Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Abstract

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.

Fig. 1.

Effects of cytokinin treatment on the proteome and phosphoproteome of Arabidopsis seedlings. (A) Average two-dimensional gel electrophoresis proteome map of 7-day-old Arabidopsis seedlings treated with cytokinin/mock buffer for 15 min. Differentially regulated protein spots are indicated. See Table 1, and Supplementary Table S1 at JXB online, for detailed information on the corresponding identified proteins. Proteins (500 μg) were separated in the first and second dimensions by IPG (18 cm strips, pH 4–7) followed by 11% SDS–PAGE then visualized by Bio-Safe Coomassie G250 staining. Isoelectric points (pI) and migrating positions of molecular mass (kDa) markers are marked. (B) Examples of spots corresponding to the differentially regulated proteins in Arabidopsis seedlings treated with 5 μM cytokinin (BA, iP, TDZ, or t-Z) or mock buffer for 15 min. For details see Materials and methods. (C) Average 2-DE phosphoproteome map of 7-day-old Arabidopsis seedlings treated with cytokinin/mock buffer for 15 min. Differentially regulated protein spots are indicated. See Table 2, and Supplementary Table S2, for detailed information on the corresponding identified proteins. Phosphoprotein fractions were obtained using a PhosphoProtein Purification Kit. Phosphoproteins (150 μg) were separated in the first and second dimensions by IPG (7 cm strips, pH 4–7) followed by 11% SDS–PAGE then visualized by Bio-Safe Coomassie G250 staining. Isoelectric points (pI) and relative migrating positions of molecular mass (kDa) markers are marked. (D) Examples of spots corresponding to the differentially regulated phosphoproteins in Arabidopsis seedlings treated with 5 μM cytokinin (BA, iP, TDZ, or t-Z) or mock buffer for 15 min. For details see Materials and methods. (This figure is available in colour at JXB online.)

Fig. 2.

Effect of calcium signalling inhibitors on regulation by cytokinin of early cytokinin response phosphoproteins. (A) Selected regions of 2D gels showing early cytokinin response phosphoproteins (indicated by arrows) whose regulation by 15 min treatment with 5 μM t-Z in Arabidopsis seedlings (t-Z) was abolished when calcium signalling inhibitors 30 μM D600 and 60 μM LaCl₃ were administered simultaneously with 5 μM t-Z (t-Z + INH). Control samples were treated with the inhibitors only (INH). For details see Materials and methods. Spot numbers as in Fig. 1C and Table 2. (B) Relative volumes of the individual spots.

Fig. 3.

(A) Classification of the early cytokinin response phosphoproteins according to their cellular functions (Bevan et al., 1998) and (B) molecular processes reportedly controlled by their phosphorylation as deduced from database entries and literature review.

Fig. 4.

Gene ontology (GO) analysis of the early cytokinin response proteins in Arabidopsis (performed in Cytoscape using BiNGO plugin version 2.3). GO categories that were significantly over-represented among the differentially expressed proteins were identified. The yellow to orange colour of the circles indicates the level of significance of over-represented categories (P=0.05, yellow; P=10⁻⁷, orange). The size of the circles is proportional to the number of proteins in each category. Links with low significance were removed manually to reduce complexity of the image.

Fig. 5.

Subcellular distribution of the early cytokinin response proteins (white) and phosphoproteins (grey) according to the UniProt database (http://www.uniprot.org). The numbers above the columns represent sums of the identified proteins and phosphoproteins located in the respective cellular compartment.