Manuel Piñeiro

The Arabidopsis E3 Ubiquitin Ligase HOS1 Negatively Regulates CONSTANS Abundance in the Photoperiodic Control of Flowering

The transcriptional and posttranslational regulation of CONSTANS (CO) expression is crucial to accurately measure changes in daylength that influence flowering time in Arabidopsis thaliana. Here, we demonstrate that ESD6/HOS1 is required to modulate precisely the timing of CO accumulation to maintain low levels of FT expression during the first part of the day and a correct photoperiodic response. The Arabidopsis thaliana early in short days6 (esd6) mutant was isolated in a screen for mutations that accelerate flowering time. Among other developmental alterations, esd6 displays early flowering in both long- and short-day conditions. Fine mapping of the mutation showed that the esd6 phenotype is caused by a lesion in the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) locus, which encodes a RING finger–containing E3 ubiquitin ligase. The esd6/hos1 mutation causes decreased FLOWERING LOCUS C expression and requires CONSTANS (CO) protein for its early flowering phenotype under long days. Moreover, CO and HOS1 physically interact in vitro and in planta, and HOS1 regulates CO abundance, particularly during the daylight period. Accordingly, hos1 causes a shift in the regular long-day pattern of expression of FLOWERING LOCUS T (FT) transcript, starting to rise 4 h after dawn in the mutant. In addition, HOS1 interacts synergistically with CONSTITUTIVE PHOTOMORPHOGENIC1, another regulator of CO protein stability, in the regulation of flowering time. Taken together, these results indicate that HOS1 is involved in the control of CO abundance, ensuring that CO activation of FT occurs only when the light period reaches a certain length and preventing precocious flowering in Arabidopsis.

EARLY IN SHORT DAYS 1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis

We have characterized Arabidopsis esd1 mutations, which cause early flowering independently of photoperiod, moderate increase of hypocotyl length, shortened inflorescence internodes, and altered leaf and flower development. Phenotypic analyses of double mutants with mutations at different loci of the flowering inductive pathways suggest that esd1 abolishes the FLC-mediated late flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. We found that ESD1 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of esd1 in a flc-3 null genetic background and the downregulation of other members of the FLC-like/MAF gene family in esd1 mutants suggest that flowering inhibition mediated by ESD1 occurs through both FLC-and FLC-like gene-dependent pathways. The ESD1 locus was identified through a map-based cloning approach. ESD1 encodes ARP6, a homolog of the actin-related protein family that shares moderate sequence homology with conventional actins. Using chromatin immunoprecipitation (ChIP) experiments, we have determined that ARP6 is required for both histone acetylation and methylation of the FLC chromatin in Arabidopsis.

Chromatin remodeling in plant development

Plant development results from specific patterns of gene expression that are tightly regulated in a spatio-temporal manner. Chromatin remodeling plays a central role in establishing these expression patterns and maintaining epigenetic transcriptional states through successive rounds of mitosis that take place within a cell lineage. Plant epigenetic switches occur not only at the embryo stage, but also during postembryonic developmental transitions, suggesting that chromatin remodeling activities in plants can provide a higher degree of regulatory flexibility which probably underlies their developmental plasticity. Here, we highlight recent progress in the understanding of plant chromatin dynamic organization, facilitating the activation or repression of specific sets of genes involved in different developmental programs and integrating them with the response to environmental signals. Chromatin conformation controls gene expression both in actively dividing undifferentiated cells and in those already fate-determined. In this context, we first describe chromatin reorganization activities required to maintain meristem function stable through DNA replication and cell division. Organ initiation at the apex, with emphasis on reproductive development, is next discussed to uncover the chromatin events involved in the establishment and maintenance of expression patterns associated with differentiating cells; this is illustrated with the complex epigenetic regulation of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Finally, we discuss the involvement of chromatin remodeling in plant responses to environmental cues and to different types of stress conditions.

WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis

This work shows that plants respond to arsenate by immediately freezing its uptake through the action of a transcriptional repressor of phosphate transporters and that the same transcription factor influences transposon expression in response to arsenate. Plants therefore have an arsenate perception mechanism that controls arsenate uptake and transposon expression, providing an integrated strategy for arsenate tolerance and genome stability. Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing.

Mutations in the Arabidopsis SWC6 gene, encoding a component of the SWR1 chromatin remodelling complex, accelerate flowering time and alter leaf and flower development

Mutations affecting the Arabidopsis SWC6 gene encoding a putative orthologue of a component of the SWR1 chromatin remodelling complex in plants have been characterized. swc6 mutations cause early flowering, shortened inflorescence internodes, and altered leaf and flower development. These phenotypic defects resemble those of the photoperiod independent early flowering 1 (pie1) and early in short days 1 (esd1) mutants, also affected in homologues of the SWR1 complex subunits. SWC6 is a ubiquitously expressed nuclear HIT-Zn finger-containing protein, with the highest levels found in pollen. Double mutant analyses suggest that swc6 abolishes the FLC-mediated late-flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. It was found that SWC6 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of swc6 in an flc null background and the down-regulation of other FLC-like/MAF genes in swc6 mutants suggest that flowering inhibition mediated by SWC6 occurs through both FLC- and FLC-like gene-dependent pathways. Both genetic and physical interactions between SWC6 and ESD1 have been demonstrated, suggesting that both proteins act in the same complex. Using chromatin immunoprecipitation, it has been determined that SWC6, as previously shown for ESD1, is required for both histone H3 acetylation and H3K4 trimethylation of the FLC chromatin. Altogether, these results suggest that SWC6 and ESD1 are part of an Arabidopsis SWR1 chromatin remodelling complex involved in the regulation of diverse aspects of plant development, including floral repression through the activation of FLC and FLC-like genes.

EARLY IN SHORT DAYS 7 (ESD7) encodes the catalytic subunit of DNA polymerase epsilon and is required for flowering repression through a mechanism involving epigenetic gene silencing

We have characterized a mutation affecting the Arabidopsis EARLY IN SHORT DAYS 7 (ESD7) gene encoding the catalytic subunit of DNA polymerase epsilon (epsilon), AtPOL2a. The esd7-1 mutation causes early flowering independently of photoperiod, shortened inflorescence internodes and altered leaf and root development. esd7-1 is a hypomorphic allele whereas knockout alleles displayed an embryo-lethal phenotype. The esd7 early flowering phenotype requires functional FT and SOC1 proteins and might also be related to the misregulation of AG and AG-like gene expression found in esd7. Genes involved in the modulation of chromatin structural dynamics, such as LHP1/TFL2 and EBS, which negatively regulate FT expression, were found to interact genetically with ESD7. In fact a molecular interaction between the carboxy terminus of ESD7 and TFL2 was demonstrated in vitro. Besides, fas2 mutations suppressed the esd7 early flowering phenotype and ICU2 was found to interact with ESD7. Discrete regions of the chromatin of FT and AG loci were enriched in activating epigenetic marks in the esd7-1 mutant. We concluded that ESD7 might be participating in processes involved in chromatin-mediated cellular memory.

Timing is everything in plant development. The central role of floral repressors

Progress in understanding the molecular basis of flowering time control has revealed that floral repressors play a central role in modulating the floral transition and are essential to prevent the precocious onset of flowering. A number of cellular processes including chromatin remodeling, selective protein degradation, and transcriptional regulation mediated by transcription factors are involved in repressing the initiation of flowering. Floral repressors interact at different levels with floral inductive pathways and prevent the premature onset of flowering that could impact negatively on the reproductive success of plants. Despite recent advances, further studies will be needed to understand how the interactions between floral repressors and the regulatory networks involved in the control of flowering time have evolved in different species. Recent data suggest that a diversity of regulatory proteins act as central floral repressors in different plants, and even in those species where regulatory modules are conserved new elements that modulate the function of these pathways have been recruited to mediate specific adaptive responses. The development of genomic tools and predictive models that can integrate large datasets related to the flowering behavior of plant species will facilitate the characterization of the repressor mechanisms underlying flowering responses, a trait with implications in the yield of crop species. In a scenario of global climate change, an in depth understanding of these gene circuits will be essential for the development of crop varieties with improved yield.

The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression

Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β-estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β-estradiol treatment. As a proof of concept for the exploitation of this resource, β-estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl-root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.

Red Light-Mediated Degradation of CONSTANS by the E3 Ubiquitin Ligase HOS1 Regulates Photoperiodic Flowering in Arabidopsis.

A complex comprising phytochrome B, the E3 ubiquitin ligase HOS1, and CONSTANS may be required to modulate a proper photoperiodic flowering response in Arabidopsis. The regulation of CONSTANS (CO) gene expression is crucial to accurately measure changes in daylength, which influences flowering time in Arabidopsis thaliana. CO expression is under both transcriptional and posttranslational control mechanisms. We previously showed that the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) physically interacts with CO in Arabidopsis. This interaction is required to precisely modulate the timing of CO accumulation and, consequently, to maintain low levels of FLOWERING LOCUS T expression during the first part of the day. The data presented here demonstrate that HOS1 is involved in the red light-mediated degradation of CO that takes place in the early stages of the daylight period. Our results show that phytochrome B (phyB) is able to regulate flowering time, acting in the phloem companion cells, as previously described for CO and HOS1. Moreover, we reveal that phyB physically interacts with HOS1 and CO, indicating that the three proteins may be present in a complex in planta that is required to coordinate a correct photoperiodic response in Arabidopsis.

Chromatin-Dependent Repression of the Arabidopsis Floral Integrator Genes Involves Plant Specific PHD-Containing Proteins

This work shows that the Arabidopsis homologs SHL and EBS play independent roles in the repression of flowering and are required to maintain an inactive chromatin configuration in floral integrator genes by preventing high levels of H3 acetylation. SHL and EBS are plant-specific proteins and could represent a fundamental difference in gene expression control between plants and other eukaryotes. The interplay among histone modifications modulates the expression of master regulatory genes in development. Chromatin effector proteins bind histone modifications and translate the epigenetic status into gene expression patterns that control development. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific proteins with a plant homeodomain (PHD) motif, SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), function in the chromatin-mediated repression of floral initiation and play independent roles in the control of genes regulating flowering. Previous results showed that repression of the floral integrator FLOWERING LOCUS T (FT) requires EBS. We establish that SHL is necessary to negatively regulate the expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), another floral integrator. SHL and EBS recognize di- and trimethylated histone H3 at lysine 4 and bind regulatory regions of SOC1 and FT, respectively. These PHD proteins maintain an inactive chromatin conformation in SOC1 and FT by preventing high levels of H3 acetylation, bind HISTONE DEACETYLASE6, and play a central role in regulating flowering time. SHL and EBS are widely conserved in plants but are absent in other eukaryotes, suggesting that the regulatory module mediated by these proteins could represent a distinct mechanism for gene expression control in plants.