4.1. Plant Material and Growth Conditions
nfu3-2[19] and the wild type Columbia genotypes were germinated and grown under long day conditions (16-h-light/8-h-dark cycle; light intensity: 120 mmoL/cm2/s). For proteomic analysis, two-week-old seedlings were grown on half-strength Murashige and Skoog medium containing 0.05%
(w/v) MES, 1% (w/v) sucrose, 0.7% (w/v) agar and 50µM iron provided as Fe(III)-EDTA as described in [21]. For histochemical GUS detection and GFP confocal observation, seedlings were grown under the same condition for 7 and 10 days, respectively. Four-week-old plants were grown on soil at 23◦C with a sunlight intensity limited to 300µmol.m−2.s−1and 16 h of light/8 h of dark.
4.2. Cloning
NFU1,NFU2,andNFU3genomic sequences from about 2 kb upstream from the start codon (2033 bp, 2579 bp, 2595 bp, respectively) and until the last coding codon (without the stop codon) were cloned into the pDONR201 vector and recombined into the pGWB3 and pGWB4 binary vectors [33].
The primers used are described in Table S6. All PCR products were obtained using high-fidelity Phusion DNA polymerase, and each construct was sequenced to ensure its integrity.
4.3. Histochemical GUS Detection and GFP Confocal Observation
Histochemical GUS detection was performed according to [34,35]. The acidified chloral hydrate–glycerol solution was prepared by dissolving 45 g chloral hydrate into a solution consisting of 25 mL 4.2% HCl and 10 mL glycerol. GFP confocal observations were carried out as described in [34]. GUS and GFP images are representative of 20 and 10 independent transgenic lines (at least 20 individuals per line were analyzed), respectively. Images shown in Figure2C were recorded with maximum Z-stack intensity projection.
4.4. Gene Expression Analysis
Total RNAs were extracted using the GenElute™MammalianTotalRNA Purification Kit(Sigma-Aldrich, Saint-Louis, MO, USA). For each sample, 1µg of total RNA treated with DNase was reverse transcribed into cDNA using the RevertAid kit (Thermo scientific, Waltham, MA, USA). qRT-PCR analyses were performed using a LightCycler®480 (Roche, Bâle, Switzerland) and TB Green Premix Ex Taq (2X) (Takara, Kusatsu, Shiga, Japan). PP2AA3 (PROTEIN PHOSPHATASE 2A SUBUNIT A3) was used as a reference gene [36]. Expression levels were calculated using the comparative threshold cycle method. The primers used are described in Table S6.
4.5. Liquid Chromatography Coupled with Mass Spectrometry (LC-MS/MS) Analysis
Protein extraction from whole seedlings, trypsin digestion and MS analysis were carried out as described in [21]. Raw mass spectrometric data were analyzed in the Maxquant environment (v.1.5.5.1, Max-Planck-Institute of Biochemistry, Planegg, Germany) [37] and Andromeda was employed for database search [38]. The MS/MS data were matched against the TAIR10 database. For protein identification and quantification, cysteine carbamidomethylation was set up as fixed modification and oxidation of methionine as a variable modification. At least two peptides were required for protein identification and quantification. Up to two missed cleavages was allowed for protease digestion.
For other characteristics, Maxquant default parameters were used. Following the quantification step
Int. J. Mol. Sci.2020,21, 8121 21 of 23
and the label free quantitative (LFQ) normalization, proteins were considered as quantifiable only if they are present in all samples. Data were log2 transformed prior analysis. For statistics, pairwise t-testsp<0.05 were carried out. Before statistical treatment, the normal distribution of the logarithmic transformed data was assessed. For the present/absent analysis, a protein was considered as absent in thenfu3-2mutant if it was identified with at least 2 peptides in at least 3 replicates achieved with WT plants and not in the 4 replicates achieved withnfu3-2plants. All raw MS data and Maxquant files generated have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020228.
Supplementary Materials:The following are available online athttp://www.mdpi.com/1422-0067/21/21/8121/s1, Figure S1.Arabidopsis thalianaNFU1, NFU2 and NFU3 protein sequences. Figure S2. Quantitative analysis of GFP fluorescence inArabidopsis thalianaProNFU:gNFU-GFP transgenic lines. Table S1. List of significantly varying proteins in theArabidopsis thaliana nfu3-2mutant relative to wild type. Table S2. List of proteins specifically present in theArabidopsis thaliana nfu3-2mutant or in the wild type. Table S3. Detailed localization of variant proteins. Table S4: Detailed localization of proteins under-accumulated specifically innfu3-2,or innfu2-1or in both mutants. Table S5: Detailed localization of proteins over-accumulated specifically innfu3-2,or innfu2-1or in both mutants. Table S6. Primers used in this study.
Author Contributions:Conceptualization, N.B., F.V., V.R., F.G., and C.D.; Formal analysis, N.B., F.V., B.T., V.R., V.D., V.S., N.R., F.G., and C.D.; Funding acquisition, N.R., F.G., and C.D.; Investigation, N.B., F.V., M.T.-B., and V.R.; Project administration, F.G. and C.D.; Resources, V.S., F.G. and C.D.; Supervision, C.D.; Validation, N.B., F.V., and V.R.; Visualization, N.B., F.V., and C.D.; Writing—original draft, N.B., F.V., N.R., and C.D. All authors have read and agreed to the published version of the manuscript.
Funding:This research was funded by the Agence Nationale de la Recherche grant number ANR-2013-BSV6-0002-01.
Acknowledgments: All confocal analyses were performed on a device of the Montpellier RIO Imaging and PHIV platform.
Conflicts of Interest:The authors declare no conflict of interest.
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