Detection and Analysis of Circadian Rhythms Via Prompt Chlorophyll Fluorescence.

Monitoring prompt chlorophyll fluorescence (F) by making consecutive pulse amplitude modulation (PAM) measurements is a noninvasive, nondestructive, potentially high-throughput technique for evaluating circadian rhythms in diverse plant species. The technique is also less labor-intensive than many others currently used and requires no transgenic procedures.

Thermal imaging as a noninvasive technique for analyzing circadian rhythms in plants.

Endogenous (˜24 circadian) rhythms control an enormously diverse range of processes in plants and are, increasingly, the target of studies aimed at understanding plant performance. Although in the previous few decades most plant circadian research has focused on Arabidopsis, there is a pressing need for low-cost, high-throughput tools for analyzing rhythms in a wider variety of species. The present contribution investigates using circadian temperature oscillations as a novel marker for assaying plant circadian rhythms. A thermal imaging platform was set up to measure diel and circadian rhythms in different plant species, in wild-type and circadian mutant plants, and in leaves and flowers. Results from the thermal imaging technique were compared with those from other established circadian assay techniques. All of the dicot and monocot species examined showed robust circadian rhythms of leaf surface temperature; the effects of circadian mutations on thermocycles were similar to those reported using other techniques. In Petunia × atkinsiana plants circadian oscillations were observed in both leaves and flowers. Thermal imaging is an extremely useful technique for analyzing circadian rhythms in plants. It is predicted that the ability to make very high temporal resolution measurements may facilitate the discovery of novel aspects of circadian control.

CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and the Circadian Control of Stomatal Aperture.

The endogenous circadian (∼24 h) system allows plants to anticipate and adapt to daily environmental changes. Stomatal aperture is one of the many processes under circadian control; stomatal opening and closing occurs under constant conditions, even in the absence of environmental cues. To understand the significance of circadian-mediated anticipation in stomatal opening, we have generated SGC (specifically guard cell) Arabidopsis (Arabidopsis thaliana) plants in which the oscillator gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1) was overexpressed under the control of the guard-cell-specific promoter, GC1. The SGC plants showed a loss of ability to open stomata in anticipation of daily dark-to-light changes and of circadian-mediated stomatal opening in constant light. We observed that under fully watered and mild drought conditions, SGC plants outperform wild type with larger leaf area and biomass. To investigate the molecular basis for circadian control of guard cell aperture, we used large-scale qRT-PCR to compare circadian oscillator gene expression in guard cells compared with the "average" whole-leaf oscillator and examined gene expression and stomatal aperture in several lines of plants with misexpressed CCA1 Our results show that the guard cell oscillator is different from the average plant oscillator. Moreover, the differences in guard cell oscillator function may be important for the correct regulation of photoperiod pathway genes that have previously been reported to control stomatal aperture. We conclude by showing that CONSTANS and FLOWERING LOCUS T, components of the photoperiod pathway that regulate flowering time, also control stomatal aperture in a daylength-dependent manner.

Correlations between Circadian Rhythms and Growth in Challenging Environments.

In plants, the circadian system controls a plethora of processes, many with agronomic importance, such as photosynthesis, photoprotection, stomatal opening, and photoperiodic development, as well as molecular processes, such as gene expression. It has been suggested that modifying circadian rhythms may be a means to manipulate crops to develop improved plants for agriculture. However, there is very little information on how the clock influences the performance of crop plants. We used a noninvasive, high-throughput technique, based on prompt chlorophyll fluorescence, to measure circadian rhythms and demonstrated that the technique works in a range of plants. Using fluorescence, we analyzed circadian rhythms in populations of wild barley (Hordeum vulgare ssp. spontaneum) from widely different ecogeographical locations in the Southern Levant part of the Fertile Crescent, an area with a high proportion of the total genetic variation of wild barley. Our results show that there is variability for circadian traits in the wild barley lines. We observed that circadian period lengths were correlated with temperature and aspect at the sites of origin of the plants, while the amplitudes of the rhythms were correlated with soil composition. Thus, different environmental parameters may exert selection on circadian rhythms.