Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

<p>Abstract</p> <p>Background</p> <p>The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR.</p> <p>Results</p> <p>In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (<it>CAC</it>), SGN-U321250 (<it>TIP41</it>), SGN-U346908 ("<it>Expressed</it>") and SGN-U316474 (<it>SAND</it>) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process.</p> <p>Conclusion</p> <p>This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability.</p

Uso de menadiona para aumentar la tolerancia al estrés salino de las plantas

Uso de menadiona para aumentar la tolerancia al estrés salino de las plantas. Esta invención está relacionada con el uso de composiciones que contienen menadiona (Vitamina K3 o Provitamina K) y/o algunos de sus derivados hidrosolubles para la mejora de la tolerancia al estrés salino de las plantas afectadas. El tratamiento se lleva a cabo mediante la aplicación de las soluciones que contengan los citados compuestos por cualquier vía que ponga en contacto las composiciones de la invención con cualquier parte de la planta y/o semillas.Peer reviewedB1 Patente con informe sobre el estado de la ténic

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis

19 páginas, 6 figuras, 2 tablas.Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.This work was partially funded by an INVESCAN, S.L. grant (No.OTT2001438) to the CSIC and by a BIO2006-02168 grant of MICINN to PT. The microarrays were funded in part by the “Genome España” Foundation. MER was supported by a research contract (ID-TF-06/002) from the Consejería de Industria, Comercio y Nuevas Tecnologías (Gobierno de Canarias). The authors thank CajaCanarias for their research support. We also thank Lorena Perales for her help in performing the bacterial growth curves, Dr. Héctor Cabrera for his useful advice on writing the manuscript, the English translation service of the Universidad Politécnica de Valencia and Mrs. Pauline Agnew whose endeavoured to edit the English translation of this paper.Peer reviewe

Caracterización de la familia multigénica ToFZY de Solanum lycopersicum y su implicación en el estrés biótico

147 páginas, 38 figuras, 5 tablas.-- Tesis Doctoral presentada por Marino Expósito para optar al grado de Dr. en Ciencias Biológicas.Peer reviewe

Uso de composiciones que contienen menadiona y/o alguno(s) de sus derivados hidrosolubles a fin de inducir en los cultivos tratados una mejora de sus frutos a la manipulación y al trasporte

Uso de composiciones que contienen menadiona y/o alguno( s) de sus derivados hidrosolubles a fin de inducir en los cultivos tratados una mejora de sus frutos a la manipulación y al transporte. Uso de composiciones que contienen menadiona y/o sus derivados hidrosolubles para bioestimular los mecanismos naturales de absorción y transporte del Ca2+ por la planta, contribuyendo por una parte, a prevenir la fisiopatía del fruto por la carencia de Ca2+, contrarrestando en parte las pérdidas en la producción por este concepto, y por otra, mejorando la resistencia a la manipulación y transporte de frutos. Estas soluciones acuosas pueden contener indistinta o conjuntamente Vitamina K3, derivados solubles [menadiona sodio bisulfito] o escasamente solubles [menadiona nicotinamida bisulfito]. Se aplican preferentemente por pulverización de la parte aérea de la planta o por el sistema de riego, pudiéndose mezclar con varios aditivos, como fertilizantes, insecticidas, nematocidas, fungicidas, bactericidas y herbicidas.Peer reviewedConsejo Superior de Investigaciones Científicas (España)B1 Patente con informe sobre el estado de la ténic

Uso de composiciones que contienen menadiona y/o algunos de sus derivados hidrosolubles a fin de inducir en los cultivos tratados una mejora de sus frutos a la manipulación y al transporte

Fecha de solicitud: 26-02-2009.- Titular: Consejo Superior de Investigaciones Científicas (CSIC)Use of compositions containing menadione and/or water-soluble derivatives thereof for biostimulating the natural mechanisms of absorption and transport of Ca 2+ by the plant, first contributing to preventing disease of the fruit due to Ca 2+ deficiency, partly counteracting production losses due to this cause, and second, improving fruit resistance during handling and transport. These aqueous soloutions can contain Vitamin K3, soluble [menadione sodium bisulphite] or barely soluble [menadione nicotinamide bisulphite] derivatives, separately or jointly. Preferably they are applied by spraying of the above-ground part of the plant or via the irrigation system, and are optionally mixed with different additives, such as fertilizers, insecticides, nematocides, fungicides, bactericides and herbicides.Uso de las composiciones que contienen el menadione y/o derivados solubles en agua de eso para biostimulating los mecanismos naturales de la absorción y del transporte de CA 2+ por la planta, primero contribuyendo a evitar la enfermedad de la fruta debida a la deficiencia del CA 2+, en parte contrarrestando las pérdidas de la producción debidas a esta causa, y en segundo lugar, mejorando resistencia de la fruta durante la manipulación y el transporte. El bote acuoso de estos soloutions contiene la vitamina K3, soluble [bisulfito del sodio del menadione] o apenas los derivados del soluble [bisulfito del nicotinamida del menadione], por separado o en común. Preferiblemente ellos son aplicado rociando de la parte sobre el suelo de la planta o vía el sistema de riego, y el son mezclaron opcionalmente con los diferentes aditivos, tales como fertilizantes, los insecticidas, los nematocides, los fungicidas, los bactericidas y los herbicidas.Peer reviewe

Priming crops against biotic and abiotic stresses: MSB as a tool for studying mechanisms

This work was partially funded by an INVESCAN Ltd. grant (No. OTT2001438) to the CSIC.Peer reviewedPeer Reviewe

Gene structure and spatiotemporal expression profile of tomato genes encoding YUCCA-like flavin monooxygenases: The ToFZY gene family

The flavin monooxygenases (FMO) encoded by plant YUCCA genes are thought to catalyze a rate-limiting step in the tryptamine pathway for indole-3-acetic acid biosynthesis. Recent experiments with different plant models have indicate that YUCCA genes play essential roles in growth and development through their contribution to the local pool of free auxin. In this study we have characterized five new genes that encode YUCCA-like FMOs in the tomato genome (ToFZY2 to ToFZY6), including gene structure, conserved motifs and phylogenetic analyses. As a first step towards clarifying the individual functions of ToFZY genes, we have used quantitative real-time RT-PCR to conduct a systematic comparison of the steady-state mRNA levels of 6 ToFZY genes, in 33 samples representing major organs and the entire tomato life cycle. We followed an absolute quantification strategy which allowed us to cross-compare transcript levels among different ToFZY genes in a given spatiotemporal coordinate. Our results indicate that expression of ToFZY genes is temporally and spatially regulated, and that the distinctive expression pattern of each ToFZY gene partially overlaps with other members of the multigenic family. We compare our data with previous results in other plant species and make some predictions about the role of tryptamine pathway in tomato growth and development.This work was partially funded by an INVESCAN, S.L. grant (No. OTT2001438) to the CSIC. The first author was supported by a research contract from the Agencia Canarias de Investigación, Inovación y Sociedad de la Información (Gobierno de Canarias). The authors thank CajaCanarias for research support

BMC Plant Biology BioMed Central

Research article Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development proces