The Plant Journal A plasmodesmata-associated b-1,3-glucanase in Arabidopsis · 2017. 10. 23. · Theenzymethat degradescallose,b-1,3-glucanase,isalso an important factor in the regulation

A plasmodesmata-associated b-1,3-glucanase in Arabidopsis

Amit Levy, Michael Erlanger, Michal Rosenthal and Bernard L. Epel*

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

Received 28 June 2006; revised 7 September 2006; accepted 10 October 2006.

*For correspondence (fax þ972 3 6409380; e-mail [email protected]).

Summary

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by

b-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodes-

mata (Pd)-rich fraction identified a b-1,3-glucanase as present in this fraction. The protein encoded by the

putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a

post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green

fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal

cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along

the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around

the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a

plasma membrane marker N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium

dibromide (FM4-64) in plasmolysed cells. In Arabidopsis T-DNA insertion mutants that do not transcribe

AtBG_ppap, functional studies showed that GFP cell-to-cell movement between epidermal cells is reduced,

and the conductivity coefficient of Pd is lower. Measurements of callose levels around Pd after wounding

revealed that callose accumulation in the mutant plants was higher. Taken together, we suggest that

AtBG_ppap is a Pd-associated membrane protein involved in plasmodesmal callose degradation, and functions

in the gating of Pd.

Keywords: Nicotiana tabacum, Arabidopsis thaliana, plasmodesmata, cell–cell communication, proteomics,

callose.

Introduction

Plasmodesmata (Pd) are co-axial membranous channels

that cross walls between adjacent plants cells, intercon-

necting the cytoplasm of these cells, thus allowing direct

cell-to-cell transport of soluble cytoplasmic macromole-

cules (proteins and RNA molecules), as well as small mol-

ecules (Heinlein and Epel, 2004; Oparka, 2004). A group of

cells that are interconnected through open Pd but sym-

plasmically isolated from other cells forms a communica-

ting ‘symplast domain’ that acts as an isolated

developmental and physiological unit (Rinne and van der

Schoot, 1998). The control of transport through Pd serves

as an important element in regulating the direct cell-to-cell

transport between cells and in the organization and func-

tioning of symplasmic domains. The fact that many plant

viruses exploit Pd as conduits for spread of infection be-

tween cells makes this transport system a crucial point for

defense against virus spread.

Although the ultrastructure of Pd is well described, their

molecular composition is still mostly unknown. The list of

proteins identified as structural or functional components of

the Pd is only partial, and includes a reversible glycosylated

polypeptide (Sagi et al., 2005), a Ca2þ-dependent protein

kinase (Yahalom et al., 1998) and the cytoskeleton proteins

actin, myosin and centrin (reviewed by Heinlein and Epel,

2004). The cell wall that surrounds the Pd has a specialized

structure devoid of cellulose and hemicellulose but contain-

ing non-esterified pectins and callose (Roy et al., 1997;

Turner et al., 1994). This specialized wall sheath, which may

be an essential part of the Pd, also requires further charac-

terization.

Pd are dynamic transport channels that can be modulated.

During the sink-to-source transition in leaf development, Pd

structure changes from a single tunnel (simple Pd) to a

branched one, and the transfer rate of proteins through the

ª 2006 The Authors 669Journal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 49, 669–682 doi: 10.1111/j.1365-313X.2006.02986.x

channels decreases (Liarzi and Epel, 2005; Oparka et al.,

1999; Roberts et al., 2001). Beyond these general develop-

mental changes, the functional state of a single plasm-

odesma is dynamic, and can change in a transient manner

from ‘closed’ to ‘open’ to ‘dilated’ (Oparka and Roberts,

2001; Zambryski and Crawford, 2000). Two different mech-

anisms are assumed to produce these focused changes in

the tunnels. The first model suggests that the conductivity

changes because of alterations in the plasmodesmal cyto-

skeleton proteins actin, myosin and centrin. This model

gains strength from the finding that actin filament disruption

increases Pd permeability, allowing dextrans up to 20 kDa

(Stokes radius ¼ 3.3 nm) to move from an injected tobacco

mesophyll cell to surrounding cells within 3–5 min, while no

movement was seen when the dextrans were injected alone

or with treatments stabilizing the actin (Ding et al., 1996).

The second model suggests that changes in the wall sheath

surrounding the Pd cause changes to its structure that alter

its conductivity. These changes are hypothesized to be

mediated by callose synthesis and hydrolysis (Olesen and

Robards, 1990; Radford and White, 2001; Radford et al.,

1998; Ruan et al., 2004; Sivaguru et al., 2000).

Callose, a poly-sugar molecule in the form of b-1,3-glucan,

is reversibly and transiently deposited in cell walls as a result

of stresses and during many developmental processes

(Kauss, 1996). These include stress responses such as

wounding (Radford et al., 1998) or aluminum toxicity (Siva-

guru et al., 2000) and developmental processes such as

cotton fiber elongation (Ruan et al., 2004) or bud dormancy

induction caused by a short photoperiod (Rinne and van der

Schoot, 1998). Callose deposition occurs in the wall sur-

rounding the Pd at both ends of the channel, compressing

the plasma membrane inward, thus creating a narrowed

neck region (Radford et al., 1998), which reduces the free

space available for the passage of molecules through the Pd.

During dormancy, callose is also deposited inside the Pd

channel, creating an inner plug (Rinne et al., 2001).

The enzyme that degrades callose, b-1,3-glucanase, is also

an important factor in the regulation of conductivity through

Pd. This was demonstrated in studies employing a tobacco

mutant with decreased levels of class I b-1,3-glucanase that

was generated by antisense transformation. In this mutant

line, higher levels of callose were found, and the suscepti-

bility to virus infection was decreased. Examining the

movement of dextrans and peptides between the cells

revealed that the size exclusion limit, which is the size of

the largest dye capable of moving through the Pd, is lower in

the mutant plants (Iglesias and Meins, 2000). When the b-1,3-

glucanase coding sequence was cloned into the TMV

replicon, the virus spread faster through the cells, and

cloning of the gene in an antisense formation led to the

opposite results (Bucher et al., 2001).

In order to characterize the molecular composition of Pd

and to understand the function of each Pd component in the

regulation of intercellular communication, a Pd isolation

protocol was developed in our laboratory, and putative Pd-

associated proteins from Arabidopsis thaliana were identi-

fied; one being a b-1,3-glucanase. We show here that this

protein, when expressed as a GFP fusion, targets to the

endoplasmic reticulum (ER) membrane and Pd. Moreover,

we present functional evidence showing that, in mutants

that lack this b-1,3-glucanase, the movement of GFP

between cells is reduced, and the amount of callose in the

Pd is elevated.

Results

Proteomic studies reveal a putative Pd-associated b-1,3-

glucanase

Proteins in an A. thaliana Pd-enriched fraction were separ-

ated by sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis (SDS–PAGE) (Figure 1). Coomassie blue-stained

bands were cut, in-gel reduced, alkylated and proteolysed

overnight with trypsin, and the tryptic peptides were re-

solved by reverse-phase chromatography on a C18 column

(see Experimental procedures). Mass spectrometry was

performed with ion-trap mass spectrometers operating in

the positive mode, using repetition of a full MS scan fol-

lowed by collision-induced dissociation (CID) of the most

dominant ion selected from the first MS. The MS data were

compared to simulated proteolysis and CID of the proteins

in the non-redundant National Center for Biotechnology

Informatic (NR-NCBI) database using the SEQUEST software

(LCQ; Thermo, www.thermo.com). One of the proteins

identified that was present in band 7 (Figure 1) was a 45 kDa,

425 amino acid long b-1,3-glucanase (At5g42100) that was

Figure 1. Detection of AtBG_ppap.

SDS–PAGE separation of the Arabidopsis Pd-rich fraction. The arrow indicates

the 45 kDa band identified as AtBG_ppap.

670 Amit Levy et al.

ª 2006 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 669–682

named AtBG_ppap (A. thaliana beta-1,3-glucanase_putative

Pd-associated protein). Five peptides were identified leading

to unique identification as At5g42100. This protein contains

the glycosyl hydrolase 17 domain (pfam00332) between

amino acids 27 and 346 (CD search http://www.ncbi.nlm.

nih.gov/Structure/cdd/wrpsb.cgi; Marchler-Bauer and

Bryant, 2004). The SOSUI computer program (http://sosui.

proteome.bio.tuat.ac.jp/sosuiframe0E.html; Hirokawa et al.,

1998) predicts that the protein contains two transmembrane

regions between amino acids 9 and 31 and between amino

acids 407 and 422. According to the SignalP program (http://

www.cbs.dtu.dk/services/SignalP; Bendtsen et al., 2004), the

protein contains an N-terminal signal peptide, with a clea-

vage site between amino acids 26 and 27.

Three different computer programs predict that

AtBG_ppap has the essential characteristics to become

glycosylphosphatidylinositol (GPI)-modified: big PI plant

predictor (http://mendel.imp.univie.ac.at/sat/gpi/plant_server.

html; Eisenhaber et al., 2003); DGPI (http://129.194.185.165/

dgpi/index_en.html; D. Buloz and J. Kronegg, University of

Geneva, unpublished data) and GPI-SOM (http://gpi.unibe.ch;

Fankhauser and Maser, 2005). This feature was recently

confirmed experimentally by cleavage of the GPI anchor with

phospholipases C and D (Elortza et al., 2003, 2006; ).

The AtBG_ppap gene (Accession number: NM_123575)

contains two exons and one intron (Figure 2). Exon 1 is

1209 bp long (403 amino acids) and ends immediately after

the predicted GPI x site (the site of the C-terminal peptide

cleavage), which is amino acid 401 (nucleotide 1203). The

intron is 431 bp long, and the second exon, which contains

the C-terminal signal, is 69 nucleotides long (23 amino

acids). The resulting spliced coding sequence is 1278 bp,

which encodes a protein containing 425 amino acids. A

second version of the protein results from retention of the

intron, which contains a stop codon 63 nucleotides after its

beginning, resulting in a 1272 bp coding sequence (Acces-

sion number: NM_203139) and a 423 amino acid protein.

This alternatively spliced protein, which was entitled

AtBG_ppapas, was not found to be GPI-anchored by the

big-PI Plant Predictor, while both the DGPI and GPI-SOM

programs predicted that it has the essential characteristics to

become GPI-modified (Figure 2).

Quantitative measurements of AtBG_ppap transcript lev-

els in the Arabidopsis tissues using real-time RT-PCR

revealed that the gene is not expressed equally in the

different tissues (Figure 3). The transcription level of

AtBG_ppap was the highest in flowers and siliques. In stem,

rosette leaves and cauline leaves, transcript accumulation

was considerably lower than in flowers and siliques. In

roots, levels were negligible compared with the flowers and

siliques (a 34-cycle RT-PCR confirmed that the gene is

transcribed in all tissues, including roots; data not shown).

Quantitative measurement of the alternatively spliced

version of AtBG_ppap (AtBG_ppapas) showed that transcript

accumulation was a few orders of magnitude lower than the

AtBG_ppap form (data not shown). We therefore focused

our research on the normally spliced form of AtBG_ppap,

and cautiously suggest that the presence of AtBG_ppapas

results from a low efficiency of normal splicing rather than a

regulated process of any biological significance, a well-

known phenomenon in plants (Lorkovic et al., 2000). How-

ever, more experiments are required to confirm this

assumption.

AtBG_ppap:GFP localizes to Pd

In order to verify plasmodesmal localization of AtBG_ppap, a

fusion between the protein and GFP was created. As

Figure 2. Schematic model of At5g42100.

The gene contains two exons (black boxes) and one intron (white box).

Splicing of the intron results in a 1278 bp coding sequence that encodes the

425 amino acid protein AtBG_ppap. For the GFP fusion, the GFP sequence was

inserted after nucleotide 1152. Retention of the intron, which contains a stop

codon 63 bp from the start, results in a 1272 bp coding sequence that encodes

the 423 amino acid alternatively spliced protein AtBG_ppapas.Figure 3. Transcription levels of AtBG_ppap in various Arabidopsis tissues.

Real-time RT-PCR measurements of AtBG_ppap, showing its relative tran-

scription levels in various Arabidopsis tissues. Transcription is highest in the

flowers and siliques, and lowest in the roots. SD is represented by vertical

lines within the bars.

A plasmodesmal b-1,3-glucanase 671


AtBG_ppap is a GPI-anchored protein and both the N- and

C-terminal ends of its preprotein are removed, it was

necessary to create a fusion protein with GFP within regions

that remain after N- and C-processing. The coding sequence

for GFP was thus inserted within the AtBG_ppap coding

sequence immediately after amino acid 384, which is

localized 17 amino acids before the x site (Figure 2). This

region into which the GFP was inserted is described as a

‘disordered/unstructured’ and ‘low complexity’ region by

the SMART program (http://smart.embl-heidelberg.de;

Letunic et al., 2004), and is suspected to serve as a linker

between the active glucanase domain and the GPI anchor.

AtBG_ppap:GFP was expressed transiently in Nicotiana

benthamiana source leaves by microprojectile bombard-

ment (Figure 4a) and stably in Nicotiana tabacum by Agro-

bacterium transformation (Figure 4b). In both cases, the

fusion protein appeared along the entire periphery of

epidermal leaf cells, and in a punctate pattern (Figure 4a,b)

or as pairs of fluorescent foci on opposite sides of the cell

wall (enlarged boxes in Figure 4b), a pattern characteristic of

Pd. Spongy mesophyll cells have regions with no cell–cell

contact, where walls face an intercellular space and are

devoid of Pd, and regions with cell–cell contact, where walls

contain Pd. AtBG_ppap:GFP fluorescence was almost unde-

tected within cell wall regions where there is no cell–cell

contact (Figure 4c,d). This differential labeling of walls

further indicates that AtBG_ppap:GFP is associated with Pd

or with the wall sheath surrounding Pd.

To verify that the AtBG_ppap:GFP fluorescence foci

inside cell walls indeed represent Pd, we stained callose

using aniline blue. Callose has been widely used as a

plasmodesmal marker (Baluska et al., 1999; Bayer et al.,

2004; Gorshkova et al., 2003; Sagi et al., 2005 and more).

When transgenic tobacco source leaves expressing AtBG_

ppap:GFP were stained by aniline blue, AtBG_ppap:GFP co-

localized with the aniline blue-stained callose present

around Pd (Figure 5). Control experiments verified that no

GFP fluorescence is seen under aniline blue conditions and

no aniline blue fluorescence is seen under GFP conditions.

AtBG_ppap:GFP is a membrane protein

As AtBG_ppap is a GPI-anchored protein, it is expected to

target both the ER and the plasma membrane (Udenfriend

and Kodukula, 1995). Indeed, when AtBG_ppap:GFP is tran-

siently expressed by Agrobacterium leaf injection, it can be

seen to accumulate in ER membranes (Figure 6a). Likewise,

as predicted, AtBG_ppap:GFP also localizes to the plasma

membrane. AtBG_ppap:GFP transgenic leaves were stained

(a) (b)

(c) (d)

Figure 4. Cellular localization of AtBG_ppap:GFP.

(a) Transiently expressed AtBG_ppap:GFP in the epidermis of Nicotiana

benthamiana 48 h after microprojectile bombardment. Fluorescence is seen

along the periphery of the wall and in a punctate pattern (arrows).

(b) Stably expressed AtBG_ppap:GFP in transgenic tobacco epidermis.

Punctate fluorescence spans the walls and is seen as paired foci. The areas

inside the white boxes are enlarged in the insets.

(c,d) Spongy mesophyll cell of AtBG_ppap:GFP-expressing transgenic

tobacco. Fluorescence is detected only in wall areas where there is cell–cell

contact, and is absent from wall areas without cell–cell contact (d). To

emphasize wall partitions, spongy mesophyll cells are also shown with the

fluorescence channel turned off (c).

Bar in (a) ¼ 10 lm, those in (b)–(d) ¼ 20 lm.

(a) (d)

(c)

(b)

(f)

(e)

Figure 5. AtBG_ppap:GFP in transgenic tobacco co-localizes with aniline

blue-stained callose present around plasmodesmata.

Aniline-blue stained callose is shown in blue (a), AtBG_ppap:GFP is shown in

red (b), and both are shown overlaid (c) in a section of cell wall between

epidermal cells. (d)–(f) are enlargements of the areas inside the boxes in (a)–

(c), respectively. Bars ¼ 20 lm.



with FM4-64 (N-[3-triethylammoniumpropyl]-4-[p-diethyl-

aminophenylhexatrienyl] pyridinium dibromide), a plasma

membrane marker (Bloch et al., 2005), and cells were

plasmolysed. Following plasmolysis, AtBG_ppap:GFP could

be seen to recede from the cell wall along with the FM4-64

fluorescently stained plasma membrane (Figure 6b–e).

GFP cell-to-cell spread is slower in AtBG_ppap mutant plants

It is hypothesized that non-selective plasmodesmal con-

ductivity is a function of the steady-state level of callose

accumulation in the wall sleeve surrounding Pd, and that

this steady-state level is the net result of glucanase versus

synthase activities (Heinlein and Epel, 2004). Assuming no

change in callose synthase activity, a lower b-1,3-glucanase

activity in the Pd is expected to increase callose levels and

reduce cell-to-cell transport.

To test this hypothesis, we measured the diffusion of GFP

between leaf epidermal cells in wild-type and AtBG_ppap

knockout mutants. As GFP movement between cells is

diffusive (Crawford and Zambryski, 2000; Liarzi and Epel,

2005; Oparka et al., 1999), it is expected that, in AtBG_ppap

knockout mutant plants, there will be less callose hydrolysis,

resulting in a narrowing of the cytoplasmic sleeve and hence

a slower diffusion of GFP.

Two independent Arabidopsis lines carrying a T-DNA

insertion that disrupts the AtBG_ppap locus (SALK_

019116.47.55.x, Alonso et al., 2003; SAIL_115_G04, Sessions

et al., 2002) were employed to test this hypothesis. PCR

analysis and sequencing of the two T-DNA lines established

that the insertion in SALK_019116.47.55.x disrupted the gene

at 344 bp downstream of the ATG start codon, and that the

insertion in SAIL_115_G04 is located 625 bp from the ATG

start (Figure 7a; data not shown). Plants of the two lines,

homozygous for the insertion, do not transcribe AtBG_ppap

RNA, as determined by RT-PCR (Figure 7b). Epidermal cells

of vegetative wild-type and mutant leaves were transfected

with pGFP by microprojectile bombardment of gold particles,

a method that wounds the leaf and induces callose formation

(Hunold et al., 1994). Forty-eight hours postbombardment,

the number of the cells in a cluster created by GFP movement

was analyzed by scanning the epidermal cells using a

confocal laser scanning microscope (CLSM; see Experimen-

tal procedures). The cells counted are those showing green

fluorescence in the nucleus and the cytoplasm (Figure 8). A

box-plot analysis, comparing the sizes of the GFP clusters in

leaves of wild-type and knockout mutants, shows that, in

wild-type plants, the diffusion of GFP is more extensive

(Figure 8). A one-way ANOVA analysis revealed no statistical

difference between SALK and SAIL mutants (P ¼ 0.949), but

showed a statistically significant difference between the

mutants and wild-type (P < 0.05).

In a second analysis, the exponential decay parameter band the coefficient of conductivity of Pd, C(Pd), were

(a) (b)

(c) (d) (e)

Figure 6. AtBG_ppap:GFP localization in the ER and plasma membranes.

(a) A projection of several optical sections of transiently expressed AtBG_ppap:GFP in N. benthamiana plasmolysed epidermal cell 48 h after Agrobacterium

infiltration, showing the localization of AtBG_ppap:GFP in the ER membrane system.

(b)–(e) AtBG_ppap:GFP fluorescence (shown in green) co-localizes with FM4-64-stained plasma membrane (shown in red) in plasmolysed transgenic tobacco

epidermal cells. (b) Overlay of all channels showing that AtBG_ppap:GFP and FM4-64 co-localize (see arrows), indicating that AtBG_ppap:GFP is a plasma

membrane protein. (c)–(e) show separate channels of (b). (c) Nomarsky differential interference contrast (DIC) showing the cell wall (CW). (d) DIC and FM4-64

fluorescence overlay. Upon plasmolysis, plasma membrane (PM) withdraws from the cell wall. (e) DIC and GFP fluorescence overlay. Upon plasmolysis,

AtBG_ppap:GFP withdraws from the cell wall as well, in the same pattern as the plasma membrane.

Bars ¼ 10 lm.



calculated for GFP spread in vegetative leaves of the wild-

type and AtBG_ppap knockout mutants, according to the

protocol described by Liarzi and Epel (2005) (Table 1). The

GFPC (Pd) value in wild-type plants was found to be higher

than the GFPC (Pd) value in the SALK and SAIL mutants (0.70,

0.59 and 0.52, respectively). A one-way ANOVA of b showed

no difference between the SALK and SAIL mutants, but

showed a statistically significant difference between the

mutants and the wild-type (P < 0.05). The results suggest

that the conductivity of Pd is downregulated in AtBG_ppap-

deficient mutants.

To test whether the decreased cell-to-cell movement of

GFP results from increased callose accumulation in Pd, we

examined the amount of Pd callose in the epidermis of

Arabidopsis vegetative source leaves 2 days after wound-

ing, employing an aniline blue fluorescence assay. Quanti-

fication of 1487 fluorescent sites in AtBG_ppap-deficient

(a)

(b)

Figure 7. Identification of AtBG_ppap T-DNA insertion lines.

(a) A schematic model showing the T-DNA insertions in SALK and SAIL

mutants (SALK_019116.47.55.x and SAIL_115_G04 respectively). Numbers

indicate the coding sequence location of the insertions.

(b) RT-PCR of the homozygous SALK mutant, homozygous SAIL mutant, wild-

type (WT) and heterozygous SAIL mutant (Het) Arabidopsis leaves. The upper

panel shows the amplification of AtBG_ppap coding sequence. No amplifi-

cation is found in the homozygous mutants. The lower panel shows the

amplification of control gene Ubiquitin10.

(a)

(b) (c) (d)

Figure 8. Movement of bombarded GFP be-

tween Arabidopsis epidermal cells in wild-type

and AtBG_ppap-deficient mutants.

(a) A box-plot diagram of the GFP cluster sizes

(determined by the number of cells in a cluster),

two days after bombardment, in SALK and SAIL

mutants, and in wild-type plants. The plot repre-

sents the statistical distribution of the results.

The thick lines across the boxes represent the

median cluster sizes, and the ends of boxes

indicate quartile results. Circles represent out-

liers. The median cluster sizes are 6.5 for the

SALK mutant (n ¼ 36), 8 for the SAIL mutant

(n ¼ 62) and 15 for wild-type (n ¼ 97).

(b)–(d) Average GFP clusters of SALK mutants

(b), SAIL mutants (c) and wild-type plants (d),

48 h after GFP bombardment. GFP is produced in

the intensely fluorescent cell and moves to the

neighboring cells that show weaker fluores-

cence. Bar ¼ 50 lm.

Table 1 Changes in Pd conductivity in AtBG_ppap-deficient mu-tants

Plant type Mean ba SEb GFPC(Pd)c

Wild-type )1.41 (31)d 0.072 0.7SALK mutant )1.66 (31) 0.068 0.59SAIL mutant )1.88 (15) 0.194 0.52

aAverage decay parameter b, also termed impedance, which des-cribes the slope of the gradient formed by differences in plasmodes-mal conductivity.bSE, SEM b value.cThe coefficient of conductivity of plasmodesmata for the cell-to-cellspread of GFP, calculated as )1/mean b value.dThe number in parentheses is the number of cells analyzed.



mutants and 779 fluorescent sites in wild-type plants

revealed that callose accumulation was 45% higher in

AtBG_ppap mutants (Figure 9). An independent-samples

t test showed the results to be significantly different at

P < 0.001. Although the background levels varied between

repeats with different leaves, the net intensity in the Pd was

the same. These results are in agreement with the hypothe-

sized role of callose in regulating Pd function, and support

the hypothesis AtBG_ppap functions as a plasmodesmal

protein.

Discussion

Identification of a Pd-associated Arabidopsis b-1,3-gluca-

nase

b-1,3-glucanases, enzymes that catalyze the hydrolysis of b-

1,3-glucan linkages, hydrolyse mostly callose in plants, and

play a role in various important physiological processes. For

example, they are found in the pistils, where callose is

known to be abundant in the growing pollen tube wall and

as plugs behind the growing tube protoplast, which pre-

sumably maintains the two nuclei in the proximity of the

tube tip (Kauss, 1996). In the style, developmentally regula-

ted b-1,3-glucanases were hypothesized to function in

regulating pollen tube growth or in defense against patho-

gen attack during fertilization (Delp and Palva, 1999; Ori

et al., 1990). In anthers, b-1,3-glucanases were found to be

expressed just before microspores are released, where they

function in degradation of the callose that surrounds the

microspore tetrad, and contribute to the release of the

microspores as pollen grains (Hird et al., 1993; Tsuchiya

et al., 1995). Some b-1,3-glucanases are involved in stress

processes through their function as ‘pathogenesis-related‘

proteins (Leubner-Metzger and Meins, 1999; Stintzi et al.,

1993). b-1,3-glucanase enzymes have been localized to the

cell wall, the intercellular space, the plasma membrane and

the vacuole (Benhamou et al., 1989; Hu and Rijkenberg,

1998; Keefe et al., 1990). During dormancy release in birch

(Betula pubescens), b-1,3-glucanases were found peripher-

ally in spherosome-like vacuoles, in close proximity to the

Pd (Rinne et al., 2001).

In Arabidopsis, five b-1,3-glucanase genes have been

studied. BG2 and BG3 are 37 and 30 kDa acidic proteins,

respectively, whose genes are upregulated after pathogen

infection. BG2 is located extracellularly, and both proteins

are purported to function as part of the plant defense system

(Dong et al., 1991; Uknes et al., 1992). A6 is a 53 kDa basic

protein whose gene is expressed in the tapetum cells in

anthers just before microspores are released (Hird et al.,

1993). BG4 and BG5 are 38 kDa proteins with an acidic and

basic pI, respectively. BG4 is expressed in the style and

septum of the ovary, and may play a role in the plant

reproductive process (Delp and Palva, 1999). In tobacco,

based on their sequences homologies, the b-1,3-glucanase

proteins have been classified into three classes. Class I are

basic proteins localized in the vacuole of mesophyll and

epidermal cells, while classes II and III are acidic, extracel-

lular isoforms (Beffa and Meins, 1996).

Here we describe the isolation and characterization of a

45 kDa b-1,3-glucanase present in an enriched Pd fraction

from Arabidopsis and termed AtBG_ppap. AtBG_ppap was

identified by MS/MS analysis of a tryptic digest of a protein

isolated from a Pd-enriched fraction. The mature form of

AtBG_ppap with its N- and C-terminal ends removed (see

below) is predicted to be a neutral protein (pI 6.18), and thus

does not fit into the tobacco basic/acidic classification

scheme.

According to the Arabidopsis Information Resource

(TAIR), the Arabidopsis genome contains 48 b-1,3-gluca-

nase genes. A bioinformatic analysis, using a sensitive

prediction program for the compatibility of plant proteins

with GPI lipid-anchoring motif requirements (big-PI Plant

Predictor) (http://mendel.imp.univie.ac.at/sat/gpi/plants/pred/

plants.athal.class.html#glyco), predicted that 18 Arabidop-

sis b-1,3-glucanase proteins, including AtBG_ppap

(At5g42100), are GPI-anchored. GPI-anchored proteins

undergo post-translational modifications, in the course

of which both their C-terminal and N-terminal ends are

spliced. The tobacco class I isoforms are also synthesized

as prepro-enzymes, which similarly undergo post-transla-

tional processing in which their C- and N-terminal ends

are removed (Beffa and Meins, 1996). However, the big-PI

Plant Predictor did not find the class I GLA protein to be

GPI-anchored. Alignment (Corpet, 1988) between the

(a) (b)

Figure 9. Callose accumulation in AtBG_ppap-deficient mutants and wild-

type Arabidopsis plants in response to wounding.

Epidermal cells of (a) an AtBG_ppap knockout mutant and (b) a wild-type

plant, stained with aniline blue two days after wounding and observed under

a fluorescent microscope. The fluorescence intensity of callose deposits

around Pd was measured by drawing a small circle around Pd foci [see circle

in (a)] and determining the minimum fluorescence intensity inside the circle

(background) and the maximum intensity of the Pd foci (maximum) using the

IMAGEJ software. Pd intensity was determined by subtracting the background

from the Pd maximum intensity [for the circle in (a), the maximum Pd intensity

is 143, the minimum intensity is 103, therefore the Pd intensity is 40]. Two

days after wounding, the mean Pd callose intensity was 40.4 � 0.42 (mean

intensity �SEM in arbitrary units) in AtBG_ppap knockouts and 27.9 � 0.404

in wild-type plants (n ¼ 1487 and 779, respectively). Bar ¼ 30 lm.



predicted GPI-anchored b-1,3-glucanases of Arabidopsis

(see Figure in Supplementary Material) shows that

AtBG_ppap shares the highest homology with At1g32860

(61% identity and 73% similarity). Both of these proteins

contain a C terminus that is rich in glycine and serine, and is

described as a ‘disordered/unstructured’ region by the

SMART program (http://smart.embl-heidelberg.de; Letunic

et al., 2004). The C terminus of both proteins is clearly

distinguishable from that of the other proteins in this group,

and may indicate a unique property of these proteins.

A novel plasmodesmal b-1,3-glucanase

Several lines of evidence presented here support the hypo-

thesis that AtBG_ppap is a Pd-associated protein. First, the

protein was identified in a Pd-enriched fraction from Ara-

bidopsis. Secondly, AtBG_ppap:GFP fluorescence appears

in a punctate pattern and as pairs of fluorescent foci on

opposite sides of the cell walls (Figure 4), a pattern similar to

that of plasmodesmal marker MPTMV:GFP or the Pd-associ-

ated protein AtRGP2:GFP (Sagi et al., 2005). Third, AtBG_

ppap:GFP fluorescence co-localizes with aniline-blue stained

callose present around Pd (Figure 5).

In addition to plasmodesmal localization, our results

indicate that AtBG_ppap also has a plasmodesmal function.

Monitoring the movement of GFP between cells in AtBG_

ppap knockout mutants and wild-type plants revealed that

GFP diffused more slowly in AtBG_ppap mutant plants. The

number of cells into which GFP diffused from a GFP-

transformed cell is approximately twice as high in wild-type

as in the AtBG_ppap-deficient mutants (Figure 8), and the

conductivity of Pd [GFPC (Pd)] in these mutants is downreg-

ulated (Table 1). These results strongly suggest that the Pd

are in a more ‘closed’ state in AtBG_ppap knockout mutants,

resulting in reduced cell-to-cell spread of GFP.

Using an assay for the direct measurement of callose

present around the Pd, we found that, after wounding

induced by cutting leaves, the level of callose around Pd was

45% higher in AtBG_ppap knockout mutant plants than in

wild-type controls (Figure 9). These results show that

AtBG_ppap deficiency enhances callose accumulation in

response to wounding. Several studies have shown that

accumulation of callose around Pd results in decreased cell-

to-cell movement of fluorescent dyes (Radford and White,

2001; Sivaguru et al., 2000). Taken together, our results

strongly suggest that AtBG_ppap functions in degrading the

callose associated with Pd, and that the reduced cell-to-cell

spread of GFP and the elevated callose levels in AtBG_ppap

mutant plants reflect a decrease in callose degradation. Our

results also support the hypothesis that the level of steady-

state callose accumulation around the Pd is a function of b-

1,3-glucan synthase versus glucanase activities.

The functional results we obtained are similar to those

described for class I b-1,3-glucanases in tobacco. In class I b-

1,3-glucanase-deficient mutant plants, reduced trafficking of

fluorescent probes between cells and induced callose

deposition was also found, although callose intensity was

not measured around the Pd (Iglesias and Meins, 2000). An

important difference, however, exists in the intercellular

localization found for these proteins. While AtBG_ppap was

found to be localized in the Pd, tobacco class I b-1,3-

glucanases were found to be vacuolar proteins (Keefe et al.,

1990). It is possible that some members of the class I family

are indeed primarily vacuolar, but other undetected mem-

bers may localize in Pd.

GPI anchoring of AtBG_ppap in the Pd

Bioinformatic analysis predicts that AtBG_ppap is a GPI-an-

chored protein. This prediction was confirmed by two dif-

ferent proteomic analyses aimed at identifying GPI-anchored

proteins in Arabidopsis (Elortza et al., 2003, 2006). In these

analyses, membrane fractions were treated with phosphati-

dylinositol phospholipase C (PI-PLC) (Elortza et al., 2003)

and phospholipase D (PLD) (Elortza et al., 2006) enzymes in

the presence of Triton X-114. These enzymes hydrolyse the

phosphatidylinositol, releasing a soluble GPI protein from

the membrane. Proteins that were enriched in the aqueous

phase upon PI-PLC or PLD treatments were identified by

liquid chromatography MS/MS. AtBG_ppap (At5g42100)

was identified as GPI-anchored in both analyses.

GPI anchoring, found in all eukaryotic organisms, is a way

to attach proteins to the outer leaflet of the plasma mem-

brane (Udenfriend and Kodukula, 1995). GPI-anchored pro-

teins have a cleavable N-terminal secretion signal for

translocation into the ER. They also have a C-terminal

transmembrane region that is thought to function as a signal

for transamidase, which cleaves the C-terminal hydrophobic

peptide and transfers the protein to a prefabricated GPI

anchor. From the ER, the GPI-anchored protein is transpor-

ted to the cell membrane via the Golgi (Udenfriend and

Kodukula, 1995). Our results confirm that AtBG_ppap:GFP

indeed localizes to the ER and the plasma membrane.

GPI anchoring can serve as a link between the plasma

membrane and the cell wall, as the protein is facing the wall

while attached to the membrane. Callose deposition is

hypothesized to occur in a collar around Pd and to push the

membrane inward, reducing the channel diameter (Olesen

and Robards, 1990; Radford et al., 1998). The proposed GPI-

anchored localization of AtBG_ppap in the outer plasma

membrane of the Pd facing the wall is therefore very

appropriate in terms of function. It will be very interesting

to determine whether GPI anchoring in the Pd is a wider

phenomenon with functional significance.

At present, the mechanism by which AtBG_ppap accu-

mulates in the Pd is unknown. This targeting may be the

result of a special targeting mechanism or an internal

property of the anchored enzyme, which holds it in the



channels. Alternatively, it may be that its accumulation in Pd

is simply the result of much lower rates of turnover in the

protected environment within the Pd.

Possible roles of AtBG_ppap in symplasmic regulation

Our results suggest that AtBG_ppap is active in degrading

callose around Pd and in widening the channels. Closure or

sealing of Pd by callose accumulation and their re-opening

by callose breakdown mediated by b-1,3-glucanase provide

an important regulation mechanism for the symplasmic

communication between cells (Rinne and van der Schoot,

2003). This regulation has a crucial role in both development

and defense, and may indicate possible roles for AtBG_ppap.

For example, in birch, a perennial plant, exposure to short

days induces dormancy and Pd closure by specific structures

around the Pd channel entrances within the shoot apical

meristem (SAM), resulting in symplasmic isolation of the

cells and the blockage of signaling networks (Rinne and van

der Schoot, 1998; Rinne et al., 2001). These structures

contain callose, as determined by immunolocalization and

tannic acid staining, which visualize putative glucan syn-

thase complexes (Rinne and van der Schoot, 1998; Rinne

et al., 2001). After adequate exposure to cold, dormancy is

broken and the SAM regains its potential. In this process,

symplasmic connection is restored and callose disappears

(Rinne et al., 2001). Rinne et al. have found that b-1,3-

glucanase proteins were upregulated during this process,

and that b-1,3-glucanases were localized in spherosome-like

vacuoles or lipid bodies in vicinity of the cell membrane and

in contact with Pd. They suggested that these b-1,3-glucan-

ases are delivered to the Pd and take part in degrading the

plasmodesmal callose sphincters. It would be of interest to

determine whether, upon release from dormancy in peren-

nial plants, an AtBG_ppap homologue is involved in callose

degradation, and whether this regulation is at the transcrip-

tional or translational level.

Symplasmic blockage also occurs during the hypersensi-

tive response to certain pathogens, with the apparent aim of

restricting the spread of the invading pathogen. During this

response, callose is deposited at the cell walls around the

infection sites (Kauss, 1996). A recent paper studying tobacco

plants constitutively expressing the movement protein (MP)

of tomato spotted wilt virus (Rinne et al., 2005) shows that, in

MP-expressing cells in source leaves, Pd are obstructed by

the formation of sphincters that contain callose deposits.

This obstruction was accompanied by a reduction in cell-to-

cell movement. Temperature shift treatments (from 22 to

32�C) restored the transport capacity through Pd, and levels

of b-1,3-glucanase were found to increase. The authors

suggest that the removal of callose from the Pd of

MP-expressing cells is mediated by b-1,3-glucanase. It

should be noted that an increase in callose accumulation as

a result of pathogen-induced stress is not necessarily due

solely to an increase in callose synthase activity alone, but

could be the result of a shift in the balance between callose

synthesis and callose hydrolysis. Thus, a role is suggested

for Pd b-1,3-glucanase during viral pathogenesis in both

symplasmic blockage and symplasmic recovery. The hypo-

thesis that the activity of b-1,3-glucanase breaks down the

callose around Pd and thus promotes cell-to-cell movement

is further supported by the finding that increased levels

of class I b-1,3-glucanase promote virus spread, while

decreased levels in tobacco mutants result in decreased

virus spread (Bucher et al., 2001; Iglesias and Meins, 2000).

The regulation of symplasmic communication by Pd is

of central importance in plant development, involving

temporal regulation of different developmental domains

(Oparka et al., 1999; Rinne and van der Schoot, 2003;

Zambryski, 2004). With the identification of AtBG_ppap as

a Pd-associated protein (henceforth to be annotated as

AtBG_pap), it will be possible to analyze its role in these

processes. Additionally, as callose accumulation is depend-

ent on the equilibrium between callose synthase and callose

hydrolysis, another major future goal will be the identifica-

tion of a Pd-associated b-1,3-glucan synthase, the suggested

‘second half’ of this plasmodesmal regulatory system.

Experimental procedures

Plant material

Nicotiana benthamiana and N. tabacum cv. Samson plants weregrown in 10 cm pots in a mix of equal volumes of potting mixtureand vermiculite (Pecka Hipper Gan, Rehovot, Israel) at 25�C underlong-day conditions (16 h light/8 h dark cycles). Arabidopsis thali-ana ecotype Columbia plants were grown in potting mixture in 6 cmpots at 22�C under long-day conditions. For measurement of calloselevels, plants were grown under short-day conditions (8 h light/16 hdark).

Cell wall preparation

Four- to six-week-old A. thaliana cry2 mutants grown under long-day conditions were harvested and stored at )80�C. The late-flowering cry2 mutant was used in order to obtain a maximumamount of plant material. Plants were pulverized in liquid nitrogen,and homogenized with 2 ml g)1 tissue of buffer A (0.25 M sucrose,4 mM EDTA, 10 mM EGTA, 20 mM Tris/HCl, pH 8.5, 0.02% azide)containing a cocktail of protease inhibitors [1.5 lM aprotonin, 0.01units/ml a2-macroglobulin, 2 mM phenylmethyl-sulfonyl fluoride(PMSF) (Roche, Mannheim, Germany), 42 lM leupeptine, 14.5 lM

pepstatine (Sigma-Aldrich, http://www.sigmaaldrich.com/), 2.5 mM

1.10 orthophenantroline (Merck, www.merck.de) and 14 lM E64(Fluka, www.sigmaaldrich.com/Brands/Fluka_Riedel_Home.html)].The homogenate was filtered through a 16 lm nylon cloth, and thewall fraction (WF) retained on the nylon filter was re-homogenizedwith 2 ml g)1 (starting tissue) of buffer B (0.25 M sucrose, 2 mM

EDTA, 10 mM EGTA, 20 mM Tris/HCl, pH 7.5, 0.02% azide) and pro-tease inhibitors as above. The homogenate was re-filtered througha 5 lm nylon cloth, and the WF was re-suspended with 2 ml g)1

tissue of buffer B and protease inhibitors as above.



The WF kept at 0–4�C was sonicated (560 W and a 0.5 inch flattip) for 10–15 min, with 5 sec on/5 sec off pulses. The WF wasthen centrifuged at 10 000 g for 2 min at 4�C, and the wall pelletwas resuspended with buffer B containing PMSF and 1.10orthophenantroline proteases at the concentrations above. Soni-cation was repeated, and the WF was pelleted at 1000 g for2 min, the supernatant discarded and the pellet compacted at10 000 g for 1 min, and supernatants discarded. The WF wasresuspended and resonicated three or four additional times asdescribed for the second sonication, until no green supernatantwas observed and the wall pellet was a grayish color. After thefourth sonication, the suspension was filtered through a plastickitchen sieve to remove large unbroken pieces of wall (mainlyvascular tissue). After the last sonication and centrifugation, thepellet was re-suspended in two volumes of buffer B withoutproteases and filtered through a 5 lm nylon cloth. The WFcaptured on the nylon filter was then centrifuged at 10 000 g for1 min to compact the wall, and stored at )80�C. All procedureswere performed at 4�C.

Pd preparation

Cellulase preparation. Digestion buffer (10 mM MES, pH 5.5,4.4% mannitol, 0.02% azide) was warmed to 55�C, and 0.6% w/v ofCellulase R10 (Karlan www.karlan.com) was added and stirred untildissolved. The mixture was heated at 55�C for 5 min, filteredthrough 6 lm nylon cloth and cooled to 4�C. Protease inhibitorswere added as described above for cell wall preparation, and thesolution was centrifuged (75 600 g for 40 min at 4�C) to clear thesolution of any particulate matter.

Wall digestion. WF was washed in 1 ml g)1 (fresh tissue) ofdigestion buffer, and centrifuged at 8000 g for 1 min at 4�C. Thewashed wall was then re-suspended in 1 ml g)1 (fresh tissue) ofheat-cured cellulase solution (the enzyme solution was prewarmedto 37�C) and vortexed until no clumps were visible. The solution washomogenized using a Teflon glass homogenizer, transferred to anErlenmeyer flask and gently shaken in a rotary shaker (100 r.p.m.) at37�C for 1.5 h. The solution was then centrifuged at 5860 g for 5 minat 4�C. Both the supernatant and the pellet were collected sepa-rately. The supernatant (Sup I) was adjusted to pH 7 with 18 ll ml)1

1 M Tris, pH 8. The pellet was resuspended in 1 ml g)1 tissue ofMMA buffer (10 mM 3-[N-morpholino] propane-sulfonic acid[MOPS], pH 7.5, 4.4% mannitol, 0.02% azide), homogenized andcentrifuged as before (5860 g, 5 min, 4�C), and the supernatantcollected (Sup II). Sup I and Sup II were combined and filteredthrough a 6 lm nylon cloth, and the filtered supernatant was cen-trifuged at 75 600 g for 40 min. The pellet was resuspended in100 ll g)1 (fresh tissue equivalent) of MOPS washing buffer (10 mM

MOPS, pH 7.5, 0.02% azide) using a Teflon glass homogenizer, andcentrifuged twice at 7 000 g for 5 min at 4�C in Corex tubes. Thesupernatant containing Pd was then centrifuged at 75 600 g for40 min at 4�C and the supernatant was discarded. The compactedPd pellet was stored at )80�C.

PAGE separation

The Pd fraction (equivalent of 50 g plant tissue) was dissolved in50 ll dissolving buffer (9 M urea, 40 mM Tris, 2% SDS, 50 mM DTTand 20% glycerol). The sample was incubated at room temperaturefor at least 1 h and separated by one-dimensional SDS–PAGE on a12.5% gel. Gels were stained with PhastGelTM BlueR (Amersham

Pharmacia, www.amershambiosciences.com) solution for 1 h on arotary shaker (25 r.p.m.). Mild distaining was carried out with 7%acetic acid in 5% methanol. Several changes of distaining solutionwere required till the background was cleared. Bands were excisedfrom the gel and used for protein sequencing.

In-gel proteolysis, chromatography and mass spectrometry

Coomassie blue-stained bands were cut and in-gel reduced with10 mM DTT, incubated at 60�C for 30 min, alkylated with 10 mM

iodoacetamide at room temperature for 30 min, and proteolysedwith trypsin overnight at 37�C, using modified trypsin (Promega,http://www.promega.com/) at a 1:100 enzyme-to-substrate ratio. Thetryptic peptides were resolved by reverse-phase chromatography ona 1 · 150 mm C18 column (Vydac, www.vydac.com). The peptideswere eluted using linear 80 min gradients from 5% to 95% acetonit-rile containing 0.1% acetic acid. Mass spectrometry was performedwusing ion-trap mass spectrometers (LCQ; Thermo) operating in thepositive mode using repetition of a full MS scan followed by CID ofthe most dominant ion selected from the first MS. The MS data warecompared to simulated proteolysis and CID of the proteins in the NR-NCBI database using SEQUEST software (LCQ; Thermo).

RT-PCR

Source rosette leaves and stems were cut from newly floweringArabidopsis plants, while cauline leaves, roots, flowers and siliqueswere cut from fully flowering plants. For each organ type, twosamples were collected, with two repeats, and stored at 80�C. RNAwas purified from 50–100 mg frozen tissue using the SV total RNAisolation system kit according to manufacturer’s instructions(Promega). cDNA first-strand synthesis was performed as previ-ously described (Caldelari et al., 2001). AtBG_ppap and Ubiquitin10coding sequences were amplified by PCR reactions using specificprimers: for AtBG_ppap, forward primer 5¢-CCGATAACCATGG-CTTCTTCTTCTCTGCAGTC-3¢ and reverse primer 5¢-CTATCATCC-TAGGTTACAACCGAAGCTTGATGATGCAAAG-3¢; for Ubiquitin10,forward primer 5¢-CGATTACTCTTGAGGTGGAG-3¢ and reverse5¢-AGACCAAGTGAAGTGTGGAC-3¢. AtBG_ppap was amplifiedusing the following program: 1 cycle of 95�C for 2 min, followed by33 cycles of 94�C for 15 sec, 58�C for 30 sec and 72�C for 1 min, anda final elongation step of 4 min at 72�C. Ubiquitin10 was amplifiedusing the following program: 1 cycle of 95�C for 4 min, followed by35 cycles of 94�C for 20 sec, 55�C for 30 sec and 72�C for 30 sec, anda final elongation step of 5 min at 72�C.

Real-time PCR

Real-time RT-PCR of AtBG_ppap was performed in a fluorescencetemperature cycler (LightCycler; Roche, www.roche.com), usingactin 2 þ 8gene primers for normalization. cDNA corresponding to50 ng of RNA served as a template in a 10 ll reaction containing 5pM gene-specific primers and 5 ll of QuantiTect SYBR green PCRmix (Qiagen, http://www.qiagen.com/). The samples were loadedinto capillary tubes and incubated in the fluorescence thermocycler(LightCycler) for an initial denaturation of 15 min at 95�C. The PCRreaction for AtBG_ppap consisted of 45 cycles of 15 sec at 95�C,20 sec at 55�C, 23 sec at 72�C and 5 sec at 82�C for measurement ofSYBR Green fluorescence. The reaction for actin 2 þ 8 consisted of45 cycles of 15 sec at 95�C, 20 sec at 55�C and 12 sec at 72�C. Theactin 2 þ 8 SYBR Green fluorescence was measured at the end ofeach cycle. The amplification of specific transcripts was confirmed



by producing melting curve profiles at the end of each run andsubjecting the amplification products to electrophoresis on anagarose gel. The primers used for AtBG_ppap were: forward 5¢-CCCCAAACACGTTTCT-3¢ and reverse 5¢-CGTATAGGCGTCCTCA-3¢,and those for actin were: forward 5¢-GGTAACATTGTGCTCAGT-GGTGG-3¢ and reverse 5¢-AACGACCTTAATCTTCATGCTGC-3¢.

Construction of plant expression plasmids

For the expression of AtBG_ppap in plants, we used the pRTL2/newGFPm (pGFP-MRC) plasmid (Rodriguez-Concepcion et al.,1999). Because AtBG_ppap is a GPI-anchored protein, and both itsN- and C-terminal ends are cleaved post-translationally, its codingsequence was divided into two parts, 1–1152 (Glu-N¢) and 1153–1278 (Glu-C¢), which were cloned at both sides of the GFP (Figure 2).Glu-C¢ was PCR-amplified using forward primer 5¢-CAT-TAGTGAGCTCTATCAGCCAGTCACGGGTAACC-3¢ containing a SacIsite, and reverse primer 5¢-CTATCATCCTAGGTTACAACCGA-AGCTTGATGATGCAAAG-3¢ containing an AvrII site, and clonedbetween the SacI and XbaI sites in pGFP-MRC, in-frame with GFP atits 3¢ end (XbaI and AvrII generate the same 5¢ protruding end). Glu-N¢ was PCR-amplified using forward primer 5¢-CCGA-TAACCATGGCTTCTTCTTCTCTGCAGTC-3¢ and reverse primer 5¢-CGAATTACCATGGAGATGCCACCACCGCTGGA-3¢, both containinga NcoI site, and cloned in-frame into the unique NcoI site present atthe 5¢ beginning of the GFP coding sequence. For expression inplants, the plasmid was digested using SphI, and the resultingcassette, containing the CaMV 35S promoter, AtBG_ppap fused toGFP, and the nos transcriptional terminator, was purified from theagarose gel and its protruding 3¢ ends cleaved with T4 polymerase(Fermentas, www.fermentas.com) to give blunt ends. This cassettewas subcloned into the SmaI site in pCAMBIA 2300 (CAMBIA,www.cambia.org) to create plasmid pCambiaAtBG_ppap:GFP.

Expression in plants

Stable expression of pCambiaAtBG_ppap:GFP in N. tabacum cv.Samson plants using a modified leaf disk method, and transientexpression of pCambiaAtBG_ppap:GFP in Nicotiana benthamianaplants using Agrobacterium tumefaciens leaf injection were bothperformed as described by Sagi et al. (2005).

Particle bombardment of pCambiaAtBG_ppap:GFP into vegetat-ive N. benthamiana source leaves was performed as describedbelow.

FM4-64 membrane staining and plasmolysis

Plasma membrane staining was performed by incubating 7 · 3 mmpieces of AtBG_ppap:GFP transgenic source leaves in 1:500 dilutedN-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl]pyridinium dibromide (FM4-64; Molecular Probes, www.probes.invitrogen.com) in double-distilled water, for 1.5 h.

For plasmolysis, the FM4-64-stained leaf discs were transferred to0.75 M mannitol solution, and incubated for 10 min. Plasmolysiswas determined by monitoring the retraction of FM4-64-stainedplasma membrane from the cell wall.

Mutant identification

Arabidopsis T-DNA insertion lines (SALK_019116.47.55.x andSAIL_115_G04) were obtained from the Arabidopsis BiologicalResource Center, Ohio State University, Columbus, OH, USA. Thepresence and location of each T-DNA insertion were determined by

PCR analysis using primers for the left border of the insertion andthe gene sequence. For the SALK mutant, the left border primerLBa1 (Alonso et al., 2003) and the Glu-N¢ forward primer (see above)were used, and for the SAIL mutant, the left border primer LB3(Sessions et al., 2002) and the Glu-N¢ reverse primer (see above)were used. Then, to identify homozygous plants, the Glu-N¢sequence was amplified (see above) and plants with no PCR productwere selected. Verification of the homozygous plants was carriedout by RT- PCR performed with leaf RNA, and PCR amplification ofthe AtBG_ppap coding sequence. Ubiquitin10 was used as a posit-ive control (see above).

Particle bombardment and GFP mobility assays

Bombardment of GFP into leaves for GFP movement assays wasperformed as described by Liarzi and Epel (2005), except that sourcerosette leaves from 3-week-old Arabidopsis plants which had notyet bolted were used (about six leaves were placed on the agar platefor each bombardment), and 1 lm gold particles were used insteadof tungsten.

To determine the number of cells into which GFP diffused, cells ofthe lower epidermis of the leaf were analyzed 48 h postbombard-ment using a CLSM (LSM 510; Zeiss, http://www.zeiss.com/). Foreach transformed cell, GFP intensity in the nucleus was set justbelow saturation by setting detector gain values, in fixed laser andamplifier gain values (5% and 1 respectively). Cell clusters wereanalyzed after amplification by amplifier gain and by laser over aninefold range. These amplifications were shown to be in the linearrange of the instrument (Liarzi and Epel, 2005). GFP movement wasdetermined by counting the cells around the transformed cell thatclearly showed fluorescence in the nucleus and the cytoplasm. Cellsthat synthesized GFP but showed no movement were not analyzedin order to avoid including seriously wounded cells.

Calculation of the coefficient of conductivity of Pd was performedin Arabidopsis as described by Liarzi and Epel (2005), with modi-fications: instead of analyzing all first-degree cells (cells with acommon cell wall with the transformed cell; also termed ‘cell 1’) andsecond-degree cells (cells with common cell wall with cell 1 but notwith the transformed cell; also termed ‘cell 2’), the analysis wasperformed on four cells of each degree. The cells selected are thoselocated above, below and on both sides of the transformed cell inthe slice. The reason for this alteration was the big variety in thesizes of cells in the Arabidopsis epidermis and its disorderedorganization, which makes the identification of clear first- andsecond-degrees cells difficult.

One-way ANOVA and box-plot analyses were performed usingSPSS 12.0.1 statistical software (SPSS Inc., Chicago, IL, USA).

Callose staining and quantification

Callose staining for co-localization of AtBG_ppap:GFP and callosewas performed by incubating AtBG_ppap:GFP transgenic tobaccosource leaf segments for 15 min in a mixture of 0.1% aniline blue(Fluka, www.sigmaaldrich.com/brands/Fluka_Riedel_home.html)in double-distilled water and 1 M glycine, pH 9.5, at a volume ratioof 2:3. For better visualization, a negative image was generatedusing ADOBEPHOTOSHOP 7.0 ME software, and the resulting GFP andaniline blue images were converted to red and blue, respectively,using the LSM 5 image browser (Zeiss).

After wounding, callose quantification was performed by meas-uring aniline blue fluorescence intensity. Leaves of wild-type and T-DNA insertion mutant plants (SALK_019116.47.55.x; see above) weregrown for 2 months under short-day conditions to prevent floweringand allow continuing leaf growth. Five leaf discs from two plants



were analyzed for each experiment. Leaf sections of 7 · 3 mm werecut, placed on a 1% agar Petri dish for 2 days, and then immersed in85% ethanol overnight until bleached, and transferred to aniline bluesolution (see above) for 5 h. Aniline blue fluorescence was viewedand photographed under UV conditions in a DMRBE fluorescencelight microscope (Leica, www.leica.com). Pictures were taken ran-domly from the center of the leaf; when a vein was present, the areanext to the vein was photographed due to its strong background.

Fluorescence intensity analysis of aniline blue in the Pd was per-formed under non-saturation excitation levels (exposure time0.5 sec) using the image processing software IMAGEJ version 1.31(http://rsb.info.nih.gov/ij). Callose fluorescence intensity associatedwith Pd was determined by subtracting the minimum fluorescencevalue in the area adjacent to the Pd (background) from themaximum fluorescent value of the Pd foci (Figure 9).

A statistical independent samples t test was performed using theSPSS 12.0.1 statistical software.

Microscopy

Fluorescence microscopy. Fluorescence was viewed with aDMRBE fluorescence light microscope (Leica); aniline blue-stainedcallose fluorescence was measured with a band-pass 340–380 nmexcitation filter, an RKP 400 dichromatic mirror, and a long-pass425 nm emission filter; GFP fluorescence was viewed with a band-pass 450–490 nm excitation filter, an RKP 510 dichromatic mirror,and a band-pass 515–560 nm emission filter (Leica).

Confocal fluorescence microscopy. Fluorescence was viewedwith a CLSM (LSM 510; Zeiss). GFP fluorescence was excited with a488 nm argon laser, and emission was detected with a 505–530 nmband-pass filter combination. FM4-64 fluorescence was excited witha 514 nm argon laser, and emission was detected with a 585 nmlong-pass filter combination. To expose spongy mesophyll forimaging, leaves were torn by hand so that the epidermis at the lowerface of leaf sections was peeled off.

Acknowledgements

We thank U. Hannania and A. Avni (Tel Aviv University, Israel) forkindly supplying pBinGFP plasmid, S Yalovsky (Tel Aviv University,Israel) for kindly supplying pRTL2/newGFPm plasmid, and theSmoler Proteomic Center, Faculty of Sciences, Tel Aviv University,for protein sequencing. This research was supported by ResourceGrant Award IS-3222-01C from the US–Israel Binational AgriculturalResource and Development Fund, by the Israel Science Foundation(grant 723/00-17.1) and by the Manna Institute for Plant Biosciences.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Alignment of Arabidopsis GPI-anchored B-1,3-gluca-nases.This material is available as part of the online article from http://www.blackwell-synergy.com

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Accession numbers: AtBG_ppap: NM_123575; AtBG_ppapas: NM_203139



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The Plant Journal A plasmodesmata-associated b-1,3-glucanase in Arabidopsis · 2017. 10. 23. · Theenzymethat degradescallose,b-1,3-glucanase,isalso an important factor in the regulation