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 Ca 2þ -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 669 Journal compilation ª 2007 Blackwell Publishing Ltd The Plant Journal (2007) 49, 669–682 doi: 10.1111/j.1365-313X.2006.02986.x
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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.
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.
A plasmodesmal b-1,3-glucanase 673
ª 2006 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 669–682
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
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.
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ª 2006 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 669–682
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
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
676 Amit Levy et al.
ª 2006 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 669–682
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.
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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
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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
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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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