A small subset of the transition phosphoproteome has protein
level changes
As the result of enrichment methods, one major question in
phosphoproteomics pertains to the quantified phosphorylation changes and
if they are the result of changes in protein levels. We therefore
performed an integrated analysis of the significantly changing proteome
and phosphoproteome to determine if and how phosphorylation and protein
abundance changes are related. Of the 226 proteins exhibiting a
significant change in phosphorylation (Table 1), 60% (136 proteins)
were quantified in our proteome data (Supplemental Table 6). These
results are not unexpected because of the phosphopeptide enrichment
strategy and indicate that 40% of the phosphorylated proteins in our
phosphoproteome dataset are of lower abundance and not amongst the 4762
total quantified proteins. Further assessment of significantly changing
phosphoproteins relative to the quantified proteome at the light
transitions found that 25% (L-D) and 7.1% (D-L) of the changing
phosphoproteins were not significantly changing at the protein level
(TOST P value ≤ 0.05, ε = 0.4).
We then directly compared the changing phosphoproteome and proteome to
identify proteins exhibiting a significant change in diurnal protein
abundance and phosphorylation. We found that a total of six
phosphorylated proteins (totaling 2.1% of all 288 proteins
significantly changing in protein abundance; Supplemental Table 6) had
concurrent abundance changes (Figure 5). These include nitrate reductase
1 (NIA1; AT1G77760) and 2 (NIA2; AT1G37130), protein kinase SnRK2.4
(AT1G10940), Rho guanyl-nucleotide exchange factor SPK1 (AT4G16340),
microtubule binding protein WDL5 (AT4G32330), and winged-helix
DNA-binding transcription factor family protein LARP1C (AT4G35890). NIA1
and 2 are directly related to nitrogen assimilation (Lillo, 2008; Lillo
et al., 2004), while WDL5 has been implicated in mitigating ammonium
toxicity through ETHYLENE INSENSITIVE 3 (EIN3) (Li et al., 2019).
SnRK2.4 binds fatty acid derived lipid phosphatidic acid to associate
with the plasma membrane (Julkowska et al., 2015) and responds to
changes in cell osmotic status (Munnik et al., 1999), while SPK1, WDL5
and LARP1C are connected to plant hormone signaling through abscisic
acid (WDL5; Yu et al., 2019), jasmonic acid (LARP1C; B. Zhang, Jia,
Yang, Yan, & Han, 2012) and auxin (SPK1; Lin et al., 2012; Nakamura et
al., 2018). Of these three proteins with concerted phosphorylation and
abundance changes only SPK1 showed a parallel increase in abundance and
phosphorylation at the same transition (Figure 5), while WDL5 and LARP1C
exhibited opposing patterns of phosphorylation and abundance changes,
suggesting that phosphorylation may regulate their turnover. Proteins
involved in phytohormone signaling are regulated by both protein
phosphorylation and turnover (Dai et al., 2013; Qin et al., 2014),
suggesting that these three proteins may represent new examples of
hormone-mediated phosphodegrons or phosphor-inhibited degrons (Vu,
Gevaert, & De Smet, 2018). To demonstrate this, further examination is
required of the ubiquitination status of these proteins and the
proximity of those ubiquitin modifications to the annotated
phosphorylation event.