Transcript Fruit-specific RNAi-mediated suppression of DET1 enhances

ESTs, cDNA microarrays, and gene
expression profiling: tools for dissecting plant
physiology and development
The Plant Journal, Volume 39, Number 5, September 2004, pp. 697-714(18)
www.pulsephotonics.com
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
= transcribed portion of a genome
expression profiling
ONE GENE AT A TIME
• RNA gel blot (=northern)
• differential display NOT QUANTITATIVE, difficult to confirm
• cDNA-AFLP CHEAP! little genomic data required
SENSITIVE TO
LOW-ABUNDANCE
TRANSCRIPTS
•NOT
sequencing
cDNA
libraries (ESTs)
EXPENSIVE AND LABOR-INTENSIVE
• SAGE
• microarrays expensive many genes at once, semi-quantitative
gene
discovery
expressio
n arrays
“transcriptom
e activity”
mapping,
coding
regions
EXPRESSED
SEQUENCE
TAGS
lowabundance
transcripts
sequence
errors
2 structure
impairs RT
human
error
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
21 RUBISCO homologs
but the pattern is robust
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)
carotenoids
• hydrophobic
• mevalonic acid
pathway
flavonoids
• hydrophilic
• acetate-malonate
pathway
free radical scavengers/antioxidants
enhance vertebrate immune system
not synthesized by animals
RANK IN NUTRIENT CONTRIBUTION
TO AVERAGE U.S. DIET
RANK IN NUTRIENT CONTENT
modified from C.M. Rick
flavonoid and carotenoid
biosythesis occurs through
separate pathways
lycopene (carotenoid)
B-carotene (carotenoid)
chlorogenic acid (phenylpropanoid)
naringenin-chalcone (flavonoid)
quercetin (flavonoid)
attempts at increasing phenolic/carotenoid production via
expression of biosynthetic enzymes/transcription factors
Niggeweg 2004:
overexpression of
HQA to increase CGA
production in tomato
Muir 2001:
overexpression
of petunia
chalconeisomerase
increases
flavonol tomato
Bovy 2002:
increased
flavonol
production
through
heterologous
expression of
maize
transcription
factor
Ye et al. 2000: production of
β-carotene in rice endosperm
via transformation with
biosynthetic enzymes from
daffodil, Erwinia
Fraser et al. 2002: fruitspecific expression of Erwinia
phytoene synthase increases
carotenoid production in
tomato
Ducreux 2005: enhanced
carotenoid production in
potato via heterologous
expression of Erwinia
phytoene synthase
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
Manipulation of DET1 expression in tomato results in
photomorphogenic phenotypes caused by post-transcriptional
gene silencing
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
WT
phenotypes consistent
with loss of function,
suggesting silencing
HIGH PIGMENT!
gene-specific
methylation
transgeneinduced
silencing
degradatio
n 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
“functional genomics”:
genomic
libraries
identification
of fruitspecific
promoters
genetic
mapping
cloning det1
expression data
homology
to
Arabidopsi
s
germplasm resources