Functional role of CAAT box element of the nopaline synthase (nos) promoter

The nopaline synthase (nos) promoter is active in a wide range of plant tissues and regulated by various environmental stimuli. It was previously found that the CAAT box region is important for nos promoter activity. In the present study, the location of the CAAT box element was determined by site specific mutation analysis. Point mutations within the conserved CAAT box element significantly reduced the promoter response in transgenic tobacco plants and calli to wounding, H₂O₂, methyl jasmonate, and 2,4-D, but not to salicylic acid. However, mutations immediately upstream from the CAAT box did not affect these responses. These results suggest that the CAAT box element is important in responding to certain stimuli.     

Plant nuclear factor ASF-1 binds to an essential region of the nopaline synthase promoter

We have characterized a tobacco nuclear factor that binds to the -118 region of the nopaline synthase (nos) promoter from the Ti plasmid of Agrobacterium tumefaciens. The binding site for this factor, identified by DNase I footprinting, encompasses the region from -138 to -103 of the nos promoter. This region, which contains a potential Z-DNA-forming sequence, was previously shown to be essential for nos promoter activity in transgenic tobacco. A synthetic 21-base pair sequence from the protected region (from -131 to -111), designated as nos-1, was sufficient for factor recognition in vitro. In transgenic tobacco, a tetramer of nos-1 can confer leaf and root expression when fused upstream of a truncated 35 S promoter from the cauliflower mosaic virus. Mutations at the two TGACG-like motifs in nos-1 abolish factor binding while preserving the potential for Z-DNA formation. A tetramer of the nos-1 mutant sequence has no significant activity above background when tested in transgenic tobacco. Competition experiments with activation sequence factor (ASF)-1 binding sites from the 35 S promoter of cauliflower mosaic virus (as-1) and the wheat histone H3 promoter (hex-1) demonstrate that ASF-1 is the factor that binds to nos-1.     

Induction of nopaline synthase promoter activity by H2O2 has no direct correlation with salicylic acid.

Transgenic tobacco (Nicotiana tabacum L.) plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (caf) were tested for their response to treatment with H2O2. The nos promoter-driven CAT activity increased significantly by addition of H2O2, reaching the maximum level at 15 mM. Kinetic analysis for CAT activity showed that induction by H2O2 was similar to that of methyl jasmonate (MJ), but was much slower than induction by salicylic acid (SA). Time-course experiments for mRNA level also revealed that the response to H2O2 treatment was similar to that of MJ. The nos promoter displayed a rapid and transient induction of mRNA with SA treatment, with the maximum levels occurring at 3 h, whereas the levels induced by H2O2 or MJ treatment increased continuously during the 11-h experimental period. The antioxidants N-acetyl-L-cysteine and catechol did not alter the SA effect. The responses of the nos promoter to H2O2, MJ, and wounding were significantly reduced by deletions of the CAAT box region and the sequence between -112 and -101. However, these deletions did not significantly alter the SA response. This suggests that H2O2 may have a different mechanism from that of SA for inducing nos promotor activity.     

Both TATA box and upstream regions are required for the nopaline synthase promoter activity in transformed tobacco cells

Summary Using a promoter expression vector system based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, we have studied the molecular structure of the nopaline synthase (nos) promoter which is active constitutively in transformed plant tissues. The system uses the sensitive and reliable chloramphenicol acetyltransferase (CAT) assay for the analysis of promoter strength in plant cells. Two sets of mutants were generated by sequential deletion of the nos promoter region from both 5 and 3 ends. These promoter fragments were linked to the cat coding sequence within the expression vector. The strength of the mutant promoters was measured in transformed tobacco calli as CAT activity. 3 deletions up to-17 bp did not significantly affect the promoter strength. Further deletions into the TATA box region reduced the promoter strength by about ten-fold. Analysis of the 5 deletion mutants showed that an upstream region is required for the nos promoter activity in addition to the TATA box and CCAAT box regions.     

Promoter elements controlling developmental and environmental regulation of a tobacco ribosomal protein gene L34

The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or -glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5 deletion analyses of the rpL34 promoter indicated that a 50 bp region located between –179 and –129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3 deletion analyses. A 3 deletion up to –28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to –104, eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.     

Three distinct regulatory elements comprise the upstream promoter region of the nopaline synthase gene

Summary Fine deletion mutants were generated in the upstream control region of the nopaline synthase (nos) promoter to define the position and role of upstream regulatory elements. The results indicated that the 8 bp sequence (CAGAAACC) at -106/-113 and its inverted repeat (GGTTTCTG) at -140/-147 are important for promoter function. The downstream element appears more important than the upstream element since deletion of the former reduced promoter activity more significantly than deletion of the latter. Deletion of the element alone, however, did not abolish promoter function, whereas, deletion of the 10 bp potential Z-DNA-forming (Z) element located between the repeat elements nullified promoter activity. Therefore, it appears that the Z element is an essential upstream regulator and the repeated elements are upstream modulators of the nos promoter. These elements are functionally distinct since alteration of stereospecificity or insertion of short oligonucleotides between the elements did not significantly influence promoter activity. These regulatory elements were unable to function from 200 bp upstream of the CCAAT-TATA box region.     

A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity

The nopaline synthase (nos) promoter is expressed in a wide range of plant cell types and regulated by various developmental and environmental factors. The nos upstream control region essential for this regulation was studied by means of synthetic oligomers using transient and stable transformation systems. Insertion of a 20 nucleotide sequence containing two hexamer motifs and a spacer region into deletion mutants lacking the upstream control region was essential for promoter activity. Mutation of one or more nucleotides of either hexamer sequence significantly altered the strength of expression of the nos promoter. Point mutations within the spacer region also strongly influenced promoter strength. Insertion of multiple copies of the 20 nucleotide sequence into the nonfunctional deletion mutants proportionally increased the promoter activity. These results suggest that this twenty nucleotide sequence is essential for the nos promoter to function. Substitution of the nos element with the ocs or 35S as-1 which contain similar hexamer motifs restored not only promoter activity but also responses to wounding, auxin, methyl jasmonate, and salicylic acid.     

Genetic analysis of the human thymidine kinase gene promoter.

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.     

Nopaline synthase promoter is wound inducible and auxin inducible.

The activity of the nopaline synthase (nos) promoter is differentially regulated in several plant organs. In this article we demonstrate that the nos promoter is wound inducible in both vegetative and reproductive organs. The induction of the nos promoter was observed in leaves, stems, cotyledons, and various reproductive organs, suggesting that the response is not organ specific. The wound response was further enhanced by addition of auxins. Other growth substances had no effect on the wound-inducible nos promoter activity. Deletion analysis of the nos promoter indicated that the 10-base pair (GCACATACGT) Z element located between -123 and -114 or an element overlapping with this sequence is essential for the wound and auxin responses.     

Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression.