| 1. |
- Guenin, Stephanie, et al.
(författare)
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Normalization of qRT-PCR data : the necessity of adopting a systematic, experimental conditions-specific, validation of references
- 2009
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 60:2, s. 487-493
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative ’housekeeping’ genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.
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| 2. |
- Gutierrez, Laurent, et al.
(författare)
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Combined networks regulating seed maturation
- 2007
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Ingår i: Trends in Plant Science. - : Elsevier BV. - 1360-1385 .- 1878-4372. ; 12:7, s. 294-300
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Forskningsöversikt (refereegranskat)abstract
- Seed maturation is an important phase of seed development during which embryo growth ceases, storage products accumulate, the protective tegument differentiates and tolerance to desiccation develops, leading to seed dormancy. The spatial and temporal regulation of all these processes requires the concerted action of several signaling pathways that integrate information from genetic programs, and both hormonal and metabolic signals. Recent genetic studies have identified some of the interactions that occur between four master regulators in Arabidopsis, increasing our knowledge of the control of the transcriptional program involved in seed maturation. Moreover, several recent breakthroughs have led to a better understanding of the role of abscisic acid signal modulation and the importance of metabolic regulation in the maternal to filial switch leading to the maturation phase.
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| 3. |
- Gutierrez, Laurent, et al.
(författare)
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Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of Auxin response factor transcripts and microRNA abundance
- 2009
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Ingår i: The Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 21:10, s. 3119-3132
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Tidskriftsartikel (refereegranskat)abstract
- The development of shoot-borne roots, or adventitious roots, is indispensable for mass propagation of elite genotypes. It is a complex genetic trait with a high phenotypic plasticity due to multiple endogenous and environmental regulatory factors. We demonstrate here that a subtle balance of activator and repressor AUXIN RESPONSE FACTOR (ARF) transcripts controls adventitious root initiation. Moreover, microRNA activity appears to be required for fine-tuning of this process. Thus, ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. The three ARFs display overlapping expression domains, interact genetically, and regulate each other's expression at both transcriptional and posttranscriptional levels by modulating miR160 and miR167 availability. This complex regulatory network includes an unexpected feedback regulation of microRNA homeostasis by direct and nondirect target transcription factors. These results provide evidence of microRNA control of phenotypic variability and are a significant step forward in understanding the molecular mechanisms regulating adventitious rooting.
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| 4. |
- Gutierrez, Laurent, et al.
(författare)
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The lack of a systematic validation of reference genes : a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.
- 2008
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.
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| 5. |
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| 6. |
- Le Hir, Rozenn, et al.
(författare)
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Gene expression profiling : keys for investigating phloem functions
- 2008
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Ingår i: Trends in Plant Science. - : Elsevier BV. - 1360-1385 .- 1878-4372. ; 13:6, s. 273-280
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Forskningsöversikt (refereegranskat)abstract
- Phloem is the major route for transport of carbohydrates, amino acids, and other nutrients from source to sink tissues. Hormones, mRNAs, small RNAs and proteins also are transported by the phloem, and potentially play pivotal roles in communication between organs to coordinate plant development and physiology. A comprehensive understanding of the mechanisms involved in phloem transport and signalling is still lacking. Recent transcript profiling in several plant species has provided new insights to phloem-specialized functions. Here, we review conclusions regarding the unique functions of the phloem and discuss putative roles for mRNAs and small RNA species in long-distance signalling.
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| 7. |
- Sorin, Céline, et al.
(författare)
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Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1
- 2005
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Ingår i: The Plant Cell. - : American Society of Plant Biologists. - 1040-4651 .- 1532-298X. ; 17:5, s. 1343-1359
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Tidskriftsartikel (refereegranskat)abstract
- Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.
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| 8. |
- Sorin, Céline, et al.
(författare)
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Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development
- 2006
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Ingår i: Plant Physiology. - : American Society of Plant Biologists. - 0032-0889 .- 1532-2548. ; 140:1, s. 349-364
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Tidskriftsartikel (refereegranskat)abstract
- A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis ( Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.
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| 9. |
- Svennerstam, Henrik, et al.
(författare)
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Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids
- 2007
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548. ; 143:4, s. 1853-1860
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Tidskriftsartikel (refereegranskat)abstract
- Plant nitrogen (N) uptake is a key process in the global N cycle and is usually considered a "bottleneck” for biomass production in land ecosystems. Earlier, mineral N was considered the only form available to plants. Recent studies have questioned this dogma and shown that plants may access organic N sources such as amino acids. The actual mechanism enabling plants to access amino acid N is still unknown. However, a recent study suggested the Lysine Histidine Transporter 1 (LHT1) to be involved in root amino acid uptake. In this study, we isolated mutants defective in root amino acid uptake by screening Arabidopsis ( Arabidopsis thaliana) seeds from ethyl methanesulfonate-treated plants and seeds from amino acid transporter T-DNA knockout mutants for resistance against the toxic D-enantiomer of alanine ( Ala). Both ethyl methanesulfonate and T-DNA knockout plants identified as D-Ala resistant were found to be mutated in the LHT1 gene. LHT1 mutants displayed impaired capacity for uptake of a range of amino acids from solutions, displayed impaired growth when N was supplied in organic forms, and acquired substantially lower amounts of amino acids than wild-type plants from solid growth media. LHT1 mutants grown on mineral N did not display a phenotype until at the stage of flowering, when premature senescence of old leaf pairs occurred, suggesting that LHT1 may fulfill an important function at this developmental stage. Based on the broad and unbiased screening of mutants resistant to D-Ala, we suggest that LHT1 is an important mediator of root uptake of amino acids. This provides a molecular background for plant acquisition of organic N from the soil., ISSN = 0032-0889, DOI = 10.1104/pp.106.092205, url = ://WOS:000245781000034, year = 2007, type = Journal Article
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