Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure

Abstract

Our understanding of the complex, transcriptional feedback loops in the circadian clock mechanism has depended upon quantitative, timeseries data from disparate sources. We measure clock gene RNA profiles in Arabidopsis thaliana seedlings, grown with or without exogenous sucrose, or in soil-grown plants and in wild-type and mutant backgrounds. The RNA profiles were strikingly robust across the experimental conditions, so current mathematical models are likely to be broadly applicable in leaf tissue. In addition to providing reference data, unexpected behaviours included co-expression of PRR9 and ELF4, and regulation of PRR5 by GI. Absolute RNA quantification revealed low levels of PRR9 transcripts (peak approx. 50 copies cell−1) compared with other clock genes, and threefold higher levels of LHY RNA (more than 1500 copies cell−1) than of its close relative CCA1. The data are disseminated from BioDare, an online repository for focused timeseries data, which is expected to benefit mechanistic modelling. One data subset successfully constrained clock gene expression in a complex model, using publicly available software on parallel computers, without expert tuning or programming. We outline the empirical and mathematical justification for data aggregation in understanding highly interconnected, dynamic networks such as the clock, and the observed design constraints on the resources required to make this approach widely accessible.

Multiple circadian clock outputs regulate diel turnover of carbon and nitrogen reserves

“…Metabolites: starch, sucrose, glucose, fructose, Glc6P, total amino acids, malate, fumarate, nitrate, protein, Chla, and Chlb (see original data in Data S1). Transcripts: LHY , CCA1 , PRR9 , PRR7 , PRR5 , TOC1 , GI , ELF3 , ELF4 , and LUX from Flis et al (); see plots in Figures S9 and S10 [Colour figure can be viewed at wileyonlinelibrary.com]…”

“…This is shown by the loading gradient for the circadian clock genes (Y variables) that changes from strongly negative for LHY and CCA1 transcripts to weakly negative for PRR9 , weakly positive for PRR7 transcript, and strongly positive for most dusk and EC transcripts. The low ELF3 loading might reflect the relatively weak circadian oscillation of its transcript (Flis et al, ; see Figure S9). On the metabolite axis, starch, malate, and total amino acids had high positive loadings, and Chla, Chlb, fumarate, and total protein had lower positive loadings.…”

Circadian, Carbon, and Light Control of Expansion Growth and Leaf Movement

“…1). Period length in prr7prr9 has been reported to be as long as 32 h (Farré et al, 2005;Salomé and McClung, 2005) but is closer to 28 h when plants growing in the conditions used in our study are transferred to LL at growth irradiance (Flis et al, 2015). The occurrence of a distinct growth phenotype in prr7prr9 that is not linked to the clock period length also is indicated by our correlation analysis, where prr7prr9 in a T28 cycle clusters more closely to prr7prr9 in a 24-h cycle than to treatments in which clock period matched T-cycle duration (Fig.…”

“…lhycca1 is deficient in the dawn clock genes and shows early expression of day, dusk, and evening genes, whereas prr7prr9 has high levels of LHY and CCA1 transcript until the end of the light period and strongly delayed expression of dusk and evening genes (Pokhilko et al, 2012;Fogelmark and Troein, 2014;Flis et al, 2015). One possible explanation for the stimulation of expansion growth after dawn would be that LHY or CCA1 restricts expansion growth at dawn and that this inhibition is relieved as the dawn components are repressed.…”

“…Sequential expression of these core clock components orchestrates successive waves of the expression of output genes, affecting up to half the transcriptome (Harmer et al, 2000;Michael et al, 2008). The clock has a period of about 24 h, but this is shortened to about 17 h in the lhycca1 double mutant (Alabadí et al, 2002;Mizoguchi et al, 2002;Salomé and McClung, 2005) and extended to 28 to 32 h in the prr7prr9 double mutant (Farré et al, 2005;Salomé and McClung, 2005;Flis et al, 2015).…”