Title: Association analysis reveals genes underlying natural variation
in circadian rhythms in Arabidopsis
Authors: Hannah Rees1,2, Ryan
Joynson1, James K.M. Brown3, Anthony
Hall1
Corresponding author: Anthony Hall
anthony.hall@earlham.ac.uk
Contact information: 1Earlham Institute, Norwich
Research Park, Norwich, NR4 7UG, UK; 2Institute of
Integrative Biology, University of Liverpool, Crown Street, Liverpool,
L69 7ZB, UK; 3John Innes Centre, Norwich Research
Park, Norwich, NR4 7UH, UK
Funding: This project was supported by the BBSRC via the Earlham
institute CSP (BB/P016774/1, AH, HR) and BBSRC Design Future Wheat
(BB/P016855/1, AH, RJ).
Abstract: Circadian clocks have evolved to resonate with external day
and night cycles. However, these entrainment signals are not consistent
everywhere and vary with latitude, climate and seasonality. This leads
to divergent selection for clocks which are locally adapted. To
investigate the genetic basis for this circadian variation, we used a
Delayed Fluorescence (DF) imaging assay to screen 191 naturally
occurring Swedish Arabidopsis accessions for their circadian
phenotypes. We demonstrate that period length co-varies with both
geography and population sub-structure. Several candidate loci linked to
period, phase and Relative Amplitude Error (RAE) were revealed by
genome-wide association mapping and candidate genes were investigated
using TDNA mutants. We show that natural variation in a single
non-synonymous substitution within COR28 is associated with a
long-period and late-flowering phenotype similar to that seen in TDNA
knock-out mutants. COR28 is a known coordinator of flowering
time, freezing tolerance and the circadian clock; all of which may form
selective pressure gradients across Sweden. We demonstrate the effect of
the COR28 -58S SNP in increasing period length through a
co-segregation analysis. Finally, we show that period phenotypic tails
remain diverged under lower temperatures and follow a distinctive
‘arrow-shaped’ trend indicative of selection for a cold-biased
temperature compensation response.
Keywords: Circadian clock, temperature compensation, Arabidopsis ,
natural variation, Sweden, 1001 genomes project, GWA-mapping,COR28 , ELF3
Acknowledgements: We thank Paul W. Goedhart (Wageningen University) for
advice about circular phase statistics using the RCIRCULAR procedure and
Grant Calder (John Innes Centre) for his assistance developing the
96-well ROI selection using FIJI. We thank Susan Duncan for help and
advice with the flowering time investigation.
We also thank the Dean Research group (JIC) for providing the 191
Swedish accessions and the Liu Research group (Shanghai Institutes for
Biological Sciences) for providing the cor28-2 , cor27-1and cor27-1/28-2 mutants. We also thank Ewan Holmes and Georgia
Scutter for help with seed harvesting.
Main text:
Introduction
Plants are highly adapted to survive and exploit the daily fluctuations
in light and temperature experienced as the earth spins on its axis. The
circadian clock plays an intrinsic role in this; integrating temporal
cues from the environment to inform photosynthetic, metabolic and
developmental processes (Covington
et al., 2008; Harmer et al., 2000). A robustly oscillating circadian
clock which is highly synchronised to external day-length contributes to
the overall fitness of the plant, giving it an edge over competitors,
predators and pathogens (Dodd et al., 2005; Green et al., 2002; Ingle et
al., 2015; Michael et al., 2003). Circadian rhythms can be quantified by
their period (the time taken to complete one cycle), their phase (the
time of day in which the rhythm peaks), their amplitude (the change in
intensity from a baseline) and their Relative Amplitude Error (RAE)
which is the amplitude error divided by the overall amplitude of the
rhythm and can be equated to rhythm robustness. In our study, circadian
phenotyping was achieved using delayed fluorescence (DF) imaging.
Delayed fluorescence levels are circadian regulated and reflect the
changes in the photosynthetic state of photosystem II (Gould et al.,
2009). DF imaging has been validated as a reliable and flexible tool to
measure circadian rhythms in a range of plant models but has not
previously been used for phenotyping of a large population of
individuals on the scale required for genome-wide association mapping.
Arabidopsis thaliana is a model plant system which has been
extensively studied in circadian biology. The Arabidopsis clock
is comprised of a series of interlocking negative transcriptional
feedback loops connected by key activators that control the oscillation
of clock gene expression(Hernando et al., 2017). Key genes within this
network include CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) andLATE ELONGATED HYPOCOTYL (LHY) which transcriptionally repressTIMING OF CAB EXPRESSION (TOC1) (Alabadi et al., 2001).
Downstream genes interact with the clock to communicate circadian
rhythmicity to physiological outputs. Examples include photoperiodic
regulation of flowering time and the temporal gating of cold acclimation
responses (Kinmonth-Schultz et al., 2013). Flowering under long days is
instigated through accumulation of the floral promoter CONSTANS (CO)
controlled by the circadian clock component GIGANTEA (Suárez-López et
al., 2001). In winter-annuals, flowering is also dependent on the
vernalization response of FLOWERING LOCUS C (FLC) andFRIGIDA (FRI) (Bastow et al., 2004; Johanson et al.,
2000). Cold tolerance is diurnally activated through alternative
splicing of CCA1 which co-regulates the expression of COLD
REGULATED (COR) genes alongside light and temperature stress pathways
(James et al., 2012; Kinmonth-Schultz et al., 2013).
The endogenous core circadian network is entrained by external stimuli;
most notably light and temperature. Day-length, light composition and
light intensity have all been shown to affect circadian rhythms
(Aschoff, 1979; Más et al., 2003; Pittendrigh & Minis, 1964; Yanovsky
& Kay, 2002). Temperature also has a well-documented effect on
entrainment of circadian rhythms (Edwards et al., 2005; Gould et al.,
2006; Salome & McClung, 2005) with a degree of period shortening
expected under higher temperatures. Circadian rhythms are said to be
‘temperature compensated’; they resist large changes in period-length in
response to temperature (Pittendrigh & Harvey, 1954). The extent of
temperature compensation has been shown to vary betweenArabidopsis accessions (Gould et al., 2006; Kusakina et al.,
2014). Rhythm robustness is also strongly affected by temperature,
although the temperature which produces the most rhythmic oscillations
appears to be dependent on the species and circadian assay used
(Kusakina et al., 2014; Rees et al., 2019). Plants with clocks which
resonate with environmental conditions are typically fittest, however it
has been suggested that in climates with large seasonality there may be
a compromise for clocks which are adaptable to rapidly changing
day-lengths (Michael et al., 2003).
Natural populations of several important model organisms that exhibit
extensive diversity in their circadian behavior have been documented.
Pupal eclosion rhythms of Drosophila are latitude dependent; with
shorter rhythms, earlier phases and less robust rhythms observed in
Northern latitudes (Allemand & David, 1974; Lankinen, 1986). InArabidopsis , Michel et al. conducted a global study of leaf
movement rhythms in 150 accessions and found that day-length of origin
country correlated positively with period, but not phase or amplitude.
They also identified several loci in the TOC1/PRR family which
determined natural variation in period, phase and amplitude
independently (Michael et al., 2003). Other investigations have shown
allelic diversity of several clock related genes including FLC(Swarup et al., 1999) GI (de Montaigu et al., 2015) andEARLY FLOWERING 3 (ELF3) (Anwer et al., 2014) which contribute to
natural circadian phenotypes without fully disrupting the clock
mechanism. The positive relationship with period and latitude has also
been observed in natural populations of Mimulus guttatus(Greenham et al., 2017) and in cultivated varieties of soybean and
tomato (Greenham et al., 2017; Müller et al., 2016), potentially due to
unintentional selection for circadian rhythms which function best at
particular latitudes.
In this study, we focused on a collection of Arabidopsisaccessions from across a large latitudinal range in Sweden; a country
with variations in climatic, anthropogenic and day-length factors all
possibly influencing clock adaption. Northern latitudes around 63°N have
permanent snow cover during winter months and the growing season is much
cooler and shorter than in the South. At the solstice, there is almost 3
hours difference in day-length between the North and South. This
divergence of climate has led to selection for ecotypes with adapted
growth and flowering strategies (Bloomer & Dean, 2017; Shindo et al.,
2005). Analysis of the global population structure of Arabidopsisaccessions has previously identified Swedish accessions as being
genetically distinct from the wider population, with further
differentiation within the country between North and South (Horton et
al., 2012; Huber et al., 2014; Long et al., 2013; Nordborg et al.,
2005). Accessions from South Sweden have high genetic diversity within a
relatively small area, perhaps suggesting a historic emigration from
central Europe following glacial retreat (Alonso-Blanco et al., 2016).
Northern Swedish accessions have lower genetic diversity but larger
genome sizes (Long et al., 2013) and also carry a surprising reservoir
of drought tolerance genes (Exposito-Alonso et al., 2018). Completion of
the 1001 genomes project in 2006 has facilitated a recent expansion inArabidopsis genome wide association (GWA) studies made possible
through the provision of high-quality re-sequenced genotype data. These
accessions are publicly available, geo-referenced and genetically inbred
making it easy for researchers to perform experiments over several
generations under a variety of conditions (Weigel & Mott, 2009). Many
of the accessions in this study have also been characterized for other
phenotypic traits such as seed dormancy (Kerdaffrec et al., 2016),
flowering time (Sasaki et al., 2015), and freezing tolerance (Horton et
al., 2016).
The objectives for this research were firstly; to investigate whether
circadian diversity exists in Swedish Arabidopsis and to
understand to what extent this variation can be explained by either
latitude or the underlying population structure. We next wanted to
identify and validate genetic variation in candidate genes which might
explain the observed circadian variation. To our knowledge, this work
provides the first example of a GWA study using circadian phenotypes in
plants. Our final objective was to measure circadian rhythms under 10°C
and 16°C to test whether divergence of circadian traits persists across
a range of temperatures.
Methods