Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence

Highlight To illustrate the hypoxia signal mediated by SnRK1.1, phosphoproteomics was used to identify not only the new SnRK1.1 putative targets related to submergence, but also to gain an insight into early hypoxia signalling events.

Flowchart of iTRAQ sample preparation and prediction of SnRK1.1 targets. Total proteins of 9-day-old seedlings with different durations of submergence treatment were TCA precipitated and resuspended in the Tris buffer. They were further processed and fractionated for total proteomics or phosphoproteomics. The processes indicated by red and green arrows were for sample preparations in phosphoproteomics and total proteomics, respectively. After LC-MS/MS, the data were analyzed by the Proteome Discoverer software (ver. 1.3, Thermo Scientific) to generate the list of SnRK1.1-dependent phosphorylated protein under submergence. To identify the SnRK1.1 potential targets, the proteins identified in the SnRK1.1 dependent list were purified and examined their phosphorylation by the SnRK1.1 using the in vitro kinase assay.  The chromatogram acquisition, detection of mass spectral peaks, and their waveform processing were performed using Thermo Xcalibur 2.1 SP1 software (Thermo Scientific, USA).

Antibody and western blot analysis
For western blot examination, protein extraction was performed according to the procedures described in (Hsieh et al., 2012

LC-MS/MS analysis
The SCX fractions were analyzed on a nanoUPLC system (nanoAcquity,

Phosphorylation motif analysis using motif X
To identify the phosphorylation motifs that were significantly enriched under submergence, all the identified phosphorylated sites of peptides, for which the pRS site probabilities were above 75%, were collected. The phosphorylation sites of the low pRS site probabilities that were caused by closely-spaced multiple phosphorylation sites in the peptide were also selected for motif analysis. Parameters were set to 13 amino acids of peptide length, an occurrence of 10, and a P value < 10 -5 . The default Arabidopsis proteomics data set was used as the background data set (which has the same motif patterns as used in the protein sequences of the whole genome Arabidopsis database TAIR 8 in the Fasta format), as described by (Reiland et al., 2009).

Recombinant protein expression and in vitro kinase assay
The coding sequences of F2KP, PENTA, eIFiso4G1, and PTP1 were cloned into pET32a (Novagen) through Gateway system (
Each sample was re-suspended with 500 ml chloroform/ACN (1:2) and 12 µg/ml 13 C-Glucose. 13 C-Glucose was used as an internal standard. The mixture was sonicated for 30 min at 4°C and the sugars were extracted twice from the organic phase by liquid-liquid extraction with 500 ml water.
Subsequently, the aqueous phase was collected and vacuum dried.

Quantitative polymerase chain reaction
RNA extraction, reverse transcription and qRT-PCR were performed as previously described (Hsu et al., 2013). Sequences of primers are listed in Table S8. TUB3 (AT5G62700) was used as an internal control for normalization. Relative expression levels were calculated by comparing the expression of the gene at a certain time point to the sample of time zero.

Accession numbers of sequences
Sequence data from this article can be found in the Arabidopsis Genome     (2) The nubmers indicate the average value and standard deviation of phosphorylation fold change of all biological repeats. The time point data that is significantly different (P < 0.05) is marked as "Y". If not, marked as "N"   Table 5. Differential phosphorylation of selected submergence-responsive genes in SnRK1.1 K48M mutants. *, indicates phosphorylation site.

Time (h)
Phospho peptide AGI number mean (±)SD P<0.05 mean (±)SD P<0.05 mean (±)SD P<0.05 mean (±)SD P<0.05 mean (±)SD P<0.05 mean (±)SD P<0.05 mean (±)SD P<0. The pRS score of these phosphorylated peptides in Col-0 was passed the criteria (>50), but that in SnRK1.1K48M-5 was not. The nubmers indicate the average value and standard deviation of phosphorylation fold change of all biological repeats. The time point data that is significantly different (P < 0.05) is marked as "Y". If not, marked as "N" The time point data that is not detected in the three batches or detected once in three batches is marked as NA (not available).