Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plantsin vivo

Hedtke, Boris; Meixner, Martin; Gillandt, Sabine; Richter, Ekkehard; Börner, Thomas; Weihe, Andreas

doi:10.1046/j.1365-313x.1999.00393.x

Cited by 57 publications

(37 citation statements)

References 18 publications

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“…The limited recognition of NEP promoters by RpoTp in vitro, which may reflect the absence of RpoTp cofactors in our assays, is contrasted by an intrinsic specificity of this enzyme for several mitochondrial promoters. This finding is unexpected, as in vitro and in vivo import experiments suggest RpoTp to strictly localize to plastids (Hedtke et al, 1997(Hedtke et al, , 1999. Remarkably, Pycf1-39, which is the only NEP promoter activating RpoTp in vitro, resembles in sequence the mitochondrial promoter Patp8-228/ 226 that is also efficiently recognized by both RpoTm and RpoTp in vitro (Figures 4 and 7, Table 1).…”

Section: Selective Promoter Recognition By Rpotm and Rpotpmentioning

confidence: 95%

“…RpoT genes have been identified in the nuclear genomes of various angiosperms (Hess and Bö rner, 1999;Weihe, 2004;Shiina et al, 2005;Liere and Bö rner, 2007) and in the moss Physcomitrella patens (Kabeya et al, 2002;Richter et al, 2002). According to in vitro and in vivo import studies, the small family of three RpoT genes in Arabidopsis thaliana encodes a mitochondrial RNAP (RpoTm), a plastidial enzyme (RpoTp), and a polypeptide suggested to be imported into both mitochondria and plastids (RpoTmp) (Hedtke et al, 1997(Hedtke et al, , 1999(Hedtke et al, , 2000. The conservation of functionally critical amino acid positions of the T7 enzyme (McAllister and Raskin, 1993;Sousa et al, 1993) in RpoTm, RpoTmp, and RpoTp as well as in other plant RpoT polypeptides (Hess and Bö rner, 1999) and nonspecific transcription activity of recombinant RpoTmp in vitro (Hedtke et al, 2000) argue for RpoT gene products to function in organellar RNA synthesis.…”

Section: Introductionmentioning

confidence: 99%

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Arabidopsis Phage-Type RNA Polymerases: Accurate in Vitro Transcription of Organellar Genes

Kühn¹,

Bohne²,

Liere

et al. 2007

The Plant Cell

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The T7 bacteriophage RNA polymerase (RNAP) performs all steps of transcription, including promoter recognition, initiation, and elongation as a single-polypeptide enzyme. Arabidopsis thaliana possesses three nuclear-encoded T7 phage-type RNAPs that localize to mitochondria (RpoTm), plastids (RpoTp), or presumably both organelles (RpoTmp). Their specific functions are as yet unresolved. We have established an in vitro transcription system to examine the abilities of the three Arabidopsis phage-type RNAPs to synthesize RNA and to recognize organellar promoters. All three RpoT genes were shown to encode transcriptionally active RNAPs. RpoTmp displayed no significant promoter specificity, whereas RpoTm and RpoTp were able to accurately initiate transcription from overlapping subsets of mitochondrial and plastidial promoters without the aid of protein cofactors. Our study strongly suggests RpoTm to be the enzyme that transcribes most, if not all, mitochondrial genes in Arabidopsis. Intrinsic promoter specificity, a feature that RpoTm and RpoTp share with the T7 RNAP, appears to have been conserved over the long period of evolution of nuclear-encoded mitochondrial and plastidial RNAPs. Selective promoter recognition by the Arabidopsis phage-type RNAPs in vitro implies that auxiliary factors are required for efficient initiation of transcription in vivo.

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Section: Selective Promoter Recognition By Rpotm and Rpotpmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Arabidopsis Phage-Type RNA Polymerases: Accurate in Vitro Transcription of Organellar Genes

Kühn¹,

Bohne²,

Liere

et al. 2007

The Plant Cell

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“…For the detection of lignin, roots were stained with 2% phloroglucinolHCl as described by Schrick et al (2004). For visualization of mitochondria, roots were stained with MitoTracker Red CMXRos (M-7512; Invitrogen) as described by Hedtke et al (1999). The membrane potential of mitochondria was assessed in hypocotyl cells using the cationic lipophilic fluorescent dye TMRM.…”

Section: Histochemical Analysesmentioning

confidence: 99%

Defense Activated by 9-Lipoxygenase-Derived Oxylipins Requires Specific Mitochondrial Proteins

et al. 2012

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9-Lipoxygenases (9-LOXs) initiate fatty acid oxygenation, resulting in the formation of oxylipins activating plant defense against hemibiotrophic pathogenic bacteria. Previous studies using nonresponding to oxylipins (noxy), a series of Arabidopsis (Arabidopsis thaliana) mutants insensitive to the 9-LOX product 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), have demonstrated the importance of cell wall modifications as a component of 9-LOX-induced defense. Here, we show that a majority (71%) of 41 studied noxy mutants have an added insensitivity to isoxaben, an herbicide inhibiting cellulose synthesis and altering the cell wall. The specific mutants noxy2, noxy15, and noxy38, insensitive to both 9-HOT and isoxaben, displayed enhanced susceptibility to Pseudomonas syringae DC3000 as well as reduced activation of salicylic acid-responding genes. Map-based cloning identified the mutation in noxy2 as At5g11630 encoding an uncharacterized mitochondrial protein, designated NOXY2. Moreover, noxy15 and noxy38 were mapped at the DYNAMIN RELATED PROTEIN3A and FRIENDLY MITOCHONDRIA loci, respectively. Fluorescence microscopy and molecular analyses revealed that the three noxy mutants characterized exhibit mitochondrial dysfunction and that 9-HOT added to wild-type Arabidopsis causes mitochondrial aggregation and loss of mitochondrial membrane potential. The results suggest that the defensive responses and cell wall modifications caused by 9-HOT are under mitochondrial retrograde control and that mitochondria play a fundamental role in innate immunity signaling.

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“…(B) Testing the GFP localization of the pTAR-GFP construct shown in (A). RpoT;3 is a positive control for targeting to plastids (Hedtke et al, 1999), whereas pOL-LT-GFP (full name, pOL-LT-GFP-L64T65; a gift from Ian Small) is the basic vector used to generate the pTAR-GFP construct in which GFP is localized to the cytosol and the nucleoplasm. GFP fluorescence was detected in plastids (p) with both pTAR-GFP and RpoT;3 and in the cytosol (c) with pOL-LT-GFP, as indicted by the arrows.…”

Section: Transgenic Arabidopsis Plants Expressing Fanes1 Produce Hydrmentioning

confidence: 99%

Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants[W]

Aharoni¹,

Giri²,

et al. 2003

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Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40-to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae , the FaNES1 -expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants.

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Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plantsin vivo

Cited by 57 publications

References 18 publications

Arabidopsis Phage-Type RNA Polymerases: Accurate in Vitro Transcription of Organellar Genes

Arabidopsis Phage-Type RNA Polymerases: Accurate in Vitro Transcription of Organellar Genes

Defense Activated by 9-Lipoxygenase-Derived Oxylipins Requires Specific Mitochondrial Proteins

Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants[W]

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