The circadian clock is an autonomous oscillator that produces endogenous biological rhythms with a period of about 24 hours. This clock allows organisms to anticipate predicted daily changes in the environment and to coordinate developmental and metabolic processes with environmental cues, such as light and temperature, that cycle with the rotation of the earth [1–5]. Conceptually, a circadian system comprises three basic components: input pathways that sense light and temperature, a core oscillator that defines the rhythm, and output pathways that control various developmental and metabolic processes, resulting in the optimal adaptation to daily changing environments. The core oscillator that generates circadian rhythms is comprised of autoregulatory interlocking positive/negative feedback loops. In the eudicot Arabidopsis, the central loop called the “core oscillator” is composed of two partially redundant Myb-like transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)  and LATE ELONGATED HYPOCOTYL (LHY) , and the pseudo response regulator (PRR) TIMING OF CAB EXPRESSION 1 (TOC1). The morning expressed CCA1 and LHY repress TOC1 by directly binding to its promoter, which results in the evening accumulation of TOC1 that in turn represses CCA1 and LHY expression . These three genes are critical to sustain rhythms as the cca1 lhy toc1 triple mutant was found to be arrhythmic . The core oscillator is further fine-tuned by a morning-phased loop and an evening-phased loop. The morning loop includes members of the pseudo response regulated gene family, PRR3, PRR5, PRR7 and PRR9 which contain a pseudo receiver domain at the N terminus and a CCT (CONSTANS, CONSTANS-LIKE, and TOC1) motif at the C terminus [10, 11]. PRR transcripts start accumulating after dawn sequentially in the order of PRR9, PRR7, PRR5, PRR3, and PRR1/TOC1, and it has been shown that PRR7 and PRR9 repress CCA1 and LHY during the day . The evening-phased loop is proposed to include GIGANTEA (GI), ZEITLUPE (ZTL), TOC1, and an unknown factor Y. GI decreases TOC1 protein level through stabilization of the ZTL protein [13, 14]. The decreased TOC1 protein tends to relieve repression of Y, increased Y expression in turn activates TOC1 expression, so that Y directly and GI indirectly activate TOC1 expression .
Output pathways from the oscillator convey circadian rhythms to the various physiological and molecular processes, which include many with agronomic importance, such as photosynthesis, growth, phytohormone signaling, and photoperiodic flowering [16, 17]. The circadian clock may thus be a key for improving agronomic performance and stress adaptation of crops. Indeed, diurnal expression analysis of field grown maize showed that ~22% of all genes in leaf tissue exhibit diurnal expression patterns . In addition, a null allele of the rice GI ortholog affected diurnal expression of 75% of all tested genes and conferred reduced seasonal adaptability in field grown rice . These studies highlight the critical role of the clock in cereals. Some orthologs of Arabidopsis core-clock genes have been identified in the monocot plants rice  and Lemna[21, 22]. The rice genome was reported to encode a single ortholog for LHY and CCA1 and five PRR orthologs designated as OsPRR1 orthologous to TOC1, OsPRR73/OsPRR37 corresponding to AtPRR7 or AtPRR3, and OsPRR59/OsPRR95 corresponding to AtPRR5 or AtPRR9. Over-expression of OsCCA1 or OsPRR1 in Arabidopsis modified circadian rhythms . Rice orthologs of TOC1 and PRR7 partially complemented the corresponding Arabidopsis toc1 and PRR7 mutants, which is consistent with the function of these proteins being conserved between monocots and Arabidopsis [20, 24]. A full compendium analysis of the monocot clock awaits to be performed.
The temperate crop barley, which includes the domesticated form Hordeum vulgare spp. vulgare and the wild subspecies Hordeum vulgare spp. spontaneum, is characterized by high genetic diversity and good adaptation to stress prone marginal environments [25, 26]. Interestingly, adaptation in barley is influenced by the photoperiod response gene Ppd-H1, also known as HvPRR37, which is orthologous to the rice gene OsPRR37 and the Arabidopsis clock gene PRR7. A natural, recessive mutation in the CCT domain of Ppd-H1 causes photoperiod insensitivity and late flowering in cultivated spring barley. In contrast, wild and cultivated winter barley genotypes harbor the photoperiod responsive Ppd-H1 allele, which induces early flowering under long photoperiods. Barley genotypes with a photoperiod responsive Ppd-H1 allele are characterized by elevated expression of Vrn-H3 (HvFT1) homologous to the Arabidopsis gene FLOWERING LOCUS T (FT). In Arabidopsis, FT is the mobile florigen hormone that moves as a protein from the leaves through the phloem to the shoot apical meristem where it induces the switch from vegetative to reproductive growth [28, 29]. FT expression is triggered by the photoperiod response gene CONSTANS (CO). CO protein is degraded in darkness and expression of the protein during the day is crucial for induction of the floral activator FT and flowering . It was suggested that the mutation in Ppd-H1 of spring barley delayed flowering time by shifting the diurnal expression peaks of the barley CO orthologs HvCO1 and HvCO2 into the dark phase, so that the proteins are not synthesized and Vrn-H3 (HvFT1) not expressed .
Winter barley is vernalization sensitive, exposure to prolonged periods of cold during winter are translated into an increased competence to flower in spring. Vernalization response is controlled by variation at the vernalization genes Vrn-H1 and Vrn-H2 and by the MADS-box transcription factors HvVrt2, HvBM1, and HvBM10, which are cereal orthologs of SHORT VEGETATIVE PHASE (SVP) in Arabidopsis [31, 32]. In winter barley, Vrn-H1, with similarity to the Arabidopsis meristem identity genes APETALA1, CAULIFLOWER, and FRUITFUL, is only expressed after exposure to cold . Insertions or deletions in the first intron of Vrn-H1 in spring barley cause up-regulation of the gene independently of vernalization . Spring barley is also characterized by a deletion of the entire Vrn-H2 locus, which includes one truncated and two full sequence ZCCT (Zinc finger and CCT domain) genes with no clear orthologs in Arabidopsis . In photoperiod-sensitive winter barley, Vrn-H2 represses Vrn-H3 (HvFT1) to counteract the Ppd-H1 dependent long day induction of Vrn-H3 prior to winter. Up-regulation of Vrn-H1 during vernalization and consequent down-regulation of Vrn-H2 transcript levels in the leaf facilitate the up-regulation of Vrn-H3 during long days mediated by Ppd-H1. HvVrt2, HvBM1, and HvBM10 are floral repressors, which may act downstream of Vrn-H1 and HvFT1 in barley, and thus, integrate light and temperature dependent regulation of flowering [31, 37]. However, the effects of variation at Ppd-H1 on circadian expression of photoperiod and vernalization response genes have not yet been analyzed. The natural mutation in the Ppd-H1 gene may affect the photoperiod and vernalization pathways either by changing circadian timing of clock genes or by direct control of flowering time genes independently from its clock function.
In this study, we analyzed whether orthologs of Arabidopsis clock genes are structurally and functionally conserved in the temperate crop and long-day plant barley. For this we 1) identified barley clock orthologs from available genomic databases and 2) analyzed their diurnal and circadian expression patterns in two barley genotypes differing at the photoperiod response gene and clock ortholog Ppd-H1. We showed that barley clock orthologs exhibit a high level of sequence similarity and conservation of expression profiles as compared to Arabidopsis and rice clock genes. The natural mutation at Ppd-H1 did not affect expression of clock genes, but caused arrhythmicity of clock output genes HvCO1, HvCO2, and Vrn-H1 under constant conditions. Our study provides a characterization of the compendium of barley clock genes under circadian conditions, and sets the basis to explore the effects of the circadian clock on performance in temperate crop species.