Different cis-Regulatory DNA Elements Mediate Developmental Stage- and Tissue-specific Expression of the Human COL2A1 Gene in Transgenic Mice

Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5′ flanking region and intron 1 are known to control tissue-specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2,388 to +2,696) in intron 1 in conjunction with 6.1-kb 5′ sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5′ sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue-specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from −90 to −1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from −1,500 to −6,100 and +2,388 to +2,855.     

In Plant Protoplasts, the Spontaneous Expression of Defense Reactions and the Responsiveness to Exogenous Elicitors Are under Auxin Control

When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of β-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of β-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating β-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced β-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 × 10⁻⁵ molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco β-glucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.     

Stress responses in alfalfa (Medicago sativa L.). 8. Cis-elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5 deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between –326 and –130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions –326 and –130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.Abbreviations CAT chloramphenicol acetyltransferase - CHS chalcone synthase (EC 2.3.1.74) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

A new bioassay for auxins and cytokinins

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.     

cis-regulatory elements involved in ultraviolet light regulation and plant defense.

An elicitor-regulated transient expression system was established in soybean protoplasts that allowed the identification of cis-regulatory elements involved in plant defense. The 5' region of an ultraviolet (UV) light-inducible and elicitor-inducible chs gene (chs1) of soybean was subjected to deletion analysis with the help of chimeric chs-nptII/gus gene constructs. This analysis delimited the sequences necessary for elicitor inducibility to -175 and -134 of the chs1 promoter. The same soybean sequences were able to direct elicitor inducibility in parsley protoplasts, suggesting a conserved function of cis-acting elements involved in plant defense. In addition, this region of the soybean promoter also promotes UV light inducibility in parsley protoplasts. However, in contrast to the elicitor induction, correct regulation was not observed after UV light induction when sequences downstream of -75 were replaced by a heterologous minimal promoter. This result indicates that at least two cis-acting elements are involved in UV light induction.     

Osmotin gene expression is controlled by elicitor synergism

Arachidonic acid (AA), a fatty-acid fungal elicitor, and a cellulase preparation from Aspergillus niger , a protein-type fungal elicitor, induced osmotin gene expression. Both elicitors activated the osmotin promoter fused to a β-glucuronidase (GUS) reporter gene in a tissue-specific manner in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38). The cellulase preparation was more effective than AA at the concentrations tested and, unlike AA, also induced the accumulation of osmotin mRNA and protein. Combinations of AA and the cellulase preparation had a greater than additive effect on the activation of the osmotin promoter and the accumulation of osmotin mRNA and protein. Both AA and the cellulase preparation, when applied separately, were virtually ineffective in the induction of the osmotin promoter in cotyledon tissues. However, together they were able to induce synergistically GUS fused to the osmotin promoter. Increases in osmotin-promoter-driven GUS activity and accumulation of osmotin mRNA induced by AA, the cellulase preparation or their combination were reversed by norbornadiene, an ethylene action inhibitor, indicating that ethylene is involved in the induction of the osmotin gene by these elicitors.     

Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts

The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26°C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with ³⁵S-methionine. Incubation at 40° and 42°C resulted in the synthesis of polypeptides not detectable at 26°C. Introduction of a chimeric maize heat shock protein 70 promoter-chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40°C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress.     

Inhibition of Sucrose Enhancer Effect of the Potato Proteinase Inhibitor II Promoter by Salicylic Acid

Effect of salicylic acid (SA) on the expression of the potato proteinase inhibitor (PI) II promoter was studied with transgenic tobacco plants (Nicotiana tabacum) carrying a gene fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) reporter. As previously observed, the PI-II promoter was inducible by wounding and the promoter activity was further enhanced by sucrose. Addition of SA did not influence the wound induction of the PI-II promoter but significantly inhibited the sucrose response. The 5′-deletion mutant −573 was unable to respond to wounding but did respond to sucrose and SA. The 3′-deletion analysis indicated the presence of a sucrose-responsive element between −574 and −520. A study of the insertion mutants revealed the function of another sucrose-responsive element between −522 and −500. Enhancer effects of these sucrose-responsive elements were inhibited by SA. These studies suggest that SA inhibits PI-II promoter activity by decreasing the sucrose response. Analysis of SA-related chemicals revealed that only acetyl-SA showed a similar inhibitory effect, and other hydroxybenzoic acids had little or no effect on the sucrose enhancer activity. Therefore, it seems that the interaction between SA and the receptor molecule is specific.     

Purification and Properties of an Elicitor of Castor Bean Phytoalexin from Culture Filtrates of the Fungus Rhizopus stolonifer

Evidence has been obtained for the presence in filtrates of 3-day-old cultures of the fungus Rhizopus stolonifer grown on potato-dextrose medium of both high molecular weight and low molecular weight elicitors of the production of the phytoalexin casbene in cell-free extracts of castor bean (Ricinus communis L.) seedlings. The high molecular weight elicitor activity was purified by means of gel filtration chromatography. Both protein and carbohydrate are associated with the most purified fraction containing elicitor activity. The elicitor is inactivated by treatments at 60 C or higher temperatures for 15 minutes. The molecular weight of the purified elicitor was estimated from gel filtration chromatography in 10 mm Na-phosphate (pH 7) to be 30,000 ± 5,000. Treatments of the purified elicitor fraction with either sodium periodate or the nonspecific protease preparation, pronase, substantially reduced its activity as an elicitor of casbene production. On the basis of these properties it is concluded that the elicitor is most likely a protein and may be a glycoprotein. It is estimated that 2 × 10⁻⁸ m elicitor gives about a 14-fold increase in casbene synthetase activity in extracts of treated split seedlings in comparison with controls. This corresponds to about 50% of the maximal activity obtainable in this assay system developed to measure elicitor activity.     

Identification of G-Box Sequence as an Essential Element for Methyl Jasmonate Response of Potato Proteinase Inhibitor II Promoter

The potato proteinase inhibitor II promoter was studied to identify cis-acting regulatory sequences involved in methyl jasmonate (MJ) response using transgenic tobacco plants carrying various lengths of the promoter fused to a chloramphenicol acetyltransferase reporter gene. An internal fragment between −625 and −520 was sufficient to confer a response to MJ, wounding, or sucrose when it was placed upstream of the nos promoter −101, which contains the CAAT-TATA region. Deletion of the proteinase inhibitor II promoter sequence upstream of −611 did not affect the MJ response, but a further deletion to −573 eliminated the response. The 3′-deletion study showed that the DNA sequence downstream from −520 is dispensable. However, 3′-deletion mutant −574 did not respond to the MJ treatment. These results indicated that an element essential for the MJ response is located at the −574/−573 region where the G-box sequence (CACGTGG) is located. The G-box sequence was not required for the sucrose enhancer effect, suggesting that the MJ response mechanism is different from that of sucrose.     

Functional Characterization of cis-Elements Conferring Vascular Vein Expression of At4g34880 Amidase Family Protein Gene in Arabidopsis

The expression of At4g34880 gene encoding amidase in Arabidopsis was characterized in this study. A promoter region of 1.5 kb on the upstream of the start codon of the gene (referred as AmidP) was fused with uidA (GUS) reporter gene, and transformed into Arabidopsis plant for determining its spatial expression. The results indicated that AmidP drived GUS expression in vascular system, predominately in leaves. Truncation analysis of AmidP demonstrated that VASCULAR VEIN ELEMENT (VVE) motif with a region of 176 bp sequence (−1500 to −1324) was necessary and sufficient to direct the vascular vein specific GUS expression in the transgenic plant. Tandem copy of VVE increased vascular system expression, and 5′- and 3′- deletions of VVE motif in combination with a truncated −65 CaMV 35S minimal promoter showed that 11bp cis-acting element, naming DOF2 domain, played an essential role for the vascular vein specific expression. Meanwhile, it was also observed that the other cis-acting elements among the VVE region are also associated with specificity or strength of GUS activities in vascular system.