PARENT SESSION

Symposium S8C C4 and CAM
Friday September 3rd, 2004 8:30 AM-10:30 AM Room 510B
Chair: Rowan Sage
Co-Chair: Howard Griffiths

Molecular dissection of the CAM circadian clock. Susanna Boxall¹, Jonathan Foster¹, Hans Bohnert^{2, 5}, John Cushman³, Hugh Nimmo⁴, James Hartwell^{*,1, 2, 3, 4}, ¹ CNAP, Department of Biology (Area 7), York, North Yorkshire, UK² Department of Biochemistry and Molecular Biophysics, Tucson, Arizona, USA⁵ Department of Plant Biology, Urbana, Illinois, USA³ Department of Biochemistry, Mailstop 200, Reno, Nevada, USA⁴ Plant Molecular Science Group, Glasgow, Strathclyde, UK

ABSTRACT- We are studying the molecular basis of the CAM CO₂ fixation circadian rhythm. We have cloned the Mesembryanthemum crystallinum orthologues of the central plant clock genes and determined that TOC1 and LHY/CCA1 are under reciprocal circadian control in a similar manner to their regulation in the Arabidopsis clock. TOC1 peaks around subjective dusk and LHY/CCA1 peak around subjective dawn. The regulation of LHY/CCA1 is not affected by CAM induction, whilst TOC1 is slightly salt-induced. ELF4 is also clock-controlled in C3 and CAM with peak transcript in the evening. We have also found that homologues of FKF, ZTL, GI and ELF3 are under circadian control in C3 and CAM leaves. Interestingly, whilst the transcript abundance of ZTL has not been found to oscillate in Arabidopsis, it does oscillate in Mesembryanthemum. Using semi-quantitative RT-PCR, we have examined changes in the transcript abundance profile for over 150 genes in C3 and CAM leaves of Mesembryanthemum under 12:12 light/dark cycles (LD) and free-running conditions (constant light and temperature, LL). This work has revealed that many genes encoding enzymes of the CAM pathway, glycolysis and starch metabolism are controlled by the circadian clock in CAM-induced leaves of Mesembryanthemum. We have cloned the promoters of the most strongly CAM-induced and clock-controlled genes in order to identify the motifs involved. We have recently identified several transcription factors that may be important in the clock control of CAM-associated genes. We are also investigating the relationship between developmental and salt induction of CAM. Finally, our efforts to transform both Mesembryanthemum and Kalanchoe fedtschenkoi will be discussed. We are generating transgenic lines that overexpress the central clock genes CCA1/LHY, TOC1, and ELF4. These transgenic lines will have an arrhythmic central clock allowing us to assess the impact of arrythmia on CO₂ fixation. Progress with the transformation will be discussed.

KEY WORDS: circadian clock, CAM, gene transcript profiling, Mesembryanthemum crystallinum