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.     

Organ-specific and developmental regulation of the nopaline synthase promoter in transgenic tobacco plants

Control regions of the nopaline synthase (nos) gene have been widely used to express foreign genes in plants since the promoter is active in a wide variety of plant tissues. We report here the characteristics of the nos promoter activity in transgenic tobacco (Nicotiana tabacum) plants at various developmental stages. The promoter was highly active in the lower parts of a plant and gradually decreased in the upper parts. This vertical gradient was maintained throughout plant growth until the flowering stage when the overall promoter strength decreased significantly in the vegetative organs. However, in various flower organs, the nos promoter activities increased dramatically. Higher activity was observed in calyx, corolla, and stamens although the maximum promoter activity in each organ was found at different stages of flower development. The promoter activity in pistils was low and gradually increased in the ovaries after anthesis. In developing fruits, the nos promoter activity was strongly induced during the mid-stage of embryogenesis. These results indicate that the expression of the nos promoter is developmentally regulated and organ specific in transgenic tobacco plants.     

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.     

Isolation of tobacco DNA segments with plant promoter activity.

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.     

Sucrose-phosphate synthase activity and yield analysis of tomato plants transformed with maize sucrose-phosphate synthase

Sucrose synthesis is a major element of the interactions between photosynthesis and plant growth and development. Tomato (Lycopersicon esculentum Mill. cv. UC82B) plants transformed with maize sucrose-phosphate synthase (SPS; EC 2.3.1.14) expressed from either a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit promoter (SSU) or the cauliflower mosaic virus 35S promoter (35S) were used to study effects of increased sucrose synthesis rates on plant growth. The plants were grown in growth chambers, field plots, and open-top chambers. The 35S plants had a 2 to 3-fold increase in young-leaf SPS activity, a 10 to 20-fold increase in young-root SPS activity and no increase in young-fruit SPS activity. The leaf SPS activity in one of the 35S lines fell to control levels by two months of age. The SSU plants had a 4 to 5-fold increase in leaf SPS activity and no significant increase in root or young-fruit SPS activity. One 35S line, which maintained high leaf SPS activity throughout development, yielded 70–80% more than controls at both normal and elevated CO₂ in open-top chambers in the field and 20–30% more than controls in two additional field trials. The other 35S line and the two SSU lines either yielded less or did not differ from controls under several growth conditions. Since only one of four transformed lines showed an increase in yield, we can not yet conclude that increased leaf SPS activity leads to increased yield. However, increased leaf SPS activity appears to result in increased fruit sugar content since all three lines with increased leaf SPS usually also had increased fruit sugars. Received: 18 November 1996 / Accepted: 22 January 1997     

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.     

Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli

Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5′-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ∼42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ∼43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ∼45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.     

Development of Q-chromosome-specific DNA markers in tobacco and their use for identification of a tobacco monosomic line

We developed seven Q-chromosome-specific DNA markers in Nicotiana tabacum by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis using two hybrid lines, and we were able to identify tobacco monosomic plants among F1 progeny derived from the cross N. tabacum Haplo-QxN. tabacum cv. Samsun NN using Q-chromosome-specific DNA markers. Based on the results, we discuss the roles of the Q chromosome in embryo sac development and embryogenesis. Here, we propose a new method for identifying DNA markers for a particular chromosome in the genus Nicotiana.     

Integrative, xylE-based promoter probe vectors for use in Streptomyces

Two promoter probe plasmid vectors, designated pIPP1 and pIPP2, were constructed from the existing plasmids pXE4 and pSET152. pIPP1 and 2 use the xylE gene of Pseudomonas putida as a reporter and can be transferred to streptomycetes by conjugation from Escherichia coli. The function of these plasmids as promoter probes was demonstrated in Streptomyces antibioticus and Streptomyces coelicolor using the phenoxazinone synthase and polynucleotide phosphorylase promoters from S. antibioticus. xylE activity could be detected in colonies on agar plates or via the in vitro assay for catechol dioxygenase. The integration into the S. antibioticus chromosome of the constructs containing the phsA promoter was verified by Southern blotting. The presence of the bla locus in pIPP1 allows the recovery of putative promoters by marker rescue.     

A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation

Agrobacterium rhizogenes, along with A. tumefaciens, has been used to affect genetic transformation in plants for many years. Detailed studies conducted in the past have uncovered the basic mechanism of foreign gene transfer and the implication of Ri/Ti plasmids in this process. A number of reviews exist describing the usage of binary vectors with A. tumefaciens, but no comprehensive account of the numerous binary vectors employed with A. rhizogenes and their successful applications has been published till date. In this review, we recollect a brief history of development of Ri-plasmid/Ri-T-DNA based binary vectors systems and their successful implementation with A. rhizogenes for different applications. The modification of native Ri plasmid to introduce foreign genes followed by development of binary vector using Ri plasmid and how it facilitated rapid and feasible genetic manipulation, earlier impossible with native Ri plasmid, have been discussed. An important milestone was the development of inducible plant expressing promoter systems which made expression of toxic genes in plant systems possible. The successful application of binary vectors in conjunction with A. rhizogenes in gene silencing and genome editing studies which are relatively newer developments, demonstrating the amenability and adaptability of hairy roots systems to make possible studying previously intractable research areas have been summarized in the present review.