Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression

In order to scan the 5 flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5 end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from –67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from –142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (–59 and –52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.     

Binding of ArsR,the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the ?35 and ?10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the ?35 and ?10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.     

Binding of ArsR, the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the –35 and –10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the –35 and –10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.     

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A P_fr/P_tot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of P_fr in order to be fully effective.Abbreviations HIR high irradiance response - P_fr far red absorhing form of phytochrome - P_tet total phytochrome - UV ultraviolet light     

Functional architecture of the light-responsive chalcone synthase promoter from parsley.

We have combined in vivo genomic footprinting and light-induced transient expression of chalcone synthase promoter derivatives in parsley protoplasts to identify cis sequences regulating light activation. The parsley chalcone synthase promoter contains two cis "units" that are light-responsive. Each unit is composed of short DNA stretches of approximately 50 base pairs, and each contains two in vivo footprints. One of the footprints in each unit covers a sequence that is highly conserved among other light- and stress-regulated plant genes. The other footprinted sequences in each unit are not related to each other. The TATA distal light-responsive unit is inherently weak but can compensate partially for the loss of the stronger TATA proximal unit. Levels of light-induced expression from either can be influenced by the presence of a region of approximately 100 base pairs located upstream of the TATA distal light-responsive unit. Combination of the light-responsive units and upstream region generates a synergistic response to light. We speculate that functional compensation generated by nonidentical, but sequence-related, cis units foreshadows combinatorial diversity of cognate trans factors.     

Regulation of phytochrome A mRNA abundance in parsley seedlings and cell-suspension cultures

A cDNA clone encoding the apoprotein of a parsley phytochrome was isolated and classified as parsley PHYA phytochrome, on the basis of a sequence homology comparison with all available phytochrome sequences. Red light pulses led to a phytochrome-dependent down-regulation of PHYA mRNA abundance in etiolated parsley seedlings to a level of 10–20% compared with the dark control. The PHYA mRNA abundance in a parsley cell suspension culture was also down-regulated by light pulses. Transient expression assays in parsley protoplasts showed light regulation of a chimeric pea PHYA promoter uidA-gene construct.     

Desensitization by red and blue light of phototropism in maize coleoptiles

The effects of pretreatments with red and blue light (RL, BL) on the fluence-response curve for the phototropism induced by a BL pulse (first positive curvature) were investigated with darkadapted maize (Zea mays L.) coleoptiles. A pulse of RL, giving a fluence sufficient to saturate phytochrome-mediated responses in this material, shifted the bell-shaped phototropic fluence-response curve to higher fluences and increased its peak height. A pulse of high-fluence BL given immediately prior to this RL treatment temporarily suppressed the phototropic fluence-response curve, and shifted the curve to higher fluences than induced by RL alone. The shift by BL progressed rapidly compared to that by RL. The results indicate (1) that first positive curvature is desensitized by both phytochrome and a BL system, (2) that desensitization by BL occurs with respect to both the maximal response and the quantum efficiency, and (3) that the desensitization responses mediated by phytochrome and the BL system can be induced simultaneously but develop following different kinetics. It is suggested that theses desensitization responses contribute to the induction of second positive curvature, a response induced by prolonged irradiation.Abbreviations BL blue light - RL red light CIW-DPB Publication No. 1001     

An insulator element 3′ to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells

Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3′-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.     

Transient reduction of responsiveness of blue-light-mediated hair-whorl morphogenesis inAcetabularia mediterranea induced by blue light

After a prolonged period of red light the formation of a new whorl of lateral hairs can be induced inAcetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva) by a pulse of blue light. It has previously been shown that the response to blue light obeys the law of reciprocity. In this paper we demonstrate that the responses to blue light are additive only within 10 min after the onset of blue-light treatment, since the responsiveness of the cells is also affected by blue light. One hour after a short blue-light pulse the response to a second blue-light pulse has come to a minimum. After that, the responsiveness is restored in a refractory period of several hours. The fluenceresponse curves for hair-whorl formation at the time of minimum responsiveness are shifted parallel to the original fluence-response curves without preirradiation. Again, the law of reciprocity applies. This indicates an increased light requirement only for the same degree of hair-formation response. The sensitivity to blue light of the reduction of responsiveness response is higher by a factor of about 50 than the induction of hairformation response.     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet