Fig 2: Purification of NifH from chloroplasts of N. benthamiana co-expressing nifH, nifM, nifU and nifS. (a) Schematic representation of the multigenic construct used in N. benthamiana-transient expression assays leading to NifH purification. (b) Western blot analysis of the agroinfiltrated tissue using antibodies targeting Strep-tagged NifH, NifM, NifU and NifS as indicated. Expected sizes are Twin-Strep NifH: 35.3 kDa, NifM: 32.3 kDa, NifU: 34.4 kDa and NifS: 44.3 kDa. (c) Chloroplast purification assays. Total proteins extract prior to chloroplast purification (CE). Intact chloroplasts were isolated and then broken to separate the soluble (S) and membrane-associated (P) fractions. Control refers to N. benthamiana plants agroinfiltrated with agrobacterium transformed with an empty vector. Proteins were resolved by SDS-PAGE and detected by Western blot. Expected sizes are RbcL: 52.7 kDa; Actin-11: 41.6 kDa and IDH: 39 kDa. The triangles in red mark the mature form of the protein. (d) Coomassie staining illustrating the Strep-Tactin purification procedure of A. vinelandii NifH expressed in leaves of N. benthamiana. CFE, cell-free extract (soluble fraction following centrifugation and filtering of disrupted leaf tissue); FT, Strep-Tactin flow-through fraction; W, wash fraction; E, biotin-eluted fraction; Ec, concentrated eluate. (e–f) Western blot analysis of the Strep-Tactin purification procedure. Antibodies targeting Strep-tag (e) or NifH (f) were used for visualization of the purified NifH protein.

Fig 3: Localization of stroma-targeted Nif proteins. (a) Schematic representation of the constructs used in N. benthamiana-transient expression assays leading to Nif localization. (b) Confocal microscopy images of Nif proteins fused to tGFP. N.C. corresponds to ‘negative control’, a non-agroinfiltrated leaf. The three columns show the individual signals for tGFP (green, on the left) and chlorophyll autofluorescence (red, in the centre). On the right, overlap of both signals (merge). (c) Chloroplast purification assays. Total proteins extract prior to chloroplast purification (CE). Intact chloroplasts were isolated and then broken to separate the soluble (S) and membrane-associated (P) fractions. Control refers to N. benthamiana plants agroinfiltrated with agrobacterium transformed with an empty vector. Proteins were resolved by SDS-PAGE and detected by Western blot. Expected sizes are Twin-Strep NifH: 35.3 kDa, NifM: 32.3 kDa, NifU: 34.4 kDa, NifS: 44.3 kDa (27 kDa more in the case of tGFP fusions), RbcL: 52.7 kDa, Actin-11: 41.6 kDa and IDH: 39 kDa. Note that the IDH blot was re-probed after striping anti-RuBisco and some signal remains (upper band).

Fig 4: Expression, purification and activity assays of NifU from N. benthamiana chloroplasts. (a) Schematic representation of the construct used in N. benthamiana-transient expression assays leading to NifU purification. pe35s: 35S mosaic cauliflower virus-enhanced promoter; SSU: CTP from the small subunit of RuBisCo; Tnos: nopaline synthase Agrobacterium terminator; p35s: 35S mosaic cauliflower virus promoter. (b) Purification of chloroplasts from NifU and NifS expressing cells. Total proteins extract prior to chloroplast purification (CE). Intact chloroplasts were isolated and then broken to separate the soluble (S) and membrane-associated (P) fractions. Control refers to N. benthamiana plants agroinfiltrated with agrobacterium transformed with an empty vector. Proteins were resolved by SDS-PAGE and detected by Western blot. Expected sizes are Twin-Strep NifU: 36.5 kDa, NifS: 43.7 kDa, RbcL: 52.7 kDa; Actin-11: 41.6 kDa and IDH: 39 kDa. Note that the IDH blot was re-probed after striping anti-RuBisco and some signal remains (upper band). (c) Coomassie staining illustrating the Strep-Tactin purification procedure of A. vinelandii NifU expressed in leaves of N. benthamiana. CFE, cell-free extract (soluble fraction following centrifugation and filtering of disrupted leaf tissue); FT, Strep-Tactin flow-through fraction; W, wash fraction; E, biotin-eluted fraction. (d–f) Western blot analysis of the Strep-Tactin purification procedure. Antibodies targeting Strep-tag (d) or NifU (e) were used for visualization of the purified NifU protein. Antibodies targeting NifS (f) were used to visualize NifS protein co-purifying with NifU. (g) A. vinelandii apo-NifH was activated either by in vitro [4Fe–4S] cluster reconstitution (Rc Apo-NifH) or by incubation with cluster-loaded NifU purified from E. coli (EcNifU) or from N. benthamiana leaves (NbNifU) in 1:1 or 1:2 NifH/NifU molar ratio. Identical reaction mixtures without NifU were used as negative control (Rc C-). Error bars represent mean ± SD (n = 2).

Fig 5: CFS1 is crucial for autophagic flux in A. thaliana. (A) Confocal microscopy images of root epidermal cells of Col-0, cfs1, or atg5 expressing pUBQ::mCherry-ATG8E under NaCl or NaCl + conA treatment. 5-d-old Arabidopsis seedlings were incubated in 1/2 MS media containing either 150 mM NaCl (NaCl) or 90 mM NaCl and 1 µM conA (NaCl + conA) for 2 h before imaging. Representative images of 10 replicates are shown. Scale bars, 5 µm. (B) Left panel, quantification of the mCherry-ATG8E puncta per normalized area (10,000 µm2) of the NaCl-treated cells imaged in A. Right panel, quantification of the mCherry-ATG8E puncta inside the vacuole per normalized area (10,000 µm2) of the NaCl + conA-treated cells imaged in A. Bars indicate the mean ± SD of 10 replicates. Two-tailed and unpaired Student t tests with Welch’s correction were performed to analyze the significance of mCherry-ATG8E puncta density differences between Col-0 and cfs1, Col-0 and atg5, or cfs1 and atg5. ***, P value <0.001. (C) Western blots showing GFP-ATG8A cleavage level and endogenous NBR1 level in Col-0 or cfs1 mutants under control, nitrogen-deficient (-N) or salt-stressed (NaCl) conditions. Arabidopsis seedlings were grown under continuous light in 1/2 MS media for 1-wk and 7-d-old seedlings were subsequently transferred to 1/2 MS media ±1 µM conA, nitrogen-deficient (-N) 1/2 MS media ±1 µM conA, or 1/2 MS media containing 150 mM NaCl ±1 µM conA for 12 h. 15 µg of total protein extract was loaded and immunoblotted with anti-GFP and anti-NBR1 antibodies. Representative images of five replicates are shown. Reference protein sizes are labeled as numbers at the left side of the blots (unit: kD). (D) Quantification of the relative autophagic flux in C. Values were calculated through protein band intensities of GFP divided by GFP-ATG8A and normalized to untreated (Control) Col-0. Results are shown as the mean ± SD of five replicates. One-tailed and paired Student t tests were performed to analyze the significance of the relative autophagic flux differences. ns, not significant. *, P < 0.05. **, P < 0.01. (E) Quantification of the relative NBR1 level in C compared to untreated (control) Col-0. Values were calculated through normalization of protein bands to Ponceau S and shown as the mean ± SD of five replicates. One-tailed and paired Student t tests were performed to analyze the significance of the relative NBR1 level differences. **, P < 0.01. ***, P < 0.001. (F) Serial sections of transmission electron microscopy micrographs showing a mitophagosome engulfing a mitochondrion. 7-d-old Arabidopsis cfs1 seedlings were incubated in 1/2 MS media containing 50 µM DNP for 1 h before cryofixation. Scale bar, 500 nm. MPh, mitophagosome. M, mitochondrion. (G) Immunoblot assay of uncoupler-induced mitochondrial protein degradation in Arabidopsis Col-0 and cfs1 seedlings. 7-d-old seedlings were incubated in 1/2 MS media containing 50 µM DNP ±1 µM conA for 4 h before protein extraction. A mitochondrial matrix protein, isocitrate dehydrogenase (IDH), was immunoblotted by anti-IDH antibodies. Actin (ACT) was immunoblotted by anti-ACT antibodies and was used as a loading control. Representative images of three replicates are shown. Reference protein sizes are labeled as numbers at the left side of the blots (unit: kD). (H) Quantification of relative IDH intensities in G compared to untreated (control) Col-0. Values were calculated via normalization of protein bands to ACT and shown as the mean ± SD of three replicates. One-tailed and paired Student’s t tests were performed to analyze the significance of the relative IDH level differences. ns, not significant. *, P < 0.05. **, P < 0.01. (I) TEM micrographs of mitochondria in Arabidopsis Col-0, cfs1 and atg5 root cells. 7-d-old seedlings were incubated in 150 mM NaCl-containing 1/2 MS media for 2 h for autophagy induction before cryofixation. Yellow arrowheads mark the distinctive electron dense precipitates of compromised mitochondria that appear after NaCl treatment. Scale bars, 500 nm. M, mitochondria. Source data are available for this figure: SourceData F4.