Rob Alba; Zhangjun Fei; Paxton Payton; Yang Liu; Shanna L. Moore; Paul Debbie;

ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development 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 www.pulsephotonics.com The Plant Journal, Volume 39, Number 5, September 2004, pp. 697-714(18)

Transcriptome = 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 NOT SENSITIVE TO LOW-ABUNDANCE TRANSCRIPTS CHEAP! little genomic data required EXPENSIVE AND LABOR-INTENSIVE expensive many genes at once, semi-quantitative

mapping, coding regions gene discovery expression arrays “transcriptome activity” EXPRESSED SEQUENCE TAGS low-abundance transcripts 2 structure impairs RT sequence errors 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 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 intomatoes 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 Biotechnology23, 890 - 895 (2005)

hydrophobic mevalonic acid pathway hydrophilic acetate-malonate pathway carotenoids flavonoids • free radical scavengers/antioxidants • enhance vertebrate immune system • not synthesized by animals

RANK IN NUTRIENT CONTENT RANK IN NUTRIENT CONTRIBUTION TO AVERAGE U.S. DIET 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 Ye et al. 2000: production of β-carotene in rice endosperm via transformation with biosynthetic enzymes from daffodil, Erwinia Muir 2001: overexpression of petunia chalcone-isomerase increases flavonol tomato Fraser et al. 2002: fruit-specific expression of Erwinia phytoene synthase increases carotenoid production in tomato Bovy 2002: increased flavonol production through heterologous expression of maize transcription factor 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 transcriptional gene silencing transgene-induced silencing degradation of gene transcripts post-transcriptional gene silencing 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 genetic mapping germplasm resources cloning det1 identification of fruit-specific promoters homology to Arabidopsis expression data Arabidopsis genome

Rob Alba; Zhangjun Fei; Paxton Payton; Yang Liu; Shanna L. Moore; Paul Debbie;