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. 2014 Jun 25;9(6):e99741.
doi: 10.1371/journal.pone.0099741. eCollection 2014.

FtsHi4 Is Essential for Embryogenesis Due to Its Influence on Chloroplast Development in Arabidopsis

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FtsHi4 Is Essential for Embryogenesis Due to Its Influence on Chloroplast Development in Arabidopsis

Xiaoduo Lu et al. PLoS One. .
Free PMC article

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Abstract

Chloroplast formation is associated with embryo development and seedling growth. However, the relationship between chloroplast differentiation and embryo development remains unclear. Five FtsHi genes that encode proteins with high similarity to FtsH proteins, but lack Zn2+-binding motifs, are present in the Arabidopsis genome. In this study, we showed that T-DNA insertion mutations in the Arabidopsis FtsHi4 gene resulted in embryo arrest at the globular-to-heart-shaped transition stage. Transmission electron microscopic analyses revealed abnormal plastid differentiation with a severe defect in thylakoid formation in the mutant embryos. Immunocytological studies demonstrated that FtsHi4 localized in chloroplasts as a thylakoid membrane-associated protein, supporting its essential role in thylakoid membrane formation. We further showed that FtsHi4 forms protein complexes, and that there was a significant reduction in the accumulation of D2 and PsbO (two photosystem II proteins) in mutant ovules. The role of FtsHi4 in chloroplast development was confirmed using an RNA-interfering approach. Additionally, mutations in other FtsHi genes including FtsHi1, FtsHi2, and FtsHi5 caused phenotypic abnormalities similar to ftshi4 with respect to plastid differentiation during embryogenesis. Taken together, our data suggest that FtsHi4, together with FtsHi1, FtsHi2, and FtsHi5 are essential for chloroplast development in Arabidopsis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Isolation and characterization of Arabidopsis ftshi4 mutant.
A, Diagram of the T-DNA insertion position in the FtsHi4 gene. Black boxes represent exons. The 5′ untranslated region (UTR) and 3′ UTR are shown in grey boxes. B, 9-DAP dissected wild-type silique. C, 9-DAP dissected ftshi4 (+/−) mutant silique. Bar  = 1 mm.
Figure 2
Figure 2. Development of ftshi4 mutant embryos.
A to C, Wild-type embryos from heterozygous ftshi plants undergoing normal development. A, Heart-shaped stage. B, Early torpedo stage. C, Mature embryo. D to G, Mutant embryos from heterozygous ftshi4-1 plants were retarded and morphologically abnormal compared to wild-type embryos from the same silique. D, Mutant embryo development was retarded when wild-type embryo developed to the heart-shaped stage in the same silique. E, Mutant embryo showing abnormalities in the regions that develop an embryo axis and radicle compared with wild-type embryos that developed to the early torpedo stage in the same silique. F, Mutant globular embryo was morphologically abnormal when wild-type embryo reached maturity in the same silique. G, Mutant embryo was arrested at the heart-stage when wild-type embryo reached maturity in the same silique.
Figure 3
Figure 3. Transmission electron microscopy analyses of chloroplast biogenesis in the ftshi4 mutant embryos.
A, Wild-type torpedo embryo from a heterozygous ftshi4-1 plant with well-developed chloroplasts showing thylakoid membranes beginning to stack into grana. B, Mutant embryo from the same heterozygous ftshi4-1 silique with development-disrupted “plastids”. C, Enlargement of an above-described chloroplast indicated by an arrow.D, Enlargement of an above-described “plastid” indicated by an arrow.
Figure 4
Figure 4. Subcellular and suborganellar localization of FtsHi4.
A, In vivo targeting of green fluorescent protein (GFP) mediated by the FtsHi4 signal peptide in protoplasts. Arabidopsis protoplasts transformed by fusion between the FtsHi4 signal peptide and GFP. GFP fluorescence, chlorophyll autofluorescence, and merged images are shown. Free GFP was used as the control. B, Suborganellar localization of FtsHi4 protein. Thylakoid membranes were prepared from wild-type plants, fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and visualized using antibodies raised against the FtsHi4 segment, PsbO (a 33-kDa luminal protein of PSII), or CP47 (a core protein of PSII). Membranes that were not subjected to salt treatment (CK) were used as controls.
Figure 5
Figure 5. D2 and PsbO protein accumulation is defective in the ftshi4 mutant.
A, Detection of the PSII core subunit D2 and FtsHi4 by immunoblotting on two-dimensional gel electrophoresis. Membrane samples were solubilized with DM and separated in the first dimension on a blue native gel followed by SDS-PAGE in the second dimension. D2 and FtsHi4 proteins were detected using appropriate antibodies. The separated complexes are designated as: I, PSII supercomplexes; II, monomeric PSI and dimeric PSII; III, monomeric PSII; IV, dimeric cytochrome b6/f dimmer; V, trimeric LHCII; and VI, monomeric LHCII. B, Immunoblot analyses for the accumulation of D2, PsbO and FtsHi4 PSII proteins in wild-type and mutant ovules from heterozygous ftshi4-1 mutant plants. The thylakoid membrane proteins were fractionated by SDS-urea-PAGE, and the blots were probed using antibodies raised against D2, PsbO, or FtsHi4.
Figure 6
Figure 6. Down regulation of FtsHi4 by RNAi leads to defects in chloroplast development.
A, 2-week-old seedlings of WT grown on half MS medium. B, 2-week-old seedlings of RNAi-FtsHi4 line grown on half MS medium. C, 5-week-old plants of WT grown in soil. D, 5-week-old plants of RNAi-FtsHi4 line grown in soil. E, Chloroplasts from a 5-week-old leaf of wild-type plant. These chloroplasts are well developed, with redundant grana interconnected by stroma thylakoids. Enlargement of such a chloroplast is shown in (G). F, Plastids from 5-week-old leaf of RNAi-FtsHi4 plants, these plastids had straight thylakoids but lacked granal lamellae. Enlargement of such a plastid is shown in (H). ItoJ, qRT-PCR and immunoblot analysis showing expression levels of FtsHi4 in leaves excised from 2-week seedlings described in A and B. Chloroplast proteins were further detected in 2-week seedlings of the RNAi mutant using a specific antibody.
Figure 7
Figure 7. Defects in the PSII complex of the RNAi-Ftshi4 mutant block energy transfer within PSII.
A, The chlorophyll concentrations of wild-type and RNAi-FtsHi4 mutant cotyledons and true leaves. B, Immunoblot analyses for the accumulation of D1, D2, CP43, CP47, PsbO, and Cyt f proteins in wild-type and RNAi-FtsHi4 mutant cotyledons and true leaves. The thylakoid membrane proteins were fractionated by SDS-urea-PAGE, and the blots were probed using antibodies raised against D1, D2, CP43, CP47, PsbO, or Cyt f, respectively.
Figure 8
Figure 8. Expression patterns of FtsHi4 gene in Arabidopsis.
Total RNA was extracted from different tissues of 40-day-old wild-type plants grown in soil. Gene-specific primers were used to detect FtsHi4 transcripts. The ACTIN2 gene was used as a control.

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Grant support

This work was supported by the National Science Foundation of China (grant number 31071077 to C. Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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