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.