Evaluation of candidate reference genes for qPCR in maize

Manoli, Alessandro; Sturaro, Alba; Trevisan, Sara; Quaggiotti, Silvia; Nonis, Alberto

doi:10.1016/j.jplph.2012.01.019

Cited by 220 publications

(170 citation statements)

References 64 publications

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“…Quantification of gene expression requires the use of appropriate reference genes and such references have been developed for some plant species [9,22,25,[43][44][45][46]. For rice, a number of genes have been used.…”

Section: Discussionmentioning

confidence: 99%

High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L. (ssp. indica and japonica) to abiotic stress conditions

2013

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Rice (Oryza sativa L.) is important to food security and is also an excellent model plant for numerous cereal crops. A functional genomics study in rice includes characterization of the expression dynamics of genes by quantitative real-time PCR (qPCR) analysis; this is a significant key for developing rice varieties that perform well in the face of adverse climate change. The qPCR analysis requires the use of appropriate reference genes in order to make any quantitative interpretations meaningful. Here, the new potential reference genes were selected from a huge public database of rice microarray experiments. The expression stability of 14 candidates and 4 conventional reference genes was validated by geNorm PLUS and NormFinder software. Seven candidates are superior to the conventionally used reference genes in qPCR and three genes can be used reliably for quantitating the expression of genes involved in abiotic stress responses. These high-quality references EP (LOC_Os05g08980), HNR (LOC_Os01g71770), and TBC (LOC_Os09g34040) worked very well in three indica genotypes and one japonica genotype. One of indica genotypes including the Jasmine rice, KDML105 developed in Thailand for which no reference genes have been reported until now.

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Section: Discussionmentioning

confidence: 99%

High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L. (ssp. indica and japonica) to abiotic stress conditions

2013

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“…Melting-curve analysis confirmed the absence of multiple products and primer dimers. Data were exported and analysed according to the method of Livak and Schmittgen (2001) and MIQE guidelines (Bustin et al 2009), using MEP (membrane proteinPB1A10.07c, primers: forward 5′-TGT ACT CGG CAA TGC TCT TG-3′ and reverse 5′-TTT GAT GCT CCA GGC TTA CC-3′), as the reference gene (Manoli et al 2012). Only transcripts showing amplification with quantification cycle (C q ) < 35 were selected for subsequent gene expression analysis.…”

Section: Reverse-transcription Quantitative Real-time Pcr (Rt-qpcr)mentioning

confidence: 99%

Identification and characterization of the BZR transcription factor family and its expression in response to abiotic stresses in Zea mays L.

et al. 2017

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Brassinosteroids (BRs) are plant specific steroidal hormones that play diverse roles in regulating a broad spectrum of plant growth and developmental processes, as well as, in responding to various biotic and abiotic stresses. Extensive research over the years has established stress-impact-mitigating role of BRs and associated compounds in different plants exposed to various abiotic and biotic stresses, suggesting the idea that they may act as immunomodulators, thus opening new approaches for plant resistance against hazardous environmental conditions. In this research the characterization of the transcriptional response of 11 transcription factors (TFs) belonging to BRASSINAZOLE-RESISTANT 1 (BZR1) TF family of Zea mays L. was analyzed in seedlings subjected to different stress conditions. Being important regulators of the BR synthesis, BZR TFs might have stress resistance related activities. However, no stress resistance related functional study of BZR TFs has been reported in maize so far. In silico analyses of the selected 11 TFs validated the features of their protein domains, where the highest degree of similarity observed with recognized BZR TFs of rice and Sorghum bicolor. Additionally, we investigated the organ-specific expression of 11 ZmBZR in maize seedlings. Five of them did not show any transcript accumulation, suggesting that ZmBZR expression might be regulated in a manner dependent on plant developmental stage. For the remaining six ZmBZR, their ubiquitous expression in the whole plant indicates they could function as growth regulators during maize development. More importantly, in response to various stress conditions, the spatial transcript accumulation of all ZmBZR varies along the plant. All six ZmBZR showed up-regulation against N starvation, hypoxia and salt stress. On the contrary, heat stress clearly down-regulated gene expression of all ZmBZR analysed. Consistently with the expression results, the distribution of stress-related cis-acting elements in the promoter of these genes inferred that the maize BZR TFs might play some roles in regulating the expression of the corresponding genes in response to multifarious stresses. In conclusion, these data reveal that BZR TFs have stress signaling activity in maize, in addition to their confirmed role in regulating plant physiology and morphology.

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“…In addition, RBCS transcripts were barely detectable in M cells, whereas rbcL transcripts accumulated to about 30% of the level observed in BS extracts. As controls for cell type cross-contamination, MDH was used as an M-specific transcript and ME as a BS-enriched transcript, and their levels were normalized to the validated control membrane protein P1A10.07c (Manoli et al, 2012), which is similarly expressed in BS and M cells based on laser-capture microdissection (Li et al, 2010). The M-to-BS ratio was found to average 3% for ME and 435% for MDH ( Fig.…”

Section: Ls Is a Controlled Epistasy Of Synthesis Subunit In Maizementioning

confidence: 99%

Ectopic Expression of Rubisco Subunits in Maize Mesophyll Cells Does Not Overcome Barriers to Cell Type-Specific Accumulation

et al. 2012

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In maize (Zea mays), Rubisco accumulates in bundle sheath but not mesophyll chloroplasts, but the mechanisms that underlie cell type-specific expression are poorly understood. To explore the coordinated expression of the chloroplast rbcL gene, which encodes the Rubisco large subunit (LS), and the two nuclear RBCS genes, which encode the small subunit (SS), RNA interference was used to reduce RBCS expression. This resulted in Rubisco deficiency and was correlated with translational repression of rbcL. Thus, as in C3 plants, LS synthesis depends on the presence of its assembly partner SS. To test the hypothesis that the previously documented transcriptional repression of RBCS in mesophyll cells is responsible for repressing LS synthesis in mesophyll chloroplasts, a ubiquitin promoter-driven RBCS gene was expressed in both bundle sheath and mesophyll cells. This did not lead to Rubisco accumulation in the mesophyll, suggesting that LS synthesis is impeded even in the presence of ectopic SS expression. To attempt to bypass this putative mechanism, a ubiquitin promoter-driven nuclear version of the rbcL gene was created, encoding an epitope-tagged LS that was expressed in the presence or absence of the Ubi-RBCS construct. Both transgenes were robustly expressed, and the tagged LS was readily incorporated into Rubisco complexes. However, neither immunolocalization nor biochemical approaches revealed significant accumulation of Rubisco in mesophyll cells, suggesting a continuing cell type-specific impairment of its assembly or stability. We conclude that additional cell type-specific factors limit Rubisco expression to bundle sheath chloroplasts.

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Evaluation of candidate reference genes for qPCR in maize

Cited by 220 publications

References 64 publications

High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L. (ssp. indica and japonica) to abiotic stress conditions

High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L. (ssp. indica and japonica) to abiotic stress conditions

Identification and characterization of the BZR transcription factor family and its expression in response to abiotic stresses in Zea mays L.

Ectopic Expression of Rubisco Subunits in Maize Mesophyll Cells Does Not Overcome Barriers to Cell Type-Specific Accumulation

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