Response to low R/FR light ratio in wt and brc1 mutant Arabidopsis axillary buds
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ABSTRACT: To study the transcriptomic profile of wt and brc1 mutant axillary buds during the shade avoidance response, we simulated a canopy shade with a low R/FR light ratio. We treated plants with white light supplemented with far-red light (Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 146 μeinstein · m-2 seg-1) for 8 hours. Control plants were left for 8 hours in white light (Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 2.2 μeinstein · m-2 seg-1) . Six biological replicates of 7-8 plants were collected for each genotype and condition (wt WL, wt FR, brc1 WL, brc1 FR). Samples were compared wt WL vs wt FR and brc1 WL vs brc1 FR.
Project description:To study the transcriptomic profile of wt and brc1 mutant axillary buds during the shade avoidance response, we simulated a canopy shade with a low R/FR light ratio. We treated plants with white light supplemented with far-red light (Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 146 μeinstein · m-2 seg-1) for 8 hours. Control plants were left for 8 hours in white light (Red light = 29 μeinstein · m-2 seg-1, Far-Red light= 2.2 μeinstein · m-2 seg-1) .
Project description:This project aimed to understand the interplay between light signalling, transzeatin signalling and nitrogen availability on hypocotyl elongation responses. To do so, mRNAseq on shoots was performed, with the following variables studied in all possible combinations: Genotypes: Wild-Type (Col-0), abcg14 (Ko et al., 2014), cypDM (cyp735a1,a2, Kiba et al., 2013) Light quality: White Light (WL, R:FR=2.5), White Light + Far Red light (WL+FR, R:FR=0.25) Nitrate availability: High Nitrate (HN, 10mM KNO3), Low Nitrate (LN, 0.2mM KNO3) Plants were grown for 4 days under WL conditions (16 h light : 8 h dark, PAR=150, 70% humidity, R:FR=2.5 at 20°C) and on the different nitrate conditions. Plants from the WL+FR (R:FR=0.25) samples were treated at ZT 8 for 90min with WL+FR prior the collection of shoots.
Project description:This study provides a comparison of gene expression in two tomato cultivars, Moneymaker and M82, focusing on first internode and its innermost cell type pith—under two light conditions: white light with far-red supplementation (WL+FR) and the control condition of white light (WL). The samples were harvested at four time points leading to the point where internode elongation could be measured in response to WL+FR. The timepoints were 6, 24, 30, and 48 hours after treatment initiation.
Project description:Plants respond to changes in the red:far red ratio (R:FR) of incident light. A reduction in this ratio (increase in FR) results in the Shade Avoidance Response (SAR) with associated changes in gene expression. The Phyotchrome-Interacting Factors (PIFs) are bHLH transcription factors known to be involved in the SAR. An analysis of changes in gene expression in WT and quadruple pif1pif3pif4pif5 (pifq; Leivar et al., 2008 (PMID 19920208)) mutant seedlings in response to an increase in FR should identify primary targets of PIF signaling. We used microarrays to examine the SAR in WT (Columbia) and pifq mutant Arabidopsis seedlings. Arabidopsis WT and pifq mutant seeds were plated on GM medium without sucrose at room temperature. During this procedure, the seeds were routinely exposed to white light (WL) for a total of 1.5 hours after imbibition. Seeds were then stratified for 5 days at 4ºC in darkness, and then grown in WL (19 umol/m2/s, R/FR ratio of 6.48) for 2 days at 21°C (WL0 samples). Two-day-old WL-grown seedlings were then maintained in the same fluence rate of WL supplemented with far-red light (WL-FR, R/FR ratio of 0.006) for 1 (FR1), 3 (FR3) or 24 (FR24) hours before harvesting. Control seedlings were also maintained in parallel in the same fluence rate of WL for 24h (WL24) before harvesting. Three different biological replicates of each treatment were grown separately and extracted, processed, and analyzed independently.
Project description:This RNA-seq was designed to help gain understanding on the genetic program behind phytochrome developmental time dependent control of leaf 3 (L3) cell proliferation and expansion phases, to ultimately regulate organ growth. Briefly, Arabidopsis thaliana Col-0 plants were grown under a light : dark (LD) 12 h : 12 h photoperiod, at a 100 µmoles/m2/s fluence rate and 21 °C of constant temperature. Plants were then exposed to either daily End of Day (EoD) far-red (FR, 40µmoles/m2/s of FR light (730nm) for 10 minutes) from day 6 and harvested L3 primordia or blade tissue at days 13, 16 and 20, or EoD FR from day 18, sampling at day 20. Plant kept under white light (WL) conditions were used as controls.
Project description:This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway. The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway. We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment. In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall. The proposed experiment will consist of: wild-type: D-pretreated (x2 biological replicates) wild-type: FR-preconditioned (x2) gun1,gun5: D-pretreated (x2) gun1,gun5: FR-preconditioned (x2) phyA: D-pretreated (x1) phyA: FR-preconditioned (x1) Experiment Overall Design: Number of plants pooled:300 seedlings
Project description:Plant architecture greatly depends on its branching patterns. Branches are formed from meristems initiated in the axils of leaves. Axillary meristems may develop immediately giving new shoots or they may become arrested after a short period of growth as dormant axillary buds. This decision is affected by endogenous and environmental factors. We are studying two Arabidopsis genes coding for TCP transcription factors, BRANCHED1 (BRC1) and BRANCHED2 (BRC2) that control this key decision. Several endogenous and environmental stimuli affect this process one of them is the quality of ambient light. Plants have developed sophisticated mechanisms that allow them to detect the presence of nearby plants and trigger responses of development to avoid the shade. This set of responses is known as shade avoidance. One response to this syndrome, with high agronomic relevance, is the suppression of branching. The genetic basis of this response is still largely unknown. Our goal is to carry out, in Arabidopsis, a systematic study of the genetic control of the removal of branching during the escape response of the shadow. We found that Arabidopsis plants produce fewer branches when grown at high density. We also found that the removal of high-density branch is accompanied by up-regulation of BRC1. On the other hand, short light treatment enriched in far-red (which simulate the shade plant) also cause an accumulation of BRC1 mRNA levels in plants grown at low density. Besides initial data off transcriptomic analysis (wt vs. brc1-2) indicate that BRC1 could be involved in signaling / response to light in the axillary buds. In this study we have identified several potential target genes of BRC1 involved in the response to light. One is PIL2 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 2), a gene that encodes a bHLH transcription factor that interacts with APRR1/TOC1. We are currently characterizing in more detail at the genetic and molecular level the BRC1 relationship with this and other potential target genes and their role in the control of branching patterns. The microarray analysis was performed using RNA samples obtained from three independent plant pools grown under identical conditions. The cDNA synthesized from RNA of wild type or brc1-2 plants grown either in white light (WT-WL, brc1-2-WL) or red+far red light (WT-FR, brc1-2-FR) was hybridized.
Project description:This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway. The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway. We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment. In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall. The proposed experiment will consist of: wild-type: D-pretreated (x2 biological replicates) wild-type: FR-preconditioned (x2) gun1,gun5: D-pretreated (x2) gun1,gun5: FR-preconditioned (x2) phyA: D-pretreated (x1) phyA: FR-preconditioned (x1) Keywords: strain_or_line_design
Project description:This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway. The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway. We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment. In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall. The proposed experiment will consist of: wild-type: D-pretreated (x2 biological replicates) wild-type: FR-preconditioned (x2) gun1,gun5: D-pretreated (x2) gun1,gun5: FR-preconditioned (x2) phyA: D-pretreated (x1) phyA: FR-preconditioned (x1)