Abstract
Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants. In addition, sucrose is a compound that serves as a signal affecting metabolic flux and development. Here we provide first results of externally induced phosphorylation changes of plasma membrane proteins in Arabidopsis. In an unbiased approach, seedlings were grown in liquid medium with sucrose and then depleted of carbon before sucrose was resupplied. Plasma membranes were purified, and phosphopeptides were enriched and subsequently analyzed quantitatively by mass spectrometry. In total, 67 phosphopeptides were identified, most of which were quantified over five time points of sucrose resupply. Among the identified phosphorylation sites, the well described phosphorylation site at the C terminus of plasma membrane H(+)-ATPases showed a relative increase in phosphorylation level in response to sucrose. This corresponded to a significant increase of proton pumping activity of plasma membrane vesicles from sucrose-supplied seedlings. A new phosphorylation site was identified in the plasma membrane H(+)-ATPase AHA1 and/or AHA2. This phosphorylation site was shown to be crucial for ATPase activity and overrode regulation via the well known C-terminal phosphorylation site. Novel phosphorylation sites were identified for both receptor kinases and cytosolic kinases that showed rapid increases in relative intensities after short times of sucrose treatment. Seven response classes were identified including non-responsive, rapid increase (within 3 min), slow increase, and rapid decrease. Relative quantification of phosphorylation changes by phosphoproteomics provides a means for identification of fast responses to external stimuli in plants as a basis for further functional characterization.
Highlights
Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants
Isolation of Plasma Membrane Phosphopeptides—To determine the effect of sucrose on phosphorylation of plasma membrane proteins, Arabidopsis seedlings were germinated and grown in continuous light in liquid Murashige/Skoog medium supplemented with 30 mM sucrose for 7 days
This study is the first analysis of phosphorylation sites in plants that resolves a time course after a stimulus; in this case it was sucrose resupply after depletion
Summary
Seedling Growth Conditions—Arabidopsis (Col-0) seedlings were germinated and grown under constant light (100 mol mϪ2 sϪ1) in liquid half-strength Murashige/Skoog medium with 30 mM sucrose for 7 days. Ion intensity sums of m/z values identifying the same peptide sequence at the same retention time in different liquid chromatography runs were used for quantitative comparison between the different samples representing different time points. If no unphosphorylated peptides were identified for a given protein, the mean of all identified non-phosphopeptides was used for normalization. Statistical Analysis of Time-dependent Phosphorylation—Within one biological experiment, significance of time-dependent changes in relative phosphorylation was tested by one-way ANOVA using the log ratios of independently quantified analytical replicates. Reproducibility of the time course responses between two biological experiments was confirmed by linear regression analysis (r2 ϭ 0.725; p Ͻ 0.01) of the log ratios of quantifiable phosphopeptides identified in both of the two independent experiments across all time points (Supplemental Fig. 2). For detection of Hϩ-ATPase antibodies raised against either the N terminus (number 762) or C terminus (number 759) of plasma membrane Hϩ-ATPase were used (1:5000) [42]
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