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The Arabidopsis minE mutation causes new plastid and FtsZ1 localization phenotypes in the leaf epidermis.

Fujiwara MT, Kojo KH, Kazama Y, Sasaki S, Abe T, Itoh RD - Front Plant Sci (2015)

Bottom Line: Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission.In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells.Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Nishina Center Saitama, Japan ; Graduate School of Science and Technology, Sophia University Tokyo, Japan.

ABSTRACT
Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission. While mesophyll chloroplasts have generally been used to study plastid division, recent studies have suggested the presence of tissue- or plastid type-dependent regulation of plastid division. Here, we report the detailed morphology of plastids and their stromules, and the intraplastidic localization of the chloroplast division-related protein AtFtsZ1-1, in the leaf epidermis of an Arabidopsis mutant that harbors a mutation in the chloroplast division site determinant gene AtMinE1. In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells. In particular, atminE1 epidermal plastids occasionally displayed grape-like morphology, a novel phenotype induced by a plastid division mutation. Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis. Further examination revealed that constriction of plastids and stromules mediated by the FtsZ1 ring contributed to the plastid pleomorphism in the atminE1 epidermis. These results illustrate that a single plastid division mutation can have dramatic consequences for epidermal plastid morphology, thereby implying that plastid division and morphogenesis are differentially regulated in epidermal and mesophyll plastids.

No MeSH data available.


Related in: MedlinePlus

Expression and function of AtMinE1-YFP in atminE1. (A) Domain structure of AtMinE1-YFP. Three regions of AtMinE1, an S/T-rich N-terminal region, an E. coli AMD (anti-MinCD domain)-like region, and an E. coli TSD (topological specificity domain)-like region, as well as a linker sequence between AtMinE1 and YFP, are indicated. (B) Quantitative RT-PCR analysis. RNAs from leaves of WT, atminE1, and transgenic atminE1 with AtMinE1p::AtMinE1-YFP (an overexpression line and a complemented line) were analyzed. Relative amounts of AtMinE1 (white bars) and AtMinD1 (black bars) transcripts compared to 18S rRNA (WT = 1) are shown. (C) Immunoblotting. Proteins from seedlings of WT (lane 1) and transgenic atminE1 plants (an overexpression line [lane 2] and a complemented line [lane 3]) were analyzed using mouse anti-GFP and anti-actin antibodies. Chemiluminescent signals of AtMinE1-YFP (arrowhead) and non-specific, extra signals (∗) are indicated at the right. (D) Complementation of plant phenotype of atminE1 by AtMinE1-YFP. One-month-old WT, atminE1, and a complemented plant are shown. (E) Complementation of plastid morphology of atminE1 by AtMinE1-YFP. Images of chlorophyll autofluorescence from epidermal (top; from 3-week-old seedlings) and cortex (bottom; from 2-week-old seedlings) plastids of WT, atminE1, and transgenic atminE1 plants are shown. Arrowheads represent epidermal plastids. Bar = 10 μm. (F) Fluorescence stereomicroscopy. A complemented atminE1 line at both the vegetative and reproductive stages was observed. Images of bright field (BF), YFP fluorescence (green), and chlorophyll autofluorescence (Chl, magenta) are shown. Arrowheads indicate accumulation of YFP signals at the shoot apices. Bars = 1 mm (single) and 200 μm (double). (G) Localization of AtMinE1-YFP signals in leaf epidermal plastids. Fluorescence images of YFP (green) and chlorophyll (magenta) in a leaf epidermal cell of the complemented atminE1 line are shown. Bar = 5 μm.
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Figure 5: Expression and function of AtMinE1-YFP in atminE1. (A) Domain structure of AtMinE1-YFP. Three regions of AtMinE1, an S/T-rich N-terminal region, an E. coli AMD (anti-MinCD domain)-like region, and an E. coli TSD (topological specificity domain)-like region, as well as a linker sequence between AtMinE1 and YFP, are indicated. (B) Quantitative RT-PCR analysis. RNAs from leaves of WT, atminE1, and transgenic atminE1 with AtMinE1p::AtMinE1-YFP (an overexpression line and a complemented line) were analyzed. Relative amounts of AtMinE1 (white bars) and AtMinD1 (black bars) transcripts compared to 18S rRNA (WT = 1) are shown. (C) Immunoblotting. Proteins from seedlings of WT (lane 1) and transgenic atminE1 plants (an overexpression line [lane 2] and a complemented line [lane 3]) were analyzed using mouse anti-GFP and anti-actin antibodies. Chemiluminescent signals of AtMinE1-YFP (arrowhead) and non-specific, extra signals (∗) are indicated at the right. (D) Complementation of plant phenotype of atminE1 by AtMinE1-YFP. One-month-old WT, atminE1, and a complemented plant are shown. (E) Complementation of plastid morphology of atminE1 by AtMinE1-YFP. Images of chlorophyll autofluorescence from epidermal (top; from 3-week-old seedlings) and cortex (bottom; from 2-week-old seedlings) plastids of WT, atminE1, and transgenic atminE1 plants are shown. Arrowheads represent epidermal plastids. Bar = 10 μm. (F) Fluorescence stereomicroscopy. A complemented atminE1 line at both the vegetative and reproductive stages was observed. Images of bright field (BF), YFP fluorescence (green), and chlorophyll autofluorescence (Chl, magenta) are shown. Arrowheads indicate accumulation of YFP signals at the shoot apices. Bars = 1 mm (single) and 200 μm (double). (G) Localization of AtMinE1-YFP signals in leaf epidermal plastids. Fluorescence images of YFP (green) and chlorophyll (magenta) in a leaf epidermal cell of the complemented atminE1 line are shown. Bar = 5 μm.

Mentions: To elucidate the relationship between the mutant phenotypes and the function of AtMinE1, it is important to examine whether AtMinE1 is expressed in leaf epidermal cells. To investigate the expression profile of AtMinE1 in planta, we previously performed GUS staining using transgenic A. thaliana plants harboring an AtMinE1-upstream genomic sequence::uidA fusion (Itoh et al., 2001), revealing that GUS activation occurred strongly in the shoot apex and moderately in green tissues and pollen, but not in roots, in most transgenic plants. One exceptional line (Itoh et al., 2001) exhibited GUS staining in whole plants including roots, and we recently considered the possibility that this exceptional transgenic line might represent AtMinE1 expression in light of a comprehensive A. thaliana transcriptome study (Winter et al., 2007) and a study of non-photosynthetic plastids of atminE1 (Kojo et al., 2009). We constructed an AtMinE1 promoter::AtMinE1-YFP fusion gene (Figure 5A) and introduced it into the nuclear genome of atminE1 via Agrobacterium-mediated transformation. The complemented atminE1 transgenic plants, if obtained, would produce almost WT levels of AtMinE1 fused to a visual reporter subjected to control at the transcriptional, splicing, translational, and post-translational levels.


The Arabidopsis minE mutation causes new plastid and FtsZ1 localization phenotypes in the leaf epidermis.

Fujiwara MT, Kojo KH, Kazama Y, Sasaki S, Abe T, Itoh RD - Front Plant Sci (2015)

Expression and function of AtMinE1-YFP in atminE1. (A) Domain structure of AtMinE1-YFP. Three regions of AtMinE1, an S/T-rich N-terminal region, an E. coli AMD (anti-MinCD domain)-like region, and an E. coli TSD (topological specificity domain)-like region, as well as a linker sequence between AtMinE1 and YFP, are indicated. (B) Quantitative RT-PCR analysis. RNAs from leaves of WT, atminE1, and transgenic atminE1 with AtMinE1p::AtMinE1-YFP (an overexpression line and a complemented line) were analyzed. Relative amounts of AtMinE1 (white bars) and AtMinD1 (black bars) transcripts compared to 18S rRNA (WT = 1) are shown. (C) Immunoblotting. Proteins from seedlings of WT (lane 1) and transgenic atminE1 plants (an overexpression line [lane 2] and a complemented line [lane 3]) were analyzed using mouse anti-GFP and anti-actin antibodies. Chemiluminescent signals of AtMinE1-YFP (arrowhead) and non-specific, extra signals (∗) are indicated at the right. (D) Complementation of plant phenotype of atminE1 by AtMinE1-YFP. One-month-old WT, atminE1, and a complemented plant are shown. (E) Complementation of plastid morphology of atminE1 by AtMinE1-YFP. Images of chlorophyll autofluorescence from epidermal (top; from 3-week-old seedlings) and cortex (bottom; from 2-week-old seedlings) plastids of WT, atminE1, and transgenic atminE1 plants are shown. Arrowheads represent epidermal plastids. Bar = 10 μm. (F) Fluorescence stereomicroscopy. A complemented atminE1 line at both the vegetative and reproductive stages was observed. Images of bright field (BF), YFP fluorescence (green), and chlorophyll autofluorescence (Chl, magenta) are shown. Arrowheads indicate accumulation of YFP signals at the shoot apices. Bars = 1 mm (single) and 200 μm (double). (G) Localization of AtMinE1-YFP signals in leaf epidermal plastids. Fluorescence images of YFP (green) and chlorophyll (magenta) in a leaf epidermal cell of the complemented atminE1 line are shown. Bar = 5 μm.
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Figure 5: Expression and function of AtMinE1-YFP in atminE1. (A) Domain structure of AtMinE1-YFP. Three regions of AtMinE1, an S/T-rich N-terminal region, an E. coli AMD (anti-MinCD domain)-like region, and an E. coli TSD (topological specificity domain)-like region, as well as a linker sequence between AtMinE1 and YFP, are indicated. (B) Quantitative RT-PCR analysis. RNAs from leaves of WT, atminE1, and transgenic atminE1 with AtMinE1p::AtMinE1-YFP (an overexpression line and a complemented line) were analyzed. Relative amounts of AtMinE1 (white bars) and AtMinD1 (black bars) transcripts compared to 18S rRNA (WT = 1) are shown. (C) Immunoblotting. Proteins from seedlings of WT (lane 1) and transgenic atminE1 plants (an overexpression line [lane 2] and a complemented line [lane 3]) were analyzed using mouse anti-GFP and anti-actin antibodies. Chemiluminescent signals of AtMinE1-YFP (arrowhead) and non-specific, extra signals (∗) are indicated at the right. (D) Complementation of plant phenotype of atminE1 by AtMinE1-YFP. One-month-old WT, atminE1, and a complemented plant are shown. (E) Complementation of plastid morphology of atminE1 by AtMinE1-YFP. Images of chlorophyll autofluorescence from epidermal (top; from 3-week-old seedlings) and cortex (bottom; from 2-week-old seedlings) plastids of WT, atminE1, and transgenic atminE1 plants are shown. Arrowheads represent epidermal plastids. Bar = 10 μm. (F) Fluorescence stereomicroscopy. A complemented atminE1 line at both the vegetative and reproductive stages was observed. Images of bright field (BF), YFP fluorescence (green), and chlorophyll autofluorescence (Chl, magenta) are shown. Arrowheads indicate accumulation of YFP signals at the shoot apices. Bars = 1 mm (single) and 200 μm (double). (G) Localization of AtMinE1-YFP signals in leaf epidermal plastids. Fluorescence images of YFP (green) and chlorophyll (magenta) in a leaf epidermal cell of the complemented atminE1 line are shown. Bar = 5 μm.
Mentions: To elucidate the relationship between the mutant phenotypes and the function of AtMinE1, it is important to examine whether AtMinE1 is expressed in leaf epidermal cells. To investigate the expression profile of AtMinE1 in planta, we previously performed GUS staining using transgenic A. thaliana plants harboring an AtMinE1-upstream genomic sequence::uidA fusion (Itoh et al., 2001), revealing that GUS activation occurred strongly in the shoot apex and moderately in green tissues and pollen, but not in roots, in most transgenic plants. One exceptional line (Itoh et al., 2001) exhibited GUS staining in whole plants including roots, and we recently considered the possibility that this exceptional transgenic line might represent AtMinE1 expression in light of a comprehensive A. thaliana transcriptome study (Winter et al., 2007) and a study of non-photosynthetic plastids of atminE1 (Kojo et al., 2009). We constructed an AtMinE1 promoter::AtMinE1-YFP fusion gene (Figure 5A) and introduced it into the nuclear genome of atminE1 via Agrobacterium-mediated transformation. The complemented atminE1 transgenic plants, if obtained, would produce almost WT levels of AtMinE1 fused to a visual reporter subjected to control at the transcriptional, splicing, translational, and post-translational levels.

Bottom Line: Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission.In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells.Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Nishina Center Saitama, Japan ; Graduate School of Science and Technology, Sophia University Tokyo, Japan.

ABSTRACT
Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission. While mesophyll chloroplasts have generally been used to study plastid division, recent studies have suggested the presence of tissue- or plastid type-dependent regulation of plastid division. Here, we report the detailed morphology of plastids and their stromules, and the intraplastidic localization of the chloroplast division-related protein AtFtsZ1-1, in the leaf epidermis of an Arabidopsis mutant that harbors a mutation in the chloroplast division site determinant gene AtMinE1. In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells. In particular, atminE1 epidermal plastids occasionally displayed grape-like morphology, a novel phenotype induced by a plastid division mutation. Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis. Further examination revealed that constriction of plastids and stromules mediated by the FtsZ1 ring contributed to the plastid pleomorphism in the atminE1 epidermis. These results illustrate that a single plastid division mutation can have dramatic consequences for epidermal plastid morphology, thereby implying that plastid division and morphogenesis are differentially regulated in epidermal and mesophyll plastids.

No MeSH data available.


Related in: MedlinePlus