Whole transcriptome analysis of germinating smoke water treated maize seedsEndre 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

OOO

Experimental layout & problems

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

up- and downregulated gene lists

Gene Ontology analysis (24h, )

Gene Ontology analysis (48h, )

Gene Ontology analysis (24h, )

Gene Ontology analysis (48h, )

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 Numbe

rDescription

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

Germination phase results3h 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

Calcineurin B-like pr. 9 -8 Putat. cyt. P450 -9 TaWIN1 -13 Unk. prot. +8

NADPH cyt. P450 -13 Put. DHHC type prot. +9 Lipid-transf. prot. like +12 TF MADS32 +8

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 results24h 27h

Adenylate translocator +18

Photosystem II OE17 +37

No description +12

Glycosyltransferase +27

No description +12

Hypothetical prot. +23

Put. cyt. P450 -9 Unknown prot. +22

Ubiquitin act. enzyme +6 ZmGR1b +20

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