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Whole transcriptome analysis of germinating smoke water treated maize seeds Endre Sebestyén ARI-HAS Department of Applied Genomics H-2462 Martonvásár, Brunszvik street 2. Introduction Smoke released from burning vegetation contains a chemical signal, which : is a germination cue breaks dormancy and promotes seed germination also improves seedling vigour Effect on plants varies Fire-prone species (Australia, South A.) Non fire-prone species Crops 3-methyl-2H-furo[2,3-c]pyran-2-one O O O Experimental layout & problems Maize microarray with 46k features from www.maizearray.org Chip annotation is incomplete Funding for the project ended Samples Germinating maize embryos (3, 6, 9, 12, 18, 24, 27h) Early postgermination phase (24, 48h) Smoke treated vs control samples The molecular background of smoke action is unknown Postgermination phase results 24h results (1121/711 up/downregulated) Sulfiredoxin-like protein, LRR-receptor like kinase 2, ubiquitin activating enzyme, etc Lots of genes without annotation 48h results (765/887 up/downregulated) Auxin regulated protein, zinc finger protein, etc Also lots of genes without function Validated by rtPCR Some of the significantly up- or downregulated genes might be good candidates for further analysis, but what about the overall process? Gene Ontology analysis Gene Ontology www.geneontology.org Biological process Molecular function Cellular component GeneMerge software Modified version to speed up analysis 1,5h -> 1-2min Search for significantly overrepresented GO terms in the upand downregulated gene lists Gene Ontology analysis (24h, ) 35 30 25 20 15 10 5 0 Gene Ontology analysis (48h, ) 8 7 6 5 4 3 2 1 0 Gene Ontology analysis (24h, ) 20 18 16 14 12 10 8 6 4 2 0 Gene Ontology analysis (48h, ) 30 25 20 15 10 5 0 Promoter analysis BLAST search with available cDNA sequences against available maize genome sequence Extract promoters up to 1500 bp upstream Search for known transcription factor binding sites (TFBS) TRANSFAC 12.1 (www.gene-regulation.com) 834 experimentally verified plant TFBS Promoter analysis (24h, ) TRANSFAC ID Number Description NT$PR1A 68 (21%) Pathogensis rel. prot. 1a, GT box PEA$IAA45_06 52 (16%) Auxin regulated gene 4/5, footprint-F element AT$WCOR413_01, etc 52 (16%) Cold regulated genes, DRE element MAIZE$DHN1_09, etc 45 (14%) DHN1 gene, ABRE element RICE$CRL1_01 42 (13%) CRL1 gene, AUXRE2 element 317 promoters available from the 24h upregulated list Other promoter elements (TATA-box, etc) Experimental validation B/1: control B/2: ABA B/3: smoke B/4 ABA+smoke Germination phase results 3-12h – germination phase 24-27h – postgermination phase 1800 1600 1400 1200 1000 Upregulated 800 Downregulated 600 400 200 0 3 6 9 12 24 27 Germination phase results 3h 6h 9h 12h Ubiquitin act. enzyme +40 Ubiquitin act. enzyme +27 Hypothetical prot. -16 Put. hASNA-I +12 Expressed prot. -30 Put. senesc. assoc. prot. -11 No description -13 Expressed prot. +10 Putat. cyt. P450 +12 Put. endonuclease -10 Put. splicing factor -13 Expressed prot. +10 Auxin reg. prot. -10 Put. xylose isomerase +9 Put. NLI intercting fact. +13 Unk. prot. +9 Putat. cyt. P450 -9 TaWIN1 -13 Unk. prot. +8 TF MADS32 +8 Calcineurin B-like pr. 9 -8 NADPH cyt. P450 -13 Put. DHHC type prot. +9 Lipid-transf. prot. like +12 No description -15 Put. glucanase +9 Put. DHHC type prot. -6 Put. chitinase +7 Hypothetical prot. -11 Put. cyt. P450 +7 Calcineurin B-like pr. 9 -7 Ubiquitin act. enzyme +6 Germination phase results 24h 27h Adenylate translocator +18 Photosystem II OE17 +3 7 No description +12 Glycosyltransferase +2 7 No description +12 Hypothetical prot. +2 3 Put. cyt. P450 -9 Unknown prot. +2 2 Ubiquitin act. enzyme +6 ZmGR1b +2 0 Gene Ontology analysis Significantly enriched GO terms 3h : response to heat, salt stress, trancription 6h : response to cold, salt stress, water depr., transcription 9h : no significantly enriched terms 12h : translation, biosynth. processes, response to cold, etc 24h : response to bacterium, growth, development 27h : growth, morphogenesis, response to bacterium K-means clustering K-means clustering Conclusions Smoke induces stress related changes Supported by Gene Ontology and promoter analysis Rapid decay of mRNAs present in embryos The hardening effect of smoke is similar to that of caused by ABA Vigour test also shows possible interaction between smoke and ABA Participants Vilmos Soós Angéla Juhász János Pintér Marnie E. Light Johannes van Staden Ervin Balázs