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

389 Evolutionary Proteomics Identifies Ligand-binding Amino Acids Of The Cytokinin Receptor
CHASE Domain
Alexander Heyl 1 , Klaas Wulfetange 1 , Birgit Pils 2 , Nicola Nielsen 1 , Georgy Romanov 3 , Thomas Schmulling 1
1
Free University Berlin, Germany, 2 Julius Maximilian University, Wurzburg, Germany, 3 Russian Academy of
Sciences, Moscow, Russia
In Arabidopsis thaliana, the plant hormone cytokinin is sensed by three cytokinin receptors (AHK2, AHK3, CRE1/
AHK4). Using a bacterial binding assay, we show that the ligand is bound by an extracellular domain, the so-called
CHASE domain (cyclases, histidine kinase associated sensory extracellular). This domain is not only found in plants,
but also in many uncharacterized receptors of bacteria and lower eukaryots. Using the wealth of the available genomic
sequence information, an modified evolutionary proteomics approach was taken to discover amino acids important for
the cytokinin binding. We searched for residues which are conserved in the plant cytokinin receptors, but not in receptors
binding other ligands and compared the differences in the evolutionary rates of the respective amino acids. Five amino
acids within the CHASE domains of plants seemingly important for cytokinin binding were identified. Site-directed
mutagenesis and in vivo binding assays confirmed the importance of four of the five selected residures. This study clearly
shows the power of this novel approach in which the computational analyis of the genomic sequence information is used
to generate an experimental verifyable hypothesis.
390 A CBL-Interacting Protein Kinase Is Involved in Early Nitrate Signaling
Heng-Cheng Hu 1, 2 , Yi-Fang Tsay 1
1
Institute of Molecular Biology, Academia Sinica, Taiwan., 2 Institute of Genetics, National Yang-Ming
University, Taiwan.
CHL1 is a dual-affinity root nitrate transporter in Arabidopsis. To elucidate the physiological impacts of CHL1,
microarray using Affymetrix ATH1 indicated that CHL1 plays important roles in regulating several metabolism pathways,
including N-assimilation, glycolysis, trehalose-6-phosphate pathway and some ion transporters. Interestly, some calcineurin
B-like protein (CBL) and CBL-interacting protein kinase (CIPK) genes are up- or down-regulated in chl1 mutant. The
CBL-CIPK networks are well-known to be involved in stress signaling. Here we report that a CIPK is also involved
in early nitrate signaling. Analysis of two independent knockout mutants indicated that CIPK can positively regulate
the nitrate-induction levels of nitrate transporter genes and nitrate assimilation genes. Similarly, nitrate in the medium
will inhibit the primary root growth of wild type, but the inhibitory effect of nitrate is eliminated in the cipk mutants. In
conclusion, the CIPK is the first kinase found to be involved in nitrate signaling. Our data also indicated that in addition
to stress, CBL-CIPK is also responsible for nitrate sensing.