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

93 Reverse Genetic Analysis of the F-box Protein Family in Arabidopsis
Xiaohua Zheng, Richard Vierstra
Department of Genetics, University of Wisconsin-Madison
The growth and development of Arabidopsis are delicately tuned by the synthesis of nascent protein as well as
degradation of undesirable protein. The breakdown of many regulatory proteins is accomplished by the ubiquitin (Ub)
26S proteasome system. Through an ATP-dependent enzymatic cascade involving E1s, E2s and E3s, Ub becomes
covalently attached to various short-lived proteins, then these ubiquitinated species are recognized and degraded by the
26S proteasome. A large family of E3s (or Ub-protein ligases) is responsible for choosing appropriate substrates for
turnover. One type of E3s, the SCF complex, is composed of cullin, Rbx1, Skp1(Ask) and F-box protein subunits, with
the F-box protein serving as the substrate recognition factor. We previously identified almost 700 loci in Arabidopsis
that encode F-box proteins, making this family one of the largest in plants. While a number have been shown to control
important developmental and physiological processes in plants, the functions of most F-box proteins are unknown.
To help define these function(s), we have generated a large collection of T-DNA insertion mutants in F-box genes that
previously have not t been studied in Arabidopsis. In total, we screened 249 Salk lines and obtained useful mutants for
104 loci. We failed to obtain homozygous mutants for only two lines, indicating that a majority of the Arabidopsis F-box
protein genes are not essential. Phenotypic analysis of the remaining 104 revealed little or no defects under standard
growth conditions. However, mild phenotypes were detected for some mutants when exposed to various conditional
treatments. Preliminary analyses of several have revealed roles for F-Box proteins in sugar metabolism and various
hormone responses. Further analysis of this population is ongoing with the aim of comprehensively understanding the
functions of key F-box proteins in plant development.
with meals
94 Purification and Biochemical Characterization of Two beta-Thioglucoside Hydrolysases
(TGG1 and TGG2) in Arabidopsis thaliana
Changhe Zhou 4 , David Bevan 2 , Michael Reichelt 3 , Asim Esen 1
1
Department of Biology, Virginia Tech, 2 Department of Biochemistry, Virginia Tech, Blacksburg, VA24061,
3
Department of Biochemistry, Max-Planck Institute, Jena, Germany, 4 Department of Biology, Virginia Tech,
Blacksburg, VA24061
The myrosinase-glucosinolate system is a major pre-formed chemical defense system involved in a range of biological
interactions between insects, pathogens and host plants of the Brassicaceae. Myrosinases (beta-thioglucoside hydrolysase)
catalyze the hydrolysis of glucosinolates, a group of nitrogen- and sulphur-containing secondary metabolites. Native myrosinase
from white mustard seed forms a dimer stabilized by a Zn2+ ion, is heavily glycosylated and folds into a (b/a)8-barrel structure.
There are several functional sites in the native myrosinase including binding sites for ascorbate, glucosinolate, water, Zn2+ and
myrosinase-binding proteins. Four myrosinase genes, namely TGG1 (At5g26000), TGG2 (At5g25980), TGG4 (At1g47600) and
TGG5 (At1g51470), as well as two pseudogenes, have been identified in Arabidopsis. There is little or no information available
about purification and biochemical characterization native myrosinases from Arabidopsis. To better understand the function
and biochemistry of myrosinases, TGG1 and TGG2 homozygous T-DNA insertion lines have been selected and confirmed with
Western analysis using peptide-specific antibodies, which showed that TGG1 and TGG2 expression were completely blocked in
T-DNA lines. In comparison with wildtype, no abnormal phenotypes were observed in TGG1 and TGG2 T-DNA lines. Western
blot analysis showed that TGG1 and TGG2 were strongly expressed in seedling, rosette leaf, cauline leaf, stem, flower and silique
while specific activity in different organs showed that TGG1 has higher activity than TGG2 in those organs. Both TGG1 and
TGG2 expression could be induced by methyl jasmonic acid and Pseudomonas syringae. The homogeneous TGG1 and TGG2
were isolated from Arabidopsis plants and characterized, including kinetics and substrate specificity. More than 90% of TGG1
in Arabidopsis plants is soluble, whereas least 60% of TGG2 is insoluble. High pH and salts enhanced the solubility of TGG2.
Native TGG1 is a dimer with a molecular mass of 150kD and a monomer with a molecular mass of 73kD. TGG1 and TGG2
exhibited a wide range of substrate specificity, they also had beta-glucosidase activity and hydrolyzed p-nitrophenyl beta-Dglucopyranoside(pNPG),
o-nitrophenyl beta-D-glucopyranoside(oNPG) and 4-methylumbelliferyl-beta-D-glucopyranoside(4-
MUG). TGG1 could be activated in the presence of ascorbate while TGG2 was inhibited by ascorbate.
This project has been supported by NSF Arabidopsis 2010