Low frequency of T‐DNA based activation tagging in <i>Arabidopsis</i> is correlated with methylation of CaMV 35S enhancer sequences

Chalfun-Júnior, Antônio; Mes, Jurriaan J.; Mlynárová, Ľudmila; Aarts, Mark G. M.; Angenent, Gerco C.

doi:10.1016/s0014-5793(03)01300-0

Cited by 31 publications

(18 citation statements)

References 37 publications

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“…The vector set was primarily anticipated for transient studies in which the 35S promoter is still a powerful driver of expression despite its inherent silencing issues when used in stable transformants (Chalfun-Junior et al, 2003). Nevertheless, the T1 generation of stably transformed Arabidopsis seedlings was analyzed: out of 74 Basta-resistant seedlings that were screened for fluorescence signals, 10% expressed both mEGFP-VAMP723 and mCherry-SYP121 (Fig.…”

Section: Dual Expression From One T-dnamentioning

confidence: 99%

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

Hecker

Wallmeroth²,

Peter³

et al. 2015

Plant Physiol.

100

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Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.Two technological advances allowed the field of modern cell biology to emerge: the development of high-resolution microscopes and the groundbreaking work in the development of fluorescent proteins (FPs) that were originally isolated from jellyfish (for review, see Day and Davidson, 2009;Zimmer, 2009). Genetically encoded FPs can be fused directly to proteins of choice, offering insights into their functional properties by allowing researchers to monitor subcellular localization, chemical environment, interaction, movement, and/or turnover rate.Research on the FPs themselves has exploded in the past two decades, and a range of technological improvements to and variations of the FPs were generated. These improvements include novel spectral properties from all areas of the color palette and from a diverse range of organisms. They also extend to a multitude of parameters, such as brightness, folding efficiency, chromophore oxidation rate, quantum yield, pH, and photostability (Day and Davidson, 2009). Taken together, these optimizations further the possibilities of available methods or prompt the development of new techniques.In 1946, the German scientist Theodor Förster laid the theoretical foundation for resonance energy transfer, now known as Förster resonance energy transfer (FRET;Forster, 1946). According to this theory, a donor chromophore in an excited state can transfer the excitation energy to a nearby acceptor chromophore via dipole-dipole coupling without the emission of a photon (for review, see Clegg, 2009;Ishikawa-Ankerhold et al., 2012). FRET depends on the spectral overlap between the emission spectrum of the donor and the absorbance spectrum of the acceptor chromophores. It also depends on the fluorescence quantum yield and the excited state lifetime of the donor in the absence of an acceptor as well as the relative orientation of the two chromophores. Most importantly, FRET ef...

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Section: Dual Expression From One T-dnamentioning

confidence: 99%

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

Hecker

Wallmeroth²,

Peter³

et al. 2015

Plant Physiol.

100

View full text Add to dashboard Cite

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“…Based on the findings described by Marsch-Martinez et al (2002) and our own observations on the methylation of T-DNA based activation tagging using the quadruple 35S enhancer (Chalfun-Junior et al, 2003), we propose a transposon-based tagging strategy as being the most promising. Hopefully this strategy, in which the described SHP2 enhancer could be used as tissue-specific enhancer, will lead to a better understanding of pod dehiscence and to novel approaches to control the shattering behaviour of crops.…”

Section: Discussionmentioning

confidence: 95%

“…Requirements for such a strategy are that: i) the used enhancer frag- 404 Chalfun-Junior et al ment shows tissue specificity, avoiding unwanted side effects in other tissues ii) the activity of the enhancer should still be functional at least at several kbs from the natural promoter or gene, increasing the efficiency that genes will be activated, and iii) the insertion in the genome should be carried out by a system that minimizes the chances of complex insertion integrations (Nacry et al, 1998), which can cause silencing of the introduced enhancer (Chalfun-Junior et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

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Analysis of the SHP2 enhancer for the use of tissue specific activation tagging in Arabidopsis thaliana

et al. 2006

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Activation tagging is a powerful tool to identify new mutants and to obtain information about possible biological functions of the overexpressed genes. The quadruple cauliflower mosaic virus (CaMV) 35S enhancer fragment is a strong enhancer, which is most commonly used for this purpose. However, the constitutive nature of this enhancer may generate lethal mutations or aberrations in different plant organs by the same overexpressed gene. A tissue-specific activation tagging approach may overcome these drawbacks and may also lead more efficiently to the desired phenotype. For this reason the SHATTERPROOF2 (SHP2) promoter fragment was analysed for enhancer activity. The SHP2 gene is involved in dehiscence zone development and expressed during silique development. The aim of the experiments described here was to identify a dehiscence zone specific enhancer that could be used for tissue-specific activation tagging. The chosen SHP2 enhancer fragment was found to be expressed predominantly in the dehiscence zone and showed enhancer activity as well as ectopic expression activity. This activity was not influenced by its orientation towards the promoter and it was still functional at the largest tested distance of 2.0 kb. Based on these results, the SHP2 enhancer fragment can potentially be used in a tissue-specific activation tagging approach to identify new Arabidopsis mutants with an altered dehiscence zone formation.

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“…The pCAMBIA1301 vector displays a hygromycin gene and a GUS gene driven by a double 35S gene from the cauliflower mosaic virus. The promoter controls the GUS reporter gene and a NOS terminator (Chalfun-Junior et al, 2003). The activity of the GPAT promoter was studied using this vector by replacing the CaMV35S promoter with the GPAT promoter.…”

Section: Deletion Analysis Of Gpat Promoter and Construction Of Recommentioning

confidence: 99%

Characterization of the promoter region of the glycerol-3-phosphate-O-acyltransferase gene in Lilium pensylvanicum

Chen¹,

Zhang²,

Qi³

et al. 2017

Turk J Biol

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Yadav et al., 2014), while associations between the promoters of these genes and resistance have not yet been reported. Plant promoters play an important role in the regulation of gene expression in plants ( Kim et al., 2013;Reynolds et al., 2013). Promoters are located on the 5'-flanking region of structural genes, recognized by RNA polymerase and combined with template DNA, ensuring that transcription starts effectively and accurately. The promoter consists of two parts: the core region and upstream regulator region (Zhu and Li, 2002). There are abundant cis-acting elements that participate in gene regulation in the promoter (Razdan et al., 2013;Sarvestani et al., 2013). Transient transformation and histochemical staining can contribute to promoter functional analysis (Koia et al., 2013). Cloning and functional studies related to the promoters of the key genes in improving resistance of plants will aid us in understanding its signal transduction pathways and gene expression patterns.According to gene expression, promoters can be divided into two categories: constitutive promoters and specific Abstract: Cold environmental conditions influence the growth and development of plants, causing crop reduction or even plant death. Under stress conditions, cold-inducible promoters regulate cold-related gene expression as a molecular switch. Recent studies have shown that the chloroplast-expressed GPAT gene plays an important role in determining cold sensitivity. However, the mechanism of the transcriptional regulation of GPAT is ambiguous. The 5'-flanking region of GPAT with length of 1494 bp was successfully obtained by chromosome walking from Lilium pensylvanicum. The cis-elements of GPAT promoters were predicted and analyzed by a plant cisacting regulatory DNA element database. There exist core promoter regions including TATA-box and CAAT-box and transcription regulation regions, which involve some regulatory elements such as I-box, W-box, MYB, MYC, and DREB. Full-length and four 5'-deletion fragments linked with GUS vectors were constructed and transformed into Nicotiana tabacum by Agrobacterium-mediated transformation. Transient transformation and histochemical staining of leaves indicated that the activity of the GPAT promoter was strong and induced by low-temperature stress. The deletion of a -294 bp region suggested that the DRE-motif was functionally an essential element for cold induction, and the deletion of -1494 bp and -1194 bp regions suggested that negative regulation exists in the promoter. Our results show that the GPAT gene promoter is a key regulator under cold stress and we think that this study will have significant impact on lily molecular breeding and improving the resistance of plants.

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Low frequency of T‐DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences

Cited by 31 publications

References 37 publications

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

Analysis of the SHP2 enhancer for the use of tissue specific activation tagging in Arabidopsis thaliana

Characterization of the promoter region of the glycerol-3-phosphate-O-acyltransferase gene in Lilium pensylvanicum

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