title: Forward genetic analysis of the circadian clock separates the multiple functions of ZEITLUPE.
abstract: The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.
authors.affiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged.
authors.forename: Eva
authors.initials: E
authors.lastname: Kevei
authors.forename: Péter
authors.initials: P
authors.lastname: Gyula
authors.forename: Anthony
authors.initials: A
authors.lastname: Hall
authors.forename: László
authors.initials: L
authors.lastname: Kozma-Bognár
authors.forename: Woe-Yeon
authors.initials: WY
authors.lastname: Kim
authors.forename: Maria E
authors.initials: ME
authors.lastname: Eriksson
authors.forename: Réka
authors.initials: R
authors.lastname: Tóth
authors.forename: Shigeru
authors.initials: S
authors.lastname: Hanano
authors.forename: Balázs
authors.initials: B
authors.lastname: Fehér
authors.forename: Megan M
authors.initials: MM
authors.lastname: Southern
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Bastow
authors.forename: András
authors.initials: A
authors.lastname: Viczián
authors.forename: Victoria
authors.initials: V
authors.lastname: Hibberd
authors.forename: Seth J
authors.initials: SJ
authors.lastname: Davis
authors.forename: David E
authors.initials: DE
authors.lastname: Somers
authors.forename: Ferenc
authors.initials: F
authors.lastname: Nagy
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Millar
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002940:Circadian Rhythm / Q000235:genetics*; D017930:Genes, Reporter; D008027:Light; D008156:Luciferases / Q000032:analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000378:metabolism; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D036226:Seedlings / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis
chemical_list: D029681:Arabidopsis Proteins; C410503:ZTL protein, Arabidopsis; D008156:Luciferases; D001616:beta-Galactosidase