Day Length Signalling and Crosstalk Between Cytoplasmic and Chloroplastic Calcium Oscillations in Arabidopsis thaliana

The production of grain, fruit and vegetables relies on flowering; without flowers, therefore, there would be no agriculture. Plants carefully control the timing of flowering to optimise pollination and resources, and thereby maximise seed production. Flowering occurs at very precise times of the year, but how this is achieved remains a mystery. Since the early 1930's it has been known that plants keep track of the seasons by comparing the ticking of an internal chronometer to the day length they experience at different times of the year. However, we still know very little about how the signals between this internal pacemaker and day length are perceived and understood. This project will research that critical process which allows plants to flower at the best possible time. I will investigate the hypothesis that calcium-signalling pathways are involved in coordinating flowering time. Calcium is an essential signalling molecule in plants. I have previously shown that the internal chronometer generates calcium oscillations that are shaped by the relative duration of days and nights. Work in other laboratories has shown that a sudden calcium increase occurs in the chloroplasts (the organelles where photosynthesis occurs), at nightfall. I propose that co-incidence, or 'crosstalk' between these two calcium signals is involved in floral timing. To investigate this possibility, I will develop new recombinant techniques for manipulating and imaging calcium in living plants. I will combine these technologies with molecular analysis of flowering time genes and classic physiological protocols to elucidate the role of calcium in flowering time. This research will lead to new, important insights into the seasonality of flowering that will have general application to agriculture. For example, the flowering of crops may be extended over a whole season to avoid gluts, reducing waste, storage costs and the need for imports. Controlling flowering time by precision agriculture may therefore result in improved crop management practices and the production of higher quality food that will benefit the farmer, the consumer and the environment.

Seed production is the most crucial element in agriculture. Maximum seed production relies on the precise timing of seasonal flowering to optimise pollination and resources. Plants track the seasons by measuring photoperiod against an endogenous rhythm generated by the circadian clock; the 'external co-incidence' model. My previous studies indicate that Ca2+, a primary second messenger in plants, is involved in photoperiod signalling, though precisely how remains a mystery. I hypothesised that coincidence or crosstalk between cytoplasmic [Ca2+] oscillations from the endogenous circadian clock and dark-induced [Ca2+] spikes in the chloroplast modulate the expression of genes that control flowering. This hypothesis will be investigated in the model angiosperm Arabidopsis thaliana by experimentally tasting the predictions that suppressing or damping cytoplasmic Ca2+ oscillations from the circadian clock and dark-induced chloroplast [Ca2+] spikes promotes flowering by altering the expression patterns of the key flowering genes CONSTANS (CO) and FLOWERING LOCUS T (FT). Ca2+ signals in the cytoplasm and in the chloroplast will be altered by mis-expression of the calreticulin C-domain or of the Ca2+ transporter PPF1, and verified using the bioluminescent Ca2+ sensor, aequorin. Crosstalk between cytoplasmic and chloroplastic Ca2+ signals will be imaged high-resolution confocal or multi-photon microscopy of newly developed cameleon-GFPs. CO and FT transcription analysis will enable us to firmly establish the link between cellular Ca2+ signalling and floral timing. By combining new methods of Ca2+ manipulation, imaging and molecular analysis, this multidisciplinary project will provide significant new data on photoperiodic signalling, challenge and enhance current molecular models of flowering, and take our understanding of floral timing in a radically new direction.