A Global Proteomic Approach Sheds New Light on Potential Iron-Sulfur Client Proteins of the Chloroplastic Maturation Factor NFU3

Abstract

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins.

Keywords: Arabidopsis; NFU1; NFU2; NFU3; chloroplast; iron-sulfur cluster; maturation factor; quantitative proteomic.

Figure 1

Analysis of NFU1, NFU2, and NFU3 gene expression and protein localization in Arabidopsis thaliana. Analysis of NFU1, NFU2, and NFU3 gene expression in roots and shoots of 7-day-old wild type Arabidopsis seedlings (A) and in leaves, stems, flowers and siliques of 4-week-old plants (B) by qRT-PCR. (A,B) Means within each condition with the same letter are not significantly different according to one-way ANOVA followed by post hoc Tukey test, p < 0.05 (n = 6 technical repeats from three biological replicates from one representative experiment). Error bars show the means ± SD. (C) NFU1, NFU2, and NFU3 protein localization revealed by GUS activity in 7-day-old seedlings and in leaves, stem, flowers, siliques, seeds, and embryos of 4-week-old plants. Bars: seedling aerial parts and roots, stems = 0.5 cm; root tips, leaves, flowers and siliques = 0.1 cm; seeds and embryos = 0.05 cm.

Figure 2

NFU1, NFU2, and NFU3 protein localization in Arabidopsis thaliana protoplasts and roots. Subcellular localization of NFU1-GFP, NFU2-GFP, and NFU3-GFP fusion proteins (green) expressed under the control of their own promoter with confocal microscopy in Arabidopsis protoplasts. Negative control: Untransformed protoplast. Bars: 10 µm (A). Subcellular localization of NFU1-GFP, NFU2-GFP, and NFU3-GFP fusion proteins expressed under the control of their own promoter with confocal microscopy in the primary (B) and secondary (C) root of 10-day-old seedlings after propidium iodide staining (magenta). RAM: Root apical meristem. Bars: 60 µm (B,C). White arrowheads indicate NFU1-GFP and NFU3-GFP weak fluorescence in roots (B,C).

Figure 3

Subcellular localization and gene ontology analysis of proteins whose abundance is modified in the Arabidopsis thaliana nfu3-2 mutant compared to the wild type. Protein localization was analyzed in silico using the multiple marker abundance profiling method of the SUBA4 bioinformatic platform (http://suba.live/) (A). Gene ontology (GO) term enrichment analysis was conducted using the PANTHER gene ontology platform (http://go.pantherdb.org) considering proteins whose abundance was significantly decreased (B) or increased (C) in nfu3-2 mutant when compared to the wild type. Significant GO term enrichment was determined using Fisher exact test with Bonferroni correction.

Figure 4

Comparison of proteins whose abundance is decreased in the Arabidopsis thaliana nfu3-2 and nfu2-1 mutants when compared to the wild type. Venn diagram comparing the proteins whose abundance is decreased in both nfu3-2 and nfu2-1 mutants (whole seedlings). Seedlings were grown as described in [21]. Venn diagram was done with Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) (A). Subcellular localization of proteins whose abundance is decreased in both nfu3-2 and nfu2-1 mutants. Protein localization was analyzed in silico using the multiple marker abundance profiling method of the SUBA4 bioinformatic platform (http://suba.live/) (B). Gene ontology (GO) analysis of proteins whose abundance is decreased in both nfu3-2 and nfu2-1 mutants. GO term enrichment analysis was conducted using the PANTHER gene ontology platform (http://go.pantherdb.org). Significant GO term enrichment was determined using Fisher exact test with Bonferroni correction (C).

Figure 5

Comparison of proteins whose abundance is increased in the Arabidopsis thaliana nfu3-2 and nfu2-1 mutants when compared to the wild type. Venn diagram comparing the proteins whose abundance is increased in both nfu3-2 and nfu2-1 mutants (whole seedlings). Seedlings were grown as described in [21]. Venn diagram was done with Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) (A). Subcellular localization of proteins whose abundance is increased in both nfu3-2 and nfu2-1 mutants. Protein localization was analyzed in silico using the multiple marker abundance profiling method of the SUBA4 bioinformatic platform (http://suba.live/) (B). Gene ontology (GO) analysis of proteins whose abundance is increased in both nfu3-2 and nfu2-1 mutants. GO term enrichment analysis was conducted using the PANTHER gene ontology platform (http://go.pantherdb.org). Significant GO term enrichment was determined using Fisher exact test with Bonferroni correction (C).

Figure 6

Analysis of DWARF27.2, FdC1, FdC2, GLT1, HCAR, NIR, PetC, and THIC gene expression in Arabidopsis thaliana wild type and nfu3-2 mutant. Analysis of DWARF27.2, FdC1, FdC2, GLT1, HCAR, NIR, PetC, and THIC gene expression in 2-week-old wild type and nfu3-2 Arabidopsis seedlings by qRT-PCR. t-test significant difference: * p < 0.05; ** p < 0.01. (n = 6 technical repeats from three biological replicates from one representative experiment). Error bars show the means ± SD.

Figure 7

Summary scheme integrating known and newly identified potential client proteins of NFU3 and redundancy with its NFU1 and NFU2 paralogs. Among the 16 NFU3 potential client proteins, eight were newly identified in this study by label-free quantitative proteomic analysis. ^N: Novel NFU3 potential targets identified in this study. *: Interactions between NFU3 and apo-targets previously confirmed by BiFC (in [21]), **: Interactions between NFU3 and apo-targets previously confirmed by Y2H (in [15,21]), ^#: Interactions between NFU3 and apo-targets not confirmed by BiFC and/or Y2H in previous studies (in [21]).