A multiple-stimuli-responsive as-1-related element of parA gene confers responsiveness to cadmium but not to copper.

The expression of parA, an auxin-regulated gene expressed during the culture of tobacco (Nicotiana tabacum L.) mesophyll protoplasts, is induced by cadmium. To identify the cadmium-responsive element, we examined the parA promoter using the GUS reporter gene. Cadmium responsiveness was retained in a 5' deletion of the parA promoter to -78 bp, but it was nullified by further deletion to -49bp, which implies that the region -49 to -78 bp contained a cadmium-responsive element. This region contains a sequence similar to as-1, an enhancer sequence from the cauliflower mosaic virus 35S RNA promoter that binds the nuclear factor ASF-1. We named the sequence in the parA promoter pas. Gel-shift assays revealed that pas and as-1 compete for the same DNA-binding nuclear protein(s). Since pentamers of either pas and as-1 were able to confer cadmium responsiveness on a minimal promoter but mutant as-1 was not, we propose that pas and as-1 are involved in cadmium-responsive gene expression. Neither pas nor as-1 conferred responsiveness to copper. The specificity of this response, involving the function of as-1-related elements including pas, is discussed.     

Interaction between two cis-acting elements,ABRE and DRE,in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.     

Characterization of a delayed early serum response region.

The proliferin (PLF) gene promoter provides a relatively simple model system for the study of growth-regulated gene expression in mouse cells. The promoter elements required for this serum-induced regulation have been identified and include an AP-1 site as well as an adjacent element comprised of three imperfect repeats that are similar in sequence to the simian virus 40 (SV40) Sph motif. Distinct protein complexes bound independently to the AP-1 and Sph elements, and both of these juxtaposed sites could be occupied simultaneously. Furthermore, serum stimulation of mouse fibroblasts resulted in similar increases in protein binding to the AP-1 and Sph elements. Consistent with this increase in AP-1 and Sph binding activity, the PLF AP-1 and Sph elements were independently able to confer serum responsiveness to a minimal promoter, and together these two elements acted synergistically in response to serum. Although several members of the AP-1 family were able to activate the PLF gene promoter in transient cotransfection experiments, the predominant AP-1 components interacting with the PLF gene promoter in serum-stimulated cells were Fra-1, JunB, and JunD. Analysis of the Sph element revealed that mutation of Sph repeats I or III abolished serum responsiveness of the PLF gene promoter, and mutation of Sph repeat III decreased protein binding to this element. Although the Sph element is similar in sequence to the SV40 element, the PLF Sph-binding factor is distinct from TEF-1, the factor that binds to the SV40 Sph motif.     

Local and differential control of vegetative storage protein expression in response to herbivore damage in Arabidopsis thaliana

Vegetative storage proteins (VSPs) are thought to fulfil important nutritional roles during plant development and stress adaptation. Plant responses to mechanical wounding and herbivore damage include an activation of VSP expression. It was recently suggested that vsp is part of the systemic response of Arabidopsis to wounding. To test this proposal, we monitored the spatial regulation of vsp mRNAs and VSP proteins. Arabidopsis contains two vsp genes and real-time quantitative PCR allowed us to characterize their differential expression. The ratio of vsp1 to vsp2 mRNA abundance increased when plants were challenged with diamondback moth larvae or Egyptian cotton worms, but not when they were mechanically wounded. We observed a dramatic increase of vsp1 and vsp2 mRNA as well as VSP protein levels in leaves that experienced herbivore damage. By contrast, there was a relatively minor increase of vsp mRNA and VSP protein levels in undamaged leaves of infested plants. These results clearly demonstrate that VSPs are part of the local plant response to herbivore attack. To obtain additional information on vsp regulation, we analysed a fusion of a soybean vspB promoter fragment to the β-glucuronidase gene in transgenic Arabidopsis plants. The vspB promoter responded to both jasmonate and herbivore treatments, suggesting that similar signals regulate its expression in both plant species.     

The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms

A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.