Post-Transcriptional Coordination of the Arabidopsis Iron Deficiency Response is Partially Dependent on the E3 Ligases RING DOMAIN LIGASE1 (RGLG1) and RING DOMAIN LIGASE2 (RGLG2)

Abstract

Acclimation to changing environmental conditions is mediated by proteins, the abundance of which is carefully tuned by an elaborate interplay of DNA-templated and post-transcriptional processes. To dissect the mechanisms that control and mediate cellular iron homeostasis, we conducted quantitative high-resolution iTRAQ proteomics and microarray-based transcriptomic profiling of iron-deficient Arabidopsis thaliana plants. A total of 13,706 and 12,124 proteins was identified with a quadrupole-Orbitrap hybrid mass spectrometer in roots and leaves, respectively. This deep proteomic coverage allowed accurate estimates of post-transcriptional regulation in response to iron deficiency. Similarly regulated transcripts were detected in only 13% (roots) and 11% (leaves) of the 886 proteins that differentially accumulated between iron-sufficient and iron-deficient plants, indicating that the majority of the iron-responsive proteins was post-transcriptionally regulated. Mutants harboring defects in the RING DOMAIN LIGASE1 (RGLG1)(1) and RING DOMAIN LIGASE2 (RGLG2) showed a pleiotropic phenotype that resembled iron-deficient plants with reduced trichome density and the formation of branched root hairs. Proteomic and transcriptomic profiling of rglg1 rglg2 double mutants revealed that the functional RGLG protein is required for the regulation of a large set of iron-responsive proteins including the coordinated expression of ribosomal proteins. This integrative analysis provides a detailed catalog of post-transcriptionally regulated proteins and allows the concept of a chiefly transcriptionally regulated iron deficiency response to be revisited. Protein data are available via ProteomeXchange with identifier PXD002126.

Fig. 1.

Identified and differentially expressed proteins and transcripts in roots and leaves of Col-0 plants. A, B, Number of proteins identified in roots, A, and leaves, B, of iron-sufficient and iron-deficient plants in three biological repeats. C, Differentially expressed proteins. D, Differentially expressed transcripts. E, F, Overlap of differentially expressed transcripts and proteins in leaves, E, and roots, F.

Fig. 2.

Comparison of gene expression changes between microarray data (this study) and RNA-seq data take from (4) (roots) and (5) (leaves).

Fig. 3.

Phenotype of the rglg1 rglg2 mutant. A, Two-week-old seedlings grown on iron-replete (40 μm FeEDTA) or low Fe (0.5 μm FeEDTA) media. B, Leaves of 2-week-old plants grown on iron-replete media. Adaxial surface (left panel) and abaxial surface (right panel). C, D, Cross-sections in the root hair zone of Col-0 plants, C, and rglg1 rglg2 mutants on Fe-replete media, D. E, Quantification of trichome density on leaves of Col-0 and rglg1 rglg2 plants. F, G, Scanning electron micrographs of the leaf surface of Col-0 plants, G, and rglg1 rglg2 mutants, F. Scale bars = 500 μm.

Fig. 4.

Identified and differentially expressed proteins and transcripts in roots and leaves of rglg1 rglg2 double mutants. A, B, Number of proteins identified in roots, A, and leaves, B, of iron-sufficient and iron-deficient plants in three biological repeats. C, Differentially expressed proteins. D, Differentially expressed transcripts. E, F, Overlap of differentially expressed transcripts and proteins in leaves, E, and roots, F.

Fig. 5.

Gene Ontology (GO) categorization of differentially expressed proteins.

Fig. 6.

Concordance between changes in the abundance of mRNAs and the encoded proteins. A–D, Correlation between protein and transcript fold-changes upon iron deficiency for significantly changed transcript/protein pairs in roots, A, B, and leaves, C, D, with mRNA data taken from mircoarray analysis, A, C, or an RNA-seq survey B, D (5).

Fig. 7.

Protein–protein interaction (PPI) network of post-transcriptionally regulated proteins. A, PPIs of differentially expressed proteins in roots. B, PPIs of differentially expressed proteins in leaves. Networks were generated using the STRING (http://string-db.org) algorithm.

Fig. 8.

RGLG-dependent regulation of transport proteins. Proton-cotransport of bicarbonate, nitrate and phosphate at the plasma membrane is post-transcriptionally down-regulated to maintain an acidic uptake pattern that facilitates the mobilization of Fe(III) oxides in the rhizosphere by ATPase-mediated proton extrusion. The transcript levels of these transporters remained unchanged. At the tonoplast, IREG2 and MTPA2 protein was decreased under iron-deficient conditions, whereas the genes were highly up-regulated at the transcriptional level. Numbers denote log₂ fold changes of the respective proteins. n.c. = not changed.

Fig. 9.

Differential expression of r-proteins. A, Changes induced by iron deficiency, B, Altered expression of r-proteins in rglg1 rglg2 double mutants.

Fig. 10.

Regulatory control of iron-responsive proteins. Processes directly related to iron homeostasis such as the acquisition, uptake, chelation, and storage of iron, as well as adaptive responses in the plastid and shoot-to-root communication are chiefly transcriptional regulated. Proteins involved in RNA-directed DNA methylation, RNA decay and changes in oxidative phosphorylation are primarily post-transcriptionally regulated. RGLG affects predominantly processes involved in protein translation and degradation, but also post-transcriptional modifications of histones. RGLG is also critical for tuning the abundance of transport proteins. Proteins listed in the figure are discussed in the text.

Fig. 11.

Estimate of the concordance between mRNA and protein expression in iron-deficient plants. Only 13% (roots) and 11% (leaves) of the proteins that are differentially expressed between iron-deficient and iron-sufficient plants (DE proteins) are associated with similarly regulated transcripts. For proteins in this subset, changes in transcript level account for circa 80% of the observed changes in protein abundance.