Establishment of integrated protocols for automated high throughput kinetic chlorophyll fluorescence analyses.

BACKGROUND: Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge. RESULTS: We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (ΦPSII). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of ΦPSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency. CONCLUSIONS: The incorporation of imaging systems suitable for kinetic chlorophyll fluorescence analysis leads to a substantial extension of the feature spectrum to be assessed in the presented high throughput automated plant phenotyping platforms, thus enabling the simultaneous assessment of plant architectural and biomass-related traits and their relations to physiological features such as PSII operating efficiency. The implemented high throughput protocols are applicable to a broad spectrum of model and crop plants of different sizes (up to 1.80 m height) and architectures. The deeper understanding of the relation of plant architecture, biomass formation and photosynthetic efficiency has a great potential with respect to crop and yield improvement strategies.

Phototropins do not alter accumulation of evening-phased circadian transcripts under blue light.

The circadian system induces rhythmic variation in a suite of biochemical and physiological processes that serve to optimise plant growth in diel cycles. To be of greatest utility, these rhythmic behaviors are coordinated with regular environmental changes such as the rising and setting of the sun. Photoreceptors, along with metabolites produced during photosynthesis, act to synchronise the internal timing mechanism with lighting cues. We have recently shown that phototropins help maintain robust rhythms of photosynthetic operating efficiency (ϕPSII or Fq'/Fm') under blue light, although rhythmic accumulation of morning-phased circadian transcripts in the nucleus was unaffected. Here we report that evening-phased nuclear clock transcripts were also unaffected. We also observe that rhythms of nuclear clock transcript accumulation are maintained in phototropin mutant plants under a fluctuating lighting regime that induced a loss of Fq'/Fm' rhythms.

Phototropins maintain robust circadian oscillation of PSII operating efficiency under blue light.

The circadian system allows plants to coordinate metabolic and physiological functions with predictable environmental variables such as dusk and dawn. This endogenous oscillator is comprised of biochemical and transcriptional rhythms that are synchronized with a plant's surroundings via environmental signals, including light and temperature. We have used chlorophyll fluorescence techniques to describe circadian rhythms of PSII operating efficiency (Fq'/Fm') in the chloroplasts of Arabidopsis thaliana. These Fq'/Fm' oscillations appear to be influenced by transcriptional feedback loops previously described in the nucleus, and are induced by rhythmic changes in photochemical quenching over circadian time. Our work reveals that a family of blue photoreceptors, phototropins, maintain robust rhythms of Fq'/Fm' under constant blue light. As phototropins do not influence circadian gene expression in the nucleus our imaging methodology highlights differences between the modulation of circadian outputs in distinct subcellular compartments.