Transcript Whole transcriptome analysis of germinating smoke water

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