Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

Paolacci, Anna Rita; Tanzarella, O. A.; Porceddu, E.; Ciaffi, M.

doi:10.1186/1471-2199-10-11

Cited by 567 publications

(486 citation statements)

References 64 publications

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“…Previous studies showed that Ta2291 and Ta54227 were most stably expressed in different wheat tissues and the normalization factor derived from these two reference genes further improved the reliability of reference expression levels as compared to the single reference genes (Paolacci et al, 2009). Therefore, the geometric mean of Ta2291 and Ta54227 was used for normalization of HYD gene and homeolog expression.…”

Section: Real-time Qpcr Analysismentioning

confidence: 99%

Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains

Qin

Zhang²,

Dubcovsky³

et al. 2012

Plant Mol Biol

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Carotenoid β-hydroxylases attach hydroxyl groups to the β-ionone rings (β-rings) of carotenoid substrates, resulting in modified structures and functions of carotenoid molecules. We cloned and characterized two genes (each with three homeologs), HYD1 and HYD2, which encode β-hydroxylases in wheat. The results from bioinformatic and nested degenerate PCR analyses collectively suggest that HYD1 and HYD2 may represent the entire complement of non-heme diiron β-hydroxylases in wheat. The homeologs of wheat HYDs exhibited major β-ring and minor ε-ring hydroxylation activities in carotenoid-accumulating E. coli strains. Distinct expression patterns were observed for different HYD genes and homeologs in vegetative tissues and developing grains of tetraploid and hexaploid wheat, suggesting their functional divergence and differential regulatory control in tissue-, grain development-, and ploidy-specific manners. An intriguing observation was that the expression of HYD1, particularly HYD-B1, reached highest levels at the last stage of tetraploid and hexaploid grain development, suggesting that carotenoids (at least xanthophylls) were still actively synthesized in mature grains. This result challenges the common perception that carotenoids are simply being turned over during wheat grain development after their initial biosynthesis at the early grain development stages. Overall, this improved understanding of carotenoid biosynthetic gene expression and carotenoid metabolism in wheat grains will contribute to the improvement of the nutritional value of wheat grains for human consumption.

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Section: Real-time Qpcr Analysismentioning

confidence: 99%

Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains

Qin

Zhang²,

Dubcovsky³

et al. 2012

Plant Mol Biol

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“…C, Expression of TaSAP5-A, TaSAP5-B, and TaSAP5-D in response to osmotic stress created through 20% PEG6000 solution treatment. The expression levels were normalized to that of the wheat b-ACTIN gene (Paolacci et al, 2009). Data are means 6 SD (n = 3).…”

Section: Tasap5 Overexpression Confers Drought Tolerance In Arabidopsmentioning

confidence: 99%

The E3 Ligase TaSAP5 Alters Drought Stress Responses by Promoting the Degradation of DRIP Proteins

et al. 2017

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In Arabidopsis (Arabidopsis thaliana) plants growing under normal conditions, DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) is present at low levels because it is ubiquitinated and destabilized by DREB2A INTERACTING PROTEIN1 (DRIP1) and DRIP2 through 26S proteasome-mediated proteolysis. Drought stress counteracts the ubiquitination and proteolysis of DREB2A, thus allowing the accumulation of sufficient amounts of DREB2A protein to activate downstream gene expression. The mechanisms leading to drought stress-mediated DREB2A accumulation are still unclear. Here, we report that the wheat (Triticum aestivum) TaSAP5 protein, which contains an A20/AN1 domain, acts as an E3 ubiquitin ligase to mediate DRIP degradation and thus increase DREB2A protein levels. Drought induces TaSAP5 expression in wheat, and TaSAP5 overexpression in Arabidopsis and wheat seedlings increased their drought tolerance, as measured by survival rate and grain yield under severe drought stress. TaSAP5 can interact with and ubiquitinate TaDRIP, as well as AtDRIP1 and AtDRIP2, leading to their subsequent degradation through the 26S proteasome pathway. Consistent with this, TaSAP5 overexpression enhances DRIP degradation and increases the levels of DREB2A protein and its downstream targets. These results suggest that TaSAP5 acts to link drought with DREB2A accumulation and illustrate the molecular mechanisms involved in this process.

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“…Transcription levels of core starch biosynthesis genes were quantified with a CFX96 Real-Time PCR Detection System (Bio-Rad) using the intercalating dye SYBR-green following the 2(-Delta Delta C(T)) method [58]. ADP-RF (ADP-ribosylation factor) was used as the reference gene according to previous report [59,60]. qRT-PCR was performed in a 20 μL volume containing 10 μL 2×SYBR®Premix Ex Taq™ (TaKaRa), 2 μL 10-fold diluted cDNA, 0.…”

Section: Qrt-pcrmentioning

confidence: 99%

Comparative proteome analysis of A- and B-type starch granule-associated proteins in bread wheat (Triticum aestivum L.) and Aegilops crassa

Cao

Xing

Chen

et al. 2015

Journal of Proteomics

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Comparative proteome analysis of A-and B-type starch granule-associated proteins in bread wheat (Triticum aestivum L.) and Aegilops crassa, Journal of Proteomics (2014Proteomics ( ), doi: 10.1016Proteomics ( /j.jprot.2014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E DM A N U S C R I P T Our results demonstrated that the large granule size is associated with higher activities of multiple starch biosynthesis enzymes. The phosphorylation of starch biosynthesis enzymes is related with the formation of B-type starch granules. Biological significanceAnalysed the proteome, transcriptome and phosphorylation of core starch granule biosynthesis enzymes and provided new insights into the differential mechanisms underlying the A-and B-type starch granules biosyntheses. A C C E P T E D

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Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

Cited by 567 publications

References 64 publications

Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains

Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains

The E3 Ligase TaSAP5 Alters Drought Stress Responses by Promoting the Degradation of DRIP Proteins

Comparative proteome analysis of A- and B-type starch granule-associated proteins in bread wheat (Triticum aestivum L.) and Aegilops crassa

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