Key plant processes involve independent changes in both proteome
and phosphoproteome
We queried the data for all measured proteins that change in their
abundance and/or phosphorylation status during the 24 h diurnal period.
This revealed predominantly proteins involved in translation, metabolism
and cell wall biosynthesis, suggesting that these cellular processes are
subject to diurnal regulation at the protein level. The translation
rates of Arabidopsis enzymes of light-induced metabolic reactions
fluctuate diurnally and this correlates with their activity (Seaton et
al., 2018). For example, several central metabolic enzymes are
synthetized at 50 to 100% higher rates during the light phase of the
photoperiod (Pal et al., 2013; Piques et al., 2009). We identified 15
proteins involved in protein translation that have diurnal changes in
abundance (Table 3; Supplemental Table 2). Although they belong to
several clusters shown in Figure 1A, nine of the proteins are grouped in
CL3 that represents a general protein increase at the onset of light. In
addition, we found 8 translation-related proteins with changes in their
phosphorylation status at L-D and D-L transitions, of which 5/8 are
eukaryotic initiation factor (eIF) proteins (Table 3; Supplemental Table
8). Phosphorylation affects eukaryotic translation at the initiation
step (Jackson, Hellen, & Pestova, 2010; Le et al., 2000; Muench et al.,
2012), and numerous eIFs and ribosomal proteins show differences in
phosphorylation levels between light and dark periods (Boex-Fontvieille
et al., 2013; Turkina, Klang Arstrand, & Vener, 2011; Uhrig et al.,
2019). Our analysis revealed additional diurnally regulated eIFs and
suggests that specific translational regulation mechanisms and ribosome
composition could be controlled by light changes (e.g. day versus night)
and also throughout the 24h photoperiod.
Several enzymes related to fatty acid, biotin, mitochondrial acetyl-CoA
and chloroplast metabolism have diurnal changes in abundance (Figure 2;
Table 3; Supplemental Table 2). Of particular interest are peroxisomal
fatty acid β-oxidation enzymes 3-ketoacyl-CoA thiolase 2 (KAT2/PKT3;
AT2G33150) and 3-hydroxyacyl-CoA dehydrogenase (MFP2/AIM1-like;
AT3G15290). KAT2 is a central enzyme in peroxisomal fatty-acid
degradation for the production of acetyl-CoA that is required for
histone acetylation, which in turn affects DNA methylation (Wang et al.,
2019), and ABA signaling (Jiang, Zhang, Wang, & Zhang, 2011), which is
essential to daily regulation of stomatal conductance. MFP2/AIM1-like is
an uncharacterized ortholog of MULTIFUNCTIONAL PROTEIN 2 (MFP2) and
ENOYL-COA ISOMERASE (AIM1), which are involved in indole-3-acetic acid
and jasmonic acid metabolism (Arent, Christensen, Pye, Norgaard, &
Henriksen, 2010; Delker, Zolman, Miersch, & Wasternack, 2007). KAT2
loss-of-function mutants require sucrose to supplement plant acetyl-CoA
production, suggesting that diurnal changes in fatty acid degradation
through KAT2 and MFP2/AIM1-like are tied to sucrose production and that
products downstream of KAT2 and MFP2/AIM1-like (e.g. hormones) are
essential to plant growth and development (Pinfield-Wells et al., 2005).
Previously, fatty acid and lipid metabolism in leaves and seedlings has
been suggested to be diurnally / circadian clock regulated (Gibon et
al., 2006; Hsiao et al., 2014; Kim, Nusinow, Sorkin, Pruneda-Paz, &
Wang, 2019; Nakamura, 2018; Nakamura et al., 2014). This includes:
diurnal changes in fatty acids and lipids (Gibon et al., 2006) in
wild-type plants as well as diurnal changes in triacylglycerol (Hsiao et
al., 2014) and phosphatidic acid (Kim et al., 2019) in the circadian
clock double mutant lhycca1 . Complementing these studies, our
findings provide a new protein-level understanding of where fatty acid
and lipid regulation may rest which differs from our current transcript
/ metabolite based knowledge, indicating that further protein-level
investigations are required.
We identified enzymes in primary metabolism that changed their
phosphorylation status at the D-L and L-D transitions (Table 3;
Supplemental Table 9). Several of these enzymes were previously
identified as phosphorylated proteins (PhosPhat 4.0) (Heazlewood et al.,
2008). Our sampling of three closely spaced time-points provides new
information about the rate of protein phosphorylation changes at each
transition. Moreover, our results demonstrate that in Arabidopsis
metabolic enzymes are subject to changes in either protein abundance or
phosphorylation, or both, which likely is of regulatory relevance for
metabolic pathway flux. This information is useful when deciding which
protein isoform would be best for engineering increased pathway flux if
two are present simultaneously. For example, in case of the
well-characterized NITRATE REDUCTASE 1 (NIA1; AT1G77760) and 2 (NIA2;
AT1G37130) proteins their abundance parallels transcript levels but
opposed to changes in protein phosphorylation, with NIA2 showing more
rapid changes in phosphorylation at the D-L transition compared to NIA1
(Table 3; Supplemental Figure 4). NIA1 and NIA2 are regulated both
transcriptionally and post-translationally by phosphorylation (Lillo,
2008; Lillo et al., 2004, Wang, Du, & Song, 2011). Our results define a
rate of change in the phosphorylation of these related isozymes at the
L-D and D-L transitions and define when peak NIA1 and NIA2 protein
levels occur relative to peak transcript levels. These new insights help
to better understand the precise functional differences between NIA1 and
NIA2. NIA1 and NIA2 have tissue-specific gene expression
profiles, with NIA1 expression generally complementing that
of NIA2 in the same organ. NIA1 was predominantly found in
leaves, while NIA2 was predominantly found in meristematic tissue (Olas
& Wahl, 2019). We analyzed whole Arabidopsis rosettes before bolting,
of which developing leaves and apical meristematic tissue comprises only
a small amount of total tissue. Therefore, it will be interesting to
determine if the observed difference in NIA1 and NIA2 phosphorylation
rates at known regulatory phosphorylation sites reflect a higher
sensitivity of NIA2 to changes in nitrate levels in meristematic and
developing tissues (Olas et al., 2019).
Plant genomes often encode multiple forms of enzymes (isozymes) in
metabolic pathways. The temporal rate at which related co-expressed
protein orthologs are modified by a post-translational modifications
such as protein phosphorylation provides more detailed information on
cellular regulation. Our analysis of the phosphoproteome at three D-L
and L-D time-points shows the dynamics of phosphorylation events at both
transitions. Further information about the temporal rate at which
different phosphorylation sites in a protein are utilized can then be
combined with e.g., enzyme kinetics to reveal how metabolic flux through
multiple enzyme reactions may be fine-tuned by PTMs versus changes in
protein abundance. NIA1 and NIA2 are a good example that resolving
differences in PTM rates helps to better understand the role of PTMs in
protein regulation.
We also find diurnal changes in both protein abundance and protein
phosphorylation for enzymes involved in carbohydrate metabolism (Table
3; Supplemental Table 2, 4). Starch biosynthesis and degradation is
diurnally regulated to manage the primary carbon store in plants
(Kotting, Kossmann, Zeeman, & Lloyd, 2010). For example, granule bound
starch synthase 1 (GBSS1; AT1G32900) levels increase preceding the D-L
transition, likely in anticipation of starch granule formation
(Szydlowski et al., 2011). Debranching enzyme 1 (DBE1, AT1G03310)
increases in abundance at the end of the light period to facilitate
effective starch degradation in the dark (Delatte, Trevisan, Parker, &
Zeeman, 2005). Other enzymes such as beta-amylase 1 (BAM1; AT3G23920)
were phosphorylated immediately after the onset of light. Although the
function of BAM1 phosphorylation is currently unknown, our results
provide information to understand its regulation in stomatal starch
degradation and sensitivity to osmotic changes in rosettes (Zanella et
al., 2016).
Cell wall metabolic enzymes involve both diurnal fluctuations in protein
abundance (Figure 2, Table 3; Supplemental Table 2) and changes in
phosphorylation status (Figure 3, Table 3; Supplemental Table 4) at the
D-L and L-D transitions. Cell wall biosynthesis is a major metabolic
activity of growing plants (Barnes & Anderson, 2017; Cosgrove, 2005).
We find that cellulose synthase enzymes CESA5 (AT5G09870) and CSLC6
(AT3G07330) were rapidly phosphorylated at the L-D transition. CESA5 has
been shown to be phosphorylated and phosphorylation memetic-mutant
enzymes increase movement of the cellulose synthase complex (CSC) in
dark-grown seedlings, indicating a photoperiod-dependent regulation cell
wall biosynthesis (Bischoff et al., 2011). Diurnal cellulose synthesis
may also be controlled by the intracellular trafficking of CSC enzymes
as a result of changes in metabolism (Ivakov et al., 2017). In
dark-grown hypocotyls the ratio of CESA5 to CESA6 phosphorylation in the
CSC complex is important for cellulose synthesis (Bischoff et al.,
2011). Our phosphoproteome results now provide additional information on
the rate of CESA5 phosphorylation at the onset of that dark period. We
also find phosphorylation of the plasma membrane
H+-ATPase HA1 (AT2G18960) at the L-D transition
(Figure 3B). Phosphorylation activates H+-ATPases
(Duby & Boutry, 2009; Sondergaard, Schulz, & Palmgren, 2004) and
implicates HA1 as a primary candidate H+-ATPase in
diurnal cell wall acidification to facilitate cell expansion during the
night (Ivakov et al., 2017).