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. 2009 Feb;21(2):581-94.
doi: 10.1105/tpc.108.060145. Epub 2009 Feb 17.

An Arabidopsis GPI-anchor Plasmodesmal Neck Protein With Callose Binding Activity and Potential to Regulate Cell-To-Cell Trafficking

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An Arabidopsis GPI-anchor Plasmodesmal Neck Protein With Callose Binding Activity and Potential to Regulate Cell-To-Cell Trafficking

Clare Simpson et al. Plant Cell. .
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Abstract

Plasmodesmata (Pds) traverse the cell wall to establish a symplastic continuum through most of the plant. Rapid and reversible deposition of callose in the cell wall surrounding the Pd apertures is proposed to provide a regulatory process through physical constriction of the symplastic channel. We identified members within a larger family of X8 domain-containing proteins that targeted to Pds. This subgroup of proteins contains signal sequences for a glycosylphosphatidylinositol linkage to the extracellular face of the plasma membrane. We focused our attention on three closely related members of this family, two of which specifically bind to 1,3-beta-glucans (callose) in vitro. We named this family of proteins Pd callose binding proteins (PDCBs). Yellow fluorescent protein-PDCB1 was found to localize to the neck region of Pds with potential to provide a structural anchor between the plasma membrane component of Pds and the cell wall. PDCB1, PDCB2, and PDCB3 had overlapping and widespread patterns of expression, but neither single nor combined insertional mutants for PDCB2 and PDCB3 showed any visible phenotype. However, increased expression of PDCB1 led to an increase in callose accumulation and a reduction of green fluorescent protein (GFP) movement in a GFP diffusion assay, identifying a potential association between PDCB-mediated callose deposition and plant cell-to-cell communication.

Figures

Figure 1.
Figure 1.
Structural Organization of PDCB1. The predicted locations of the N- and C-terminal signal cleavage sites (arrows), the conserved X8 domain (underlined), and the insertion point for YFP (triangle) are marked on the complete PDCB1 amino acid sequence. The consensus sequence for the X8 domain for all Arabidopsis X8 proteins is shown below.
Figure 2.
Figure 2.
YFP-PDCB1 Shows Targeting to Pds. (A) Transgenic expression of 35SproYFP-PDCB1 in Arabidopsis showing fluorescence as punctate spots on the walls of epidermal pavement cells. (B) and (C) Transgenic expression of PDCB1pro:YFP-PDCB1 in Arabidopsis showing similar patterns of fluorescence as in (A), in the absence of overexpression. (D) to (F) Similar patterns of fluorescence for YFP-PDCB1 were seen in spongy mesophyll cells where the fluorescent punctae were restricted to regions of wall–wall contact between adjacent cells and were stably located following plasmolysis; (F) is the same as (E) except with combined differential interference contrast microscopy. Arrows indicate YFP-PDCB1 fluorescence on adjoining walls. Dotted lines indicate periphery of retracted protoplast and dashed lines the position of the cell wall. Fluorescence is shown for GFP in green and chlorophyll autofluorescence in magenta. (G) to (I) YFP-PDCB1 labeling on new anticlinal division walls in newly divided root epidermal cells immediately behind the root meristem. (J) to (L) Colocalization (e.g. arrows) of YFP-PDCB1 (J) and aniline blue staining for callose (K) supporting the identification of the fluorescent punctae as Pds; merged images are shown in (L). Aniline blue fluorescence is shown using magenta (K) false color. Bars = 10 μm.
Figure 3.
Figure 3.
Immunogold Localization of YFP-PDCB1 to Pds. Thin sections of YFP-PDCB1-transgenic Arabidopsis leaves were subjected to immunogold labeling using anti-PDCB1 antiserum. (A) A length of cell wall on which two branched Pds are labeled at the cytoplasmic ends of the structures (arrows). Note that there is little labeling with the antibody in the central cavity region of the Pds. Bar = 500 nm. (B) A proportional quantification of the immunogold distribution where the majority of gold particles are associated with Pds and a minority scattered over other areas; total particle count from randomized electron micrographs is 1136. The latter labeling corresponded with the weak labeling observed with the preimmune serum; total particle count is 389. (C) The general organization of Pds with the location of callose at the peripheral neck regions. ER, endoplasmic reticulum; PM, plasma membrane.
Figure 4.
Figure 4.
The Larger PDCB Family of Pd Proteins. (A) Phylogenetic tree of the X8 domains of Arabidopsis PDCB-related proteins. The analysis, which includes X8 domains from 30 PDCB structural homologs, is presented as a midpoint rooted phylogenetic tree of proteins containing X8 as their only identifiable functional domain; 1,3-β-glucanases are excluded from this analysis. X8 domains from proteins with (bold) and without GPI anchors are listed as Arabidopsis Genome Initiative numbers. Olive OLE-e10 is included and shows closer similarity to the clade of non-GPI X8 proteins than to the GPI-anchored proteins. Only bootstrap values >70% are shown. The PDCB subfamily is indicated. Proteins tested for their subcellular targeting as YFP fusion proteins are underlined. The sequences used to generate this phylogeny are presented in Supplemental Data Set 1 online. (B) Transient expression of 35SproYFP-PDCB2 in N. benthamiana leaves showing fluorescence as punctate spots on the walls of epidermal pavement cells. (C) Transgenic expression of 35SproYFP-PDCB3 in Arabidopsis showing fluorescence as punctate spots on the walls of epidermal pavement cells. (D) Transgenic expression of 35SproYFP-At1g69295 in Arabidopsis showing fluorescence as punctate spots on the walls of epidermal pavement cells. (E) Transgenic expression of 35SproYFP-At3g58100 in Arabidopsis showing fluorescence as punctate spots on the walls of epidermal pavement cells. Bars in (B) to (E) = 10 μm.
Figure 5.
Figure 5.
Congruent Tertiary Structures for PDCB1 and OLE-e9 X8 Domains. The sequence of the PDCB1 X8 domain was analyzed by threading onto the tertiary structure of OLE-e9 determined by nuclear magnetic resonance (Trevino et al., 2008). The parent OLE-e9 structure is shown in magenta; the PDCB1 X8 domain is shown in gray. The disulphide bridges (arrows) are highlighted (OLE-e9, green; PDCB1 X8, yellow).
Figure 6.
Figure 6.
In Vitro Callose Binding Properties of PDCB Proteins. Soluble purified thioredoxin-PDCB1, -2, or -3 fusion proteins were assessed for ability to bind to polysaccharides in a nondenaturing gel retardation assay. (A) Callose binding was seen as retarded migration of the fusion protein (P) in the presence of the 1,3-β-glucan laminarin ligand when compared with the gel lacking laminarin (control) and to the unchanged migration of BSA (B) or thioredoxin alone (T). M, molecular weight markers. (B) Bar chart showing a positive correlation between gel retardation and substrate concentration. Bars indicate ±sd from triplicate assays. (C) to (E) PDCB1-poysaccharide binding specificity tested against hexalaminarin (1,3-β-glucan), carboxymethyl (CM) cellulose (derivatized 1,4-β-glucan), and lichenan (mixed 1,4;1,3-β-glucan), respectively. Binding was only observed with hexalaminarin. (F) Assay for the binding of thioredoxin-PDCB2 and thioredoxin-PDCB3 to laminarin in a gel retardation assay. In comparison to the negative control, BSA (B), thioredoxin-PDCB2 (P2) shows retarded migration. It was not possible to assay binding for thioredoxin-PDCB3 (P3) due to aggregation and low migration in both test and control gels. M, molecular weight markers.
Figure 7.
Figure 7.
Tissue-Specific Expression of PDCB1, -2, and -3. Predicted promoter sequences for PDCB1, -2, and -3 were isolated from the Arabidopsis genome and fused to a GUS reporter gene. Transgenic lines were stained for GUS activity as 11-d-old seedlings. Bars = 0.5 cm in (A), (C), and (E) and 0.2 cm in (B), (D), and (F). (A) and (B) Views of a whole seedling (A) and an isolated leaf (B) from a PDCB1pro:GUS transgenic line after staining for GUS activity to reveal the distribution of gene-specific expression. (C) and (D) As in (A) and (B) except for PDCB2pro:GUS. (E) and (F) As in (A) and (B) except for PDCB3pro:GUS.
Figure 8.
Figure 8.
PDCB1 Transgenic Plants Show Increased Callose Accumulation and Decreased Plasmodesmal Molecular Diffusion. (A) Crude protein extracts from two independent 35Spro:PDCB1 overexpressing hemizygous transgenic Arabidopsis Col-0 lines (OE1 and OE2) were assessed for PDCB1 accumulation by immunoblot analysis using anti-PDCB1 antibody compared with wild type (wt) Columbia-0 (Col-0) (top). Equal protein loading is shown from the equivalent section of the protein gel stained with Coomassie blue (bottom). MW, protein markers in kilodaltons. (B) Cell-to-cell movement potential in the OE1 plants was assessed in a GFP diffusion assay where diffusion of soluble GFP from a bombarded epidermal cell to surrounding cells was measured. Representative examples of restricted and permitted GFP diffusion following bombardment of 35Spro:GFP into the leaves of a PDCB1 overxpressing (OE1) or a wild-type plant. GFP recipient epidermal cells are indicated (arrows). Bars = 10 μm. (C) Quantification of the GFP diffusion was determined after 24 and 48 h, at which times the overexpressing line showed significantly less (P < 0.05 and P < 0.0001, respectively) plasmodesmal flux; numbers in parentheses record the numbers of bombardment foci counted for each treatment. The experiment was repeated twice with similar results. Bars indicate ±se. (D) GFP diffusion assay applied to leaves of a 35Spro:YFP-PDCB3 transgenic plant. Data from the 48 h time point are presented. GFP diffusion in the transgenic line was significantly (P < 0.05) less than in wild-type plants. Bars indicate ±se. (E) and (F) Confocal micrographs of leaf tissue from transgenic PDCB1 overexpressing (OE1) and wild-type Col-0 plants, respectively, stained with aniline blue to reveal callose accumulation. Images were collected using identical confocal microscope settings. Bars = 20 μm. (G) and (H) Quantification of the callose fluorescence on representative optical sections achieved using image intensity analysis with IMAGEJ software. Either the size of individual aniline blue–stained foci (in pixels) were compared (G), or gray values of individual foci were compared (H). For each treatment, 100 data sets were collected. As the data did not show a normal distribution, statistical analysis was done using a Mann-Whitney Hugh test. In either case, the data sets between the treatments are statistically different at P < 0001. Bars indicate ±se.

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