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

407 Functional Requirements for PIF3 in the De-etiolation Response
Bassem Al-Sady 1 , Weimin Ni 1 , Stefan Kircher 2 , Eberhard Schafer 2 , Peter Quail 1
1
Department of Plant and Microbial Biology, U. C. Berkeley, CA, 94720 and USDA/ARS, Plant Gene
Expression Center, 800 Buchanan Street, Albany, CA 94710, USA, 2 Albert-Ludwigs-Universitat Freiburg,
Institut fur Biologie II/Botanik, Schanzlestrasse 1, 79104 Freiburg, Germany
In order to gain an understanding of the mechanisms of phytochrome (phy) signaling, our laboratory has used yeast
two-hybrid screens and other assays to identify proteins that interact directly and specifically with the biologically
active Pfr form of the phy molecule. Our current focus is on defining the functional role and mechanism of action of
phytochrome-interacting factor 3 (PIF3), a previously identified bHLH transcriptional regulator, and other closely related
PIFs in the bHLH family. Reverse-genetic disruption of these loci indicates that each of the PIFs thus far examined
appears to have a differential role in regulating phy-induced seedling deetiolation and, that PIF3 specifcally, is involved
positively in controlling early phy-mediated gene expression and acts negatively, under prolonged red-light irradiation,
on the phy-mediated hypocotyl growth response.
In trying to uncover the role of the interaction of PIF3 with photoactivated phys and with its G-box DNA target site
towards PIF3’s in vivo function, we have sought to specifically disrupt these interactions by targeted mutagenesis in
the PIF3 protein. We have been able to dissect phyA and phyB binding sites in PIF3 and found that they are located in
separate and unrelated domains of the PIF3 protein. In vivo mutant rescue analysis indicates that the requirements for
phy and DNA binding towards PIF3’s in vivo function can be temporally separated. We have further been interested in
understanding how phys control the observed rapid light induced PIF3 degradation. Recent evidence indicates that light
induces rapid phy-dependent phosphorylation of the PIF3 protein in vivo as a prelude to proteosomal degradation. This
finding may provide insight into the biochemical mechanism of phy signal transfer to target proteins in the cell.
408 Evidence for Functional Conservation, Sufficiency and Proteolytic Processing of the
CLAVATA3 CLE Domain
Jun Ni, Steven Clark
Department of Molecular, Cellular and Developmental Biology, University of Michigan
Members of the CLE (CLV3/ESR-related) protein family are small proteins broadly present in land plants. This
family is defined by their similarity to Arabidopsis CLAVATA3 (CLV3) C-terminal sequence, a conserved domain termed
CLE. This motif is also shared by several parasitism proteins from plant nematode species. Other than the CLE domain,
CLV3 and the CLE proteins are not related in the rest of the proteins. CLV3 is a secreted protein. It has been hypothesized
to act as a ligand for the CLV1/CLV2 receptor complex in the regulation of stem cell specification at shoot and flower
meristems. Mutations within the CLE domain can disrupt CLV3 function. We have tested the ability of 13 Arabidopsis
CLEs to replace CLV3 in vivo and found a significant variability, ranging from complete to no complementation. The best
rescuing CLE depends on CLV1 for function, while other CLEs act independently of CLV1. Domain-swap experiments
indicate that differences in function can be traced to the CLE domain within these proteins. Indeed, when the CLE
domain of CLV3 is placed downstream of an unrelated signal sequence, it is capable of fully replacing CLV3 function.
Interestingly, we have detected proteolytic activity in extracts from cauliflower (Brassica oleracea), Arabidopsis and
tobacco that process CLV3, CLE1 and other CLE-containing proteins at their C termini. For CLV3, processing appears
to occur at the absolutely conserved arginine-70 located at the beginning of the CLE domain. We propose that CLV3
and the other CLE proteins are C-terminally processed to generate an active CLE peptide.