8 research outputs found
Functional analysis of orthologous transcription factors FLOWERING LOCUS C and PERPETUAL FLOWERING 1 in A. thaliana and A. alpina
The 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.
Genome-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 overrepresented 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
Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina
Arabis 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
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Divergence of regulatory networks governed by the orthologous transcription factors FLC and PEP1 in Brassicaceae species.
Genome-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 overrepresented 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
Phosphorylation of CONSTANS and its COP1-dependent degradation during photoperiodic flowering of Arabidopsis
Seasonal flowering involves responses to changes in day length. In Arabidopsis thaliana, the CONSTANS
(CO) transcription factor promotes flowering in the long days of spring and summer. Late flowering in short
days is due to instability of CO, which is efficiently ubiquitinated in the dark by the CONSTITUTIVE PHOTOMORPHOGENIC
1 (COP1) E3 ligase complex. Here we show that CO is also phosphorylated. Phosphorylated
and unphosphorylated forms are detected throughout the diurnal cycle but their ratio varies, with the relative
abundance of the phosphorylated form being higher in the light and lower in the dark. These changes
in relative abundance require COP1, because in the cop1 mutant the phosphorylated form is always more
abundant. Inactivation of the PHYTOCHROME A (PHYA), CRYPTOCHROME 1 (CRY1) and CRYPTOCHROME 2
(CRY2) photoreceptors in the phyA cry1 cry2 triple mutant most strongly reduces the amount of the phosphorylated
form so that unphosphorylated CO is more abundant. This effect is caused by increased COP1
activity, as it is overcome by introduction of the cop1 mutation in the cop1 phyA cry1 cry2 quadruple
mutant. Degradation of CO is also triggered in red light, and as in darkness this increases the relative abundance
of unphosphorylated CO. Finally, a fusion protein containing truncated CO protein including only the
carboxy-terminal region was phosphorylated in transgenic plants, locating at least one site of phosphorylation
in this region. We propose that CO phosphorylation contributes to the photoperiodic flowering
response by enhancing the rate of CO turnover via activity of the COP1 ubiquitin ligase.Peer reviewe
Gibberellins act downstream of Arabis PERPETUAL FLOWERING1 to accelerate floral induction during vernalization
Regulation 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
Plant science : A regulatory circuit conferring varied flowering response to cold in annual and perennial plants
The reproductive strategies of plants are highly variable. Short-lived annuals flower abundantly soon after germination, whereas longer-lived perennials postpone and spatially restrict flowering. We used CRISPR/Cas9 and interspecies gene transfer to understand divergence in reproductive patterns between annual and perennial crucifers. We show that in perennial Arabis alpina, flowering in response to winter cold depends on the floral integrator SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15), whose activity is limited to older shoots and branches during cold exposure. In annuals, this regulatory system is conserved, but cold-induced flowering occurs in young shoots, without requirement for SPL15, through the photoperiodic pathway when plants return to warm. By reconstructing the annual response in perennials, we conclude that characteristic patterns of reproduction in annuals and perennials are conferred through variation in dependency on distinct flowering pathways acting in parallel.</p