Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations

Remans, Tony; Smeets, Karen; Opdenakker, Kelly; Mathijsen, Dennis; Vangronsveld, Jaco; Cuypers, Ann

doi:10.1007/s00425-008-0706-4

Cited by 309 publications

(237 citation statements)

References 32 publications

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“…For normalization of the PAA2 transcript level we used the two control genes yellow-leaf-specific 8 (YLS8) and the gene of a SAND family protein (see supplemental Table S1). Neither transcript is affected by Cu in Arabidopsis (26). The result for YLS8 is presented in Fig.…”

Section: Rna Extraction Quantitative Real-time Pcr (Qrt-pcr) Analysimentioning

confidence: 91%

Plastocyanin Controls the Stabilization of the Thylakoid Cu-transporting P-type ATPase PAA2/HMA8 in Response to Low Copper in Arabidopsis

Tapken

Ravet

Pilon

2012

Journal of Biological Chemistry

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Background:The P 1B -type ATPase PAA2/HMA8 transports Cu into the thylakoid lumen of the chloroplast for delivery to plastocyanin. Results: PAA2/HMA8 stability is affected by plastocyanin and by the Cu content within the chloroplast. Conclusion: The abundance of PAA2/HMA8 is subject to feedback control. Significance: A newly identified layer of Cu-homeostasis via a novel regulatory mechanism of an organellar Cu-transporter.

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Section: Rna Extraction Quantitative Real-time Pcr (Qrt-pcr) Analysimentioning

confidence: 91%

Plastocyanin Controls the Stabilization of the Thylakoid Cu-transporting P-type ATPase PAA2/HMA8 in Response to Low Copper in Arabidopsis

Tapken

Ravet

Pilon

2012

Journal of Biological Chemistry

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“…The full-length AtYchF1 cDNA clone was amplified from total cDNA of Col-0 by gene-specific primers. The relative gene expression was calculated by using the 2 -ΔΔCT method (28) and normalized with the A. thaliana UBQ10 gene (29). The G4 motif of OsYchF1 or AtYchF1 was converted from the noncanonical sequence (NMSE) to the canonical sequence (NKSD) by using PCR site-directed mutagenesis with specially designed primers.…”

Section: Methodsmentioning

confidence: 99%

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

Cheung

Miao

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life.G TP-binding proteins (G proteins) play important roles in diverse fundamental biological processes in living organisms, including signal transduction, cell division, development, intracellular transport, translation, and others (1, 2). The functions and working mechanisms of some ancestral G proteins, which are believed to play essential roles in cellular processes, are still unknown. One such group is the Obg family, which features an N-terminal glycine-rich sequence, followed by a G domain for nucleotide binding and a C-terminal TGS (ThrRS, GTPase, and SpoT) domain that has nucleic acid binding affinity (3).A unique subgroup within the Obg family, the YchF proteins, exhibit relaxed nucleotide-binding specificities (4-6). All G proteins contain a G domain (composed of the G1-G5 motifs) for GTP binding and hydrolysis (4). The G4 motif (canonical sequence: NKxD) confers the specificity for binding GTP (4). In YchFs, the noncanonical G4 sequence (NxxE) is correlated with the loss of nucleotide-binding specificities (4, 5). In some cases, ATP is the preferred ligand (4, 5). However, the physiological significance of the ancient and evolutionarily conserved YchF proteins having the relaxed binding specificities for both ATP and GTP is still unknown.Only a few reports touch on the biological functions of YchF proteins and most focus on human and microorganisms (7-9). The YchF proteins may play a role in iron utilization and regulation of the Ton system in Brucella melitensis (7), and in the growth of the procyclic forms of Trypanosoma cruzi (5). The human YchF homolog hOLA...

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“…The primer efficiencies were calculated as E = 10 -1/slope on a standard curve generated using a 4-or 2-fold dilution series over at least five dilution points of cDNA. The expression analysis of GAPC genes was performed by the Pfaffl method, using EF1a as the reference gene (Pfaffl, 2001;Remans et al, 2008).…”

Section: Rna Analysismentioning

confidence: 99%

Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Cadmium-Stressed Arabidopsis Roots

et al. 2013

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NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in the glycolytic pathway. It has been widely demonstrated that mammalian GAPDH, in addition to its role in glycolysis, fulfills alternative functions mainly linked to its susceptibility to oxidative posttranslational modifications. Here, we investigated the responses of Arabidopsis (Arabidopsis thaliana) cytosolic GAPDH isoenzymes GAPC1 and GAPC2 to cadmium-induced stress in seedlings roots. GAPC1 was more responsive to cadmium than GAPC2 at the transcriptional level. In vivo, cadmium treatments induced different concomitant effects, including (1) nitric oxide accumulation, (2) cytosolic oxidation (e.g. oxidation of the redox-sensitive Green fluorescent protein2 probe), (3) activation of the GAPC1 promoter, (4) GAPC1 protein accumulation in enzymatically inactive form, and (5) strong relocalization of GAPC1 to the nucleus. All these effects were detected in the same zone of the root tip. In vitro, GAPC1 was inactivated by either nitric oxide donors or hydrogen peroxide, but no inhibition was directly provided by cadmium. Interestingly, nuclear relocalization of GAPC1 under cadmium-induced oxidative stress was stimulated, rather than inhibited, by mutating into serine the catalytic cysteine of GAPC1 (C155S), excluding an essential role of GAPC1 nitrosylation in the mechanism of nuclear relocalization, as found in mammalian cells. Although the function of GAPC1 in the nucleus is unknown, our results suggest that glycolytic GAPC1, through its high sensitivity to the cellular redox state, may play a role in oxidative stress signaling or protection in plants.

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Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations

Cited by 309 publications

References 32 publications

Plastocyanin Controls the Stabilization of the Thylakoid Cu-transporting P-type ATPase PAA2/HMA8 in Response to Low Copper in Arabidopsis

Plastocyanin Controls the Stabilization of the Thylakoid Cu-transporting P-type ATPase PAA2/HMA8 in Response to Low Copper in Arabidopsis

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Cadmium-Stressed Arabidopsis Roots

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