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A novel chloroplast-localized protein EMB1303 is required for chloroplast development in Arabidopsis
Xiaozhen Huang, Xiaoyan Zhang and Shuhua Yang
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Figure 1.
Growth phenotypes of the emb1303 mutants. Wild-type and the emb1303 mutant seeds were grown at 22 °C under long-light conditions for 5 days (A), and 35 days (C). (B) Leaves and inflorescences of wild type (top) and emb1303-1 (bottom). (D, E) Cross-sections of leaves from the wild type (D) and emb1303-1 (E). Bar: 100 
m. (F, G) Pavement cells from true leaves of wild type (F) and emb1303-1 (G) stained by propidium iodide. Bar: 100 m.
Figure 2.
Scanning electron microscope images of leaf epidermis of the emb1303 mutant. Adaxial (A, C), and abaxial (B, D) leaves from wild-type Col and emb1303-1 mutant are shown. Plants were grown on 1/2 MS medium supplemented with 2% sucrose for 14 days and the second leaves are shown. Bar: 100 m.
Figure 3.
Isolation of the emb1303 mutants. (A) A schematic diagram of genomic structure of the EMB1303 gene. Filled boxes and lines indicate exons and introns, respectively. Positions of T-DNA insertions are indicated by triangles with left border indicated by arrows. The T-DNA insertion site in emb1303-1 spans EMB1303 gene, leading to a deletion of the gene. The T-DNA in emb1303-2 is located at 5'-UTR of EMB1303. (B) RT-PCR analysis of EMB1303 transcripts in the wild type and emb1303 mutants. 
-tubulin gene TUB8 was used as a control. (C) Complementation of the emb1303-1 mutant with genomic fragment of EMB1303 gene.
Figure 4.
Spacial expression pattern of the EMB1303 gene. Spacial expression pattern of the EMB1303 gene. (A) RT-PCR analysis of EMB1303 transcripts in various organs of Arabidopsis plants. Total RNA was isolated from the following tissues: roots (R), stems (S), leaves (L), young flowers (YF), maturing flowers (MF), and mature siliques (SL) of Col plants grown under long-day conditions. RT-PCR was performed with EMB1303-specific primers (top gel) and TUB8-specific primers (bottom gel). (B-H) GUS expression in seedlings at the cotyledon (B), four-leaf (C) and eight-leaf (D) stages, stem and cauline leaf (E), inflorescence and flowers (F, G), and mature silique (H) of pEMB1303::GUS transgenic plants.
Full figure and legend (28K)
Figure 5.
Subcellular localization of the EMB1303 protein in N. bethamiana leaves. p35S::EMB1303-GFP and p35S::GFP were transformed into N. benthamiana by A. tumefaciens-mediated infiltration. Signals were detected by a laser confocal-scanning microscope from intact leaf mesophyll cells (A). (B) Protoplasts prepared from the same leaves as in (A) , and (C) broken protoplasts from (B). Green fluorescence signals, chlorophyll red autofluorescence, an overlay of green and red signals, and bright-field images are shown in panels (in the order from left to right). Bar: 20 m. (D) Immunoblot assay of proteins. Total proteins (T) and chloroplast proteins (Chl) were extracted from protoplasts prepared from the same leaves of (A) and analyzed by immunoblotting with polyclonal antibodies raised against GFP.
Figure 6.
Phenotypes of emb1303-1 embryos during seed development. (A) Wild-type seeds. (B-D) Seed segregation in siliques from an emb1301/+ heterozygous plant at different stages. Approximately, one-quarter of albino embryos segregated at early stages. (E-P) Cleared seeds observed under Nomarski optics. Embryo development of wild type (E, G, I, K, M, O) and emb1303-1 (F, H, J, L, N, P) at globular, heart, torpedo and early cotyledon, and mature cotyledon stages. Bar: 100 m.
Figure 7.
Transmission electron microscopy of plastids from emb1301-1 seedlings. Plants were grown on 1/2 MS medium supplemented with 2% sucrose for 14 days and the second leaf of a representative plant for each phenotype category was fixed for transmission electron microscope analysis. (A, B) An overview of parenchyma cells. Bar: 10 m. (C, D, E) Enlarged views of chloroplasts. Bar: 500 nm. chl, chloroplast; mit, mitochondrion; st, starch granule.
Figure 8.
Gene expression and chloroplast protein expression in emb1303-1 plants. (A) Expression of nuclear- and chloroplast-encoded genes. Total RNA prepared from 3-week-old seedlings of Col and emb1303-1 grown on 1/2 MS media containing 2% sucrose was used for semi-quantitative RT-PCR. The -tubulin gene (TUB8) was used as a quantitation control. (B) Coomasssie brilliant blue staining of total proteins. Total proteins were extracted from seedlings used in (A) and separated by SDS-PAGE. RBCL refers to the large subunit of Rubisco. (C) Immunoblot analysis of chloroplast proteins. Total proteins were analyzed by immunoblotting with antibodies against D1, D2, LHCII, PsbO, and AtpB proteins.
