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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01092

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Chloroplast division site placement requires dimerization of the ARC11/AtMinD1 protein in Arabidopsis

Makoto T. Fujiwara^1,2,, Ayako Nakamura², Ryuuichi Itoh^2,*, Yukihisa Shimada², Shigeo Yoshida² and Simon Geir Møller^1,

¹ Department of Biology, University of Leicester, University Road, Leicester, LE1 7RH, UK
² Plant Functions Laboratory and Plant Science Center, RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan

View larger version (111K): [in a new window]   Fig. 1. The elongated and multiple-arrayed dividing chloroplasts in developing seedlings of Arabidopsis arc11. Chloroplasts in primary leaf petioles of 7-day light-grown wild-type (WT, Ler) and arc11 seedlings were observed by CLSM. (A) Imaging of chlorophyll autofluorescence of WT and the arc11. (B) Differential interference contrast (DIC)-single optical sections of dividing chloroplasts in WT (inset) and arc11. Membrane constriction sites of dividing chloroplasts are indicated by black arrowheads. Mini-chloroplasts (2 µm in diameter) in a population of expanding and dividing chloroplasts in arc11 are indicated by white arrowheads. Bars, 10 µm.

View larger version (44K): [in a new window]   Fig. 2. Structure and the mutation point of AtMinD1 in the arc11. (A) Chromosomal location of AtMinD1 and domain structure of its product. (B) Sequence alignment of MinD proteins from Arabidopsis (At; database accession number AB030278), Oryza sativa (Os; AP001129), Chlorella vulgaris (Cv; AB001684), Escherichia coli (Ec; J03153) and Pyrococcus furiosus (Pf; NC_003413), with secondary structure elements based on structural (Hayashi et al., 2001; Sakai et al., 2001) and membrane localization analyses (Szeto et al., 2002; Hu and Lutkenhaus, 2003), and the PSIPRED secondary structure prediction program (http://bioinf.cs.ucl.ac.uk/psipred/). A single base substitution of AtMinD1 at position Ala 296 in 11 helix, changing Ala(GCG) to Gly(GGG), is indicated by arrowheads (A,B) and boxed in (B). (C) Assignment of the AtMinD1 N-terminal region responsible for chloroplast targeting by localization analysis of nonfused and AtMinD1 N-terminus-fused (AtMinD1(1-64)) GFP. CLSM images of GFP (green), chlorophyll autofluorescence (Chl, red) and DIC are shown. Bar, 5 µm.

View larger version (15K): [in a new window]   Fig. 3. Quantification of endogenous and total AtMinD1 transcripts in Arabidopsis plants. Total RNAs from whole seedlings of Arabidopsis WT, arc11 and transgenic arc11 plants (11HA38 and 11HA42 as complemented lines, 11HA2 and 11HA7 as division-inhibited lines, 11HA44 as a partially complemented line, T4 generation) harboring the AtMinD1-dHA::Tnos transgene were analyzed by TaqMan real-time quantitative RT-PCR system. Primer sets specific to the coding region and 3'-UTR of AtMinD1 were employed to monitor total (white bars) and endogenous (black bars) AtMinD1 transcript levels, respectively. Relative amounts of AtMinD1 transcripts to 18S ribosomal RNA are shown as the means±s.e.m. (with WT=1) from three different plant samples.

View larger version (85K): [in a new window]   Fig. 4. Complementation of the arc11 mutant with appropriate expression of wild-type AtMinD1-dHA. Chloroplasts in leaf petioles of 15-day seedlings were microscopically observed. (A) WT. (B) arc11 mutant. A minichloroplast is indicated by an arrowhead. (C) Complemented arc11 transgenic plant (11HA38, T4 generation, see Fig. 3). (D) Division-inhibited arc11 transgenic plant (11HA2, T4 generation, see Fig. 3). (E-H) Partially complemented transgenic arc11 plants containing slightly expanded and surface-rugged chloroplasts compared to WT and complemented plants. (E) WT. (F) Complemented plant (11HA38, identical to (C)). (G) A segregated plant of 11HA38 in the T2 generation showing a partially complemented phenotype. (H) Partially complemented plant (11HA44, T4 generation, see Fig. 3). Bars, 10 µm.

View larger version (69K): [in a new window]   Fig. 5. Inhibition of chloroplast division by overexpression of AtMinD1(A296G) in transgenic Arabidopsis. (A) Relative amounts of total (white bars) and endogenous (black bars) AtMinD1 transcripts to 18S ribosomal RNA analyzed by quantitative RT-PCR (see Fig. 3). Data are shown as the means±s.e.m. (with WT=1) from three different plant samples. (B) Images of chloroplasts in leaf petioles of WT and CaMV35S-AtMinD1(A296G) (11S) plants. Some populations of chloroplasts look vacuolated internally (arrowheads, see Fig. 4D). Bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 6. Aberrant distribution of an AtMinD1(A296G)-YFP fusion protein inside chloroplasts. Expression vectors were introduced into young tobacco leaves by particle bombardment, and intraplastidic fluorescent patterns of full-length AtMinD1 and AtMinD1(A296G) proteins were analyzed. To visualize outlines of leaf epidermal chloroplasts, an expression vector for transit peptide-fused CFP was cotransformed. (A) Leaf epidermal chloroplasts containing YFP and CFP fluorescence. (B) Single chloroplast images at a higher magnification. Bars, 5 µm (A) and 1 µm (B).

View larger version (33K): [in a new window]   Fig. 7. Effect of the A296G mutation on AtMinD1 protein-protein interaction in the yeast two-hybrid system. AH109 cells harboring two expression vectors were grown on selection media plates at 30°C. (A) A 3-day plate lacking Leu and Trp (left) and a 5-day plate lacking His, Leu and Trp (right). (B) Growth of yeast cells on -His plates. Controls and classification of yeast growth are described in Materials and Methods.

View larger version (40K): [in a new window]   Fig. 8. FRET assay for AtMinD1 protein-protein interaction in chloroplasts. AtMinD1-CFP and/or AtMinD1-YFP were expressed in tobacco leaves by particle bombardment. Fluorescence of CFP and YFP in leaf epidermal chloroplasts was detected by epifluorescence microscopy. In the FRET channel, emission of YFP was detected upon CFP excitation. (A) Single and dual expression of fluorescent protein-tagged AtMinD1 proteins. (B) Moderate photobleaching of the acceptor YFP leading to the increased emission of CFP. All fluorescent images were taken at the same exposure time (200 milliseconds) using a 60x objective lens (Nikon). Emission signals of CFP (light blue) and YFP (yellowish green) are pseudo-colored. Bars, 5 µm (A) and 1 µm (B).

View larger version (31K): [in a new window]   Fig. 9. A possible working model for AtMinD1-mediated chloroplast division site placement in chloroplasts. (A) In WT AtMinD1 displays functional dimerization and binds to a putative MinC-like protein (?) followed by appropriate polar localization. This ensures correct division machinery placement resulting in a single central constriction site. (B) In AtMinD1(A296G) overexpressing plants, the mutated protein is unable to form dimers but can `activate' a putative MinC-like protein (?) resulting in FtsZ polymerization inhibition and division arrest. (C) In arc11 chloroplasts AtMinD1(A296G) does not dimerize but binds a putative MinC-like protein (?) and due to its mislocalization inappropriate division machinery placement takes place, resulting in multiple constriction site formation.

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