Microsoft PowerPoint - 070531_KDChang_E4_Gene-to-metabolite_networks_for_terpenoid_indole_alkaloid_biosynthesis_in_Catharanthusroseus cells
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Rischer, Heiko et al. (2006) Proc. Natl. Acad. Sci. USA 103, 5614-5619
1. Introduction
4. Integration 5. Discussion
terpenoid indole alkaloids
ajmalicine - antihypertensive alkaloid
vinblastine and vincristine - antineoplastic bisindole alkaloids
• Plant Secondary Metabolism • no apparent role in the growth and development of the plant
• important as toxins or feeding deterrents
• exhibit strong pharmacological activities
• transcription factors binding to promoters of TIA biosynthetic genes
• transcript (boxes) and metabolite (circles)
• left and right jasmonate and auxin
Biosynthesis of C. roseus TIAs
• 35 intermediates and 30 enzymes • seven different (sub)cellular compartments • extensive transport of intermediates
Biosynthesis of C. roseus TIAs
strictosidine
Biosynthesis of C. roseus TIAs
late steps of vindoline formation
epidermal layers only in the early phases of TIA biosynthesis take place
cDNA-amplified fragment length polymorphism (AFLP) +
targeted and nontargeted liquid chromatography-mass spectrometry
regulatory system governing TIA metabolism &
metabolic engineering of alkaloid biosynthesis
Why not microarray?
Metabolite Analysis tabersonine
2. matching mass and retention time ajmalicine, tabersonine, catharanthine, yohimbine, cathenamine, secologanine, lochnerinine, 16-methoxy-
2,3-dihydro-3-hydroxytabersonine, and desacetoxyvindoline
monomeric TIA metabolites
presence and absence of auxin (NA) and MeJA (MJ) and the time points (in hours)
retention time and the m/z value
stimulated accumulation of metabolites
repressed accumulation of metabolites
43 metabolites
53 metabolites
• The differential auxin mediated regulation of some of these metabolites might be enforced by
or dependent on the presence of MeJA. • Not all monomeric TIAs identified are found within
the same auxin-modulated subcluster, indicating
divergent regulation of metabolite biosynthesis and accumulation within the TIA pathway.
eg. tabersonine and catharanthine
Breyne, P., ey al. (2003) Mol. Genet. Genomics 269, 173–
179.
• good-quality sequencing 417 fragments (74%)
• Blasting with the 236 C. roseus European Molecular Biology Laboratory 10% gave a (near)perfect match
• 37% of the CR tags displayed no sequence similarity to any known plant genes
Transcript Profiling
Functional Catalog of the Munich Information Center for Protein Sequences
Transcript Profiling
MeJA 42.8%
growth 33.8%
auxin 28.5%
Information Center for Protein
Metabolism and Energy TIA genes
Eg. genes associated with terpenoid moiety, indole moiety, monomer TIAs, TFs
Transcript Profiling
• close similarity (70%) to 10HGO, STR, T16H, and DAT
• in plant secondary metabolism even close sequence similarity is not sufficient to indicate a correct functional annotation
without a (near) perfect sequence match
activation
repression
• 178 metabolite peaks + 417 transcripts • principal component analysis with mean scaling was performed
first to explore the variability structure of the data.
PC1: auxin treated and untreated
cultures
response to elicitation
• The Pearson correlation
variables
(i) the presence of the known early
pathway TIA genes and the
ORCA transcription factors
genes with as-yet-unspecified
only gene or metabolite pairs or
triplets) containing,ajmalicine,
value of correlation coefficient C 0.8
this network centered on tabersonine includes numerous tags
that are, thus, likely to code for some of the missing links in the
biosynthesis of tabersonine or other monomeric TIAs.
Discussion
• the lack of large sequence repertoires serial analysis of gene expression or microarray analysis are notapplicable
• genome-wide cDNA-AFLP transcript profiling build a substantial collection of known and previously undescribed genes
• The scarcity of gene sequences from either C. roseus or other species belonging to the Apocynaceae family 37% ‘‘no-hit’’ tags in BLAST searches
Discussion
• 2D PAGE proteomic analysis of TIA-producing identified 58 proteins, only two are known to be involved in TIA biosynthesis
• cytosolic mevalonate pathway or transcription factors steady-state levels are constant throughout the different hormone or elicitor treatments
• Upon overlay of the gene expression &metabolite accumulation profiles on the pathway, the findings extend earlier observations
• Additionally, other metabolic pathways such as the S-adenosyl methionine (SAM) cycle .
• These gene found around the tabersonine node
Discussion