27 research outputs found
Functional analysis of orthologous transcription factors FLOWERING LOCUS C and PERPETUAL FLOWERING 1 in A. thaliana and A. alpina
No full textThe orthologous transcription factors PEP1 and FLC repress flowering before vernalization in perennial A. alpina and annual A. thaliana, respectively. In A. alpina, PEP1 also represses flowering after vernalization to ensure vegetative growth after flowering, a crucial step in the perennial life-cycle. Genome-wide comparison of PEP1 and FLC targets showed high divergence. Conserved target genes were mainly associated with the control of flowering time and flower development, the main function of PEP1 and FLC. Species-specific target genes in both species were associated with responses to environmental stimuli and hormones like gibberellins. Phenotypic analysis revealed that the pep1 mutant displays several phenotypes that resemble a gibberellin-treated plant, suggesting that PEP1 affects the gibberellin response. We found that gibberellins promote floral induction during vernalization, however gibberellin levels were not increased during vernalization in wild-type or pep1. Before vernalization, pep1 had increased levels of gibberellins that might cause phenotypic changes in the mutant. In addition, we found that PEP1 represses gibberellin signaling which might also contribute to phenotypic changes in pep1 and is likely to delay floral induction in early stages of vernalization. Thus, PEP1 links environmental and developmental responses within the perennial life cycle of A. alpina by modifying the gibberellins response
Divergence of regulatory networks governed by the orthologous transcription factors FLC and PEP1 in Brassicaceae species
No full textGenome-wide landscapes of transcription factor (TF) binding sites (BSs) diverge during evolution, conferring species-specific transcriptional patterns. The rate of divergence varies in different metazoan lineages but has not been widely studied in plants. We identified the BSs and assessed the effects on transcription of FLOWERING LOCUS C (FLC) and PERPETUAL FLOWERING 1 (PEP1), two orthologous MADS-box TFs that repress flowering and confer vernalization requirement in the Brassicaceae species Arabidopsis thaliana and Arabis alpina, respectively. We found that only 14% of their BSs were conserved in both species and that these contained a CArG-box that is recognized by MADS-box TFs. The CArG-box consensus at conserved BSs was extended compared with the core motif. By contrast, species-specific BSs usually lacked the CArG-box in the other species. Flowering-time genes were highly over-represented among conserved targets, and their CArG-boxes were widely conserved among Brassicaceae species. Cold-regulated (COR) genes were also overrepresented among targets, but the cognate BSs and the identity of the regulated genes were usually different in each species. In cold, COR gene transcript levels were increased in flc and pep1-1 mutants compared with WT, and this correlated with reduced growth in pep1-1. Therefore, FLC orthologs regulate a set of conserved target genes mainly involved in reproductive development and were later independently recruited to modulate stress responses in different Brassicaceae lineages. Analysis of TF BSs in these lineages thus distinguishes widely conserved targets representing the core function of the TF from those that were recruited later in evolution.Fil: Mateos, Julieta Lisa. Max Planck Institute Fur Zuchtungsforschung; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Tilmes, Vicky. Max Planck Institute Fur Zuchtungsforschung; AlemaniaFil: Madrigal, Pedro. Polish Academy of Sciences; ArgentinaFil: Severing, Edouard. Max Planck Institute Fur Zuchtungsforschung; AlemaniaFil: Richter, René. Max Planck Institute Fur Zuchtungsforschung; AlemaniaFil: Rijkenberg, Colin W.M.. Max Planck Institute Fur Zuchtungsforschung; AlemaniaFil: Krajewski, Pawel. Polish Academy of Sciences; ArgentinaFil: Coupland, George. Max Planck Institute Fur Zuchtungsforschung; Alemani
Gibberellins Act Downstream of Arabis PERPETUAL FLOWERING1 to Accelerate Floral Induction during Vernalization
No full textRegulation of flowering by endogenous and environmental signals ensures that reproduction occurs under optimal conditions to maximize reproductive success. Involvement of the growth regulator gibberellin (GA) in the control of flowering by environmental cues varies among species. Arabis alpina Pajares, a model perennial member of the Brassicaceae, only undergoes floral induction during vernalization, allowing definition of the role of GA specifically in this process. The transcription factor PERPETUAL FLOWERING1 (PEP1) represses flowering until its mRNA levels are reduced during vernalization. Genome-wide analyses of PEP1 targets identified genes involved in GA metabolism and signaling, and many of the binding sites in these genes were specific to the A. alpina lineage. Here, we show that the pep1 mutant exhibits an elongated-stem phenotype, similar to that caused by treatment with exogenous GA, consistent with PEP1 repressing GA responses. Moreover, in comparison with the wild type, the pep1 mutant contains higher GA4 levels and is more sensitive to GA prior to vernalization. Upon exposure to cold temperatures, GA levels fall to low levels in the pep1 mutant and in wild-type plants, but GA still promotes floral induction and the transcription of floral meristem identity genes during vernalization. Reducing GA levels strongly impairs flowering and inflorescence development in response to short vernalization treatments, but longer treatments overcome the requirement for GA. Thus, GA accelerates the floral transition during vernalization in A. alpina, the down-regulation of PEP1 likely increases GA sensitivity, and GA responses contribute to determining the length of vernalization required for flowering and reproduction.Fil: Tilmes, Vicky. Max-planck-institute For Plant Breeding Research; AlemaniaFil: Mateos, Julieta Lisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Madrid, Eva. Max-planck-institute For Plant Breeding Research; AlemaniaFil: Vincent, Coral. Max-planck-institute For Plant Breeding Research; AlemaniaFil: Severing, Edouard. Max-planck-institute For Plant Breeding Research; AlemaniaFil: Carrera, Esther. Universidad Politécnica de Valencia; EspañaFil: López Díaz, Isabel. Universidad Politécnica de Valencia; EspañaFil: Coupland, George. Max-planck-institute For Plant Breeding Research; Alemani
Selection and validation of reference genes for quantitative Real-Time PCR in <i>Arabis alpina</i>
No full textArabis alpina is a perennial arctic-alpine plant and an upcoming model organism for genetics and molecular biology for the Brassicaceae family. One essential method for most molecular approaches is the analysis of gene expression by reverse-transcription quantitative Real-Time PCR (RT-qPCR). For the normalisation of expression data in RT-qPCR experiments, it is essential to use reliable reference genes that are not affected under a wide range of conditions. In this study we establish a set of 15 A. alpina reference genes that were tested under different conditions including cold, drought, heat, salt and gibberellic acid treatments. Data analyses with geNORM, BestKeeper and NormFinder revealed the most stable reference genes for the tested conditions: RAN3, HCF and PSB33 are most suitable for cold treatments; UBQ10 and TUA5 for drought; RAN3, PSB33 and EIF4a for heat; CAC, TUA5, ACTIN 2 and PSB33 for salt and PSB33 and TUA5 for gibberellic acid treatments. CAC and ACTIN 2 showed the least variation over all tested samples. In addition, we show that two reference genes are sufficient to normalize RT-qPCR data under our treatment conditions. In future studies, these reference genes can be used for an adequate normalisation and thus help to generate high quality RT-qPCR data in A. alpina.</div
RT-qPCR Cq values of candidate reference genes in all treatments.
No full textThe box includes all data points between the 25% quantile and the 75% quantile. The whiskers span all values below and above, excluding outliers which are displayed as dots. The median, which marks the 50% quantile, is displayed as a line.</p
Primer efficiencies and correlation of reference gene candidates.
No full textPrimer efficiencies and correlation of reference gene candidates.</p
Ranking of gene expression stability under stress conditions and hormone stimuli.
No full textGenes were ranked using the three commonly used statistical algorithms NormFinder, BestKeeper and geNorm. The stability value describes the variance (NormFinder and geNorm) or standard deviation (BestKeeper).</p
Abiotic stress and hormone responsive genes, primers and amplicons.
No full textAbiotic stress and hormone responsive genes, primers and amplicons.</p
Comparison of specific stress response genes normalised with different reference genes.
No full textNormalisation of the stress response genes RD29A (RESPONSIVE TO DESICCATION 29A, cold and drought responsive), HSP81.2/90 (heat responsive), TSPO (OUTER MEMBRANE TRYPTOPHAN-RICH SENSORY PROTEIN-RELATED, salt responsive) and GA3ox1 (GIBBERELLIN 3-OXIDASE 1, GA responsive) was carried out with one or two reference genes (RG1 and RG2): cold–RAN3 and HCF, drought–UBQ10 and TUA5, GA and salt–PSB33 and TUA5, heat–RAN3 and PSB33.</p
Optimal number of reference genes for various conditions.
No full textThe geNorm algorithm was used to determine the pairwise variation (V) between the reference genes for treatments with cold, drought, heat, salt and gibberellic acid. The threshold for adequate normalisation is V≤0.15, indicated by the green dashed line.</p
