Investigation Title Transcription profiling of Arabidopsis irradated with cFR (far-red) light for 18 hr and treated with 20 nM of flumioxazin Comment[Submitted Name] Moller: Identification of nuclear genes regulated by protoporphyrin IX-mediated retrograde signalling. Experimental Design compound_treatment_design individual_genetic_characteristics_design strain_or_line_design transcription profiling by array Experimental Design Term Source REF mo mo EFO Comment[ArrayExpressReleaseDate] Comment[AEMIAMESCORE] 3 Comment[ArrayExpressAccession] E-NASC-42 Comment[MAGETAB TimeStamp_Version] 2010-08-11 19:13:05 Last Changed Rev: 13058 Experimental Factor Name compound light Experimental Factor Type compound_treatment_design light Experimental Factor Term Source REF Person Last Name unknown Moller Person First Name unknown Simon Geir Person Mid Initials Person Email sgm5@le.ac.uk Person Phone +44 (0)115 951 3091 0116 252 5302 Person Fax +44 (0)115 951 3297 0116 252 3330 Person Address Plant Science Division, School of Biosciences, University of Nottingham, Sutton Bonnington Campus, Loughborough LE12 5RD, UK Department of Biology University of Leicester University Road Leicester LE1 7RH UK Person Affiliation University of Leicester Person Roles biosource_provider;investigator;submitter Person Roles Term Source REF mo Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description Plastids communicate with the nucleus by means of retrograde plastid signals. The far-red (FR) light insensitive Arabidopsis mutant laf6 disrupted in a plastid-localised ABC-like protein (atABC1) accumulates the plastid signal protoporphyrin IX (proto IX) and has attenuated nuclear gene expression (Moller et al.2001 Genes Dev. 15:90-103). Our data suggests that proto IX accumulation results in hypocotyl elongation in response to FR light and we have demonstrated that by inhibiting the plastid localised protoporphyrinogen IX oxidase (PPO) using flumioxazin wild-type plants phenocopy laf6 by accumulating proto IX with a concomitant loss of hypocotyl growth inhibition in a dose-dependent manner. It is at present unclear what effect increased proto IX has on nuclear gene expression and how this is integrated with photomorphogenic responses such as hypocotyl elongation. Protocol Name NASCGrowProt:67 NASCGrowProt:66 NASCExtractProtocol:1 NASCLabelProtocol:1 Affymetrix:Protocol:Hybridization-EukGE-WS2v4 Affymetrix:Protocol:Percentile Affymetrix:Protocol:ExpressionStat Protocol Type nucleic_acid_extraction labeling hybridization feature_extraction bioassay_data_transformation Protocol Description Plants were grown in ambient temperature, atmosphere and humidity. The lighting regime was normal photoperiod followed by 4 days of darkness; followed by irradaition with cFR (far-red) light for 18 hr. The medium was Murashige and Skoog. Plants were grown upto growth stage (Boyes key) 1.0 and whole seedlings were harvested for RNA extraction. Plants were grown in ambient temperature, atmosphere and humidity. The lighting regime was normal photoperiod followed by 4 days of darkness; followed by irradaition with cFR (far-red) light for 18 hr. The medium was Murashige and Skoog with the addition of 20 nM of flumioxazin. Plants were grown upto growth stage (Boyes key) 1.0 and whole seedlings were harvested for RNA extraction. Protocol: Qiagen RNAeasy kit protocol RNA samples were quality controlled using the Agilent 2100 Bioanalyzer. 100 pmol T7-(dT)24 primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3') was hybridized with the Total RNA ( 8 - 12ug) at 70°C for 10 minutes. First strand cDNA was synthesized by reverse transcription using 400 units SuperScript II Reverse Transcriptase in a reaction containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.5 mM dNTPs at 42C for 1 hour. The reaction was then put on ice for 2 mins, followed by second strand cDNA synthesis. 130ul of the second strand reaction mix was added to the 20ul first strand synthesis reaction. The second strand reaction mix contained 40 units E. coli DNA polymerase I, 10 units E. coli DNA ligase, 2 units E. coli RNase H and 0.2 mM dNTPs in a buffer containing 20 mMTris-HCl (pH 6.9), 90 mM KCl, 4.6 mM MgCl2, 10mM (NH4)SO4 and 0.15 mM ß-NAD+. The reaction was incubated at 16 C for 2 hours. 10 units T4 DNA Polymerase was added and the reaction incubated for a further 5 minutes at 16 C, then the reaction was terminated using EDTA. The double-stranded cDNA was cleaned up using the cDNA Cleanup Spin Column supplied in the Affymetrix GeneChip Sample Cleanup Module. The double-stranded cDNA was used as a template for in Vitro transcription of biotin-labeled cRNA using the ENZO BioArray RNA Transcript Labeling Kit supplied by Affymetrix. The Transcript Labeling kit produces large amounts of hybridizable biotin-labeled RNA targets by in Vitro transcription from bacteriophage T7 RNA polymerase promoters. The reaction mixture containing Biotin-Labeled ribonucleotides (ATP, GTP, CTP, UTP with Bio-UTP and Bio-CTP), DTT, RNase inhibitor mix and T7 RNA polymerase was incubated at 37 C for 5 hrs, gently mixing every 30 minutes during the incubation. This produced an average of 40ug biotin-labeled cRNA . The biotin-labeled cRNA was cleaned up using the cRNA Cleanup Spin Column supplied in the Affymetrix GeneChip Sample Cleanup Module. The quantity and quality of the cRNA was measured using the Agilent 2100 Bioanalyzer. The total amount of cRNA was calculated, the starting quantity of RNA subtracted to give the quantity of adjusted cRNA. The cRNA was fragmented by metal-induced hydrolysis to break down full-length cRNA to 35-200 base fragments. 15ug of adjusted cRNA was used to prepare 300ul of hybridization cocktail of which 200ul was hybridized with the GeneChip. Title: Fluidics Station Protocol. Description: Title: Affymetrix CEL Analysis (Percentile). Description: Title: Affymetrix CHP Analysis (ExpressionStat). Description: Protocol Parameters Temperature;Light;Medium;Atmosphere;Humidity; Humidity;Atmosphere;Medium;Light;Temperature; Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF The MGED Ontology SDRF File E-NASC-42.sdrf.txt Term Source Name NCI_thesaurus nasc ncbitax mo ArrayExpress mo EFO The MGED Ontology Term Source File ncithesaurus.obo.alt http://arabidopsis.info/catalogue.html http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version