Analysis of light-dark transitions in a diurnal cycle reveal
dynamic fluctuation in the Arabidopsis phosphoproteome
Protein phosphorylation is often associated with changing environmental
conditions (Li et al., 2017; S. Zhang et al., 2019; Zhao et al., 2017).
Therefore, we examined time-points before (30 min) and after (10 min,
30 min) the D-L and L-D transitions for changes in the phosphoproteome
(Supplemental Figure 1). We identified 1776 phosphopeptides from 1091
proteins (phosphorylation site probability score ≥ 0.8) and quantified
1056 of these phosphopeptides from 725 proteins at the two light
transitions (Table 1, Supplemental Table 3). We found that 176
phosphopeptides from 153 proteins at the D-L transition and 164
phosphopeptides from 144 proteins at the L-D transition had significant
changes in abundance (Supplemental Figure 2 and 3; Supplemental Table
4). We then benchmarked the quality of our dataset by querying it for
proteins known to be diurnally regulated by protein phosphorylation
(Supplemental Table 5). This revealed phototropin 1, nitrate reductase
(NIA1 and NIA2) and CF1 ATP synthase. Phototropin 1 is phosphorylated in
the light (Sullivan, Thomson, Kaiserli, & Christie, 2009; Sullivan,
Thomson, Lamont, Jones, & Christie, 2008), while the NIA1, NIA2 and the
CF1 ATP synthase beta-subunit are phosphorylated in the dark (Kanekatsu,
Saito, Motohashi, & Hisabori, 1998; Lillo, Meyer, Lea, Provan, &
Oltedal, 2004; G. Moorhead et al., 1999; Reiland et al., 2009). Our
quantitation of NIA1 and 2 protein phosphorylation changes across
time-points revealed that NIA2 was more rapidly dephosphorylated on
Ser534 at the D-L transition than NIA1, potentially
relating to regulatory differences between NIA1 and 2. Additionally, we
found a new NIA2 phosphorylation site at Ser63 with
opposing diurnal changes in phosphorylation at the same transition
(Supplemental Figure 4).
We next performed a GSEA of all significantly changing phosphoproteins
(P value ≤ 0.01, FDR ≤ 0.05, gene set size ≥ 2) at each transition.
Enriched biological processes at the D-L transition include
phosphoproteins involved in light detection, nitrogen metabolism, cell
wall-related processes and phosphorylation signaling, while
phosphoproteins identified at the L-D transition are involved in light
detection, vesicle-mediated transport, auxin signaling and nucleus
organization (Table 2). We then generated a hierarchical heat map of the
phosphopeptides to identify clusters of proteins at each light
transition with similar phosphorylation dynamics (Supplemental Figure 2
and 3). When compared to datasets of phosphorylated proteins previously
identified in Arabidopsis growing under free-running cycle conditions
(Choudhary et al., 2015; Krahmer et al., 2019), or at the ED and EN
time-points of a 12-hour photoperiod (Reiland et al., 2009; Uhrig et
al., 2019), our data reveals proteins that have diurnal changes in their
phosphorylation status and also novel rate-of-change information for
these phosphorylation events (Supplemental Figure 2 and 3). For example,
the L-D cluster I has phosphoproteins involved in nitrogen metabolism
and the cell cycle (AD10 and AD30) and the L-D cluster III (BD30) has
phosphoproteins involved in plastid organization (Supplemental Figure
2). In contrast, the D-L cluster II (AL10 and AL30) has phosphoproteins
involved in central and carbohydrate metabolism (Supplemental Figure 3).
Interestingly, parallel phosphorylation changes in L-D cluster I occur
on proteins involved in nitrogen metabolism and the cell cycle. Nitrogen
is acquired by plants primarily in the form of nitrate or ammonium, and
is an essential macronutrient for plant growth. Nitrate signaling is
linked to cell cycle progression through the TEOSINTE BRANCHED 1/
CYCLOIDEA/PROLIFERATING CELL FACTOR 20 (TCP20) – NIN-LIKE PROTEIN 6/7
(NLP6/7) regulatory network. TCP20 positively regulates genes encoding
proteins involved in nitrate assimilation and signaling and
downregulates the expression of CYCB1;1 , which encodes a key
cell-cycle protein involved in the G2/M transition (Guan, 2017). Our
data suggests that in addition to TCP20 transcriptional regulation,
reversible protein phosphorylation may also play a role in this
regulatory intersection between nitrate signaling and the cell cycle.
Similar to our analysis of protein abundance changes, we built
association networks using STRING-DB to complement the GSEA analysis of
the phosphoproteome (Figure 4). Association networks were generated
based on phosphopeptide quantification data and in silicosubcellular localization information to examine relationships between
the significantly changing phosphoproteins at both the D-L and L-D
transitions. Most of the node clusters overlap between both the D-L
(Figure 4A) and L-D (Figure 4B) networks, with larger clusters
consisting of proteins involved in light detection and signaling, carbon
and nitrogen metabolism, protein translation, hormone signaling, ion
transport, cell wall related processes and protein phosphorylation. L-D
transition-specific node clusters include RNA processing, transcription
and secretion, and protein transport (Figure 4B). Similar to our
proteome analyses, network association and GSEA analyses showed a high
degree of overlap, indicating that the two approaches revealed the same
cell processes in which proteins show differences in phosphorylation.
The dynamics of the measured Arabidopsis proteome during the diurnal
period suggests that the proteins affected by abundance changes have
significant functions in cellular processes. But as discussed above,
protein abundance changes are generally not as widespread and extensive
as transcriptome-level changes during the 24 h period. Comparison of our
diurnal proteome time-series to reported transcriptome time-series
substantiates data from earlier reports of ED vs EN studies examining
Arabidopsis rosettes (Baerenfaller et al., 2012; Seaton et al., 2018;
Uhrig et al., 2019) and circadian clock mutants (Graf et al., 2017) that
transcript changes often do not coincide with protein abundance
fluctuations (Baerenfaller et al., 2012; Graf et al., 2017). Changes in
protein phosphorylation could be dependent or independent of protein
abundance fluctuations. Our results show that the majority of
significantly changing diurnal phosphorylation events occur
independently of protein abundance changes, indicating that they most
likely regulate protein function (Duby & Boutry, 2009; Le, Browning, &
Gallie, 2000; Lillo et al., 2004; Muench, Zhang, & Dahodwala, 2012).
Further research is required to elucidate the roles of the
phosphorylation events on the Arabidopsis proteins we have identified.
Based on our results it will be interesting to investigate which of the
seemingly stable proteins / phosphoproteins and significantly changing
phosphoproteins are in fact undergoing changes in their translation and
turnover, but maintain their overall abundance (Li et al., 2017).