Cell autonomous and cell-type specific circadian rhythms in Arabidopsis.

The circadian system of plants regulates a wide range of rhythmic physiological and cellular output processes with a period of about 24 h. The rhythms are generated by an oscillator mechanism that, in Arabidopsis, consists of interlocking feedback loops of several components including CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and CCA1 HIKING EXPEDITION (CHE). Over recent years, researchers have gained a detailed picture of the clock mechanism at the resolution of the whole plant and several tissue types, but little information is known about the specificities of the clock mechanism at the level of individual cells. In this paper we have addressed the question of cell-type-specific differences in circadian systems. Using transgenic Arabidopsis plants with fluorescence-tagged CCA1 to measure rhythmicity in individual leaf cells in intact living plants, we showed that stomatal guard cells have a different period from surrounding epidermal and mesophyll leaf cells. By comparing transcript levels in guard cells with whole plants, we identified differences in the expression of some oscillator genes that may underlie cell-specific differences in clock properties. In addition, we demonstrated that the oscillators of individual cells in the leaf are robust, but become partially desynchronized in constant conditions. Taken together our results suggest that, at the level of individual cells, there are differences in the canonical oscillator mechanism that has been described for plants.

Over-expression of CONSTANS-LIKE 5 can induce flowering in short-day grown Arabidopsis.

To ensure that the initiation of flowering occurs at the correct time of year, plants need to integrate a diverse range of external and internal signals. In Arabidopsis, the photoperiodic flowering pathway is controlled by a set of regulators that include CONSTANS (CO). In addition, Arabidopsis plants also have a family of genes with homologies to CO known as CO-LIKE (COL) about which relatively little is known. In this paper, we describe the regulation and interactions of a novel member of the family, COL5. The expression of COL5 is under circadian and diurnal regulation, but COL5 itself does not appear to affect circadian rhythms. COL5, like CO, is regulated by GIGANTEA. Furthermore, COL5 is expressed in the vascular tissue. Using COL5 over-expressing lines we show that, under short days, constitutive expression of COL5 affects flowering time and the expression of the floral integrator genes, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO 1. Constitutive expression of COL5 partially suppresses the late flowering phenotype of the co-mutant plants. However, plants with loss of COL5 function do not show altered flowering. Taken together, our results suggest that COL5 has COL activity, but may either not have a role in regulating flowering in wild-type plants or may act redundantly with other flowering regulators.

Posttranslational regulation of CIRCADIAN CLOCK ASSOCIATED1 in the circadian oscillator of Arabidopsis.

As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian (approximately 24 h) clocks. In plants, the circadian clock regulates a diverse range of cellular and physiological events from gene expression and protein phosphorylation to cellular calcium oscillations, hypocotyl growth, leaf movements, and photoperiod-dependent flowering. In Arabidopsis (Arabidopsis thaliana), as in other model organisms, such as Drosophila (Drosophila melanogaster) and mice, circadian rhythms are generated by molecular oscillators that consist of interlocking feedback loops involving a number of elements. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYLS (LHY) are closely related single myb transcription factors that have been identified as key elements in the Arabidopsis oscillator. Research in other model organisms has shown that posttranslational regulation of oscillator components plays a critical role in the generation of the approximately 24-h cycles. To examine the role of posttranslational regulation of CCA1 and LHY in the Arabidopsis oscillator, we generated transgenic plants with tagged CCA1 and LHY under the control of their own promoters. We have shown that these tagged proteins are functional and can restore normal circadian rhythms to CCA1- and LHY-null plants. Using the tagged proteins, we demonstrate that CCA1 can form both homodimers and heterodimers with LHY. Furthermore, we also show that CCA1 is localized to the nucleus in vivo and that there is no significant delay between the translation of CCA1 and its translocation to the nucleus. We discuss our findings in the context of the functioning of the Arabidopsis oscillator.

Mutations in CHLOROPLAST RNA BINDING provide evidence for the involvement of the chloroplast in the regulation of the circadian clock in Arabidopsis.

The Arabidopsis circadian system regulates the expression of up to 36% of the nuclear genome, including many genes that encode photosynthetic proteins. The expression of nuclear-encoded photosynthesis genes is also regulated by signals from the chloroplasts, a process known as retrograde signaling. We have identified CHLOROPLAST RNA BINDING (CRB), a putative RNA-binding protein, and have shown that it is important for the proper functioning of the chloroplast. crb plants are smaller and paler than wild-type plants, and have altered chloroplast morphology and photosynthetic performance. Surprisingly, mutations in CRB also affect the circadian system, altering the expression of both oscillator and output genes. In order to determine whether the changes in circadian gene expression are specific to mutations in the CRB gene, or are more generally caused by the malfunctioning of the chloroplast, we also examined the circadian system in mutations affecting STN7, GUN1, and GUN5, unrelated nuclear-encoded chloroplast proteins known to be involved in retrograde signaling. Our results provide evidence that the functional state of the chloroplast may be an important factor that affects the circadian system.