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Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of a lack of characterized insulators. In this study, we took a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between an enhancer and a promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation property observed across the CTCF insulator in the mammalian genome. Taken together, our findings support that CW198 acts as an enhancer-blocking insulator in both Arabidopsis and tobacco. The significance of the present findings and their relevance to the mitigation of mutual interference between enhancers and promoters, as well as multiple promoters in transgenes, is discussed. [ABSTRACT FROM AUTHOR]

The genetic engineering of agronomic traits requires an array of highly specific and tightly regulated promoters that drive expression in floral tissues. In this study, we isolated and characterized two tobacco APETALA1-like ( AP1-like) promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using the GUS reporter system, along with tissue-specific ablation analyses. Our results demonstrated that the two promoters are active in floral inflorescences but not in vegetative apical meristems or other vegetative tissues, as reflected by strong GUS staining and DT- A-mediated ablation of apical shoot tips during reproductive but not vegetative growth. We also showed that the NtAP1Lb1 promoter was more active than NtAP1La in inflorescences, as the former yielded higher frequencies and greater phenotypic evidence of tissue ablation compared to the latter. We further revealed that both promoters were uniformly expressed in the meristems of stage 1 and 2 floral buds, but were differentially expressed in floral organs later during development. While NtAP1La was found to be active in stage 4-5 carpels, later becoming confined to ovary tissue from stage 9 onwards, NtAP1Lb1 activity was apparent in all floral organs from stages 3 to 7, becoming completely absent in all floral organs from stage 11 onward. Therefore, it seems that the two tobacco promoters have acquired similar but distinct inflorescence-, floral meristem- and floral organ-specific and development-dependent regulatory features without any leaky activity in vegetative tissues. These features are novel and have rarely been observed in other flower-specific promoters characterized to date. The potential application of these promoters for engineering sterility, increasing biomass production and modifying flower architecture, as well as their putative use in flower-specific transgene excision, will be discussed. [ABSTRACT FROM AUTHOR]

The potential for pollen-mediated transgene flow into wild or closely related species has provoked unease in terms of transgenic modification of agricultural plant species. One approach to remedy this situation in species whose seeds and fruits are not of particular value is to engineer male sterility into the transgenic lines. In this study, three meiosis-critical genes, namely AHP2, AtRAD51C and SWITCH1 ( SWI), were chosen as silencing targets to test the feasibility of incorporating sterility into plants using an RNAi-based approach. Our results indicated that the silencing of each of these genes via hairpin RNA (termed AHPi, RAD51Ci and SWIi lines) in Arabidopsis thaliana yielded a proportion of transgenic plants exhibiting a similar 'partially sterile' phenotype in which less than 50% of pollen was viable. In addition, a 'sterile' phenotype was also evident in a minority of RAD51Ci and SWIi, but not AHPi, lines in which plants yielded no seeds and either produced inviable pollen ( RAD51Ci lines) or displayed a complete absence of pollen ( SWIi lines). This suggests that AtRAD51C and SWI may function at distinct stages of meiosis. Further analyses of SWIi lines demonstrated that the 'sterile' phenotype was associated with a substantial reduction in the level of targeted gene transcript in floral tissues and resulted from sterility of the male, but not female gametes. This work demonstrates that generating male sterility through the silencing of key genes involved in the regulation of meiosis is feasible, and its advantages and potential applications for transgene containment are discussed. [ABSTRACT FROM AUTHOR]

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the AtAGIP promoter derived from the Arabidopsis AGAMOUS ( AG) second intron/enhancer specifies a carpel- and stamen-specific expression in its native host species, but not in heterologous species such as tobacco, which restricts its application in the engineering of male and female sterility. These findings also imply that the AG regulatory mechanism that has evolved in Arabidopsis may, to some extent, have diverged from that of tobacco. To test whether a similar chimeric promoter created using the AG second intron/enhancer can overcome this barrier of evolutionary divergence in closely related species, we generated forward- and reverse-oriented chimeric promoters, fPtAGIP and rPtAGIP, from the petunia AG second intron/enhancer ( PtAGI) fragment and tested them in tobacco, which, like petunia, belongs to the Solanaceae family. Our results demonstrate that both fPtAGIP and rPtAGIP confer similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues in tobacco as revealed by tissue-specific gene expression and tissue ablation. This pattern resembles that driven by the AtAGIP in Arabidopsis and indicates that the AG regulatory mechanism is more conserved between tobacco and petunia than between tobacco and Arabidopsis. The petunia-derived promoters also exhibited petal-specific activity, and their activities in floral organs were substantially influenced by the orientation of the PtAGI enhancer, with reverse-oriented enhancers displaying approximately double the effectiveness of forward-oriented enhancers. These two properties are novel and have not been observed previously with AtAGIP promoters. Furthermore, we found that PtAGIP promoter-driven tissue ablation is effective for engineering complete sterility in plants, and the resulting sterile trait is stable for at least three mitotic generations at various temperature regimes, which is important for the complete containment of seed-, pollen-, and fruit-mediated gene flow in field conditions. [ABSTRACT FROM AUTHOR]

Enhancers are known to be capable of overriding the specificity of nearby promoters in a distance-dependent manner, which is problematic when multiple promoters coexist in a single transgene unit. In an attempt to determine whether enhancer activation function is inversely related to its distance from the target promoter, we inserted 1-, 2-, and 4-kb bacteriophage λ fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter ( AGIP) fused to the β-glucuronidase ( GUS) coding region. In the absence of an insert sequence, the 35S enhancer activates AGIP-driven GUS expression in vegetative tissues of transgenic Arabidopsis thaliana lines. Moreover, neither the 2-kb nor the 4-kb λ fragment was able to block GUS expression in transgenic leaves, implying that the 35S enhancer can override a distance barrier of at least 4 kb in our system. Unexpectedly, insertion of the 1-kb λ insert into the same site resulted in diminished GUS expression in transgenic leaves. Our data indicate that this fragment functions as a true enhancer-blocking insulator that could potentially be utilized to minimize enhancer–promoter interference between multiple transcriptional units within a plasmid vector during plant transformation experiments. [ABSTRACT FROM AUTHOR]

The UVH6 gene from Arabidopsis thaliana is predicted to function in transcription regulation, based on known activities of its human and yeast homologs. In this study, we show that uvh6-1 mutants are ultra-sensitive to cold and suggest that this defect results from reduced expression of cold-stress genes. Comparison of mRNA levels in cold-treated wild-type and uvh6-1 plants reveals that expression of two cold-stress genes ( Cor6.6 and Cor15a) is impaired in the mutant. In contrast, the mutant shows normal cold induction of three transcription factor genes ( CBF1, 2, 3), which regulate these Cor genes, and normal induction of several additional CBF-targeted genes. Thus, we propose that UVH6 promotes cold resistance by specifically regulating transcription of Cor6.6 and Cor15a genes. We further find features among the regulatory sites in the Cor6.6 and Cor15a promoters which suggest unique regulation of these genes. [ABSTRACT FROM AUTHOR]

Gain-of-function studies have shown that ectopic expression of the BABY BOOM ( BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis ( Arabidopsis thaliana (L.) Heynh) and Brassica napus ( B. napus L.) seedlings. Here we examined the effect of ectopic BBM expression on the development and regenerative capacity of tobacco ( Nicotiana tabacum L.) through heterologous expression of Arabidopsis and B. napus BBM genes. 35S::BBM tobacco lines exhibited a number of the phenotypes previously observed in 35S::BBM Arabidopsis and B. napus transgenics, including callus formation, leaf rumpling, and sterility, but they did not undergo spontaneous somatic embryogenesis. 35S::BBM plants with severe ectopic expression phenotypes could not be assessed for enhanced regeneration at the seedling stage due to complete male and female sterility of the primary transformants, therefore fertile BBM ectopic expression lines with strong misexpression phenotypes were generated by expressing a steroid-inducible, post-translationally controlled BBM fusion protein (BBM:GR) under the control of a 35S promoter. These lines exhibited spontaneous shoot and root formation, while somatic embryogenesis could be induced from in-vitro germinated seedling hypocotyls cultured on media supplemented with cytokinin. Together these results suggest that ectopic BBM expression in transgenic tobacco also activates cell proliferation pathways, but differences exist between Arabidopsis/ B. napus and N. tabacum with respect to their competence to respond to the BBM signalling molecule. [ABSTRACT FROM AUTHOR]

Abstract. We have created a DNA construct, TREGED (transposon-and recombinase-mediated genome deletion), that will automatically induce deletions in plant genomes. TREGED contains the maize Ac/Ds transposon, the yeast R-RS site-specific recombination system, the bacterial tetR repression systems, a novel artificial superintron, and the marker genes GUS and Lc. The novelty of TREGED is that only one cross is required to trigger a sequence of events leading to deletion and, simultaneously, to a color assay to detect the deletion. Crossing is done to introduce Ac transposase which activates Ds transposition from TREGED to a nearby chromosome region. Ds transposition, in turn, activates recombination between an engineered RS site on TREGED and an RS site on the transposed Ds fragment, thus deleting the genome segment between TREGED and Ds. The recombination event also deletes Lc or GUS and part of tetR, which triggers expression of GUS or Lc color genes for an upstream or downstream deletion respectively. Each TREGED insertion site will produce multiple kinds of deletions identifiable by inspecting a single F[sub 1] plant and its progeny for colored tissue. The color markers can also be used to differentiate between deletion and other more rare events such as translocation and inversion. We anticipate TREGED will greatly simplify the steps required to obtain useful deletions - eventually allowing the creation of detailed deletion libraries. Such libraries will be particularly useful for analysis of gene and chromatin function in plant species with large genomes. [ABSTRACT FROM AUTHOR]

Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5′ or 3′ end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5′ end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region. [ABSTRACT FROM AUTHOR]