Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.     

Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene.

The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.     

MicroRNAs transcriptionally regulate promoter activity in Arabidopsis thaliana

Micro RNAs(mi RNAs) are vital regulators that repress gene expression in the cytoplasm in two main ways: m RNA degradation and translational inhibition. Several animal studies have shown that mi RNAs also target promoters, thereby activating expression.Whether this mi RNA action also occurs in plants is unknown. In this study, we demonstrated that several mi RNAs regulate target promoters in Arabidopsis thaliana. For example, mi R5658 was predominantly present in the nucleus and activated the expression of AT3 G25290 directly by binding to its promoter. Our observations suggest that this mode of action may be a general feature of plant mi RNAs, and thus provide insight into the vital roles of plant mi RNAs in the nucleus.     

A cluster of disease resistance genes in Arabidopsis is coordinately regulated by transcriptional activation and RNA silencing

The RPP5 (for recognition of Peronospora parasitica 5) locus in the Arabidopsis thaliana Columbia strain contains a cluster of paralogous disease Resistance (R) genes that play important roles in innate immunity. Among the R genes in this locus, RPP4 confers resistance to two races of the fungal pathogen Hyaloperonospora parasitica, while activation of SNC1 (for suppressor of npr1-1, constitutive 1) results in the resistance to another race of H. parasitica and to pathovars of the bacterial pathogen Pseudomonas syringae through the accumulation of salicylic acid (SA). Here, we demonstrate that other Columbia RPP5 locus R genes can be induced by transgenic overexpression of SNC1, which itself is regulated by a positive amplification loop involving SA accumulation. We also show that small RNA species that can target RPP5 locus R genes are produced in wild-type plants and that these R genes can be cosuppressed in transgenic plants overexpressing SNC1. Steady state expression levels of SNC1 increase in some mutants (dcl4-4, ago1-36, and upf1-5) defective in RNA silencing as well as in transgenic plants expressing the P1/Helper Component-Protease viral suppressor of RNA silencing. However, steady state levels of small RNA species do not change in mutants that upregulate SNC1. These data indicate many Columbia RPP5 locus R genes can be coordinately regulated both positively and negatively and suggest that the RPP5 locus is poised to respond to pathogens that disturb RNA silencing.     

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.     

Characterization of the yeast copper-inducible promoter system in Arabidopsis thaliana

Inducible promoters or gene-switches are used to both spatially and temporally regulate gene expression. Such regulation can provide information concerning the function of a gene in a developmental context as well as avoid potential harmful effects due to overexpression. A gfp construct under the control of a copper-inducible promoter was introduced into Arabidopsis thaliana (L.) Heynh. and the regulatory parameters of this inducible promoter were determined. Here, we describe the time-course of up- and down-regulation of GFP expression in response to copper level, the optimal regulatory levels of copper, and the tissue specificity of expression in three transgenic lines. We conclude that the copper-inducible promoter system may be useful in regulating the time and location of gene expression in A. thaliana.     

Correlation between the induction of a gene for Δ1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress

The isolation and characterization is reported of a cDNA for Δ¹-pyrroline-5-carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana . Southern blot analysis suggested that only one copy of the corresponding gene ( AtP5CS ) is present in A. thaliana . The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia . Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana . A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.     

High diversity due to balancing selection in the promoter region of the Medea gene in Arabidopsis lyrata

Molecular imprinting is the differential expression and/or silencing of alleles according to their parent of origin [1, 2]. Conflicts between parents, or parents and offspring, should cause "arms races," with accelerated evolution of the genes involved in imprinting. This should be detectable in the evolution of imprinting genes' protein sequences and in the promoter regions of imprinted genes. Previous studies, however, found no evidence of more amino acid substitutions in imprinting genes [1, 3]. We have analyzed sequence diversity of the Arabidopsis lyrata Medea (MEA) gene and divergence from the A. thaliana sequence, including the first study of the promoter region. In A. thaliana, MEA is imprinted, with paternal alleles silenced in endosperm cells [4, 5], and also functions in the imprinting machinery [4, 6]; MEA protein binding at the MEA promoter region indicates self-regulated imprinting [7-9]. We find the same paternal MEA allele silencing in A. lyrata endosperm but no evidence for adaptive evolution in the coding region, whereas the 5' flanking region displays high diversity, with distinct haplotypes, suggesting balancing selection in the promoter region.     

The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots

Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity. The genomic region spanning nucleotides 5999–7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana. In A. sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive. In A. thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique. The transgene was inherited and active in progeny plants of both A. sativa and A. thaliana.