.
The authors have developed a sensitive bioassay that can be used to detect auxins as well as cytokinins. The bioassay is based on the expression in transformed tobacco (Nicotiana tabacum) mesophyll protoplasts of a chimeric gene, consisting of the upstream sequences of the Agrobacterium tumefaciens gene 5, coupled to the coding sequence of the β-glucuronidase. The expression of this gene is induced by the presence of both auxin and cytokinin in the culture medium. Using this assay, indole-3-acetic acid was detected at 5 × 10−8 molar, whereas trans-zeatin could be detected at 5 × 10−11 molar. The assay can be performed in microtiter plates, allowing numerous samples to be analyzed simultaneously. Only 2.5 × 105 protoplasts are required for one individual assay in 250 microliters of culture medium and for qualitative results, the reaction is readily visualized by ultraviolet light. 相似文献