Light- and Phytochrome-Dependent Regulation of Hypocotyl Elongation in Arabidopsis thaliana
ABSTRACT: To identify and characterize genes required for tissue-specific phytochrome responses during hypocotyl development in far-red-light grown bvr lines, we performed gene transcriptional profiling using bvr lines with mesophyll-specific phytochrome inactivation (cab3: :pBVR2). We identified several candidate genes whose expression is significantly altered in lines with mesophyll tissue-specific BVR expression (Cab3::pBVR2), compared to constitutive phytochrome inactivation lines, i.e. 35S-driven BVR lines (35S::pBVR3). No-0 is used as wild-type (WT) Seeds of No-0 WT, 35S::pBVR3 and CAB3::pBVR2 on MS plates were exposed to Red (R) light of 75 µmol m-2 s-1 for 5 min and imbibing seeds were cold-stratified at 4 °C in darkness for 3 d. Seedlings were grown under continuous far-red illumination for 7 d. Seven-day-old vegetative whole seedlings (300 – 500 mg) were quickly (<1 min) harvested and immediately frozen in liquid nitrogen inside the FR chamber. Seedlings were grown under continuous far-red illumination for 7 d.
Project description:This SuperSeries is composed of the following subset Series: GSE35057: Phytochrome Interacting Factor 4 and 5 regulate different set of genes in high and low red/far-red light GSE35059: ChIP-Seq analysis of Phytochrome Interacting Factor 5 DNA binding in low R/FR condition Refer to individual Series
Project description:Global transcriptome patterns were performed using ORE1-IOE-2h (2h after Estradiol and Mock treatment) as well as transiently (6h) overexpressed Arabidopsis mesophyll cell protoplasts To identify genes more rapidly responding to elevated ORE1 expression we here repeated the previous experiment (Balazadeh et al., 2010), but shortened the EST induction time to 2 h (ORE1-IOE-2h dataset). Furthermore, to exclude potential misinterpretation due to EST treatment we also included an experiment where we transiently expressed a 35S::ORE1 construct in Arabidopsis mesophyll cell protoplasts and extracted RNA 6 h after the transfection (35S::ORE1-6h dataset);
Project description:PIL5 is a key negative regulator of phytochrome mediated seed germination and PIL5 protein is degraded by red light irradiation through phytochrome. The microarray aimed to find various red light-regulated genes and PIL5-regulated genes in the imbibed seeds. Keywords: genetic modification Overall design: Col-0 and pil5 seeds were irradiated by far-red light or far-red/red light and then, incubated in the dark for 12 hours. Total three biological replicates were used for the microarray.
Project description:PIL5 is a key negative regulator of phytochrome mediated seed germination and PIL5 protein is degraded by red light irradiation through phytochrome. The microarray aimed to find various red light-regulated genes and PIL5-regulated genes in the imbibed seeds. Experiment Overall Design: Col-0 and pil5 seeds were irradiated by far-red light or far-red/red light and then, incubated in the dark for 12 hours. Total three biological replicates were used for the microarray.
Project description:Phytochromes are red/far red photosensors regulating numerous developmental programs in plants. Among them phytochrome A (phyA) is essential to enable seedling de-etiolation in continuous far-red (FR) light a condition mimicking the environment under a dense canopy. The ecological relevance of this response is demonstrated by the high mortality rate of phyA mutants germinating in deep vegetational shade. phyA signaling involves a direct interaction of the photoreceptor with members of the bHLH transcription factor family, PIF1 and PIF3 (Phytochrome Interacting Factor). Here we investigated the involvement of PIF4 and PIF5 in phyA signaling and found that they redundantly control de-etiolation in FR light. The pif4pif5 double mutant is hypersensitive to low fluence rates of FR light. This phenotype is dependent on FR light perception by phyA but does not rely on alterations of the phyA level. Our microarrays analysis shows that PIF4 and PIF5 are part of an inhibitory mechanism repressing the expression of some light-responsive genes in the dark and are also needed for full expression of several growth-related genes in the light. Unlike PIF1 and PIF3, PIF4 and PIF5 are not degraded in response to FR light indicating that they are light-regulated by a different mechanism. Our genetic analysis suggests that this is achieved through the sequestration of these PIFs by the closely related bHLH transcription factor HFR1 (long Hypocotyl in FR light). Experiment Overall Design: he pif4pif5 double mutant were compared to wild-type plants when kept in the dark or subjected to 1 or 24 hours of 0.5 or 5 µmol/m2/s far-red light respectively.
Project description:The absorption of visible light in aquatic environments has led to the common assumption that aquatic organisms sense and adapt to penetrative blue/green light wavelengths, but show little or no response to the more attenuated red/far-red wavelengths. Here we show that two marine diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana, possess a bona fide red/far-red light sensing phytochrome (DPH) that uses biliverdin as a chromophore and displays accentuated red-shifted absorbance peaks compared to other characterized plant and algal phytochromes. Exposure to both red and far-red light causes changes in gene expression in P. tricornutum and the responses to far-red light disappear in DPH knockout cells, demonstrating that P. tricornutum DPH mediates far-red light signaling. The identification of DPH genes in diverse diatom species widely distributed along the water column further emphasizes the ecological significance of far-red light sensing, raising questions about the sources of far-red light. Our analyses indicate that, although far-red wavelengths from sunlight are only detectable at the ocean surface, chlorophyll fluorescence and Raman scattering can generate red/far-red photons in deeper layers. This study opens up novel perspectives on phytochrome-mediated far-red light signaling in the ocean and on the light sensing and adaptive capabilities of marine phototrophs. Overall design: Phaeodactylum tricornutum cultures growing in 12L/12D cycles were exposed, at the end of the 12 h dark period, to 30 minutes of low fluence of red or far-red light and harvested immediately after the light treatment. Cells were also collected before the light treatment as reference point and, culture kept in dark for 30 minutes represented the control condition. Two biological replicates were sampled for each condition.
Project description:FAR-RED ELONGATED HYPOCOTYL 3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE 1 (FAR1) are two transposase-derived transcription factors initially identified as the key components in phytochrome A signaling and recently shown to function in the circadian clock. However, whether FHY3 and FAR1 are involved in other processes of plant development remains largely unknown. Here, we explored chromatin immunoprecipitation-based sequencing (ChIP-seq) analysis to identify 1745 and 1171 FHY3 direct binding target genes in darkness and far-red light conditions, respectively in the Arabidopsis thaliana genome. This analysis revealed that FHY3 preferentially binds to the gene promoters through the previously identified typical FHY3/FAR1 binding motif. Interestingly, FHY3 also binds to two novel motifs in the 178-bp repeats of the Arabidopsis centromere regions in vivo. Comparison between the ChIP-seq and microarray data indicates that FHY3 regulates the expression of 196 and 85 genes in dark and far-red respectively by directly binding to their promoters. FHY3 also co-regulates a number of common target genes with PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIL5) and ELONGATED HYPOCOTYL 5 (HY5). Moreover, our genome-wide identification of FHY3 direct target genes ultimately led to the discovery and validation of a new role of FHY3 in controlling chloroplast development, by directly activating the expression of ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5), a key gene regulating chloroplast constriction and division. Taken together, our data suggest that FHY3 is involved in regulating multiple facets of plant development, thus providing new insights into the functions of this type of transposase-derived transcription factors. We used the FHY3p:FHY3-GR fhy3-4 transgenic lines expressing FHY3-glucocorticoid receptor (GR) fusion proteins under the control of the FHY3 native promoter, in which, without dexamethasone (DEX), the GR-fusion proteins localize in the cytoplasm, whereas, when treated with DEX, the fusion proteins translocate into the nucleus. The seedlings were grown in D or continuous FR light for 4 days, then treated with DEX or mock (equal volume of ethanol), and grown in the same conditions for a further 2 hours before the samples were harvested. RNA samples were extracted from these samples, and then hybridized with Affymetrix Arabidopsis ATH1 genome arrays. Four independent biological replicates for each treatment were used for the microarray analysis.
Project description:To incorporate the far-red light (FR) signal into a strategy for optimizing plant growth, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) mediates the nuclear translocation of the FR photoreceptor phytochrome A (phyA) and facilitates the association of phyA with the promoters of numerous associated genes crucial for the response to environmental stimuli. However, whether FHY1 plays additional roles following FR irradiation remains elusive. Here, by the global identification of FHY1 chromatin association sites through ChIP-seq analysis and by the comparison of FHY1-associated sites with phyA- associated sites, we demonstrated that nuclear FHY1 can either act independently of phyA or act in association with phyA to activate the expression of distinct target genes. We also determined that phyA can act independently of FHY1 in regulating phyA-specific target genes. Furthermore, we determined that the independent FHY1 nuclear pathway is involved in crucial developmental aspects, as in the case of inhibited seed germination under FR during salt-stress. Notably, the differential presence of cis-elements and transcription factors in common and unique FHY1 and/or phyA associated genes are indicative of the complexity of the independent and coordinated FHY1 and phyA pathways. Our study uncovers new aspects of FHY1 function beyond its currently recognized role in phyA-dependent photomorphogenesis To determine whether phyA and FHY1 associate with the same genes upon FR irradiation, four-day old etiolated 35S: GFP-FHY1 fhy1-1 transgenic seedlings were exposed to 3hr FR. The treated plants were utilized for ChIP-seq analysis with an anti-FHY1 antibody that exhibits high specificity in the detection of FR-induced FHY1-association events. FHY1 associated-DNA samples from three distinct biological replicates and an input DNA sample (as a negative control) were subjected to high-throughput Solexa (Illumina) sequencing.
Project description:Phytochromes mediate a profound developmental shift when dark-grown seedlings are exposed to light. Here we show that a subset of genes is up regulated in phytochrome B (phyB) mutants even before dark-grown seedlings are exposed to light. Most of these genes bear the RY cis motif, which is a binding site of the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3), and the phyB mutation also enhanced ABI3 expression. These changes in transcriptome have physiological consequences as seedlings of the abi3 mutant showed enhanced responses to pulses of far-red light, while ABI3 overexpressers exhibited the opposite pattern. Seedlings of the wild type derived from seeds germinated in full darkness showed enhanced expression of genes bearing the RY cis motif and reduced responses to far-red light. We propose that, via changes in ABI3 expression, light, perceived mainly by phyB in the seed, generates a downstream trans-developmental phase signal that pre-conditions the seedling to its most likely environment. , ,
Project description:ZFP3, a nuclear C2H2 zinc finger protein acts as a negative regulator of ABA- suppressed germination. Regulated over-expression of ZFP3 and closely related ZFP1, ZFP4, ZFP6 and ZFP7 confers ABA insensitivity to seed germination while the zfp3,zfp4 double mutant displays enhanced ABA susceptibility. Reduced expression of a large set of ABA-induced genes such as RAB18 and transcription factors ABI3, ABI4, ABI5 and RGL2 in ZFP3ox seedling suggests that ZFP3 indeed negatively regulates ABA signaling. Analysis of ZFP3ox plants revealed multiple phenotypic alterations such as semidwarf growth habit, defects in fertility and enhanced sensitivity of hypocotyl elongation to red but not to far-red or blue light. Analysis of genetic interactions with phytochrome and abi mutants suggested that ZFP3 amplifies red light signals perceived by photoreceptors other than phyB, and controlled by ABI5 downstream of ZFP3. Comparison of ZFP3 overexpressing and wild type Arabidopsis seedlings