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Physiological Functions of the COPI Complex in Higher Plants.

Ahn HK, Kang YW, Lim HM, Hwang I, Pai HS - Mol. Cells (2015)

Bottom Line: Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing.In tobacco BY-2 cells, DEX-inducible RNAi of β'-COP caused aberrant cell plate formation during cytokinesis.Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seoul 120-749, Korea.

ABSTRACT
COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β'-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β'-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β'-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β'-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

No MeSH data available.


Related in: MedlinePlus

Defective cell plate formation in DEX-inducible β′-COP RNAi BY-2 cells. (A) Real-time quantitative RT-PCR was performed to determine the β′-COP mRNA level in the RNAi BY-2 cells after 2 days (2 d) or 4 days (4 d) of 15 µM DEX treatment. The transcript levels were expressed relative to that of BY-2 cells ethanol-treated for 4 days [(−)DEX-4 d]. Data points represent means ± SD of three replicate experiments; *P ≤ 0.05; **P ≤ 0.01. (B) BY-2 cells were double-labeled with calcofluor staining (pseudo-colored red) and anti-α-tubulin antibodies to visualize the cell plate and the phragmoplast, respectively, after 4 days of ethanol (-DEX) or DEX treatment. The right-most panels show the calcofluor fluorescence intensity profiles spanning the cell plate (white lines). Scale bars = 5 μm. (C) BY-2 cells were double-labeled with aniline blue and DAPI to visualize the cell plate and the nuclei, respectively. Representative telophase cells are shown. BY-2 cells were treated with 20 µM BFA as positive control. Scale bars = 5 μm. (D) Telophase cells with (Telo + CP) or without the newly forming cell plate (Telo - CP) shown in (C) were quantified (n = ∼80 for each sample). Values are expressed as percentage of total telophase cells counted.
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f6-molce-38-10-866: Defective cell plate formation in DEX-inducible β′-COP RNAi BY-2 cells. (A) Real-time quantitative RT-PCR was performed to determine the β′-COP mRNA level in the RNAi BY-2 cells after 2 days (2 d) or 4 days (4 d) of 15 µM DEX treatment. The transcript levels were expressed relative to that of BY-2 cells ethanol-treated for 4 days [(−)DEX-4 d]. Data points represent means ± SD of three replicate experiments; *P ≤ 0.05; **P ≤ 0.01. (B) BY-2 cells were double-labeled with calcofluor staining (pseudo-colored red) and anti-α-tubulin antibodies to visualize the cell plate and the phragmoplast, respectively, after 4 days of ethanol (-DEX) or DEX treatment. The right-most panels show the calcofluor fluorescence intensity profiles spanning the cell plate (white lines). Scale bars = 5 μm. (C) BY-2 cells were double-labeled with aniline blue and DAPI to visualize the cell plate and the nuclei, respectively. Representative telophase cells are shown. BY-2 cells were treated with 20 µM BFA as positive control. Scale bars = 5 μm. (D) Telophase cells with (Telo + CP) or without the newly forming cell plate (Telo - CP) shown in (C) were quantified (n = ∼80 for each sample). Values are expressed as percentage of total telophase cells counted.

Mentions: Cell plate formation is a plant-specific process that requires deposition of vesicles derived from the Golgi in the middle of the division zone (Jürgens, 2005a). The γ-COP protein was shown to localize near the cell plate in maize roots based on immunofluorescence (Couchy et al., 2003). In order to examine the effects of COPI depletion in cell plate formation, DEX-inducible β′-COP RNAi transgenic BY-2 cell lines were generated. The β′-COP mRNA level of the RNAi BY-2 cells was reduced to about 57% of the level of ethanol-treated cells (−DEX) after 2 days of 15 μM DEX treatment, and the level was further reduced to less than 10% of the (−)DEX level after 4 days of DEX treatment (Fig. 6A). To observe cell plate formation, the β′-COP RNAi BY-2 cells were fixed and immunolabeled with anti-α-tubulin antibodies, and then stained with calcofluor-white, which stains cellular structures containing cellulose, for confocal laser scanning microscopy. The calcofluor fluorescence intensity profile spanning the cell plate (marked with white lines) is also shown. (−)DEX BY-2 cells at telophase exhibited newly forming cell plates (calcofluor staining) and normal assembly of phragmoplasts (α-tubulin labeling) (Fig. 6B). However, cell plates were missing or defective in DEX-treated cells despite normal patterns of phragmoplasts (Fig. 6B; Supplementary Fig. S6).


Physiological Functions of the COPI Complex in Higher Plants.

Ahn HK, Kang YW, Lim HM, Hwang I, Pai HS - Mol. Cells (2015)

Defective cell plate formation in DEX-inducible β′-COP RNAi BY-2 cells. (A) Real-time quantitative RT-PCR was performed to determine the β′-COP mRNA level in the RNAi BY-2 cells after 2 days (2 d) or 4 days (4 d) of 15 µM DEX treatment. The transcript levels were expressed relative to that of BY-2 cells ethanol-treated for 4 days [(−)DEX-4 d]. Data points represent means ± SD of three replicate experiments; *P ≤ 0.05; **P ≤ 0.01. (B) BY-2 cells were double-labeled with calcofluor staining (pseudo-colored red) and anti-α-tubulin antibodies to visualize the cell plate and the phragmoplast, respectively, after 4 days of ethanol (-DEX) or DEX treatment. The right-most panels show the calcofluor fluorescence intensity profiles spanning the cell plate (white lines). Scale bars = 5 μm. (C) BY-2 cells were double-labeled with aniline blue and DAPI to visualize the cell plate and the nuclei, respectively. Representative telophase cells are shown. BY-2 cells were treated with 20 µM BFA as positive control. Scale bars = 5 μm. (D) Telophase cells with (Telo + CP) or without the newly forming cell plate (Telo - CP) shown in (C) were quantified (n = ∼80 for each sample). Values are expressed as percentage of total telophase cells counted.
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Related In: Results  -  Collection

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f6-molce-38-10-866: Defective cell plate formation in DEX-inducible β′-COP RNAi BY-2 cells. (A) Real-time quantitative RT-PCR was performed to determine the β′-COP mRNA level in the RNAi BY-2 cells after 2 days (2 d) or 4 days (4 d) of 15 µM DEX treatment. The transcript levels were expressed relative to that of BY-2 cells ethanol-treated for 4 days [(−)DEX-4 d]. Data points represent means ± SD of three replicate experiments; *P ≤ 0.05; **P ≤ 0.01. (B) BY-2 cells were double-labeled with calcofluor staining (pseudo-colored red) and anti-α-tubulin antibodies to visualize the cell plate and the phragmoplast, respectively, after 4 days of ethanol (-DEX) or DEX treatment. The right-most panels show the calcofluor fluorescence intensity profiles spanning the cell plate (white lines). Scale bars = 5 μm. (C) BY-2 cells were double-labeled with aniline blue and DAPI to visualize the cell plate and the nuclei, respectively. Representative telophase cells are shown. BY-2 cells were treated with 20 µM BFA as positive control. Scale bars = 5 μm. (D) Telophase cells with (Telo + CP) or without the newly forming cell plate (Telo - CP) shown in (C) were quantified (n = ∼80 for each sample). Values are expressed as percentage of total telophase cells counted.
Mentions: Cell plate formation is a plant-specific process that requires deposition of vesicles derived from the Golgi in the middle of the division zone (Jürgens, 2005a). The γ-COP protein was shown to localize near the cell plate in maize roots based on immunofluorescence (Couchy et al., 2003). In order to examine the effects of COPI depletion in cell plate formation, DEX-inducible β′-COP RNAi transgenic BY-2 cell lines were generated. The β′-COP mRNA level of the RNAi BY-2 cells was reduced to about 57% of the level of ethanol-treated cells (−DEX) after 2 days of 15 μM DEX treatment, and the level was further reduced to less than 10% of the (−)DEX level after 4 days of DEX treatment (Fig. 6A). To observe cell plate formation, the β′-COP RNAi BY-2 cells were fixed and immunolabeled with anti-α-tubulin antibodies, and then stained with calcofluor-white, which stains cellular structures containing cellulose, for confocal laser scanning microscopy. The calcofluor fluorescence intensity profile spanning the cell plate (marked with white lines) is also shown. (−)DEX BY-2 cells at telophase exhibited newly forming cell plates (calcofluor staining) and normal assembly of phragmoplasts (α-tubulin labeling) (Fig. 6B). However, cell plates were missing or defective in DEX-treated cells despite normal patterns of phragmoplasts (Fig. 6B; Supplementary Fig. S6).

Bottom Line: Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing.In tobacco BY-2 cells, DEX-inducible RNAi of β'-COP caused aberrant cell plate formation during cytokinesis.Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seoul 120-749, Korea.

ABSTRACT
COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β'-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β'-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β'-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β'-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

No MeSH data available.


Related in: MedlinePlus