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In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm. 总被引:14,自引:0,他引:14
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The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting. 相似文献
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Wu C Washida H Onodera Y Harada K Takaiwa F 《The Plant journal : for cell and molecular biology》2000,23(3):415-421
The -197 bp promoter of the rice seed storage protein gene, GluB-1, is capable of conferring endosperm-specific gene expression. This proximal 5' flanking region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the -46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the -197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm-specific expression and that the AACA, ACGT and Prolamin-box are involved in quantitative regulation of the GluB-1 gene. A set of gain-of-function experiments using transgenic rice showed that either the Prolamin-box or AACA, although often coupled with GCN4 in many genes, is insufficient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found sufficient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes. 相似文献
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The general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH4 + to the medium triggers inactivation and degradation of the permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin–protein ligase. In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the permease resistant to NH4 + -induced inactivation. An in vivo isolated mutation ( gap1 pgr ) causes a single Glu→Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast α-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 permease against NH4 + -induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins. These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the permease. In contrast, deletion of the last eleven amino acids of Gap1p, a region conserved in other amino acid permeases, conferred resistance to NH4 + inactivation. Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4 + -insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases. 相似文献
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Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione S-transferase gene expression in maize (Zea mays L.) 总被引:10,自引:0,他引:10
The role of hydrogen peroxide (H2 O2 ) and various antioxidants in the regulation of expression of the three Cat and Gst1 genes of maize ( Zea mays L.) has been investigated. Low concentrations of H2 O2 appeared to inhibit Cat1 , Cat3 , and Gst1 gene expression, while higher doses strongly induced these genes. Time course experiments indicated that high concentrations of H2 O2 induced Cat1 , Cat2 , and Gst1 gene expression to higher levels, and in less time, than lower H2 O2 concentrations. Induction of Cat3 was superimposed on the circadian regulation of the gene. These results demonstrate a direct signaling action of H2 O2 in the regulation of antioxidant gene responses in maize.The effects of the antioxidant compounds N-acetylcysteine, pyrrolidine dithiocarbamate, hydroquinone, and the electrophile antioxidant responsive element (ARE)-inducer β -naphthoflavone were quite different and specific for each gene/compound/concentration combination examined. The response of each gene to each antioxidant compound tested was unique, suggesting that the ability of these compounds to affect expression of the maize Cat and Gst1 genes may not be the result of a common (antioxidant) mode of action. A putative regulatory ARE motif involved in the regulation of antioxidant and oxidative stress gene responses in mammalian systems is present in the promoter of all three maize catalase genes and we tested its ability to interact with nuclear extracts prepared from 10 days post-imbibition senescing scutella. Protein-DNA interactions in the ARE motif and the U2 snRNA homologous regions of the Cat1 promoter were observed, suggesting that ARE may play a role in the high induction of Cat1 in a tissue which, due to senescence, is under oxidative stress. 相似文献
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Vesna Hadi-Takov ukalovi 《Physiologia plantarum》1990,80(2):238-242
Glutamate dehydrogenase (EC 1.4.1.3) activity was assayed in homogenates of maize ( Zea mays L. inbred lines Oh43 and Oh43o2 ) endosperm during development. During the period 20–35 days after pollination anabolic (aminative) activities were higher than catabolic (deaminating) ones. In order to study the regulation of GDH activity, glutamine or glutamate were injected into the ear peduncle before sample harvesting. The amination and deamination reactions showed similar behaviour with different nitrogen sources: glutamine increased, whereas glutamate decreased, both aminative and deaminative reactions. Partially purified enzyme was active with NADH and NADPH in a ratio 9:1. In Tris-HCl buffer a broad optimum at pH 7.6–8.9 and pH 6.8–8.9 was observed with NADH and NADPH, respectively, NADH activity was activated by Ca2+ . Saturation curves for (NH4 )2 SO4 and NADH showed normal Michaelis-Menten kinetics in the presence of 1 m M Ca2+ , but substrate inhibition occurred without Ca2+ . The enzyme was inactivated by EDTA. The effect of EDTA was reversed by Ca2+ and Mn2+ , but not by Cu2+ and Mg2+ . 相似文献
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Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source 总被引:2,自引:0,他引:2
The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m−1 , containing nitrogen (0.5 or 4.5 m M ) in the form of NH4 NO3 or NaNO3 Salinity-treated (11.2 dS m−1 ) plants produced less biomass and more organic nitrogen while accumulating more NO− 3 than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO− 3 accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH4 NO3 or NaNO3 . Similar effects were observed in roots of plants grown in 4.5 m M NaNO3 . Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors. 相似文献
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Claudine Hein Jean-Yves Springael Christiane Volland Rosine Haguenauer-Tsapis Bruno André 《Molecular microbiology》1995,18(1):77-87
When yeast cells growing on a poor nitrogen source are supplied with NH4 + ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH4 + of the Gap1 permease is accompanied by its degradation. A functional NPI1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPI1 gene showed that it is identical to RSP5 . The RSP5 product is a ubiquitin—protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C2 domain that may be a Ca2+ -dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPI1/RSP5 product. Chromosomal deletion of NPI1/RSP5 showed that this gene is essential for cell viability. In the viable np1/rsp5 strain, expression of NPI1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins. 相似文献