Enzyme co-localization in pea leaf chloroplasts: glyceraldehyde-3-P dehydrogenase, triose-P isomerase, aldolase and sedoheptulose bisphosphatase

Anderson, Louise E.; Gatla, Nandita; Carol, Andrew A.

doi:10.1007/s11120-005-0790-2

Cited by 26 publications

(11 citation statements)

References 46 publications

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“…PGK1 also plays important roles in the glycolytic pathway, and PGK1 and GAPDH are potentially co-regulated [56]. In our data, however, their potential co-regulation was not significant.…”

Section: Discussioncontrasting

confidence: 78%

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

Skinner

Bennett

2012

BMC Mol Biol

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BackgroundThe selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata.ResultsThe expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments.ConclusionsWe recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.

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“…PGK1 also plays important roles in the glycolytic pathway, and PGK1 and GAPDH are potentially co-regulated [56]. In our data, however, their potential co-regulation was not significant.…”

Section: Discussioncontrasting

confidence: 78%

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

Skinner

Bennett

2012

BMC Mol Biol

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“…1-3). Both subunits are also colocalized with the Calvin cycle enzymes P-glycerate kinase (Anderson et al, 2003), triose-P isomerase, and aldolase (Anderson et al, 2005), but only the B subunit appears to be colocalized with transketolase (Anderson et al, 2006). Both subunits are co-localized with thioredoxin m, but only the A subunit is co-localized with thioredoxin f (Anderson and Carol, unpublished).…”

Section: Discussionmentioning

confidence: 99%

Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

Negi

Carol

Pandya

et al. 2008

Journal of Structural Biology

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In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH 2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A 2 B 2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex.

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“…Anderson and Carol (2004) found that labeling for RCA occurred in the chloroplast of pea plants. Hong et al (2005) also demonstrated that RCA protein was distributed in the chloroplast of bundle sheath and mesophyll cells of the C 4 plant Amaranthus tricolor.…”

Section: Discussionmentioning

confidence: 99%

Inoculation with Xanthomonas oryzae pv. oryzae induces thylakoid membrane association of Rubisco activase in Oryza meyeriana

Yang

Chen

Wang

et al. 2011

Journal of Plant Physiology

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Enzyme co-localization in pea leaf chloroplasts: glyceraldehyde-3-P dehydrogenase, triose-P isomerase, aldolase and sedoheptulose bisphosphatase

Cited by 26 publications

References 46 publications

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

Inoculation with Xanthomonas oryzae pv. oryzae induces thylakoid membrane association of Rubisco activase in Oryza meyeriana

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