Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi...

Protoplasma (1998) 203:26-34 :IOTOPt $MA �9 Springer-Veflag 1998 Printed in Austria

Immunolocalization of and [5-(l 6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

Mait6 Vicr6 x'**, Alain Jauneau 1, J. Paul Knox 2, and Azeddine Driouich 1,*

1Unit6 Propre de Recherche et d'Enseignement Sup6rieur Associe6 au CNRS 6037, Universit6 de Rouen, Mont Saint-Aignan, and 2Centre for Plant Biochemistry and Biotechnology, University of Leeds, Leeds

Received December 31, 1997 Accepted March 23, 1998

Summary. Most cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to [3-(1--.6) and [3-(1-*4)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that [3-(1-~4)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, [3- (1-~6)-D-galactau epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of [3-(1--*4)-D-galactan epitopes are high- ly regulated in developing flax roots and that different [3-1inked D- galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.

Keywords: Flax; Cell wall; Golgi apparatus; [3-Galactans; Immuno- cytochemistry,

Introduction

The plant cell wall is a dynamic structure that plays important roles in the growth and development as

well as in the interactions of plants with the environ-

*Correspondence and reprints: Unit6 Propre de Recherche et d'Enseignement Sup6rieur Associe6 au CNRS 6037, Universit6 de Rouen, F-76821 Mont Saint-Aignan Cedex, France. **Current address: Department of Botany, University of Cape Town, Rondebosh, South Africa.

ment and other organisms. It is composed of cellulose

microfibrils embedded in a matrix of polysaccharides

and proteins (glycoproteins and proteoglycans). Cel-

lulose is known to be synthesized at the plasma membrane by cellulose-synthesizing complexes, whereas

wall matrix components are assembled in the Golgi

apparatus and transported to the cell surface in Golgi- derived secretory vesicles (Delmer and Amor 1995,

Moore and Staehelin 1988, Moore et al. 1991, Dri- ouich and Staehelin 1997). The cell wall-matrix components consist of pectin and hemicellulose polysac-

charides, and glycoproteins including hydroxypro-

line-rich glycoproteins (i.e., extensin) and arabino-

galactan proteins (AGPs) (Carpita and Gibeaut 1993).

However, unlike extensins that are present only in the

cell wall, proteoglycans of the AGP class occur both

in the cell wall and in the plasma membrane (Knox et al. 1991, Komalavalis et al. 1991, Serpe and Noth- nagel 1996). These components are essential in deter- mining the structural and functional properties of the

plant cell surface, and it is therefore important to elu- cidate their distribution and localization within differ-

ent tissues and within the wall of individual cells in relation to growth and development.

The recent generation and use of immunological

probes recognizing different plant carbohydrate epi-

topes of cell wall components have proven extremely useful in locating these polysaccharides in various plant species (for a review, see Knox 1997). Several immunocytochemical studies have used antibodies specific to epitopes associated with arabinogalactan

M. Vicr6 et al.: Localization of S-linked D-galactan epitopes in flax roots 27

p ro te ins to s h o w the i r ce l l t ype - spec i f i c d i s t r i b u t i o n

in d i f fe ren t p l an t cel ls ( K n o x et al. 1991, S tacey et al.

1990, S c h i n d l e r et al. 1995, D o l a n et al. 1995). S i m i -

larly, a n t i b o d i e s speci f ic to c o m p l e x p o l y s a c c h a r i d e s

h a v e also b e e n u s e d to loca te d i f f e ren t ep i topes no t

o n l y to d i f f e ren t t i ssues , b u t also to d i f f e ren t ce l l wa l l

d o m a i n s ( M o o r e et al. 1986; V ian a n d R o l a n d 1991;

L y n c h and S t a e h e l i n 1992, 1995; M a r t y et al. 1995;

F r e s h o u r et al. 1996; His et al. 1997; J a u n e a u et al.

1997) and to d i f fe ren t c i s t e rna l c o m p a r t m e n t s of the

G o l g i appa ra tus ( Z h a n g an d S t a e h e l i n 1992, L y n c h

and S t a e h e l i n 1992). Together , these f i n d i n g s d e m o n -

strate that the syn thes i s , secre t ion , an d l o c a l i z a t i o n o f

cel l wa l l c o m p o n e n t s are r e g u l a t e d in a ce l l t y p e - s p e -

cif ic m a n n e r . H o w e v e r , n o n e of these s tud ies has c o n -

s ide red two ep i topes o f wa l l p o l y s a c c h a r i d e s c o n t a i n -

ing the s a m e suga r r e s idues d i f f e r ing o n l y in the i r

l inkage .

In this s tudy, we repor t on the spa t ia l d i s t r i b u t i o n o f

two D - g a l a c t a n ep i topes , ~ - ( 1 - ~ 4 ) - a n d [3-(1---~6)-

l i nked , f o u n d on cel l wa l l p o l y s a c c h a r i d e s ( Jones

et al. 1997, K i k u c h i et al. 1993) in f lax roo t t ips b y

i m m u n o c y t o c h e m i c a l t e ch n i q u es . O u r resul t s show

that u n l i k e [3-(1--~6)-D-galactans , w h i c h are de tec ted

in all t i ssues , 1 3 - ( l - * 4 ) - D - g a l a c t a n ep i topes are o n l y

f o u n d in pe r iphe ra l cel ls . ~ - ( 1 - ~ 4 ) - D - G a l a c t a n s are

n o t de tec ted in c o l u m e l l a an d m e r i s t e m a t i c cel ls . In

cont ras t , bo th ep i topes exh ib i t s imi l a r l o c a l i z a t i o n

w i t h i n i n d i v i d u a l wa l l s an d G o l g i s tacks of d i f f e ren t

roo t t i ssues . To our k n o w l e d g e this is the f irst r epor t

in w h i c h two d i f f e ren t ly ~ - l i n k e d D - g a l a c t a n s o f

i m p o r t a n t wa l l c o m p o n e n t s are s h o w n to be d i f f e ren -

t ia l ly exp re s sed in p l an t roo t t i ssues .

Material and methods Plant material

Seeds of flax (Linum usitatissimum L. var. Ariane) were received as a gift from La coop6rative de Fontaine Cany, Normandie, France. Seeds were germinated on moist filter paper in the dark for 36 h. Roots (ca. 2 mm from the tip) were excised and then used for different experiments.

Electron microscopy

Root tips were fixed with 1% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2). After rinsing (3 • 5 rain) in the same buffer, the root tips were post-fixed in 1% OsO4 in distilled H20 for 1 h, and dehydrated at room temperature in a graded aqueous ethanol series (10, 20, 40, 60, 80, 100%; 20 rain each step). The dehydrated root tips were then infiltrated either in Spurr (for structural observations) or in LR White (for immunogold labeling experiments) embedding resin. Infiltration with LR White was performed according to the following schedule: 2 : 1; 1 : 1; 1 : 2 (v/v) ethanol : LR White resin for 2 h each step, and 100% LR White

overnight, then 24 h with a change of resin every 8 h. The infiltrated samples were embedded in gelatin capsules and allowed to polymer- ize for 24 h at 60 ~ Infiltration with Spurr resin was performed as follows: after dehy- dration, root tips were placed in Spurr : acetone 2 : 1; 1 : 1; 1 : 2 (v/v) for 2 h each step and then 24 h pure Spurr at room temperature with a change of resin every 8 h. The polymerization was accom- plished at 60 ~ for 24 b.

Immunolabeling procedures

The antibodies used in this study are: (1) the monoclonal antibody LM5, specific to ~-(1 ~4)-D-galactans (Jones et al. 1997) and (2) the anti-Gal4 antibodies recognizing (3-(1-*6)-linked D-galactosyl resi- dues (Kikuchi et al. 1993). All incubations were carried out at room temperature. Thin sections (90 nm) mounted on gold grids were incubated in a blocking solution of 3% low-fat dried milk in PBST (10 mM Na-phosphate, 500 nm NaC1, 0.1% Tween-20) for 30 rain. After the grids were blotted dry, they were incubated overnight with the primary antibodies LM5 (diluted 1 : 5 in PBST) and anti-Gal4 (diluted 1 : 1000 in PBST). The grids were then washed with PBS containing 0.5% Tween 20 for 1-2 rain before their transfer to a droplet of goat anti-rat IgG conjugated to 10 nm colloidal gold diluted 1 : 40 in PBST (for LM5-1abeled sections) or to a goat anti-rabbit IgG conjugated to 10 nm colloidal gold solution diluted 1 : 40 in PBST (for anti-Gal4-1abeled sections) for 45 rain. The sections were washed with PBS containing 0.5% Tween-20 followed by distilled-water wash. After immunolabeling, the sections were stained with 2% uranyl acetate for l0 min and Reynolds (1963) lead stain for 30 to 60 s and observed at 80 kV in a Zeiss electron microscope (EM 109). Control experiments were performed in each case by omission of primary antibodies. Double immunolabeling experiments have been performed as previously described by Moore et al. (1991). Sections destined for ultrastructural examinations were directly stained and observed.

Light microscopy

Light microscopy was carried out on an Axioskop microscope (Zeiss) equipped with epifluorescence optics. Epifluorescence was performed on the same processed tissue used for electron microscopy. Thicker (250 to 500 nm) sections were cut from LR White-embedded samples and mounted on glass microscope slides. Immunostaining was done as described for immunogold labeling except that the secondary antibody was conjugated to fluorescein isothiocyanate (FFI'C) instead of gold particles and used at a concen- tration of 1 : 50. Epifluorescence of the immunostained tissue sections was observed with a Zeiss filter set (excitation filter 485, barri- er filter 515-565). In some cases, sections were cut from Spurt- embedded roots, etched with NaOH/methanol, 6% (w/v), as previously described (Jauneau et al. 1997) before use for immunostaining.

Results

General description of the anatomy of f lax root apex

The ex te rna l a n a t o m y o f the f lax root tip 36 h after ger-

m i n a t i o n is s h o w n in Fig. 1. This i l lus t ra tes the char-

acter is t ic o rgan iza t ion o f d i f fe rent t i ssues , the stele,

the cortex, the apical mer i s t em, and the roo t cap w h i c h

is c o m p o s e d o f c o l u m e l l a and pe r iphera l cells .

28

Fig. 1. Longitudinal section of a flax root tip stained with toluidine blue showing location of apical meristem (m), stele (s), cortex (c), and the root cap composed of columella (Co) and peripheral (p) cells. Bar: 100 btm

Electron micrographs of root cap cells, columella and peripheral cells, and meristematic ceils are shown in Fig. 2. Numerous Golgi stacks are seen in the cytoplasm of meristematic cells (Fig. 2 a). In flax roots, these stacks are compact and small in size (ca. 0.6 ~tm in diameter; Fig. 2 b) and have small vesicles com- pared to those of peripheral cells (Fig. 2 f). Cis-to- trans polarity is clearly distinguished and most, but not all, of the Golgi stacks exhibit an associated trans-

M. Vicr6 et al,: Localization of [3-1inked D-galactan epitopes in flax roots

Golgi network (TGN). Columella cells, which are derived from the apical meristem, are characterized by the presence of numerous amyloplasts in the cytoplasm (Fig. 2 c). The morphological features of Golgi stacks in columella cells resemble those of meristematic cells with a very distinct cis-to-trans polarity. However, the diameter of the Golgi vesicles in columella ceils is usually larger (Fig. 2 d). Peripheral cells are typified by the presence of numerous slime- containing vesicles and hypertrophied Golgi stacks (Fig. 2 e). Golgi-derived vesicles, as shown in Fig. 2 f, are larger in size than Golgi vesicles of columella and meristematic cells. Together these findings show that the process of differentiation of meristematic cells into peripheral cells is accompanied by marked changes in Golgi morphology that may reflect changes in the secretory state of this organelle.

Distribution of[3-(1-~6)-D-galactan epitopes in flax root tips

To examine the tissue distribution of ~-(I---~6)-D- galactans in developing flax roots, we have used immunofluorescence microscopy in conjunction with the polyclonal antibody anti-Gal4 generated by Kikuchi et al. (1993). Immunofluorescent labeling of a longitudinal section of root tips with this antibody is shown on Fig. 3 a and b. Labeling is associated with the cell wall of all cells of the root tip with apparent equal intensity. The three-way junctions are not labeled (Fig. 3 b). Examination of the labeling by immunogold electron microscopy demonstrates that gold particles are mainly found over that part of the wall close to the plasma membrane in different cell types (Fig 3 c, d). No labeling is observed within the central part of the cell wall distant from the plasma membrane (Fig. 3 c). In addition, some of the labeling appears to be over the plasma membrane. Figure 3 c and 3 e shows that secretory Golgi-derived vesicles and Golgi cisternae (mostly trans-cisternae) of different cell types are also labeled, thus indicating that galactan-containing molecules of the cell surface are synthesized and secreted via the Golgi apparatus.

Fig. 2 a-f. Micrographs illustrating the general morphology of different cell types (a, c, and e) and Golgi stacks (b, d, and f) of flax root tip. a A meristematic cell with a relatively large nucleus (n) and numerous small vacuoles (v) within the cytoplasm, c A columella cell characterized by the presence of several amyloplasts containing large starch granules (s). e A peripheral cell typified by the presence of numerous Golgi- derived, slime-containing vesicles, b, d, and f Golgi stacks in meristematic cells (b), columella (d), and peripheral (t') cells. Note the large size of the secretory vesicles (sv) in peripheral ceils and their filamentous-slime contents. The cis-to-trans structural polarity is clearly distinguished in all cell types. Cw Cell wall; er endoplasmic reticulum; m mitochondria; TGN trans-Golgi network. Arrowheads point to Golgi stacks. Bar: in a, c, and e, 1.5 lxm; in b and d, 0.15 gin; in f, 0.2 ~tm

M. Vicr6 et al.: Localization of [3-1inked D-galactan epitopes in flax roots 29

30 M. Vicr6 et al.: Localization of B-linked D-galactan epitopes in flax roots

Fig. 3 a-e. Immunolabeling with anti-Gal4 antibodies, a Immunofluorescence labeling of a longitudinal section of a flax root tip. Labeling is associated with all cell types of the root apex including the stele, the meristem, columella, and peripheral cells, b Higher-magnification image showing immunofluorescence staining in the stellar tissues in the zone of the onset of root elongation. Note that the three-way junctions are not stained (arrowhead). e Immunogold labeling of a peripheral cell with anti-Gal4 antibodies. The labeling is mostly associated with that portion of the wall adjacent to the plasma membrane. Note the labeling of Golgi-derived secretory vesicles (arrowhead). d Similar labeling is seen over the cell wall of stellar cells, e Immunogold labeling of a Golgi stack of a meristematic cell. Labeling is mostly associated with trans-type of cis- temae and vesicles (arrowheads). Cw Cell wall; g Golgi stack. Bar: in a, 50 txm; in b, 100 pxm; in c, 0.25 ~xm; in d and e, 0.15 gm

M. Vicr6 et al.: Localization of [3-linked D-galactan epitopes in flax roots 31

Fig. 4 a-d. lmmunolabeling with the monoclonal antibody LM5. a lmmunofluorescence staining of a longitudinal section of flax roots. Label- ing is associated exclusively with peripheral ceils of the root cap. b Detail from a of labeled peripheral cells showing immunofluorescence staining close to the plasma membrane and no labeling of the middle lamellae (arrowhead). c Immunogold labeling of the cell wall of peripheral ceils. Note that most of the labeling is over the zone of the cell wall immediately adjacent to the plasma membrane, d Immunogold labeling of Golgi stacks and secretory vesicles (arrowheads) in a young peripheral cell. cw Cell wall; v vacuole. Bar: in a, 100 ~tm; in b, 50 ~tm; in c, 0.i5 ~tm; ind, 0.2 ~tm

Localization of [3-(1 ~4)-D-galactans with the monoclonal antibody LM5

Figure 4 a and b shows the i m m u n o f l u o r e s c e n t reac-

t ion o f the L M 5 an t ibody with the root apex t issues.

In the root cap region , f luorescen t l abe l ing is

obse rved exc lus ive ly over pe r iphe ra l cel ls , and no

labe l ing is seen over co lume l l a or mer i s t ema t i c cel l

wal ls , or in any cel l wi th in a p p r o x i m a t e l y 1.5 m m

from the root t ip (Fig. 4 a). As wi th an t i -Ga l 4 an t ibod-

ies, no l abe l ing is seen with L M 5 in midd l e l ame l l a

and cel l corners in the root cap cel ls (Fig. 4 b).

A t the subce l lu la r level , i m m u n o g o l d l abe l ing

demons t ra tes that L M 5 an t ibody labels mos t l y that

par t o f the wal l ad jacen t to the p l a s m a m e m b r a n e

(Fig. 4 c). In contrast , no l abe l ing has been o b s e r v e d

in the wal l or Go lg i s tacks o f mer i s t ema t i c cel ls or

co lume l l a cel ls (not shown). L ike ant i -Gal4 an t ibod-

ies, L M 5 labels secre tory ves ic les and Go lg i s tacks in

pe r iphe ra l cel ls (Fig. 4 d) sugges t ing that ~-(1--*4)-D-

32 M. Vicr6 et al.: Localization of B-linked D-galactan epitopes in flax roots

galactan-containing molecules are also generated within the Golgi apparatus. Moreover, double immunoIabeling with both antibodies, anti-Gal4 and LM5, shows that both epitopes co-localize not only in the cell wall but also in Golgi vesicles (Fig. 5 a). Con- trol experiments performed with the omission of the primary antibodies show no labeling (Fig. 5 b, c).

Discussion

The immunolabeling studies described in this report demonstrate that galactan epitopes are distributed in distinct patterns in flax root ceils. The most important finding here is that whilst 13-(l~6)-D-galactans are present in all cell types, ~-(l~4)-D-galactan epitopes, recognized by the monoclonal antibody LM5, are exclusively restricted to peripheral root cap cells. These results further demonstrate that root cells can regulate the structure of their walls in a cell type-specific and developmental manner.

[3-(1-+4)-D-GaIactans as markers of differentiation of peripheral cells in flax roots

In plant cells, ~-(1--*4)-D-galactans are mostly found attached to side chains of cell wall pectins, but they can also exist as independent and free molecules (Brett and Waldron 1990). Both types of molecules can be recognized by LM5 antibodies. At the root cap region of flax, we found that ~-(1-*4)-D-galactan epitopes are restricted to peripheral cells. The exclusive presence of [3-(l~4)-D-galactans in these cells but not in meristematic and columella cells might reflect functional differences between these cell types. Peripheral cells function in water absorption and it is therefore not surprising that ~-(1-*4)-D-galactan- containing-molecules (e.g., pectins of RGI type), which are known to have water-binding capabilities (Guinel and McCully 1986), are present in the wall and the slime molecules secreted by these cells. In addition, either free galactans and galactan-containing polysaccharides secreted by peripheral cells may function with other molecules to lubricate the outer surface of the root as it pushes through the soil. Peripheral ceils of plant roots are known to differenti-

Fig, 5, a Double immunolabeling with both anti-Gal4 and LM5 anti-

bodies of a peripheral cell. Note the co-localization of gold particles

of both sizes (10 nm for anti-Gal4 and 20 nm for LM5) in the cell wall (mostly close to the plasma membrane). Labeling is also seen

over Golgi vesicles (arrowheads). b and e Control experiments: none or few gold particles are seen over the walls of control samples in which primary antibodies LM5 (b) or anti-Gal4 (e) are omitted, c w

Cell wall; g Golgi stack. Bar: in a and b, 0.22 urn; in c, 1 ~m

M. Vicr6 et al.: Localization of [3-1inked D-galactan epitopes in flax roots

ate from graviperceptive columella cells of the inner root cap which themselves originate from the apical meristem. In flax, this differentiation coincides with the expression of ~-(l~4)-D-galactan epitopes by peripheral cells which may result from an in muro unmasking of the epitope due to a structural and/or conformational re-organization of wall polysaccharides. It would thus be possible that differentiation of peripheral cells in flax roots is accompanied by cell wall changes which make the epitope accessible to the antibody. However, the fact that Golgi stacks in meristematic and columella cells are not labeled with LM5 antibodies, which suggests that [3-(1-~4)-D- galactans are not synthesized in these cells, does not support this explanation. We thus favor the interpreta- tion in which Golgi stacks of peripheral cells, but not columella or meristematic cells, synthesize large amounts of [3-(l~4)-D-galactan-containing polysaccharides and secrete them to the cell surface. Evi- dence for such a cell type-specific functional organization of the Golgi apparatus has come from different studies on various plant species (Staehelin et al. 1990; Lynch and Staehelin 1992, 1995). For instance, it has been shown that the differentiation of columella ceils into peripheral cells in clover is marked by an increase in the synthesis and secretion of pectic polysaccharides recognized by PGA/RGI antibodies (Lynch and Staehelin 1992). Such a cell type-specific macromolecular organization of plant Golgi stacks has been shown to be related to marked changes in their structure (Staehelin et al. 1990, Moore etal. 1991). In flax roots, we have also observed distinct differences in the morphology of the Golgi between meristematic, columella, and peripheral cells (Fig. 2), which reflect most probably the secretory state of these cells. Golgi stacks in meristematic cells from which columella and peripheral cells originate are small in size and produce small vesicles. In contrast, Golgi stacks of peripheral cells are larger in diameter and have very large vesicles filled with slime polysaccharides (Fig. 2 f). These vesicles are labeled with LM5 antibodies indicating that 6-(1-~4)-D-galactans are among the secreted material of peripheral cells. Thus, our findings may not only reflect an alteration of the structure and function of Golgi stacks in flax root cap cells as in clover roots (Lynch and Staehelin 1992), but most importantly demonstrate that differentiation of peripheral cells in flax is marked by an active synthesis and secretion of 6-(1-,4) galactans (either free or associated with pectins) by the Golgi apparatus.

33

Expression of[J-(1 ---*6 )-D-galactan-containing molecules

Unlike ~-(l~4)-D-galactans, ~-(l~6)-D-galactans epitopes recognized by anti-Gal4 antibodies are found in the wall of all cell types of flax roots. These antibodies have been previously shown to be specific toward galactan side chains of AGPs and have been used to localize these molecules in radish roots (Kikuchi et al. 1993). AGPs are a class of plant cell surface proteoglycans composed of a high proportion of carbohydrate (up to 95%), consisting of a ~-(1-~3)- D-galactan backbone to which ~-(l~6)-D-galactan side chains are attached (Fincher et al. 1983). The use of antibodies recognizing AGPs has shown an exten- sive regulation of AGP carbohydrate epitopes in plant cells (Knox et al. 1989, 1991; Pennell et al. 1991; Schindler eta1. 1995) and several studies have demonstrated the tissue-specific expression of AGPs during root development (Knox et al. 1989, 1991; Dolan et al. 1995). Using the anti-Gal4 antibodies, we have found no tissue-specific expression in the developing roots of flax. In contrast, in radish roots, ~- (l~6)-D-galactan epitopes are exclusively expressed in peripheral and epidermal cells of the root tip, but not in other cells (Kikuchi et al. 1993), indicating a strict developmental regulation of the associated AGPs in this plant. The abundant production of ~- (l~6)-D-galactan-containing AGPs by peripheral and epidermal cells in radish probably reflects a specific function of these molecules in these cells. AGPs are known for their water-holding capabilities and stickiness and may thus be involved in water retention and/or adhesion between the root cap and soil micro- organisms. These epitopes could also serve as markers of the differentiation of peripheral and epidermal cells in radish and might even be required to establish this differentiation. Expression of particular AGP epitopes has been shown to precede differentiation of flowers in pea and oilseed rape (Pennell and Roberts 1990, Pennell et al. 1991), as well as differentiation of somatic embryos in carrot suspension cells (Stacey et al. 1991). However, the exact mechanisms and to what extent AGPs are involved are not known. Our finding that there is no tissue-specific location of the ~-(1-~6)-D-galactan epitopes in flax roots further demonstrates a variation in the regulation of AGPs expression and distribution between different plant species. These and other observed variations raise the question to how the expression of AGPs epitopes is regulated in different plants and tissues. Since all of

34 M. Vicr6 et al.: Localization of [3-1inked D-galactan epitopes in flax roots

the antibodies used to date are directed against the carbohydrate moiety of AGPs, the essential of the regulation is likely to involve changes in the glycosylation pattern via glycosyltransferase or glycosidase activities associated with the Golgi apparatus. Future studies will help to determine the relationship between AGP expression in different plant tissues and the glycosylation patterns of these molecules.

Acknowledgements We thank Dr. Y. Tsumuraya (Saitama University, Japan) for provid- ing anti-Gal4 antibodies.

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Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi...

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Transcript of Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi...