Gyula, Péter

English

Gyula Péter

University of Szeged

Volume 46(1-2):41, 2002Acta Biologica Szegediensishttp://www.sci.u-szeged.hu/ABSDISSERTATION SUMMARYInstitute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, HungaryIsolation of circadian clock mutants in Arabidopsis thalianaPéter GyulaSupervisor: Ferenc NagyE-mail: gyulap@nucleus.szbk.u-szeged.hu41It is a well known phenomenon that some biological proc-esses exhibit periodic changes on a daily time-scale. Someof these maintain periodicity even in the absence of externalperiodic stimuli which suggests that an internal pacemakeror “clock” exists in the organism. Indeed, genetic mutationswere identified which cause defects in the mechanismproducing circadian rhytmicity. In several model organismsthe molecular basis for circadian clockwork is described indetail. Surprisingly, circadian clock seems to be a goodexample of convergent evolution: there are no conservedclock elements among organisms in different taxa. However,it is generally accepted that in all organisms special clockproteins generate circadian rhythms through a transcription/translation feedback loop, parts of which are unrelated, butthe functions and the architecture of the system is similar.The rhythm generated by the circadian clock is received byseveral output elements like photosynthetic activity or geneexpression. To keep in phase with the external 24 h cycle ofthe day, the clock can be reset by several environmentalfactors which percepted and transmitted to the clock by inputreceptors.Despite of the large amount of information available onthe molecular nature of circadian pacemaker in other or-ganisms, little is known about the clock mechanism in plants.Therefore we decided to identify the elements of the clocksystem in Arabidopsis thaliana by searching for mutantsdisplaying altered circadian phenotype. We used transgeniclines carrying a Cab2:luc reporter gene for mutagenesis byEMS and T-DNA. We screened for mutants by continuouslymeasuring the luciferase enzyme activity in individual plantsover two days in constant darkness after a 7-day longentrainment by 12 h dark-12 h light period. Progeny ofindividual mutants was tested for herited mutant phenotypeeither in constant darkness and red light. Mutants exhibitedany kind of circadian phenotype were kept for furtheranalysis and mapping. We started the genetic mapping of themutants showing the most robust phenotype.After screening of 60,000 EMS mutagenized seedlingswe obtained 37 potential mutants. Eight of these wereselected for genetic mapping. From the T-DNA screen 5 ofthe 5,000 line tested were selected as a putative mutant. Innone of the 5 cases could we show cosegregation of theT-DNA tag with the mutant phenotype, therefore thesemutants will be mapped. After obtaining a rough mapposition from some EMS mutants we found that it is the samein 3 of our mutants and it is the position where an alreadydescribed clock gene –  ZTL – is located. Therefore, wedecided to sequence that gene in the 3 mutants. All of themproved to be new alleles of the ZTL clock gene. ZTL is anF-box protein, which probably plays a role in the ubi-quitin-mediated destruction of clock proteins. It contains aLOV/PAS domain, which is tought to bind flavin cofactorsor mediate protein-protein interactions and can be found inseveral clock-associated protein. It contains the F-box whichdocks target proteins to the E3 ubiquitin ligase complex, anda Kelch-repeat domain which is a common protein-interact-ing domain and expected to bind the target protein. In one ofour mutants, there is an early STOP codon in the last Kelch-repeat, and probably it causes a loss-of-function phenotype.The second ZTL mutant contains a mutation in the 4.Kelch-repeat which causes a non-conservative Gly->Aspamino-acid change, and it is likely a loss-of-function allele,also. The third ZTL mutant mutation is located in the LOV/PAS-domain causing a Leu->Ser amino-acid change. It hasan interesting phenotype, because like other ZTL mutants ithas a long-period but the difference in period length isconstant over a large scale of light intensity which is not truein the case of null-alleles of ZTL. This indicates that in thismutant light-sensitivity remains unaffected, but the functionof the molecule is damaged in some extent. Several mutantshave not been mapped yet, but have interesting phenotypesand therefore we can expect them to have mutations inunknown clock genes.

Isolation of circadian clock mutants in Arabidopsis thaliana

http://acta.bibl.u-szeged.hu/22462/1/4641.pdf

Isolation of circadian clock mutants in Arabidopsis thaliana

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