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Prospective function of FtsZ proteins in the secondary plastid of chlorarachniophyte algae.

Hirakawa Y, Ishida K - BMC Plant Biol. (2015)

Bottom Line: FtsZ homologs were encoded by the nuclear genomes and carried an N-terminal plastid targeting signal.Immunoelectron microscopy revealed that both FtsZD-1 and FtsZD-2 formed a ring-like structure at the midpoint of bilobate plastids with a projecting pyrenoid in Bigelowiella natans.The ring was always associated with a shallow plate-like invagination of the two innermost plastid membranes.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan. hirakawa.yoshi.fp@u.tsukuba.ac.jp.

ABSTRACT

Background: Division of double-membraned plastids (primary plastids) is performed by constriction of a ring-like division complex consisting of multiple plastid division proteins. Consistent with the endosymbiotic origin of primary plastids, some of the plastid division proteins are descended from cyanobacterial cell division machinery, and the others are of host origin. In several algal lineages, complex plastids, the "secondary plastids", have been acquired by the endosymbiotic uptake of primary plastid-bearing algae, and are surrounded by three or four membranes. Although homologous genes for primary plastid division proteins have been found in genome sequences of secondary plastid-bearing organisms, little is known about the function of these proteins or the mechanism of secondary plastid division.

Results: To gain insight into the mechanism of secondary plastid division, we characterized two plastid division proteins, FtsZD-1 and FtsZD-2, in chlorarachniophyte algae. FtsZ homologs were encoded by the nuclear genomes and carried an N-terminal plastid targeting signal. Immunoelectron microscopy revealed that both FtsZD-1 and FtsZD-2 formed a ring-like structure at the midpoint of bilobate plastids with a projecting pyrenoid in Bigelowiella natans. The ring was always associated with a shallow plate-like invagination of the two innermost plastid membranes. Furthermore, gene expression analysis confirmed that transcripts of ftsZD genes were periodically increased soon after cell division during the B. natans cell cycle, which is not consistent with the timing of plastid division.

Conclusions: Our findings suggest that chlorarachniophyte FtsZD proteins are involved in partial constriction of the inner pair of plastid membranes, but not in the whole process of plastid division. It is uncertain how the outer pair of plastid membranes is constricted, and as-yet-unknown mechanism is required for the secondary plastid division in chlorarachniophytes.

No MeSH data available.


Related in: MedlinePlus

Immunoblot analyses and immunofluorescence microscopy of BnFtsZD-1 and BnFtsZD-2 proteins. Immunoblot analyses of anti-BnFtsZD-1 (a) and anti-BnFtsZD-2 (b) antibodies against the whole proteins extracted from B. natans cells. (c, d) Confocal images of immunofluorescence labeling of BnFtsZD-1 and BnFtsZD-2 with FITC (green) in B. natans cells. Pre-immune serums were used as negative controls. The chlorophyll autofluorescence of plastids is shown by red. Py, pyrenoid. The scale bars represent 2 μm
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Fig3: Immunoblot analyses and immunofluorescence microscopy of BnFtsZD-1 and BnFtsZD-2 proteins. Immunoblot analyses of anti-BnFtsZD-1 (a) and anti-BnFtsZD-2 (b) antibodies against the whole proteins extracted from B. natans cells. (c, d) Confocal images of immunofluorescence labeling of BnFtsZD-1 and BnFtsZD-2 with FITC (green) in B. natans cells. Pre-immune serums were used as negative controls. The chlorophyll autofluorescence of plastids is shown by red. Py, pyrenoid. The scale bars represent 2 μm

Mentions: To determine the detailed subcellular localization of chlorarachniophyte FtsZD proteins, we first examined the full-length of AaFtsZD-1 (486 amino acids) and AaFtsZD-2 (514 amino acids) fused with GFP. Unfortunately their localization was ambiguous and we observed no obvious accumulation of GFP at the plastid division site, probably due to an artifact of high expression of the GFP tagged proteins. Therefore, we generated polyclonal antibodies against B. natans FtsZD proteins (anti-BnFtsZD-1 and anti-BnFtsZD-2) to perform immunolocalization experiments. B. natans typically has a single plastid in the tiny cell, which has an advantage in ultrastructural studies by electron microscopy. The B. natans plastids typically possess a projecting pyrenoid with a plate-like invagination of the two innermost membranes, and a nucleomorph is located near the pyrenoid base [47]. The specificity of two antibodies was examined by immunoblot analyses against the whole proteins extracted from B. natans cells; the detected bands were consistent with the predicted size of mature proteins of BnFtsZD-1 (44 kDa) and BnFtsZD-2 (50 kDa) (Fig. 3a, b). We first performed immunofluorescence microscopy. Fluorescein isothiocyanate (FITC) signals were observed at the midpoint of the bilobate plastids in both of the anti-BnFtsZD-1 and anti-BnFtsZD-2 (Fig. 3c, d), and cytoplasmic signals would be a nonspecific background, because such signals were also observed in negative control cells treated with pre-immune serums (Fig. 3c, d). Furthermore, we carried out immunoelectron microscopy using these two antibodies. In both cases, conjugated gold particles were observed near the tip of the shallow invagination of the two innermost plastid membranes within projecting pyrenoids (Fig. 4a, e, f), on opposite sides of the pyrenoid on horizontal sections (Fig. 4b, g), on the innermost membrane in the central narrow part of the bilobate plastids near the nucleomorph (Fig. 4c, h), and in a vertical line on the bottom of the bilobate plastids (Fig. 4d, i). Immunogold signals for both BnFtsZD proteins were almost always detected at the plastid midpoints in observed cells. Taken together, these findings indicated that BnFtsZD-1 and BnFtsZD-2 assembled into a gourd-shaped ring structure on the stromal side of the innermost membrane at the midpoint of bilobate plastids, and the ring always associates with a shallow plate-like invagination of the inner pair of plastid membranes (Fig. 4j). In our electron microscopic observation, electron-dense PD-rings were not detected at the putative plastid division sites where immunogold particles were located.Fig. 3


Prospective function of FtsZ proteins in the secondary plastid of chlorarachniophyte algae.

Hirakawa Y, Ishida K - BMC Plant Biol. (2015)

Immunoblot analyses and immunofluorescence microscopy of BnFtsZD-1 and BnFtsZD-2 proteins. Immunoblot analyses of anti-BnFtsZD-1 (a) and anti-BnFtsZD-2 (b) antibodies against the whole proteins extracted from B. natans cells. (c, d) Confocal images of immunofluorescence labeling of BnFtsZD-1 and BnFtsZD-2 with FITC (green) in B. natans cells. Pre-immune serums were used as negative controls. The chlorophyll autofluorescence of plastids is shown by red. Py, pyrenoid. The scale bars represent 2 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4641359&req=5

Fig3: Immunoblot analyses and immunofluorescence microscopy of BnFtsZD-1 and BnFtsZD-2 proteins. Immunoblot analyses of anti-BnFtsZD-1 (a) and anti-BnFtsZD-2 (b) antibodies against the whole proteins extracted from B. natans cells. (c, d) Confocal images of immunofluorescence labeling of BnFtsZD-1 and BnFtsZD-2 with FITC (green) in B. natans cells. Pre-immune serums were used as negative controls. The chlorophyll autofluorescence of plastids is shown by red. Py, pyrenoid. The scale bars represent 2 μm
Mentions: To determine the detailed subcellular localization of chlorarachniophyte FtsZD proteins, we first examined the full-length of AaFtsZD-1 (486 amino acids) and AaFtsZD-2 (514 amino acids) fused with GFP. Unfortunately their localization was ambiguous and we observed no obvious accumulation of GFP at the plastid division site, probably due to an artifact of high expression of the GFP tagged proteins. Therefore, we generated polyclonal antibodies against B. natans FtsZD proteins (anti-BnFtsZD-1 and anti-BnFtsZD-2) to perform immunolocalization experiments. B. natans typically has a single plastid in the tiny cell, which has an advantage in ultrastructural studies by electron microscopy. The B. natans plastids typically possess a projecting pyrenoid with a plate-like invagination of the two innermost membranes, and a nucleomorph is located near the pyrenoid base [47]. The specificity of two antibodies was examined by immunoblot analyses against the whole proteins extracted from B. natans cells; the detected bands were consistent with the predicted size of mature proteins of BnFtsZD-1 (44 kDa) and BnFtsZD-2 (50 kDa) (Fig. 3a, b). We first performed immunofluorescence microscopy. Fluorescein isothiocyanate (FITC) signals were observed at the midpoint of the bilobate plastids in both of the anti-BnFtsZD-1 and anti-BnFtsZD-2 (Fig. 3c, d), and cytoplasmic signals would be a nonspecific background, because such signals were also observed in negative control cells treated with pre-immune serums (Fig. 3c, d). Furthermore, we carried out immunoelectron microscopy using these two antibodies. In both cases, conjugated gold particles were observed near the tip of the shallow invagination of the two innermost plastid membranes within projecting pyrenoids (Fig. 4a, e, f), on opposite sides of the pyrenoid on horizontal sections (Fig. 4b, g), on the innermost membrane in the central narrow part of the bilobate plastids near the nucleomorph (Fig. 4c, h), and in a vertical line on the bottom of the bilobate plastids (Fig. 4d, i). Immunogold signals for both BnFtsZD proteins were almost always detected at the plastid midpoints in observed cells. Taken together, these findings indicated that BnFtsZD-1 and BnFtsZD-2 assembled into a gourd-shaped ring structure on the stromal side of the innermost membrane at the midpoint of bilobate plastids, and the ring always associates with a shallow plate-like invagination of the inner pair of plastid membranes (Fig. 4j). In our electron microscopic observation, electron-dense PD-rings were not detected at the putative plastid division sites where immunogold particles were located.Fig. 3

Bottom Line: FtsZ homologs were encoded by the nuclear genomes and carried an N-terminal plastid targeting signal.Immunoelectron microscopy revealed that both FtsZD-1 and FtsZD-2 formed a ring-like structure at the midpoint of bilobate plastids with a projecting pyrenoid in Bigelowiella natans.The ring was always associated with a shallow plate-like invagination of the two innermost plastid membranes.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan. hirakawa.yoshi.fp@u.tsukuba.ac.jp.

ABSTRACT

Background: Division of double-membraned plastids (primary plastids) is performed by constriction of a ring-like division complex consisting of multiple plastid division proteins. Consistent with the endosymbiotic origin of primary plastids, some of the plastid division proteins are descended from cyanobacterial cell division machinery, and the others are of host origin. In several algal lineages, complex plastids, the "secondary plastids", have been acquired by the endosymbiotic uptake of primary plastid-bearing algae, and are surrounded by three or four membranes. Although homologous genes for primary plastid division proteins have been found in genome sequences of secondary plastid-bearing organisms, little is known about the function of these proteins or the mechanism of secondary plastid division.

Results: To gain insight into the mechanism of secondary plastid division, we characterized two plastid division proteins, FtsZD-1 and FtsZD-2, in chlorarachniophyte algae. FtsZ homologs were encoded by the nuclear genomes and carried an N-terminal plastid targeting signal. Immunoelectron microscopy revealed that both FtsZD-1 and FtsZD-2 formed a ring-like structure at the midpoint of bilobate plastids with a projecting pyrenoid in Bigelowiella natans. The ring was always associated with a shallow plate-like invagination of the two innermost plastid membranes. Furthermore, gene expression analysis confirmed that transcripts of ftsZD genes were periodically increased soon after cell division during the B. natans cell cycle, which is not consistent with the timing of plastid division.

Conclusions: Our findings suggest that chlorarachniophyte FtsZD proteins are involved in partial constriction of the inner pair of plastid membranes, but not in the whole process of plastid division. It is uncertain how the outer pair of plastid membranes is constricted, and as-yet-unknown mechanism is required for the secondary plastid division in chlorarachniophytes.

No MeSH data available.


Related in: MedlinePlus