15th International Conference on Arabidopsis Research - TAIR

T07-041
A Systems Approach to Nitrogen Networks
Coruzzi, G.(1), Gutierrez, R.(1), Lejay, L.(2), Shasha, D.(3), Palenchar, P.(1),
Cruikshank, A.(1)
1-New York University, Dept of Biology, 100 Washington Sq East. NY NY 10003
2-INRA, Biochimie et Physiologie Moleculaire, Montpellier, Cedex 1
3-New York University, Courant Institute of Mat & Computer Sciences
Our long-term goal is to identify networks in the Arabidopsis genome that
are transcriptionally controlled by C and N metabolic sensing and signaling
pathways. Visualizing and modeling such metabolic regulatory networks
should enable us to devise predictive models that may permit intervention for
agricultural traits. As a proof of principle, we have begun to use genomic,
bioinformatic and systems biology approaches to model and visualize
metabolic regulatory networks controlled in response to carbon and nitrogen
metabolites. We have used several math tools to design systematic spaces
that enable us to cover a large experimental space of treatment conditions
with a small number of experiments. One approach we have used to do this
is called Combinatorial Design. We have used Affymetrix whole genome
chips to identify genome wide responses to this matrix of C:N treatment conditions.
Hierarchical cluster analysis of the treatments reveals three types of
genome-wide responses to C and/or N treatments. We have identified gene
clusters that are transcriptionally regulated by N sensing or CN sensing. We
have used several approaches to analyze these clusters to define biologically
relevant processes controlled by N and CN networks. In one network analysis
we queried the clusters for biological themes using GO or MIPS functional
annotations, performed statistical tests for significance of the response
and display the results in a color-coded network graph. In another type of
network analysis, we used Cytoscape to build a metabolic and regulatory
network model of Arabidopsis using the information from KEGG, AraCyc and
Transfac databases. These network models enabled us to define several
biologically relevant subnetworks within the N-regulated gene networks. This
analysis has revealed that a number of cell biological, metabolic and regulatory
processes are part of a N-regulatory network in Arabidopsis. This work
has also generated biological hypothesis for mechanisms of CN sensing/signaling
which we are testing using Arabidopsis mutants.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T07-042
Elevated plastid-derived isoprenoid synthesis in the
prl1 mutant of Arabidopsis thaliana
Doris Albinsky(1), Hiroyuki Kasahara(1), Juan M. Estevez(1), Kazumi
Nakabayashi(1), Yuji Kamiya(1), Shinjiro Yamaguchi(1)
1-Plant Science Center, RIKEN, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama-shi, Kanagawa 230-
0045, Japan
Isoprenoids comprise a wide spectrum of primary and secondary plant
metabolites. Primary metabolites are present in all plants and essential for
their survival, e.g. as part of the photosynthetic machinery (phytol moiety of
chlorophylls and carotenoids), whereas the dispensable secondary metabolites
provide the plant with benefits for the interaction with its environment.
Both groups of metabolites can be synthesized either in the cytosol through
the MVA (mevalonate) pathway or in the plastid via the MEP (methylerythtritol
phosphate) pathway. Although enzymes in both pathways have been
identified in bacteria and plants, little is known about the interaction between
the two pathways and their spatial and temporal regulation. We performed
a resistance-screen in Arabidopsis thaliana activation-tagged lines using
fosmidomycin, an inhibitor of the DXR (1-deoxy-D-xylulose 5-phosphate
reductoisomerase) of the MEP pathway. In the presence of fosmidomycin,
wild-type Arabidopsis seedlings cannot develop green cotyledons due to a
block in the synthesis of pigments through the MEP pathway in plastids. We
reasoned that the MEP pathway and/or the cross-talk between the MEP and
the MVA pathways (Kasahara et al., 2002) might be modulated in fosmidomycin-resistant
(fre) mutants.
Here, we report the isolation and characterisation of a fre mutant
with a T-DNA insertion in the PRL1 (pleiotropic regulatory locus 1) gene.
PRL1 is a regulatory protein with conserved WD motifs, and is proposed
to function as a pleiotropic regulator of glucose and hormone responses
in Arabidopsis (Nemeth et al., 1998). The mutants are not only resistant to
fosmidomycin, but also to ketoclomazone inhibiting the DXS (1-deoxy-D-xylulose
5-phosphate synthase) of the MEP pathway and to fluridone, inhibiting
the phytoene desaturase in carotenoid biosynthesis. We show that the
resistance of the prl1 mutants to various herbicides blocking the plastid-derived
isoprenoid synthesis is, at least in part, due to an upregulation of protein
levels of the MEP-pathway enzymes and provide biochemical evidence for an
elevated flux through the MEP pathway. A possible regulatory role of PRL1 in
isoprenoid synthesis is discussed.
Kasahara et al. (2002) J. Biol. Chem. 277, 45188-45194
Nemeth et al. (1998) Genes Dev. 12, 3059-3073
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)