Barley Hv CIRCADIAN CLOCK ASSOCIATED 1 and Hv PHOTOPERIOD H1 Are Circadian Regulators That Can Affect Circadian Rhythms in Arabidopsis.

Circadian clocks regulate many aspects of plant physiology and development that contribute to essential agronomic traits. Circadian clocks contain transcriptional feedback loops that are thought to generate circadian timing. There is considerable similarity in the genes that comprise the transcriptional and translational feedback loops of the circadian clock in the plant Kingdom. Functional characterisation of circadian clock genes has been restricted to a few model species. Here we provide a functional characterisation of the Hordeum vulgare (barley) circadian clock genes Hv circadian clock associated 1 (HvCCA1) and Hv photoperiodh1, which are respectively most similar to Arabidopsis thaliana circadian clock associated 1 (AtCCA1) and pseudo response regulator 7 (AtPRR7). This provides insight into the circadian regulation of one of the major crop species of Northern Europe. Through a combination of physiological assays of circadian rhythms in barley and heterologous expression in wild type and mutant strains of A. thaliana we demonstrate that HvCCA1 has a conserved function to AtCCA1. We find that Hv photoperiod H1 has AtPRR7-like functionality in A. thaliana and that the effects of the Hv photoperiod h1 mutation on photoperiodism and circadian rhythms are genetically separable.ZR is grateful to the National Institute of Agricultural Botany for the Award of Scholarship. We acknowledge funding from a Marie Curie Early Stage Training project MEST-CT-2005-020526 for JK and the BBSRC-DTP for funding SC. AARW and MCM are grateful to the BBSRC for the award of BBSRC Grant BB/M006212/1, which supported aspects of the study.This is the final published version. It first appeared at http://dx.doi.org/10.1371/journal.pone.012744

Functional analysis of Casein Kinase 1 in a minimal circadian system

The Earth's rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components appear to be shared across higher species, Casein Kinase 1 (CK1) is known to affect timekeeping across metazoans and fungi, but has not previously been implicated in the circadian clock in the plant kingdom. We now show that modulation of CK1 function lengthens circadian rhythms in Ostreococcustauri, a unicellular marine algal species at the base of the green lineage, separated from humans by ~1.5 billion years of evolution. CK1 contributes to timekeeping in a phase-dependent manner, indicating clock-mediated gating of CK1 activity. Label-free proteomic analyses upon overexpression as well as inhibition revealed CK1-responsive phosphorylation events on a set of target proteins, including highly conserved potentially clock-relevant cellular regulator proteins. These results have major implications for our understanding of cellular timekeeping and can inform future studies in any circadian organism

m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5’ splice site

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5’ splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here, we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5’ splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3’ splice site usage. We generalise these findings to reveal two major classes of 5’ splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5’ splice sites crucial to alternative splicing

m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5’ splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N(6) methylation, but the role of this m(6)A modification is poorly understood. Here, we show that m(6)A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m(6)A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5’ splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m(6)A influences 3’ splice site usage. We generalise these findings to reveal two major classes of 5’ splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m(6)A modification contributes to the selection of degenerate 5’ splice sites crucial to alternative splicing

Over expression of <i>HvCCA1</i> causes circadian arrhythmia in Arabidopsis.

<p>Normalised delayed chlorophyll fluorescence (a) and period estimates vs R.A.E (b) for Ws-2, Col-0, <i>AtCCA1</i>-ox (<i>AtCCA1</i>-ox 038) and two independent <i>HvCCA1-</i>ox transgenic lines (n = 8). Period estimates vs R.A.E for leaf movement in LL or individual leaves Col-0 and <i>AtCCA1</i>-ox (<i>AtCCA1</i>-ox 038) (c) and Ws-2 and two independent transgenic lines of <i>HvCAA1-ox</i> (d). <i>n</i> = 30. All experiments were independently repeated at least twice. Ws-2 (closed squares), Col-0 (closed triangles) <i>AtCCA1</i>-ox (open triangles) and two independent <i>HvCCA1-</i>ox transgenic lines (8–3 and 18–1) (open squares and diamonds).</p

Estimates of period of circadian rhythms of (a) photosynthesis and (b) stomatal conductance in <i>HvPpd-H1</i> (filled circles) and <i>Hvppd-H1</i> (open circles) in constant white light of 100 μmol m^-2 s^-1 (black outlined symbols) or red light (red symbols).

<p>Each data point is derived from one individual seedling. Triangles represent the mean values.</p

Summary of circadian period estimates for leaf movement in Col-0, <i>prr7</i>-11 and <i>prr7-</i>11 transformed with either <i>pPRR7</i>::<i>Ppd-H1</i> or <i>pPRR7</i>::<i>ppd-H1</i>.

<p>* indicates significant difference at 5% level compared to background.</p><p>The background for <i>prr7</i>-11 is Col-0. The background for the complemented lines is <i>prr7</i>-11. SEM = standard error of the mean. %Rh = Percentage of rhythmic seedlings.</p><p>Summary of circadian period estimates for leaf movement in Col-0, <i>prr7</i>-11 and <i>prr7-</i>11 transformed with either <i>pPRR7</i>::<i>Ppd-H1</i> or <i>pPRR7</i>::<i>ppd-H1</i>.</p