Approaches

Different resources used to identify regulatory promoter motifs:

Transcript data from detailed lab studiesTranscript data from past experiments reveals co-expression.

Comparative genomics: Inter-species promoter comparisonComparison of promoter sequences of a gene between different species can identify elements highly conserved since divergence.

Large transcript data-setsClustering of array data over development, biotic/abiotic stresses in order to discover co-regulation through co-expression.

Network discovery driven by large-scale transcriptional profiling

Vast publicly-available Arabidopsis transcript data sets

Identify tightly co-expressed mitochondrial genes and predict regulatory motifs

Test activity of motifs

Cluster genes with a motif of interest based on motif organisation and expression

Test motif activity and importance of organisation

O2 H2O

NAD(P)H

NAD(P)+

NAD(P)H

NAD(P)+

ADP + Pi ATP

O2 H2O

succinate

fumarate

H+ H+ H+

Respiratory chain

MATRIX

III IV V

Number of genes in Arabidopsis:• cytochrome pathway – approx. 150• AOX – 5• alternative NAD(P)H DHs - 7

The alternative respiratory pathway was induced

ModuleFinder - Biclustering

Salicylic acid (100M)Salicylic acid (10M)Rotenone (3hr)Rotenone (12hr)Chloramphenicol (3 hr)Chloramphenicol (12 hr)CysteineH2O2

Oligomycin (1.25M)Oligomycin (0.125M)Anoxia (N2)

0% 50%

Identification of regulatory motifs

Model of module promoters:

Models of regulation - testable

Leaf development transcript profiles

0 1 2 3 4 5 60 1 2 3 4 5 60 1 2 3 4 5 6

QRT-PCR to measure transcripts over 2˚ rosette leaf development

Identification of Import Component Regulatory Elements

Leaf Development

0 1 2 3 4 5 6

Normalised Transcript Abundance

Leaf age (Weeks)

Tom20-2 / Tom20-3

Tom9-1 / Tom20-4

0 1 2 3 4 5 6

Tom20-2 / Tom20-3

Tom9-1 / Tom20-4

Test predictions

Measure reporter gene

Testing functionality

Phylogenetic analysis AtAox1c and GmAox2b motif

AtAox1a

Distance from TSS

motifs identified in the common co-expression environmentmotifs identified in the AOX1a co-expression environmentmotifs identified in the upstream regions of AOX1a and NDB2

motif identified oncemotif identified twicemotif identified three timesmotif identified four timesmotif identified five timesmotif identified six times

Colour key: Motifs predicted by co-expression analysis

Line width key:

1000 GTTATGGTTTTGTATAAAAACCCATTTGATTGTAATTTTTATTTAAGTTTTGGTTTAGGTTTGGTTTGATTAAAAACCGTAAAACCGAACGTTTTTTTTG

900 TTTTTGATTTAAATTAAAAATAATTGTATATATATATATATAATGTTCATTTGATAACATGATATCTATCAAACTATCGAAAAACAAAACCCTAACTGTA

800 ACCTAAACTAAAATTCTATATAAATTACATGCCGTCATTTAGGATTTGAGTTTACAAATTAGATTTTGATTTTATTTATGCATCACACTTATAATTTTTT

700 TTGGTAAAAACATGAAAAAACCGGAACCAAACCGGAACCGATCCGAACCAAAATACATATGGTTTTTAAATGGTTTTAATTTTTTAAAACCAAAAACTGT

600 AAAACTGTTAAAACCGAACCGTAACCAAACCGAATTTTATATGGTTTTTATATGGTTTTACTTTTCTTAAAATCGAAAAACCGTAAAACCTAAAACCGAA

500 ACGAAACCAAACCGAAAAACTGAACACCCAGCCCTTAAATATAATGAAAATCGAATAAATTTGTTTGAAAGAATCGAACAAAATTGACAATAAAATCTAA

400 TTAGGACTATTTTCGTCTAATTTTGACTTAGTTGAAACAGAATATTAGCAAAAATACTAAAACACCACAACGCGTAATAATACCCACACACGATATCATT

300 AAATTTGACCAATAAGAATCTAGCTCTTGGCGACCACGCAAGTATCTTCCATCTTGCTCTCCAAGAAAAATCTACACCGGCTTTAAATTTACATAAACAC

200 CCTCAGTCAAAGAAAAGTCGTAAACATAGTCTCTCTCATGACCACAAGGGTAACACAGTCATCCTAAATATAAACCACACAAGAAAACTGTTATACTTTA

100 TACACGTGTCATAGTCTCATTACATCTACGTGAAGAGTTTCGATCATCAACCGTTCGTTTTCTTACTATATAAACCTTGCTCGAGACCTGCGTGTGAAGC TSS

A1 B1 A2 B2

GGAGACTTGAAGCTGATGCGACGCTTCGTGATGTCGTCATGGTGGTCCGTGCTGATGAAGCGCATCACCGTGATGTTAACCACTATGCATCCGTAAGTAG

CTTGAGGAAGAAGCAATCCACTCTTACACTGAGTTTCTTAAAGAACTCGATAATGGTAACATCGAAAATGTGCCTGCACCGGCTATTGCCATTGATTACT

motifs identified by comparison to soybean Aox

motifs identified by bi-clustering