ABSTRACT
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.
Subject(s)
Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Mixed Function Oxygenases/metabolism , Solanum lycopersicum/enzymology , Solanum lycopersicum/genetics , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , Flavins/metabolism , Flowers/genetics , Flowers/metabolism , Genes, Plant/genetics , Indoleacetic Acids/metabolism , Solanum lycopersicum/growth & development , Mixed Function Oxygenases/genetics , Molecular Sequence Data , Multigene Family , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , Seeds/genetics , Seeds/metabolism , Sequence Analysis, DNA , Tryptamines/metabolismABSTRACT
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.
Subject(s)
Arabidopsis/genetics , Plant Diseases/genetics , Pseudomonas syringae/pathogenicity , Vitamin K 3/pharmacology , Arabidopsis/drug effects , Arabidopsis/immunology , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation, Plant , Genome, Plant , Immunity, Innate , Oligonucleotide Array Sequence Analysis , RNA, Plant/genetics , Stress, PhysiologicalABSTRACT
BACKGROUND: 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. RESULTS: 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 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) 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. CONCLUSION: 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.
Subject(s)
Gene Expression Regulation, Developmental , Genes, Plant , Reverse Transcriptase Polymerase Chain Reaction/standards , Solanum lycopersicum/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant , RNA, Messenger/genetics , RNA, Plant/genetics , Reference Standards , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
Using an authentic sample of 2-hydroxy-9-(p-hydroxyphenyl)-phenalen-1-one, a banana phenalenone-type phytoalexin, we studied its dynamic of accumulation during pathogenesis of banana plants (Musa acuminata (AAA), Grand Nain) inoculated with Fusarium oxysporum f.sp. cubense (FOC), Race 4, the causal agent of Panama disease. The results obtained demonstrate that banana plants treated prior inoculation with menadione sodium bisulfite (MSB), an inducer of plant defenses, are capable of changing the dynamic of accumulation (higher amount and speed of biosynthesis) of this banana phytoalexin, biosynthesized by the banana plant during pathogenesis.
