Quantitative phosphoproteome profiling of iron-deficient Arabidopsis roots

Abstract

Iron (Fe) is an essential mineral nutrient for plants, but often it is not available in sufficient quantities to sustain optimal growth. To gain insights into adaptive processes to low Fe availability at the posttranslational level, we conducted a quantitative analysis of Fe deficiency-induced changes in the phosphoproteome profile of Arabidopsis (Arabidopsis thaliana) roots. Isobaric tags for relative and absolute quantitation-labeled phosphopeptides were analyzed by liquid chromatography-tandem mass spectrometry on an LTQ-Orbitrap with collision-induced dissociation and high-energy collision dissociation capabilities. Using a combination of titanium dioxide and immobilized metal affinity chromatography to enrich phosphopeptides, we extracted 849 uniquely identified phosphopeptides corresponding to 425 proteins and identified several not previously described phosphorylation motifs. A subset of 45 phosphoproteins was defined as being significantly changed in abundance upon Fe deficiency. Kinase motifs in Fe-responsive proteins matched to protein kinase A/calcium calmodulin-dependent kinase II, casein kinase II, and proline-directed kinase, indicating a possible critical function of these kinase classes in Fe homeostasis. To validate our analysis, we conducted site-directed mutagenesis on IAA-CONJUGATE-RESISTANT4 (IAR4), a protein putatively functioning in auxin homeostasis. iar4 mutants showed compromised root hair formation and developed shorter primary roots. Changing serine-296 in IAR4 to alanine resulted in a phenotype intermediate between mutant and wild type, whereas acidic substitution to aspartate to mimic phosphorylation was either lethal or caused an extreme dwarf phenotype, supporting the critical importance of this residue in Fe homeostasis. Our analyses further disclose substantial changes in the abundance of phosphoproteins involved in primary carbohydrate metabolism upon Fe deficiency, complementing the picture derived from previous proteomic and transcriptomic profiling studies.

Figure 1.

Experimental strategy for the enrichment, analysis, and quantification of phosphopeptides. [See online article for color version of this figure.]

Figure 2.

Functional classification of Arabidopsis root phosphoproteins according to the Munich Information Center for Protein Sequences. The BioMap tool package provided at http://virtualplant.bio.nyu.edu/ was used for the analysis. [See online article for color version of this figure.]

Figure 3.

Interaction network of the identified phosphoproteins. Mapping of the network was performed with the STRING system (http://string.embl.de) based on known and predicted interactions. Lines of different colors represent different types of evidence for the associations. Four major clusters associated with mRNA processing (A), translation (B), carbohydrate metabolism (C), and proton transport (D) were derived from the associations of the proteins. Proteins without interactions have been removed from the graph. [See online article for color version of this figure.]

Figure 4.

Fe deficiency-induced alterations in carbohydrate metabolism at different regulator levels. Up-pointing arrows indicate up-regulated processes, and arrows pointing downward denote down-regulated processes. See text for details. Asterisks indicate genes that were significantly up-regulated more than 1.5-fold but less than 2-fold. Protein data were taken from Lan et al. (2011); transcript changes from Yang et al. (2010). OAA, Oxaloacetate; 2-OG, 2-oxoglutarate; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway.

Figure 5.

IAR4 is critical in root development, root hair formation, and auxin response. A, Phenotypes of the wild type (Col-0), the iar4 mutant, an iar4/IAR4 complementation line, and substitution lines in which Ser-296 was changed to Ala (iar4/S296A) or Asp (iar4/S296D). Analysis was performed on 6-d-old plants. B, Root hair phenotypes of Col-0, iar4, iar4/IAR4, and iar4/S296A. C to E, Quantification of primary root length (C), root hair number (D), and root hair length (E) of the investigated lines. Different letters indicate significant differences at P < 0.05. F, DR5-GUS expression in Col-0 plants and iar4 mutants (iar4-3[DR5-GUS]) under control, Fe-deficient, and phosphate-deficient conditions. Thirteen-day-old plants were analyzed for GUS expression.