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

331 A Putative Bifunctional Wax Ester Synthase / Acyl-CoA:Diacylglycerol Acyltransferase
WSD10 is Involved in Stem Wax Ester Synthesis in Arabidopsis
Xuemin Wu, Patricia Lam, Reinhard Jetter, David Bird, Lacey Samuels, Ljerka Kunst
Department of Botany, University of British Columbia, 6270 University Blvd, Vancouver, BC, V6T 1Z4,
Canada
Neutral lipids, including wax esters and triacylglycerols, have very important dietetic, technical and pharmaceutical
applications. A bifunctional wax ester synthase (WS)/ acyl-CoA:diacylglycerol acyltransferase (DGAT) was identified
in Acinetobacter calcoaceticus via mutant analysis. This enzyme catalyzes the transfer of an acyl group onto a very-long
chain alcohol acceptor. On the basis of homology to the WS/DGAT Acinetobacter sequence, a gene family of 11 members
(WSD family) was found in Arabidopsis. To date none of the WSD genes has been characterized. It is likely, however, that
one or more of these genes encode enzymes responsible for the formation of wax esters found in the cuticle, a protective
lipid structure deposited on shoot surfaces of all land plants. The cuticle is composed of cutin polymer matrix and waxes.
These waxes are arranged into an intracuticular layer in close association with cutin, and an epicuticular film exterior to
this, which often includes epicuticular wax crystals.
In this report we provide evidence that one gene member of the Arabidopsis WSD family, the WSD10, is required for
the production of cuticular wax esters. Wax analyses of two independent T-DNA insertion knock-out mutants of WSD10
demonstrated that there were no detectable wax esters on the stems of these lines. The cryo-SEM examination of the
surface of the wsd10 mutant stem showed altered shape of epicuticular wax crystals. WSD10promoter::GUS activity in
transgenic Arabidopsis lines revealed that, in addition to the stem, this gene is highly expressed in the pedicel, suggesting
that WSD10 may also perform acyltransferase functions at this location.
332 Characterization of K + -dependent and –independent L-asparaginases from Arabidopsis
Frederic Marsolais, Luanne Bruneau, Ralph Chapman
Agriculture and Agri-Food Canada
L-asparaginases (EC 3.5.1.1) are hypothesized to play an important role in nitrogen supply to sink tissues, especially
in legume developing seeds. Two plant L-asparaginase subtypes have been previously identified according to their K + -
dependence for catalytic activity. An L-asparaginase homologous to Lupinus K + -independent enzymes with activity
towards β-aspartyl dipeptides, At5g08100, has been previously characterized as a member of the N-terminal nucleophile
amidohydrolase superfamily in Arabidopsis. In this study, a K + -dependent L-asparaginase from Arabidopsis, At3g16150,
is characterized. Recombinant At3g16150 and At5g08100 share a similar subunit structure and conserved auto-proteolytic
pentapeptide cleavage site, commencing with the catalytic Thr nucleophile, as determined by ESI-MS. The catalytic activity
of At3g16150 was enhanced approximately 10-fold in the presence of K + . At3g16150 was strictly specific for L-Asn, and
had no activity towards β-aspartyl dipeptides. At3g16150 also had an approximately 80-fold higher catalytic efficiency
with L-Asn relative to At5g08100. Among β-aspartyl dipeptides tested, At5g08100 had a preference for β-aspartyl-His,
with catalytic efficiency comparable to that with L-Asn. Phylogenetic analysis revealed that At3g16150 and At5g08100
belong to two distinct subfamilies. Transcript levels of At3g16150 and At5g08100 were highest in sink tissues, especially
in flowers and siliques early in development, as determined by quantitative RT-PCR. The overlapping spatial patterns of
expression argue for a partially redundant function of the enzymes. However, the high catalytic efficiency suggests that
the K + -dependent enzyme may metabolize L-Asn more efficiently under conditions of high metabolic demand for N.