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

341 Genetic Dissection of Histidine Biosynthesis in Arabidopsis
Colleen Sweeney 1 , Rosanna Muralla 1 , Devin Camenares 2 , Patricia Nugent 2 , Christopher DiFraia 2 , Asya Stepansky 2 ,
Thomas Leustek 2 , David Meinke 1
1
Oklahoma State University, Stillwater, OK, USA, 2 Rutgers University, New Brunswick, NJ, USA
The biosynthesis of histidine in microorganisms, long studied through the isolation and characterization of auxotrophic
mutants, has emerged as a paradigm for the regulation of metabolism and gene expression. Much less is known about
histidine biosynthesis in flowering plants. One limiting factor has been the absence of large collections of informative
auxotrophs. We describe here the results of a systematic screen for histidine auxotrophs of Arabidopsis thaliana. Nine
insertion mutants disrupted in four different biosynthetic genes (HISN2, HISN3, HISN4, HISN6A) were identified through
a combination of forward and reverse genetics and were shown to exhibit an embryo-defective (emb) phenotype that could
be rescued by watering heterozygous plants with histidine. Male transmission of the mutant allele was in several cases
reduced. Another mutant blocked in the final step of the pathway (hisn8) and a double mutant altered in the redundant
first step (hisn1a, hisn1b) exhibited an ovule abortion (ova) phenotype in heterozygotes. Homozygous mutant plants and
callus tissue produced from rescued seeds appeared normal when grown in the presence of histidine but typically senesced
after continued growth in the absence of histidine. These mutants document the importance of histidine biosynthesis
for plant growth and development, provide valuable insights into amino acid transport and source-sink relationships
during seed development, and represent a significant addition to the limited collection of well-characterized auxotrophs
in flowering plants.
342 Proteomic Analysis of the Flavonoid Biosynthetic Machinery in Arabidopsis thaliana
Nileshwari Vaghela 1 , Iuliana Lazar 1, 2 , Brenda S. Winkel 1
1
Department of Biological Sciences, Virginia Tech, 2 Virginia Bioinformatics Institute (VBI), Virginia Tech
Work on a wide variety of metabolic pathways indicates that these systems are often, if not always, organized as multienzyme
complexes. Enzyme complexes have the potential to increase catalytic efficiency and provide unique mechanisms for the
regulation of cellular metabolism. The flavonoid biosynthetic pathway of Arabidopsis is an excellent model for studying the
organization, localization, and regulation of enzyme complexes at the cellular level [1]. Flavonoids are specialized metabolites
that perform many important physiological roles in plants. Protein interactions among several key flavonoid enzymes have been
described [2]. Moreover, at least two of the flavonoid enzymes have a dual cytoplasmic/nuclear localization [3]. These results
indicate that flavonoid enzymes assemble into one or more distinct complexes at different intracellular locations.
The current study integrates a new technology, mass spectrometry, with well-established affinity chromatography methods
to further characterize the organization and composition of the Arabidopsis flavonoid enzyme complex. Two key flavonoid
enzymes, chalcone synthase (CHS) and chalcone isomerase (CHI), are being used in these experiments to detect interacting
enzymes. Recombinant thioredoxin (TRX), TRX-CHS, or TRX-CHI is produced in E. coli [4,5], then purified by metal affinity
chromatography, and covalently coupled to an activated resin, Affi-Gel 10 [2]. Extracts prepared from 5-day-old wild type
or CHS- or CHI-deficient lines of Arabidopsis are then passed over the column and the bound proteins are eluted with SDS.
This eluate is then subjected to a liquid chromatography (LC) - mass spectrometry (MS) protocol developed for the analysis of
complex peptide mixtures [6]. An Agilent LC system coupled with an LTQ-MS instrument (Thermo Finnigan, San Jose, CA) is
being used for this purpose. Data analysis is being performed with BioWorks II software. This project offers a new prospect for
confirming previously-described interactions among flavonoid enzymes and for identifying novel interacting proteins, thereby
enhancing our understanding of the flavonoid enzyme complex as a whole.
1. Winkel B. 2004. Ann. Rev. Plant Biol. 55: 85
2. Burbulis IE, Winkel-Shirley B. 1999. Proc. Natl. Acad. Sci. USA 96: 12929
3. Saslowsky et al. 2005. J Biol Chem. 24;280(25):23735
4. Pelletier MK, Burbulis IE, Shirley BW. 1999. Plant Mol. Biol. 40: 45
5. Dana CD, Martins R, Krueger S, Winkel BSJ. Submitted.
6. Sarvaiya H, Yoon JH, Lazar IM. Submitted.