Quantitative Phosphoproteome Profiling of Iron-Deficient Arabidopsis Roots 1[C][W] Ping Lan, Wenfeng Li, Tuan-Nan Wen, and Wolfgang Schmidt* Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan (P.L., W.L., T.-N.W., W.S.); Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan (W.S.); and Genome and Systems Biology Degree Program, College of Life Science, National Taiwan University, Taipei 10617, Taiwan (W.S.) 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. Iron (Fe) often limits plant growth because of its tendency to form complexes of low solubility. To im- prove the acquisition of Fe from pools of limited availability, plants have developed a suite of responses that readjust cellular homeostasis, comprising changes in developmental programs, metabolism, and expres- sion of transporters mediating the uptake and distri- bution of Fe. In Arabidopsis (Arabidopsis thaliana), Fe acquisition is controlled by two basic helix-loop- helix (bHLH) transcription factors, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) and POPEYE (PYE), regulating nonoverlapping subsets of genes with various roles in Fe uptake and metabolism (Colangelo and Guerinot, 2004; Bauer et al., 2007; Long et al., 2010; Schmidt and Buckhout, 2011). Disruption of FIT or PYE function leads to severe growth reduction and chlorosis under Fe-limited con- ditions, indicating that the function of these genes is critical for regulating Fe homeostasis. FIT forms heter- odimers with bHLH38 and bHLH39 and positively regulates a subset of Fe-responsive genes, including two key genes required for Fe acquisition that encode the ferric reductase FERRIC REDUCTION OXIDASE2 and the Fe transporter IRT1 (Eide et al., 1996; Robinson et al., 1999; Vert et al., 2002; Colangelo and Guerinot, 2004; Yuan et al., 2008). PYE is preferentially expressed in the pericycle and aids in maintaining Fe homeostasis by positively regulating a separate cluster of genes. The expression of BRUTUS (BTS), encoding a putative E3 ligase protein that negatively regulates the Fe deficiency responses, is tightly correlated with PYE gene activity. Both proteins interact with the PYE homologs IAA- LEU-RESISTANT3 (ILR3) and bHLH115, suggesting a complex and dynamic regulatory circuit that adapts plants to fluctuating availability of Fe. The signaling processes that are upstream of or parallel to FIT, PYE, and BTS are largely unknown. All three genes are regulated by the plant’s Fe status, indicating that other components are involved in Fe sensing and signaling. A study of Fe deficiency- induced protein profile changes revealed that many of the differentially expressed proteins were not associated 1 This work was supported by Academia Sinica (grant no. 02234 to W.S.). * Corresponding author; e-mail wosh@gate.sinica.edu.tw. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Wolfgang Schmidt (wosh@gate.sinica.edu.tw). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.112.193987 Plant Physiology Ò , May 2012, Vol. 159, pp. 403–417, www.plantphysiol.org Ó 2012 American Society of Plant Biologists. 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