75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

337 Interaction-dependent localization of flavonoid enzymes in Arabidopsis
Melissa Ramirez, Brenda S. Winkel
Virginia Polytechnic Institute and State University
As early as the 1940’s, the idea of multienzyme complexes began surfacing as a way the cell might organize enzymes to
enhance the efficiency of metabolism. It has now become evident that macromolecular interaction is a fundamental aspect
of cellular biochemistry. The Winkel laboratory uses the flavonoid pathway of Arabidopsis as a model to understand the
assembly and regulation of enzyme complexes. This pathway is a well-characterized specialized plant metabolic system,
the products of which comprise a diverse set of compounds that are critical for plant survival and reproduction. While
the central steps of flavonoid biosynthesis are well detailed, a full understanding of the biochemistry of the pathway is
complicated by the fact that many of the enzymes can utilize multiple substrates and may also physically interact with
each other.
Until recently, it was believed that flavonoid biosynthesis occurred exclusively in the cytoplasm and that the products
were then transported to various sites of action within the cell. However, new evidence indicates the presence of at least
two flavonoid enzymes in the nucleus, suggesting that the synthesis of nuclear flavonoids may occur in situ. Additional
studies are needed to explore the possibility that subcellular localization of flavonoid enzymes is affected by or dependent
upon specific protein-protein interactions and that this localization determines the types and cellular locations of end
products that are produced in response to diverse biotic and environmental cues.
The study presented here begins to dissect the molecular basis of the observed dual localization of chalcone synthase
(CHS) and chalcone isomerase (CHI), the first two enzymes of the flavonoid pathway. Epi-fluorescence and confocal
laser scanning microscopy are being used to examine the localization of these enzymes expressed as fusions to green
fluorescent protein (GFP) in protoplasts and stably-transformed plants. Analyses are being performed in both wild type
cells and mutants that are depleted in various flavonoid enzymes in order to explore the possible effects of specific
protein-protein interactions on this localization. Site-directed mutagenesis is being used in parallel experiments to test
the functionality of a putative nuclear localization signal in CHS, which overlaps with the predicted CHI interaction
interface. This work represents an essential step in elucidating the mechanisms that organize related metabolic enzymes
within the crowded environment of the cell interior.
338 FAC1-Directed Herbicide Toxicity Correlates With Expanded Adenine Nucleotide Pools
Richard Sabina
Medical College of Wisconsin
EMBRYONIC FACTOR 1 (FAC1) is an early expressed plant gene and knockout lines reveal that it is essential for the zygote
to embryo transition in Arabidopsis thaliana (Plant J 42:743,2005). FAC1 encodes an AMP deaminase (AMPD), which is also the
intracellular target for a class of modified nucleosides produced by fungal pathogens that are converted in plant cells to transitionstate
inhibitors (monophosphates) of this enzyme (Plant Physiol 114:119,1997;Bioorg Med Chem Lett 9:1985,1999). Exposure
to these herbicides results in a rapid 2-3 fold increase in ATP levels, but reportedly not ADP or AMP, and eventual cessation of
seedling growth with paling and necrosis at the apical meristem. The x-ray crystal structure of Arabidopsis FAC1 bound to a
transition-state analog has provided a glimpse of the complete active site (J Biol Chem 281:In Press,2006). However, the mechanistic
basis for lethality associated with a genetic or herbicide-induced limitation in FAC1 catalytic activity is unknown. OBJECTIVE:
Explore the underlying mechanisms for FAC1-directed toxicity by considering the immediate metabolic consequences of an
inability to deaminate AMP in plant cells. APPROACH: Monitor relative growth and quantify intracellular adenine nucleotides
(AXP) and IMP in treated and untreated control Arabidopsis seedlings following systemic exposure to sublethal and lethal doses
of deaminoformycin (DF) and attempt rescue with purine nucleosides and bases. METHODS: 5 to 7 d.o. seedlings were placed
on M&S salt + 1% sucrose agar in 24-well plates with and without DF (300 nM or 22 uM) and/or a purine nucleoside or base
(0.5-1 mM). Plants were grown under long-day conditions (16 h light/8 h dark) for 7-9 days, roots were excised and the remaining
tissue was weighed and ground to a powder under liquid nitrogen. Ice-cold 10% (w/v) TCA was added and the frozen tissue was
homogenized on ice for 2 min with a motorized pestel. The homogenate was centrifuged at 14,000Xg for 2 min at 4C and the
supernatant was neutralized with an equal volume of 0.5 M tri-n-octylamine in freon. All samples were frozen immediately in
dry ice and stored at -80C. Metabolites were separated by anion-exchange HPLC. RESULTS: DF inhibits plant growth and wet
weight is inversely correlated with the levels of ALL adenine nucleotides and the AMP/IMP ratio, an in vivo index of AMPD
activity. Downstream catabolites do not rescue seedlings from a lethal dose of DF. The correlations between decreased AMPD
activity, increased AXP pools, and reduced seedling growth point to mechanisms related to upstream effects of FAC1 inhibition.
This may involve hormonal imbalance due to increased cytokinin synthesis or disruption of 14-3-3 protein function by AMP.
This work was supported by a grant from the Research Affairs Committee (RAC) at the Medical College of Wisconsin and through a
cooperative agreement with Bayer CropScience GmbH, Frankfurt am Main, Germany.