An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobacco

Pathogenesis-related proteins of group 1 (PR-1) are strongly induced in plants by pathogen attack, exposure of the plants to (acetyl)salicylic acid (ASA, SA), and by developmental cues. Functional analysis of the PR-1a promoter identified a region of 139 bp (from -691 to -553) mediating expression of the GUS reporter gene in response to ASA. Inspection of this region revealed two TGACG elements reminiscent of activation sequence-1 (as-1). Recently, as-1 has been reported to be responsive to SA in the context of the CaMV 35S RNA promoter. To address the question of whether the as-1-like sequence may be of functional significance for the expression of the PR-1a gene, gel shift assays were performed with TGA1a, a protein been shown to interact with as-1 in vitro. TGA1a was found to bind to the PR-1a as-1-like sequence with similar specificity and affinity as to as-1. Furthermore, mutations were introduced in the as-1-like sequence in the context of the inducible 906 bp PR-1a promoter which are impaired in binding TGA1a in vitro. Significantly reduced levels of GUS reporter gene activity were obtained with the mutant promoter regions as compared to the wild-type PR-1a promoter in response to all stimuli in transgenic tobacco plants. Yet, mutation of the as-1-like sequence did not abolish induction of reporter gene expression. Taken together, these results suggest that the level of expression of the tobacco PR-1a gene is controlled by an as-1-like sequence motif in the PR-1a upstream region, possibly interacting with a factor related to TGA1a.     

Rice ubiquitin promoters: deletion analysis and potential usefulness in plant transformation systems

Strong constitutive promoters form a cornerstone for basic and applied research using transgenic plants. GUS (beta-glucuronidase) expression levels from constructs containing RUBQ1 or RUB2 rice ubiquitin promoters were 8- to 35-fold higher in transgenic rice [Oryza sativa (L.)] plants, respectively, when compared to the 35S promoter. Deletion analysis of the 5'-upstream region of RUBQ2 revealed a putative enhancer region that produced a 2.4-fold increase in transient GUS expression. Southern blot analysis showed that three to seven copies of the GUS gene were stably inserted into R0 and R1 plants and inherited in a monogenic fashion.     

A novel high-throughput genetic screen for stress-responsive mutants of Arabidopsis thaliana reveals new loci involving stress responses

Activation sequence-1 (as-1) cognate promoter elements are widespread in the promoters of plant defense-related genes as well as in plant pathogen promoters, and may play important roles in the activation of defense-related genes. The as-1-type elements are highly responsive to multiple stress stimuli such as jasmonic acid (JA), salicylic acid (SA), H(2)O(2), xenobiotics and heavy metals, and therefore provide a unique opportunity for identifying additional signaling components and cross-talk points in the various signaling networks. A single as-1-type cis-element-driven GUS reporter Arabidopsis line responsive to JA, SA, H(2)O(2), xenobiotics and heavy metals was constructed for mutagenesis. A large-scale T-DNA mutagenesis has been conducted in the reporter background, and an efficient high-throughput mutant screen was established for isolating mutants with altered responses to the stress chemicals. A number of mutants with altered stress responses were obtained, some of which appear to identify new components in the as-1-based signal transduction pathways. We characterized a mutant (Delta8L4) with a T-DNA insertion in the coding sequence of the gene At4g24275. The as-1-regulated gene expression and GUS reporter gene expression were altered in the Delta8L4 mutant, but there was no change in the expression of genes lacking as-1 elements in their promoters. The phenotype observed with the Delta8L4 mutant was further verified using RNAi plants for At4g24275 (8L4-RNAi), suggesting the feasibility of use of this high-throughput mutant screening in isolating stress-signaling mutants.     

ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.     

Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea. This revised version was published online in June 2006 with corrections to the Cover Date.