Comment[ArrayExpressAccession] E-GEOD-46318 MAGE-TAB Version 1.1 Public Release Date 2013-04-23 Investigation Title A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves Comment[Submitted Name] A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves Experiment Description This SuperSeries is composed of the SubSeries listed below. Refer to individual Series Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Moore Person First Name Jonathan Person Mid Initials David Person Email jonathan.moore@warwick.ac.uk Person Affiliation University of Warwick Person Address Systems Biology, University of Warwick, Senate House, Coventry, United Kingdom Person Roles submitter Protocol Name P-GSE46318-1 P-GSE46318-2 P-GSE46318-3 P-GSE46318-7 P-GSE46318-12 P-GSE46318-8 P-GSE46318-4 P-GSE46318-13 P-GSE46318-10 P-GSE46318-5 P-GSE46318-11 P-GSE46318-6 P-GSE46318-9 Protocol Description Scanned data were quantified using Imagene 7.5.0 software (BioDiscovery, Inc.). Quantified microarray data were Lowess normalied within pintip groups and within arrays, then variation due to arrays and dyes was removed by a random effects model, and averaged, in log space, using R/MAANOVA (MicroArray ANalysis Of VAriance) (Wu et al. 2003). ID_REF = VALUE = Lowess normalized log2 signals Scanned data were quantified using Imagene 7.5.0 software (BioDiscovery, Inc.). Analysis of expression differences between Col-0 and myb2 and Col-0 and myb108 under each condition was performed using the R Bioconductor package limmaGUI (Wettenhall and Smyth, 2004). Raw data were normalized within arrays using a PrintTip lowess transformation and normalized between arrays using the quantile-normalization. The data were fitted to a linear model using a least squares method. P-values were adjusted for multiple testing using Benjamini and Hochberg method to control the false discovery rate (Benjamini and Hochberg, 1995). ID_REF = VALUE = log2 KO/wild type Scanned data were quantified using Imagene 7.5.0 software (BioDiscovery, Inc.). Analysis of expression differences between Col-0 and myb2 and Col-0 and myb108 under each condition was performed using the R Bioconductor package limmaGUI (Wettenhall and Smyth, 2004). Raw data were normalized within arrays using a PrintTip lowess transformation and normalized between arrays using the quantile-normalization. The data were fitted to a linear model using a least squares method. P-values were adjusted for multiple testing using Benjamini and Hochberg method to control the false discovery rate (Benjamini and Hochberg, 1995). ID_REF = VALUE = log2 KO/wild type INV_VALUE = log2 Cy3/Cy5 Cy3 and Cy5 labelled cDNA probes were prepared by reverse transcribing 5µg of aRNA with Cy3- or Cy5- dCTP (GE Healthcare) and a modified dNTP mix (10mM each dATP, dGTP and dTTP; 2mM dCTP) using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with the inclusion of RNase inhibitor (RNaseOUT, Invitrogen) and DTT. Labelled probes were purified using QiaQuick PCR Purification columns (Qiagen), freeze-dried, and resuspended in 50µl hybridization buffer (25% formamide, 5xSSC, 0.1% SDS, 0.5µg/µl yeast tRNA [Invitrogen]). Cy3 and Cy5 labelled cDNA probes were prepared by reverse transcribing 5µg of aRNA with Cy3- or Cy5- dCTP (GE Healthcare) and a modified dNTP mix (10mM each dATP, dGTP and dTTP; 2mM dCTP) using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with the inclusion of RNase inhibitor (RNaseOUT, Invitrogen) and DTT. Labelled probes were purified using QiaQuick PCR Purification columns (Qiagen), freeze-dried, and resuspended in 50µl hybridization buffer (25% formamide, 5xSSC, 0.1% SDS, 0.5µg/µl yeast tRNA [Invitrogen]). Four biological replicates were pooled and labelled twice with each dye giving four technical replicates. Comparisons were made pairwise between WT and mutant under each condition. The microarray experiments were carried out using the CATMA (version 4) microarray (Allemeersh et al., 2005; http://www.catma.org). Labelled samples were hybridized to slides overnight at 42oC. Leaf 7 was tagged with cotton 18 days after sowing (DAS) and harvested from five randomly selected plants, 8 h into the light period, at 23, 29, 31, 33 and 35 DAS (full senescence). Nine 3-week old Col-0, myb2 (genotype IM24) and myb108 (genotype BOS1) rosettes, cut from the root, were transferred to 3 Petri dishes containing water-saturated filter paper and stored at 20oC in complete darkness. Plates were photographed daily and RGB values calculated for leaf 5 of each rosette using the Color Histogram function in ImageJ. RGB intensities were normalized using a white-background reference point and average red/green ratios provided a quantitative measure of leaf yellowing. A red-green ratio of around 0.8 indicates the initiation of senescence. When the average ratio of Col-0 samples was >0.8, leaf 5 for Col-0, myb2 and myb108 lines was harvested (4 biological replicates). The same sampling procedure was then performed on consecutive days to sample as senescence progressed. B. cinerea pepper strain spores (Denby et al., 2004) were prepared and Arabidopsis leaves treated as described in Windram et al., Plant Cell 2012 Sep;24(9):3530-57. PMID: 23023172. Col-0, myb2 and myb108 leaves were inoculated with 4-6 (depending on leaf size) evenly spaced 10μl droplets of B. cinerea spores. Infected leaves were harvested to provide 4 biological replicates. Samples for the comparison between myb108 and Col-0 were harvested at 26 and 30 hpi. Samples for the comparison between myb2 knockout line and Col-0 or myb2 knockout line and Col-0 were harvested at 26 and 30 hpi or 24 and 30 hpi respectively. Arabidopsis plants were grown under a 16:8 hr light:dark cycle (lights on 04:00 to 20:00) at 20°C, 60% humidity and light intensity of 100 μmol photons.m-2.s-1. Arabidopsis seed was stratified for three days in 0.1% agar at 4°C before sowing onto Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1:1). The anac055 line (genotype IM4, col0 background) was T-DNA insertion line Salk_011069 (obtained from the Nottingham Arabidopsis Seed Centre). The anac019 dSpm insertion mutant (genotype 9C11, col5 background) was identified in a pool of SLAT line DNA screened with gene specific primers (Tissier et al., 1999). Arabidopsis plants Col-0, myb2 and myb108 were grown under a 16:8 hr light:dark cycle (lights on 04:00 to 20:00) at 20°C, 60% humidity and light intensity of 100 μmol photons.m-2.s-1. Arabidopsis seed was stratified for three days in 0.1% agar at 4°C before sowing onto Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1:1). The myb2, myb108 lines were T-DNA insertion lines Salk_045455, Salk_024059 respectively (obtained from the Nottingham Arabidopsis Seed Centre). Total RNA was isolated from four individual leaves from each sampled time point (arbitrarily labelled as biological replicates A, B, C and D) using TRIzol reagent (Invitrogen), purified with RNeasy columns (Qiagen) and amplified using the MessageAmp II aRNA Amplification kit (Ambion) in accordance with the kit protocol with a single round of amplification. Following hybridization, slides were washed and scanned using an Affymetrix 428 array scanner at 532nm (Cy3) and 635nm (Cy5). Protocol Type normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol labelling protocol labelling protocol hybridization protocol sample treatment protocol sample treatment protocol sample treatment protocol growth protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TIME DAYS_AFTER_SENESCENCE GENOTYPE Experimental Factor Type time days_after_senescence genotype Publication Title A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves. Publication Author List Hickman R, Hill C, Penfold CA, Breeze E, Bowden L, Moore JD, Zhang P, Jackson A, Cooke E, Bewicke-Copley F, Mead A, Beynon J, Wild DL, Denby KJ, Ott S, Buchanan-Wollaston V PubMed ID 23578292 Publication DOI 10.1111/tpj.12194 Comment[SecondaryAccession] GSE46318 Comment[GEOReleaseDate] 2013-04-23 Comment[ArrayExpressSubmissionDate] 2013-04-23 Comment[GEOLastUpdateDate] 2013-04-25 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-46318.sdrf.txt