15th International Conference on Arabidopsis Research - TAIR

T13-013
Genetic and molecular dissection of the role of CPD
steroid hydroxylase in regulation of brassinosteroid
hormone biosynthesis and signalling in Arabidopsis
Marcel Lafos(1), Zsuzsanna Koncz(1), Csaba Koncz(1)
1-Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln
The Arabidopsis CPD (Constitutive Photomorphogenic Dwarf) locus encodes
a cytochrome P450 steroid C23-hydroxylase enzyme, CYP90A1, implicated
in both early and late C6-oxidation pathways of brassinosteroid (BR) hormone
biosynthesis. We found that overexpression of CYP90A1 by the mannopine
synthase promoter in the cpd knockout mutant results in activation of pathogenesis
related PR genes, and that CYP90A1 shows interaction with several
signaling factors, including a MAP kinase (MAPK), an oxysterol-binding (OBP)
and an IAP (inhibitor of apoptosis-like) RING protein in the yeast two-hybrid
system. To explore the expression pattern, regulation, cellular localization,
and in vivo interactions of the CPD gene product, we expressed GFP and
epitope tagged forms of CYP90A1 in the cpd mutant. CYP90A1 was localized
in the plasma membrane, ER and nuclear envelope. In addition to using the
native CPD promoter, we expressed CYP90A1 with various tissue specific
promoters, as well as using a chemically inducible system, in order to obtain
tools for studying the temporal and spatial control of BR biosynthesis and
transport. Whereas several P450 enzymes in BR biosynthesis seem to utilize
NADPH P450 reductases as electron donors, our data indicate that these
enzymes fail to interact with and donate electrons to CYP90A1. Therefore, biochemical
and cross-linking studies with complemented cpd knockout lines,
producing GFP or epitope labeled CYP90A1, were initiated to identify the yet
unknown CYP90A1 reductase and confirm in vivo interaction of CYP90A1
with the MAPK, OBP and IAP proteins. This biochemical approach is supported
by genetic studies, in which we use T-DNA insertion mutations, as well
as inducible overexpression and siRNA constructs, to examine the functions
of CYP90A1-interacting MAPK, OBP and IAP proteins. This approach is supplemented
by similar functional analysis of other members of small OBP and
IAP families, which code for additional steroid carrier proteins and putative E3
ubiquitin ligase enzymes, respectively.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T13-014
Functional Analysis of Arabidopsis At4g23740 GeneF
X. Zhang(1), J. Heinz(1), J. Choi(2), C. Chetty(1)
1-Department of Natural Sciences and Mathematics, Savannah State University
2-School of Biology, Georgia Institute of Technology
In this study, Arabidopsis gene F9D16.210 (At4g23740), a putative member
of the LRR RLK gene family, is selected to characterize its possible biological
function. The LRR RLK protein coded by this gene contains all three characteristic
functional domains &#8211; an extracellular domain, a transmembrane
domain, and an intracellular kinase domain. Its extracellular domain
contains seven leucine-rich repeats. We found that this gene is expressed
in root, stem, leaf, flower, and silique of Arabidopsis plants grown under
normal conditions by using RT-PCR. Five mutant lines in this gene have been
identified from the available T-DNA mutagenized Arabidopsis lines created by
Salk Institute Genomic Laboratory (San Diego, CA). Two lines, Salk_004412
and Salk_092792, carry a T-DNA insertion in the promoter region; one line
Salk_005132 contains an insert into the second exon in the coding region,
and other two lines, Salk_096386 and Salk_047106 lines, contain an insert
in 3&#8217; untranslated region (3&#8217;UTR). These lines were verified
by polymerase chain reaction (PCR) using the appropriated combination of
a gene-specific primer and a T-DNA&#8211;specific primer. Both heterozygotes
and homozygotes for the T-DNA insert were found from each insertion
line. Currently, these verified homozygotes are being systematically screened
for phenotypes to determine the consequences of the mutations on growth
and development relative to the wild type that does not contain the insertion.
T13 Others