INTRODUCTION
Plant growth and biomass production are direct functions of the diurnal
cellular carbon balance, which is regulated by a combination of light
responses and the circadian clock. Light responses are triggered by a
change in regime (i.e., presence or absence of light), while the
circadian clock is comprised of transcriptional regulators that operate
in anticipation of a change (e.g. transition from light to dark) and
whose activities span the 24 hour (h) photoperiod (Nohales & Kay, 2016;
Oakenfull & Davis, 2017; Seluzicki, Burko, & Chory, 2017). The clock
transcriptional regulators include CCA1/LHY, PRR5, PRR7 and PRR9, which
form the morning loop, and TOC1, ELF3, LF4 and LUX, which form the
evening loop (Flis et al., 2015; Staiger, Shin, Johansson, & Davis,
2013). More than 30% of all Arabidopsis genes are regulated by the
circadian clock at the transcript level (Blasing et al., 2005;
Covington, Maloof, Straume, Kay, & Harmer, 2008). However, less is
known about how the resulting diurnal transcriptome relates to protein
abundance (Abraham et al., 2016; Choudhary, Nomura, Wang, Nakagami, &
Somers, 2015; Graf et al., 2017) and post-translational protein
modifications (Choudhary et al., 2015; Uhrig, Schlapfer, Roschitzki,
Hirsch-Hoffmann, & Gruissem, 2019), both of which may also affect
protein function at light-dark transitions and throughout the diurnal
cycle. Transcript and protein abundance changes are often disconnected
because changes in transcript levels show no corresponding change in
protein abundance (Baerenfaller et al., 2012; Seaton et al., 2018). For
example, this was found, in the circadian clock mutants CCA1/LHY,
PRR7/PRR9, TOC1 and GI (Graf et al., 2017) or for the variability in the
timing of peak protein levels relative to the cognate transcript
expression (translational coincidence) as a function of the
photoperiod-dependent coordination between transcriptome and proteome
changes (Seaton et al., 2018). Variable delays between peak transcript
and protein abundance have implicated post-transcriptional regulation
(e.g. splicing), translational regulation (e.g. translation rate) as
well as post-translational regulation (e.g. protein phosphorylation) as
possible mechanisms to explain the temporal differences in RNA and
protein abundance. Recent studies of plant protein-level regulation have
found extensive variability in protein turnover (Li et al., 2017; Seaton
et al., 2018). This adds further regulatory complexity because
quantitative proteome workflows cannot easily account for protein
turnover. Although RNA and protein synthesis, stability and turnover all
contribute to the coordination of RNA and protein abundance, how these
mechanisms are integrated is currently not well understood. Insights
into this regulatory complexity requires both time-course experiments
and multi-Omics analysis. We used a large-scale quantitative proteomics
approach to determine the extent of diurnal abundance and/or
phosphorylation changes of Arabidopsis leaf rosette proteins involved in
cellular and metabolic processes and how these protein level changes
relate to cognate transcript levels.
Reversible protein phosphorylation is the most abundant
post-translational modification (PTM) in eukaryotes (Adam & Hunter,
2018; Rao, Thelen, & Miernyk, 2014). In non-photosynthetic eukaryotes
phosphorylation is found to modulate more than 70% of all cellular
processes (Olsen et al., 2006), including the circadian clock itself
(Robles, Humphrey, & Mann, 2017). This is likely similar in land plants
because they have a significantly larger kinome (Lehti-Shiu & Shiu,
2012) compared to humans, which encode 518 protein kinases (Manning,
Whyte, Martinez, Hunter, & Sudarsanam, 2002). In contrast, both plants
and humans have an equally comparable small number of protein
phosphatases (Kerk, Templeton, & Moorhead, 2008). However, most protein
phosphatases require association with regulatory subunits to achieve
their specificity (Moorhead et al., 2008; Uhrig, Labandera, & Moorhead,
2013), suggesting that similar complexity in protein dephosphorylation
across plants and humans has evolved through the expansion of protein
phosphatase regulatory subunits.
In plants, diurnal protein phosphorylation is regulated either in
response to light, by the circadian clock (Choudhary et al., 2015), or
both (Uhrig et al., 2019), while the clock itself is regulated by
phosphorylation (Kusakina & Dodd, 2012; Uehara et al., 2019). Recent
studies of the circadian phosphoproteome combining the analysis of a
free-running cycle and the circadian clock mutant elf4 (Choudhary
et al., 2015) or CCA1-OX over-expression (Krahmer et al., 2019) have
revealed temporally modified phosphorylation sites related to casein
kinase II (CKII) and sucrose non-fermenting kinase 1 (SnRK1). SnRKs are
likely involved in the regulation of the circadian phosphoproteome
because the transcription of genes encoding multiple SnRK and
calcinuerin B-like (CBL) interacting kinases (CIPK) was mis-regulated in
the Arabidopsis circadian clock mutants cca1/lhy1 ,prr7prr9 , toc1 and gi201 mutants at end-of-day (ED)
and end-of-night (EN) (Graf et al., 2017). Similarly, studies
quantifying changes in the phosphoproteome at ED and EN in Arabidopsis
rosette leaves, roots, flowers, siliques and seedlings have revealed a
large number of diurnally changing phosphorylation events corresponding
to diverse protein kinase motifs (Reiland et al., 2009; Uhrig et al.,
2019).
Considering the physiological and metabolic changes at the light-dark
(L-D) and dark-light (D-L) transitions (Annunziata et al., 2018; Gibon
et al., 2009; Usadel et al., 2008), we performed a quantitative
phosphoproteome analysis of proteins that are phosphorylated immediately
before and after the L-D and D-L transitions during a 12 h light : 12 h
dark photoperiod and asked how these phosphorylation events intersect
with protein abundance changes. Together with the 24 h photoperiod
time-course proteome data, our systems-level analysis provides new
insights into diurnal protein and phosphorylation regulation in
Arabidopsis rosette leaves.
MATERIALS AND
METHODS
Arabidopsis Col-0 wild‑type plants were
grown at the Forschungszentrum Jülich (Germany) in an environmentally
controlled chamber (GrowScreen Chamber;
https://eppn2020.plant-phenotyping.eu/EPPN2020_installations#/tool/30;
Barboza-Barquero et al., 2015) under a 12 h light:12 h dark photoperiod
and controlled conditions as described in Baerenfaller et al.(2012), including air temperature of 21ºC during the day and 20ºC during
the night, air humidity of 70%, and an incident light intensity of
~220 mmol/m2/s at the plant level.
Whole rosettes were harvested at 31 days after sowing (DAS) prior to
flowering. Four whole rosettes were pooled into one sample and 4
biological replicates were taken at each time point. For total proteome
analyses, samples were taken every 2 h during 24 h, starting at
Zeitgeber time 1 (ZT1, i.e. 1 h after lights turned on). For protein
phosphorylation analyses, samples were 30 min before, 10 min after and
30 min after the L-D and D-L transitions. Samples were snap-frozen in
liquid N2 and stored at -80ºC until protein extraction.