Rob Alba; Zhangjun Fei; Paxton Payton; Yang Liu; Shanna L. Moore; Paul Debbie; Jonathan Cohn; Mark D'Ascenzo; Jeffrey S. Gordon; Jocelyn K. C. Rose; Gregory Martin; Steven D. Tanksley; Mondher Bouzayen; Molly M. Jahn; Jim Giovannoni

The Plant Journal, Volume 39, Number 5, September 2004, pp. 697-714(18)

ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant

physiology and development

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= transcribed portion of a genome

expression profiling

• RNA gel blot (=northern)

• differential display

• cDNA-AFLP

• sequencing cDNA libraries (ESTs)

• SAGE

• microarrays

ONE GENE AT A TIME

NOT QUANTITATIVE, difficult to confirm

CHEAP! little genomic data required

EXPENSIVE AND LABOR-INTENSIVE

NOT SENSITIVE TO LOW-ABUNDANCE TRANSCRIPTS

many genes at once, semi-quantitative expensive

low-abundance transcripts sequence

errors

2 structure impairs RT

human error

gene discovery “transcriptom

e activity”

expression arrays

mapping, coding regions

EXPRESSEDSEQUENCE

TAGS

the TOM1 array cDNA microarray based on EST library 12,899 features representing 8500 tomato

genes protocols and confirmatory data available at The Tomato Expression Database:

http://ted.bti.cornell.edu

differential expression between tomato and

pepper pericarp

microarray pitfalls

• cross-hybridization with related sequences• non-detection of sequence not included in the

array• data handling is complex, therefore prone to

human error (transformation, normalization, visualization, interpretation)

• poor replication/experimental design• cDNA microarrays:

– chimeric clones– inconsistent hybridization due to non-uniformity of

microarray features

artifacts occur but the pattern is robust

21 RUBISCO homologs photosynthesis-associated genes

data visualization

interpretation is problematic

expression profiling simultaneously measures as much of the transcriptome as is represented on the chip

this provides a valuable resource for studying regulatory and metabolic networks

massive quantities of data are generated (and need to be analyzed)

high costs and statistical difficulties encourage more focused approaches, but you only find what you’re looking for!

Fruit-specific RNAi-mediated suppression of DET1 enhances

carotenoid and flavonoid content in

tomatoes

Ganga Rao Davuluri, Ageeth van Tuinen, Paul D Fraser, Alessandro Manfredonia, Robert Newman, Diane Burgess, David A Brummell, Stephen R King, Joe Palys, John Uhlig,

Peter M Bramley, Henk M J Pennings & Chris Bowler

Nature Biotechnology  23, 890 - 895 (2005)

• hydrophobic• mevalonic acid

pathway

• hydrophilic• acetate-malonate

pathway

carotenoids flavonoids

free radical scavengers/antioxidantsenhance vertebrate immune systemnot synthesized by animals

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modified from C.M. Rick

quercetin (flavonoid)

flavonoid and carotenoid biosythesis occurs through

separate pathways

chlorogenic acid (phenylpropanoid)

B-carotene (carotenoid)

lycopene (carotenoid)

naringenin-chalcone (flavonoid)

attempts at increasing phenolic/carotenoid production via expression of biosynthetic enzymes/transcription factors

Ye et al. 2000: production of β-carotene in rice endosperm

via transformation with biosynthetic enzymes from

daffodil, Erwinia

Fraser et al. 2002: fruit-specific expression of Erwinia phytoene synthase increases

carotenoid production in tomato

Ducreux 2005: enhanced carotenoid production in potato via heterologous expression of Erwinia phytoene synthase

Niggeweg 2004: overexpression of

HQA to increase CGA production in tomato

Muir 2001: overexpression

of petunia chalcone-isomerase increases

flavonol tomato

Bovy 2002: increased flavonol

production through

heterologous expression of

maize transcription

factor

Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1.A C Mustilli, F Fenzi, R Ciliento, F Alfano, and C Bowler

Plant Cell. 1999 February; 11(2): 145–157.

de-etiolated 1:in A. thaliana, display light-grown phenotype when grown in the dark

tomato hp-2 shows no phenotype in dark, but is hyper-responsive to light

and has elevated pigment

Ganga Rao Davuluri, Ageeth van Tuinen , Diane Burgess, David A. Brummell, Stephen R. King, Joe Palys, John Uhlig, Henk M. J. Pennings, Chris Bowler, Anna Chiara Mustilli, Alessandro Manfredonia Robert Newman

Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencing

WT

HIGH PIGMENT!phenotypes consistent with loss of function, suggesting silencing

post-transcriptional gene silencing

transcriptional gene silencing

transgene-induced silencing

gene-specific methylation

degradation of gene

transcripts

Davuluri et al. (2005) apply a post-tanscriptional silencing

approach (RNAi) under a fruit-specific promoter

dimunition of TDET1 transcript in fruit but not

other tissues

identification of TDET1 degradation products in

fruit but not leaves

lycopene

B-carotene

fruit weight

brix

Arabidopsis genome

germplasm resources

“functional genomics”:

identification of fruit-specific

promoters

cloning det1

genetic mapping

homology to

Arabidopsis

genomiclibraries

expressio

n d

ata