FZL, an FZO-like protein in plants, is a determinant of thylakoid and chloroplast morphology

Abstract

FZO is a dynamin-related membrane-remodeling protein that mediates fusion between mitochondrial outer membranes in animals and fungi. We identified a single FZO-like protein in Arabidopsis, FZL, a new plant-specific member of the dynamin superfamily. FZL is targeted to chloroplasts and associated with thylakoid and envelope membranes as punctate structures. fzl knockout mutants have abnormalities in chloroplast and thylakoid morphology, including disorganized grana stacks and alterations in the relative proportions of grana and stroma thylakoids. Overexpression of FZL-GFP also conferred defects in thylakoid organization. Mutation of a conserved residue in the predicted FZL GTPase domain abolished both the punctate localization pattern and ability of FZL-GFP to complement the fzl mutant phenotype. FZL defines a new protein class within the dynamin superfamily of membrane-remodeling GTPases that regulates organization of the thylakoid network in plants. Notably, FZL levels do not affect mitochondrial morphology or ultrastructure, suggesting that mitochondrial morphology in plants is regulated by an FZO-independent mechanism.

Fig. 1.

FZL is an FZO-like protein in plants. (A) Domain structure of the mouse FZO ortholog Mitofusin 1 (Mfn1) and predicted domains of Arabidopsis FZL. (B) Alignment of the FZL and Drosophila melanogaster FZO GTPase domains. Black, identical residues; gray, similar residues. Dashes indicate gaps. An arrow indicates a mutated residue in FZL(K362M)-GFP. (C) Unrooted phylogenetic tree of dynamin superfamily proteins based on alignment of GTPase domains. Bootstrap values from neighbor-joining analysis are shown at nodes with >50% bootstrap support. “At” denotes A. thaliana. GenBank accession numbers for proteins shown are as follows: FZL, NM_100198; human Dynamin 1, NP_004399; human Dynamin II, NP_004936; yeast Dnm1p, NP_013100; AtDRP3B, NP_565362; AtDRP1A, NP_851120; glycine phragmoplastin, AAB05992; AtDRP2A, AAF22291; AtDRP2B, NP_176170; AtDRP5A, NP_175722; AtDRP5B, AY212885; Drosophila FZO, AAF56110; rice FZL, XP_475185; mouse Mitofusin 1, AAH56641; mouse Mitofusin 2, AAM88577.

Fig. 2.

FZL affects chloroplast morphology, plant growth, and thylakoid ultrastructure. (A) Gene structure of FZL. Black, exons; gray, introns. T-DNA insertion sites in the two fzl Salk mutants are indicated by arrowheads. (B) fzl mutants look pale and flower later than WT. (Left) WT. (Center) fzl mutant. (Right) fzl mutant complemented by FZL-GFP. (C–E) Chloroplast morphology and division phenotypes in mesophyll cells from 4-week-old expanded leaves. (C) Single cell from WT. (D) Two cells from an fzl mutant. (E) Single cell from an fzl mutant complemented by FZL-GFP. (F–M) Ultrastructure of chloroplasts from plants expressing different levels of FZL-GFP. (F and G) WT. (H and I) fzl. (J and K) fzl mutant complemented by FZL-GFP (comp). (L and M) fzl mutant expressing FZL-GFP above levels required to complement fzl (OE). Black and white arrows mark grana and stroma thylakoids, respectively. Arrowheads denote inflated lamellae of unevenly stacked fzl grana. ∗, vesicle-like traits in fzl. (Scale bars: 10 μm in C–E; 2 μm in F, H, J, and L; 500 nm in G, I, K, and M.)

Fig. 3.

FZL-GFP localizes to chloroplasts in a punctate pattern. (A–I) FZL has a chloroplast transit peptide. Single cells imaged by fluorescence microscopy are shown. (A–C) Plant transformed by 35S-tpFZL-GFP. (D–F) Plant transformed by 35S-GFP. (G–I) WT. (J–O) FZL-GFP localizes to punctate structures. Extended-focus (J–L) or single-section (image depth, 0.5 μm) (M and N) confocal images are shown. (K) fzl complemented by FZL-GFP. Arrowheads show GFP signals at edges of chloroplasts. (L) fzl with a high level of FZL-GFP. (O Upper) FZL-GFP detected by immunoblotting in fzl transgenic lines expressing different FZL-GFP levels. (O Lower) Equal loading is shown by Coomassie staining. Lanes 1 and 2, fzl; lanes 3 and 4, fzl partially complemented by FZL-GFP; lanes 5 and 6, fzl fully complemented by FZL-GFP; lanes 7 and 8, fzl plants with high FZL-GFP levels. (Scale bars: 10 μm in A–I and 5 μm in J–N.)

Fig. 4.

FZL is localized in envelope and thylakoid membranes. (A) Isolated intact (Left) or lysed (Right) chloroplasts were incubated with thermolysin and trypsin. (B Left) Isolated chloroplasts were lysed osmotically and separated into pellet and supernatant fractions. (B Right) FZL-GFP is solubilized by 1% Triton X-100. (C) Isolated chloroplasts were lysed osmotically and separated into thylakoid and envelope fractions. In A–C, crude proteins representing approximately equivalent proportions of the chloroplast from each fraction were analyzed by SDS/PAGE and immunoblotting with antibodies against GFP, the inner-envelope protein Tic110, the soluble stromal protein FtsZ1, or the thylakoid membrane protein LHCb. cp, chloroplast. (D) Predicted topology of FZL in the envelope and thylakoid membranes. OM, outer-envelope membrane; IM, inner-envelope membrane.

Fig. 5.

Mutations in the GTPase domain abolish FZL-GFP function and punctate localization. (A) Immunoblot of leaf extracts from fzl (lanes 1–5) or WT plants (lanes 6 and 7) expressing FZL-GFP (lanes 1 and 2) or FZL(K362M)-GFP (lanes 4–7). Protein extracted from 2 mg of fresh weight was loaded in each lane. Lane 1, fzl partially complemented by a low level of FZL-GFP; lane 2, fzl fully complemented by FZL-GFP; lanes 3–5, fzl plants expressing FZL(K362M)-GFP at different levels; lanes 6 and 7, WT plants expressing FZL(K362M)-GFP. Samples in lanes 3 and 6 correspond to plants shown in B and C, respectively. (B) FZL(K362M)-GFP cannot complement the fzl phenotype. (C) FZL(K362M)-GFP in a WT plant. The punctate pattern of localization is not observed, as in Fig. 3 K–N. (Scale bar in B: 5 μm for B and C.)