1. Whole transcriptome analysis of germinating smoke water treated maize seeds Endre Sebestyén ARI-HAS Department of Applied Genomics H-2462 Martonvásár, Brunszvik street 2.

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

3. Experimental layout & problems  Maize microarray with 46k features from www.maizearray.org 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

4. 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?

5. Gene Ontology analysis  Gene Ontology www.geneontology.orgwww.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 up- and downregulated gene lists

6. Gene Ontology analysis (24h,  )

7. Gene Ontology analysis (48h,  )

8. Gene Ontology analysis (24h,  )

9. Gene Ontology analysis (48h,  )

10. 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)www.gene-regulation.com  834 experimentally verified plant TFBS

11. Promoter analysis (24h,  ) TRANSFAC IDNumberDescription NT$PR1A68 (21%)Pathogensis rel. prot. 1a, GT box PEA$IAA45_0652 (16%)Auxin regulated gene 4/5, footprint-F element AT$WCOR413_01, etc 52 (16%)Cold regulated genes, DRE element MAIZE$DHN1_09, etc45 (14%)DHN1 gene, ABRE element RICE$CRL1_0142 (13%)CRL1 gene, AUXRE2 element  317 promoters available from the 24h upregulated list  Other promoter elements (TATA-box, etc)

12. Experimental validation B/1: control B/2: ABA B/3: smoke B/4 ABA+smoke

13. Germination phase results  3-12h – germination phase  24-27h – postgermination phase

14. Germination phase results 3h6h9h12h Ubiquitin act. enzyme+40Ubiquitin act. enzyme+27Hypothetical prot.-16Put. hASNA-I+12 Expressed prot.-30Put. senesc. assoc. prot.-11No description-13Expressed prot.+10 Putat. cyt. P450+12Put. endonuclease-10Put. splicing factor-13Expressed prot.+10 Auxin reg. prot.-10Put. xylose isomerase+9Put. NLI intercting fact.+13Unk. prot.+9 Calcineurin B-like pr. 9-8Putat. cyt. P450-9TaWIN1-13Unk. prot.+8 NADPH cyt. P450-13Put. DHHC type prot.+9Lipid-transf. prot. like+12TF MADS32+8 No description-15Put. glucanase+9Put. DHHC type prot.-6Put. chitinase+7 Hypothetical prot.-11Put. cyt. P450+7Calcineurin B-like pr. 9-7Ubiquitin act. enzyme+6

15. Germination phase results 24h27h Adenylate translocator+18Photosystem II OE17+3 7 No description+12Glycosyltransferase+2 7 No description+12Hypothetical prot.+2 3 Put. cyt. P450-9Unknown prot.+2 2 Ubiquitin act. enzyme+6ZmGR1b+2 0

16. 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

17. K-means clustering

19. 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

20. Participants  Vilmos Soós  Angéla Juhász  János Pintér  Marnie E. Light  Johannes van Staden  Ervin Balázs