The Circadian Oscillator of the Cerebellum: Triiodothyronine Regulates Clock Gene Expression in Granule Cells in vitro and in the Cerebellum of Neonatal Rats in vivo

The central circadian clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, but an SCN-dependent molecular circadian oscillator is present in the cerebellar cortex. Recent findings suggest that circadian release of corticosterone is capable of driving the circadian oscillator of the rat cerebellum. To determine if additional neuroendocrine signals act to shape cerebellar clock gene expression, we here tested the role of the thyroid hormone triiodothyronine (T3) in regulation of the cerebellar circadian oscillator. In cultured cerebellar granule cells from mixed-gender neonatal rats, T3 treatment affected transcript levels of the clock genes Per2, Arntl, Nr1d1, and Dbp, suggesting that T3 acts directly on granule cells to control the circadian oscillator. We then used two different in vivo protocols to test the role of T3 in adult female rats: Firstly, a single injection of T3 did not influence clock gene expression in the cerebellum. Secondly, we established a surgical rat model combining SCN lesion with a programmable micropump infusing circadian physiological levels of T3; however, rhythmic infusion of T3 did not reestablish differential clock gene expression between day and night in SCN lesioned rats. To test if the effects of T3 observed in vitro were related to the developmental stage, acute injections of T3 were performed in mixed-gender neonatal rats in vivo; this procedure significantly affected cerebellar expression of the clock genes Per1, Per2, Nr1d1, and Dbp. Developmental comparisons showed rhythmic expression of all clock genes analyzed in the cerebellum of adult rats only, whereas T3 responsiveness was limited to neonatal animals. Thus, T3 shapes cerebellar clock gene profiles in early postnatal stages, but it does not represent a systemic circadian regulatory mechanism linking the SCN to the cerebellum throughout life.

Wheat EARLY FLOWERING3 is a dawn-expressed circadian oscillator component that regulates heading date

Optimising the seasonal control of flowering in the major crops is an important component of breeding to match crop adaptation to the target environment. Using an eight parent Multiparent Advanced Generation Inter-Cross (MAGIC) population we investigated the contribution of variation at circadian clock-associated genes to the regulation of heading date (flowering) in UK and European winter wheat varieties. We identified homoeologues of EARLY FLOWERING 3 (ELF3) as candidate genes for the Earliness per se (Eps) D1 and B1 loci in field conditions. We confirmed that a SNP within the coding region of TaELF3-B1 is the likely causal polymorphism underlying the Eps-B1 locus. We also identified that a reported deletion at the Eps-D1 locus encompassing TaELF3-D1, is in fact a novel allele that lies within an introgression region that contains an inversion relative to the Chinese Spring D genome. Our findings that ELF3 might be associated with the regulation of heading date prompted us to investigate whether ELF3 is a circadian oscillator gene in wheat, as it is in Arabidopsis. Using T. turgidum cv. Kronos carrying loss of function alleles for both copies of TtELF3 we found that circadian rhythms were severely disrupted. Furthermore, in T. aestivum, we found that loss of functional LUX ARRHYTHMO (LUX), an orthologue of the protein partner of ELF3 in Arabidopsis, also severely disrupted circadian rhythms. Whilst these data suggest a function for both ELF3 and LUX in the wheat circadian oscillator, that oscillator might be structured differently to that of Arabidopsis because wheat ELF3 and LUX transcripts are maximal at the end of the night and day respectively, rather than co-expressed at dusk as they are in Arabidopsis. We conclude that there is sufficient allelic diversity within the three wheat ELF3 homoeologues for selection to delay or advance heading, and that this can be achieved without pleiotropic deleterious alterations to circadian rhythms.

A central circadian oscillator confers defense heterosis in hybrids without growth vigor costs

Plant immunity frequently incurs growth penalties, which known as the trade-off between immunity and growth. Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits but rarely for disease resistance. Here, we report that the central circadian oscillator, CCA1, confers heterosis for bacterial defense in hybrids without growth vigor costs, and it even significantly enhances the growth heterosis of hybrids under pathogen infection. The genetic perturbation of CCA1 abrogated heterosis for both defense and growth in hybrids. Upon pathogen attack, the expression of CCA1 in F1 hybrids is precisely modulated at different time points during the day by its rhythmic histone modifications. Before dawn of the first infection day, epigenetic activation of CCA1 promotes an elevation of salicylic acid accumulation in hybrids, enabling heterosis for defense. During the middle of every infection day, diurnal epigenetic repression of CCA1 leads to rhythmically increased chlorophyll synthesis and starch metabolism in hybrids, effectively eliminating the immunity-growth heterosis trade-offs in hybrids.

Superoxide is promoted by sucrose and affects amplitude of circadian rhythms in the evening

Plants must coordinate photosynthetic metabolism with the daily environment and adapt rhythmic physiology and development to match carbon availability. Circadian clocks drive biological rhythms which adjust to environmental cues. Products of photosynthetic metabolism, including sugars and reactive oxygen species (ROS), are closely associated with the plant circadian clock, and sugars have been shown to provide metabolic feedback to the circadian oscillator. Here, we report a comprehensive sugar-regulated transcriptome of Arabidopsis and identify genes associated with redox and ROS processes as a prominent feature of the transcriptional response. We show that sucrose increases levels of superoxide (O2–), which is required for transcriptional and growth responses to sugar. We identify circadian rhythms of O2–-regulated transcripts which are phased around dusk and find that O2– is required for sucrose to promote expression of TIMING OF CAB1 (TOC1) in the evening. Our data reveal a role for O2– as a metabolic signal affecting transcriptional control of the circadian oscillator in Arabidopsis.

Post-Translational Mechanisms of Plant Circadian Regulation

The molecular components of the circadian system possess the interesting feature of acting together to create a self-sustaining oscillator, while at the same time acting individually, and in complexes, to confer phase-specific circadian control over a wide range of physiological and developmental outputs. This means that many circadian oscillator proteins are simultaneously also part of the circadian output pathway. Most studies have focused on transcriptional control of circadian rhythms, but work in plants and metazoans has shown the importance of post-transcriptional and post-translational processes within the circadian system. Here we highlight recent work describing post-translational mechanisms that impact both the function of the oscillator and the clock-controlled outputs.