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

335 Analysis of the Complex BIO3 / BIO1 Locus of Arabidopsis
Rosanna Muralla 1 , Colleen Sweeney 1 , Elve Chen 2 , Libuse Brachova 2 , Basil Nikolau 2 , David Meinke 1
1
Oklahoma State University, Stillwater, OK, USA, 2 Iowa State University, Ames, IA, USA
The biosynthesis and utilization of biotin in plants has been elucidated in part through the analysis of two auxotrophic
mutants of Arabidopsis (bio1 and bio2) first identified 15-20 years ago using a forward genetic screen for embryo-defective
mutants rescued by growth of arrested embryos on enriched media. We demonstrate here through reverse genetics that
the bio3 mutant, which is disrupted in a region predicted to encode an enzyme that functions in an intermediate step in
the pathway, has a phenotype similar to that of the bio1 and bio2 mutants. The surprising discovery is that the BIO3 and
BIO1 loci are positioned adjacent to each other on the chromosome, in the same orientation as found in many bacterial
and fungal species, and that differential splicing results in the production of two types of transcripts, one with the potential
to encode two separate proteins, and the other capable of producing a fusion protein that catalyzes two different steps in
the pathway. The existence of the fusion protein in plant extracts is being tested using antibodies directed against each
of the monocistronic gene products produced in E. coli. The results obtained to date have provided important clues to
the genomic organization of biotin biosynthetic genes in Arabidopsis, the intracellular localization of biotin synthesis,
and the evolutionary remnants of a prokaryotic operon in a flowering plant.
336 Characterization of GDU1 and GDU1-Like Genes Involved in the Regulation of Amino Acid
Metabolism and Transport
Rejane Pratelli 2 , Wolf Frommer 1 , Guillaume Pilot 2
1
Carnegie Institution, 260 Panama Street, Stanford, CA 94305-4101 USA, 2 IZMB Universitaet Bonn,
Kirschallee 1, 53115 Bonn, Germany
The GDU1 (Glutamine Dumper 1) gene was identified by the study of an activation tagged mutant from Arabidopsis (gdu1-
1D). This gene encodes an uncharacterized, 158 amino acid-long protein, expressed in the vascular tissues, in both phloem and
xylem parenchyma cells. GDU1 protein contains a single transmembrane spanning region and seems to be targeted to the plasma
membrane and the membrane of yet undefined vesicles (1). Arabidopsis genome encodes five proteins sharing between 32 and
76% overall similarity with GDU1 and showing strong sequence conservations in two domains that are specific of this family
of plant proteins. The promoters of the GDU1-like genes are active in various organs of the plant, each gene showing a distinct
expression pattern. This suggests that the GDU genes present similar functional properties but have different roles due in part to
their specific expression pattern and possibly to the sequence differences they display outside of the two conserved domains.
The activation-tagged gdu1-1D mutant over-expresses GDU1. These plants are smaller than the wild type, secrete glutamine
and sodium at the hydathodes and display necrosis spots on the older leaves. The leaf content of each of the free amino acids
is increased in the mutant compared to the wild type, resulting in a doubling of the overall amount of free amino acids. The
concentration of amino acids in the phloem and the xylem saps is also increased twofold in the mutant. It is thought that the
glutamine secreted from the hydathodes originates from the xylem sap, as glutamine constitutes about 90% of xylem sap amino
acids. The hydathode tissues from the mutant would thus be deficient in reabsorbing the xylem amino acids. Wild type plants are
unable to grow on media containing high concentrations of several amino acids (e.g. Met, Thr, Phe, Tyr) because of the feedback
inhibition of biosynthetic pathways induced by these amino acids. Interestingly, the gdu1-1D mutant is unaffected by these high
concentrations, reminiscent of the phenotype of the pig1-1 mutant (2), which is supposed to be altered in the regulation of amino
acid metabolism. These data suggest that the transport and the metabolism of amino acids are altered in the gdu1-1D mutant.
(1) Pilot, G., Stransky, H., Bushey, D.F., Pratelli, R., Ludewig, U., Wingate, V.P., and Frommer, W.B. (2004). Overexpression of GLUTAMINE
DUMPER1 leads to hypersecretion of glutamine from hydathodes of Arabidopsis leaves. Plant Cell 16, 1827-1840.
(2) Voll, L.M., Allaire, E.E., Fiene, G., and Weber, A.P. (2004). The Arabidopsis phenylalanine insensitive growth mutant exhibits a deregulated
amino acid metabolism. Plant Physiol. 136, 3058-3069.