Abstract
BackgroundA robust circadian clock has been implicated in plant resilience, resource-use efficiency, competitive growth and yield. A huge number of physiological processes are under circadian control in plants including: responses to biotic and abiotic stresses; flowering time; plant metabolism; and mineral uptake. Understanding how the clock functions in crops such as Triticum aestivum (bread wheat) and Brassica napus (oilseed rape) therefore has great agricultural potential. Delayed fluorescence (DF) imaging has been shown to be applicable to a wide range of plant species and requires no genetic transformation. Although DF has been used to measure period length of both mutants and wild ecotypes of Arabidopsis, this assay has never been systematically optimised for crop plants. The physical size of both B. napus and T. aestivum led us to develop a representative sampling strategy which enables high-throughput imaging of these crops.ResultsIn this study, we describe the plant-specific optimisation of DF imaging to obtain reliable circadian phenotypes with the robustness and reproducibility to detect diverging periods between cultivars of the same species. We find that the age of plant material, light regime and temperature conditions all significantly effect DF rhythms and describe the optimal conditions for measuring robust rhythms in each species. We also show that sections of leaf can be used to obtain period estimates with improved throughput for larger sample size experiments.ConclusionsWe present an optimized protocol for high-throughput phenotyping of circadian period specific to two economically valuable crop plants. Application of this method revealed significant differences between the periods of several widely grown elite cultivars. This method also identified intriguing differential responses of circadian rhythms in T. aestivum compared to B. napus; specifically the dramatic change to rhythm robustness when plants were imaged under constant light versus constant darkness. This points towards diverging networks underlying circadian control in these two species.
Highlights
A robust circadian clock has been implicated in plant resilience, resource-use efficiency, competitive growth and yield
Measurements of Delayed fluorescence (DF) have been correlated with the photosynthetic state of photosystem II (PSII) [17] and the amount of DF production is regulated by the circadian clock
DF can be measured with a low-light imaging system identical to that used for luciferase imaging and output rhythms have been shown to oscillate with a comparable period to those estimated from luciferase reporter experiments [14]
Summary
A robust circadian clock has been implicated in plant resilience, resource-use efficiency, competitive growth and yield. This research has been conducted by studying leaf movement rhythms or by following luciferase gene expression under the control of a circadian regulated promoter [11,12,13]. Rhythm robustness was assessed by: the percentage of samples classified as rhythmic; the relative amplitude error (RAE); the period coefficient of variation (CV) and the average period error threshold (all defined in Additional file 1). Together, these parameters allow the effects of different imaging conditions to be quantified
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