Delayed fluorescence as a universal tool for the measurement of circadian rhythms in higher plants

Abstract

SUMMARYThe plant circadian clock plays an important role in enhancing performance and increasing vegetative yield. Much of our current understanding of the mechanism and function of the plant clock has come from the development of Arabidopsis thaliana as a model circadian organism. Key to this rapid progress has been the development of robust circadian markers, specifically circadian-regulated luciferase reporter genes. Studies of the clock in crop species and non-model organisms are currently hindered by the absence of a simple highthroughput universal assay for clock function, accuracy and robustness. Delayed fluorescence (DF) is a fundamental process occurring in all photosynthetic organisms. It is luminescence-produced post-illumination due to charge recombination in photosystem II (PSII) leading to excitation of P680 and the subsequent emission of a photon. Here we report that the amount of DF oscillates with an approximately 24-h period and is under the control of the circadian clock in a diverse selection of plants. Thus, DF provides a simple clock output that may allow the clock to be assayed in vivo in any photosynthetic organism. Furthermore, our data provide direct evidence that the nucleus-encoded, three-loop circadian oscillator underlies rhythms of PSII activity in the chloroplast. This simple, high-throughput and non-transgenic assay could be integrated into crop breeding programmes, the assay allows the selection of plants that have robust and accurate clocks, and possibly enhanced performance and vegetative yield. This assay could also be used to characterize rapidly the role and function of any novel Arabidopsis circadian mutant.

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoë fedtschenkoi

“…The imaging system for DF was identical to the luciferase and delayed fluorescence imaging system described previously (Gould et al, 2009). DF was quantified using Imaris (Bitplane) to measure mean intensity for specific regions within an image.…”

“…KfGI was particularly noteworthy for the fact that the phasing of its transcript peaks and troughs shifted late relative to the wild type in line rPPCK1-3 on the second and third 24-h period of the LL experimental run ( Figure 6L), which contrasted strikingly with the earlier phase/ shorter period of the LL CO 2 fixation rhythm for rPPCK1-3 ( Figure 3A). The level of delayed fluorescence (DF) has previously been reported to be under robust circadian control in K. fedtschenkoi and provides a simple high-throughput assay for measuring circadian period, robustness, and accuracy (Gould et al, 2009). DF was measured in the wild type, rPPCK1-1, and rPPCK1-3 under LL free-running conditions (Figure 7), and the data were analyzed to calculate key circadian rhythm statistics using Biodare Zielinski et al, 2014).…”

“…DF was quantified using Imaris (Bitplane) to measure mean intensity for specific regions within an image. Background intensities were calculated for each image and subtracted, to give a final DF measurement (Gould et al, 2009). …”

“…The Petri dish was placed in the imaging system at 22°C in constant red-blue light (LL). DF images were collected every hour for 108 h as described previously (Gould et al, 2009). The DF images were processed as described by Gould et al (2009).…”

“…DF images were collected every hour for 108 h as described previously (Gould et al, 2009). The DF images were processed as described by Gould et al (2009). The luminescence was normalized by subtracting the Y value of the best straight line from the raw Y value.…”