Spatial and temporal variability of xylan distribution in differentiating secondary xylem of hybrid...

ORIGINAL ARTICLE

Spatial and temporal variability of xylan distributionin differentiating secondary xylem of hybrid aspen

Jong Sik Kim • David Sandquist • Bjorn Sundberg •

Geoffrey Daniel

Received: 14 November 2011 / Accepted: 13 December 2011 / Published online: 30 December 2011

� Springer-Verlag 2011

Abstract Xylans occupy approximately one-third of the

cell wall components in hardwoods and their chemical

structures are well understood. However, the microdistri-

bution of xylans (O-acetyl-4-O-methylglucuronoxylans,

AcGXs) in the cell wall and their correlation with func-

tional properties of cells in hardwood xylem is poorly

understood. We demonstrate here the spatial and temporal

distribution of xylans in secondary xylem cells of hybrid

aspen using immunolocalization with LM10 and LM11

antibodies. Xylan labeling was detected earliest in fibers at

the cell corner of the S1 layer, and then later in vessels and

ray cells respectively. Fibers showed a heterogeneous

labeling pattern in the mature cell wall with stronger

labeling of low substituted xylans (lsAcGXs) in the outer

than inner cell wall. In contrast, vessels showed uniform

labeling in the mature cell wall with stronger labeling of

lsAcGXs than fibers. Xylan labeling in ray cells was

detected much later than that in fibers and vessels, but was

also detected at the beginning of secondary cell wall for-

mation as in fibers and vessels with uniform labeling in the

cell wall regardless of developmental stage. Interestingly,

pit membranes including fiber–, vessel– and ray–vessel pits

showed strong labeling of highly substituted xylans

(hsAcGXs) during differentiation, although this labeling

gradually disappeared during pit maturation. Together our

observations indicate that there are temporal and spatial

variations of xylan deposition and chemical structure of

xylans between cells in aspen xylem. Differences in xylan

localization between aspen (hardwood) and cedar (soft-

wood) are also discussed.

Keywords Aspen � Immunolocalization � LM10 and

LM11 antibodies � Populus � Xylan � Xylem development

Abbreviations

AGXs Arabino-4-O-methylglucuronoxylans

G lignin Guaiacyl lignin

hsAcGXs Highly substituted O-acetyl-4-O-

methylglucuronoxylans

IL Intercellular layer

lsAcGXs Low substituted O-acetyl-4-O-

methylglucuronoxylans

MLcc Middle lamella cell corner

PL Protective layer

PW Primary cell wall

S lignin Syringyl lignin

Introduction

Xylans represent one of the main hemicelluloses of wood

cell walls and have a complex structure with variations in

the concentration and structure between wood species. In

hardwoods (angiosperms), O-acetyl-4-O-methylglucuron-

oxylans (AcGXs) are the main hemicellulose, occupying

about 30% of total cell wall components. AcGXs are

composed of a backbone of xylose units with several

substitutions, including 4-O-methylglucuronic acid with

a-(1-2)-glycosidic linkages and O-acetyl groups at C-2 and

C-3 of xylose units (Fengel and Wegener 1989). In

J. S. Kim � D. Sandquist � G. Daniel (&)

Wood Science, Department of Forest Products,

Swedish University of Agricultural Sciences,

P.O. Box 7008, SE 750 07 Uppsala, Sweden

e-mail: [email protected]

B. Sundberg

Umea Plant Science Center, Swedish University

of Agricultural Sciences, SE 901 83 Umea, Sweden

123

Planta (2012) 235:1315–1330

DOI 10.1007/s00425-011-1576-8

contrast, softwood xylans known as arabino-4-O-methyl-

glucuronoxylans (AGXs) occupy around 10% of cell wall

components and contain arabinose units linked by a-(1-3)-

glycosidic bonds to the xylan backbone without acetyl

groups (Fengel and Wegener 1989).

Xylans are basically considered as linked with cellulose

and lignin to enhance the mechanical strength of cell walls

and several advanced functions of xylans in wood cell wall

formation have been suggested. From immunolabeling of

xylans, Vian et al. (1986) suggested that xylans may play an

important role as a twisting agent for cellulose microfibrils in

the transition zone between the S1 and S2 layers. More

recently, in vitro studies with bacterial cellulose have indi-

cated that xylans may influence the assembly of cellulose

microfibrils and cellulose crystal size (Tokoh et al. 2002a, b).

In association with lignin, xylans are often considered as a

primer providing binding sites for lignin monomers in the

cell wall (Atalla 2005; Terashima et al. 2004, 2009; Vian

et al. 1992). From neutral sugar analysis, Yoshinaga et al.

(1993) suggested that xylans are closely associated with

guaiacyl (G) lignin and play an important role for water

conductance. However, the roles of xylans, apart from their

basic function as coupling agents between cellulose and

lignin in the formation of wood cell wall, is still disputed.

Populus is an important angiosperm tree as a model for

experimental research and used frequently for genomic

studies of woody plants (reviewed by Mellerowicz et al.

2001 and Taylor 2002). Populus xylem is composed of

fibers, vessel elements (vessels), ray parenchyma (ray cells)

and axial parenchyma, with different proportions between

cell types (Ilvessalo-Pfaffli 1994). Each cellular element of

Populus xylem also shows different structural properties

depending on its different cell wall layers, including

intercellular layer, primary and multilayered secondary cell

walls and pits. Chemically, normal mature Populus xylem

contains approximately 42–49% cellulose, 16–23% hemi-

cellulose and 21–29% lignin (Sannigrahi et al. 2010) of

which 15–22% of the cell wall components are xylans

(Sannigrahi et al. 2010; Willfor et al. 2005). However, the

spatial and temporal correlation between cell types and

different cell wall layers regarding xylan distribution is

poorly understood since chemical data are mostly derived

through the fractionation and analysis of whole wood.

In an effort to understand the microdistribution of cell

wall components in hardwood cell walls, many microscopic

studies have investigated xylan distribution in hardwood

xylem applying a variety of methods including chemical

and enzymatic extraction (Parameswaran and Sinner 1979;

Parameswaran and Liese 1982) and immunolabeling in

combination with different immunological probes (Awano

et al. 1998, 2000, 2002; Filonova et al. 2007; Kaneda et al.

2010; Ruel et al. 2006; Vian et al. 1986, 1992). However,

the temporal and spatial distribution of xylans in

differentiating hardwood xylem cells is still poorly under-

stood since previous studies have focused mainly on a

particular cell type, i.e. fibers, or specific developmental

stage of the cell walls. Even in Populus, although several

studies have shown xylan distribution in differentiating and

mature xylem cells (Filonova et al. 2007; Kaneda et al.

2010; Ruel et al. 2006), these studies have only shown xylan

distribution in some developmental stages of xylem cells

and thus the full process of xylan deposition in differenti-

ating xylem cells in Populus is still unclear.

To extend our understanding of the spatial and temporal

distribution of xylans in hardwoods, the present work

investigated the distribution of xylans in differentiating

Populus xylem cells using immuno-microscopic methods

in combination with monoclonal antibodies (LM10 and

LM11) specific to b-(1-4)-linked xylopyranosyl residues

(McCartney et al. 2005). Spatial and temporal variations of

xylan distribution and structure regarding its substitution

among cell types and different cell wall layers were

investigated. Finally, the results are also compared with

previous studies on xylan distribution in softwood per-

formed using a similar approach (Kim et al. 2010, 2011).

Materials and methods

Plant materials

Small cross-sections were taken at a stem height of 20 cm

above ground level from 3-month-old hybrid aspens

(Populus tremula L. 9 P. tremuloides Michx., clone T89)

grown in the greenhouse with 18 h day length, 22/15�C

(day/night) temperature. Small sectors were cut from the

cross-sections and fixed with 3% v/v glutaraldehyde ? 2%

v/v paraformaldehyde in 0.1 M sodium cacodylate buffer

(pH 7.2) for 9 h at room temperature. After dehydration

through a graded ethanol series, sectors were infiltrated in a

mixture of LR White resin (London Resin Co., UK) and

ethanol with gradual increase of resin concentration to

100% resin over a week. The sectors were then embedded

in pure LR White resin and polymerized at 65�C overnight.

Immunofluorescence labeling

Labeling was conducted according to procedures described

previously (Kim et al. 2010). Semi-thin sections (ca 1 lm)

mounted on slides coated with silane (Sigma, USA) were

treated with 50 mM glycine/phosphate-based saline (PBS)

solution for 15 min, followed by washing with PBS buffer

for 5 min, and then suspended in blocking buffer (pH 7.2,

PBS buffer containing 3% w/v bovine serum albumin

(BSA)) for 30 min at room temperature. After washing

with PBS buffer for 5 min, sections were incubated in

1316 Planta (2012) 235:1315–1330

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LM10 or LM11 monoclonal antibodies (PlantProbes, UK;

1:20 dilution in PBS buffer) for 2 days at 4�C. The LM10

antibody binds unsubstituted or only low substituted

xylans, whereas LM11 binds both low and highly substi-

tuted xylans (McCartney et al. 2005).

After three washes with PBS buffer for 10 min each,

sections were incubated with anti-rat IgG Alexa Fluor 488

(Invitrogen, USA; 1:100 dilution in PBS buffer) for 2 h at

35�C. For control, sections were incubated with anti-rat

IgG Alexa Fluor 488 only. After three washes with PBS

buffer for 10 min each, sections were examined under a

fluorescence microscope (Leica DMRE, Germany) with I3

filter cube (excitation 450–490 nm, emission [515 nm).

Lignin autofluorescence was completely eliminated

through a short exposure time (not shown). For observa-

tions of general anatomy, serial sections were stained with

1% w/v toluidine blue in 0.1% borax buffer and observed

using a Leica DMBL light microscope. The results reflect

observations on four different sectors prepared from four

cross-sections (i.e. four trees).

Immunogold labeling

Immunogold labeling was observed on four cross-sections

prepared from the four separate sectors, two for differentiating

xylem (i.e. two trees) and two for mature xylem (i.e. two

trees). Labeling was conducted according to procedures

described by Kim et al. (2010) with minor modifications in the

dilution rate and incubation time. Briefly, transverse ultrathin

sections (ca 90 nm) prepared from LR white embedded blocks

were mounted on nickel grids and incubated in blocking buffer

(pH 8.2, Tris-buffered saline (TBS) containing 1% w/v BSA

and 0.1% w/v NaN3) for 30 min at room temperature. Grids

were then incubated with LM10 or LM11 antibodies (1:20

dilution in blocking buffer) for 2 days at 4�C. Thereafter, grids

were incubated with goat anti-rat secondary antibody conju-

gated with 10-nm colloidal gold (BBInternational, UK) for

4 h at 35�C for the LM10 (1:50 dilution in blocking buffer)

and at room temperature for the LM11 (1:100 dilution in

blocking buffer). After poststaining with 4% w/v uranyl ace-

tate for 10 min, grids were examined using a Philips CM12

transmission electron microscope (TEM, USA) operated at

80 kV. Negative TEM films were scanned using an Epson

Perfection Pro 750 film scanner.

Results

Immunofluorescence localization of xylans

in differentiating xylem cells

Xylan labeling was not observed in the cambial and radial

expansion zones (Fig. 1a–c), but was first detected at the

corner of the fiber cell wall at the early stage of secondary cell

wall formation (inserts d–2 and e–2 in Fig. 1d, e) and

increased gradually during fiber maturation (Fig. 1d, e). No

xylan labeling was detected in ray cell walls during early fiber

secondary cell wall formation regardless of antibody type

(inserts d–1 and e–1 in Fig. 1d, e). Xylan labeling in the early

stage of vessel formation was not clearly recognized using

fluorescence microscopy (Fig. 1d, e). In mature xylem, strong

xylan labeling was detected in all cell types (Fig. 1f, g), but

showed different distribution patterns in the cell wall with the

LM10 and LM11 antibodies (Fig. 1h, i). LM11 showed

almost uniform labeling across fiber cell walls, while LM10

revealed an uneven labeling pattern with stronger labeling in

the outer than inner secondary wall layers (Fig. 1h, i). Vessels

labeled with LM10 showed stronger labeling than fibers,

whereas almost identical labeling was observed between cells

using LM11 (Fig. 1h, i). In addition, although xylan labeling

was absent in vessel pit membranes regardless of antibody

type (arrows in Fig. 1f, g), LM11 showed some weak labeling

in ray–vessel pit membrane regions (insert g–1 in Fig. 1 g;

arrowheads) that may not reflect the membrane, but rather the

protective layer (PL, see below).

Immunogold localization of xylans in differentiating

xylem cells

Fiber cell walls

Neither LM10 nor LM11 (not shown) antibodies showed

xylan labeling in cambium cells and fibers in radial

expansion zone (Fig. 2a, b), that are composed of inter-

cellular (IL) and primary cell wall (PW) layers. Xylan

labeling was initially detected in the corner of the cell wall

at the beginning of S1 formation, regardless of antibody

type (Figs. 2c, 3a). During S1 formation, xylan labeling

increased gradually in the cell wall, but showed different

distribution patterns in the cell wall between the two

antibodies. LM10 showed stronger labeling in the outer S1

layer than the inner layer (Fig. 2d), whereas LM11 showed

almost uniform distribution of labeling in the S1 layer

(Fig. 3b). Even during S2 formation, LM10 (Fig. 2d–g)

showed more heterogeneous distribution patterns of xylan

labeling in the cell wall than LM11 (Fig. 3c–e). LM10

showed strong labeling in the innermost cell wall (arrow-

heads in Fig. 2d–g) and then a gradual increase of labeling

in the outer cell wall layer during secondary cell wall

formation (Fig. 2d–g). In contrast, xylan labeling by LM11

was detected across the whole cell wall from the outermost

to innermost layer during secondary cell wall formation

(Fig. 3c–e). In mature fibers, LM10 showed much stronger

xylan labeling in the outer layer of the secondary cell wall

than the inner layer (Fig. 2h), while LM11 displayed an

uniform distribution of xylan labeling in the whole

Planta (2012) 235:1315–1330 1317

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secondary cell wall (Fig. 3f). In addition, both antibodies

showed strong labeling in the middle lamella cell corner

(MLcc) regions from the early stage of secondary cell wall

formation, although it tend to be limited to less dense

regions of the MLcc (Figs. 2d, e, 3c–e). However, it was

not clear if the less dense regions represent the tips of fibers

or electron lucent regions that are less lignified (Daniel

et al. 1991).

Fig. 1 Immunofluorescence localization of xylans in differentiating

xylem with LM10 and LM11 antibodies. Strong xylan labeling was

detected in differentiating xylem (a–c). Note the absence of xylan

labeling in the cambial (CA) and radial expansion (RE) zones.

d, e Enlargement of cells marked with squares in b and c,

respectively. Xylan labeling was first detected in the corner of the

fiber cell wall at the early stage of secondary cell wall formation

(inserts d–2 and e–2 in d, e; arrowheads). Note no xylan labeling in

ray cell walls (R, inserts d–1 and e–1 in d, e; asterisks). f, g Mature

xylem. Strong xylan labeling was observed in all cell types. Note

weak labeling in ray–vessel pit membrane regions by LM11 (insert

g–1 in g; arrowheads). Note also the absence of labeling in ray–vessel

pit membrane regions by LM10 (arrowheads in f) and vessel pit

membranes (arrows in f, g) regardless of antibody type. h, i Enlarge-

ment of cells marked with squares in f and g, respectively. LM10

(h) showed stronger labeling in the outer (SWou) than inner fiber

secondary cell walls (SWin), whereas LM11 (i) revealed almost

uniform labeling across the whole cell wall. Note stronger labeling in

the vessel (V) than fiber (F) by LM10 (arrowheads in h) and almost

identical labeling in fibers and vessels by LM11 (arrowheads in i). SWsecondary cell wall; TB toluidine blue staining; Bar 100 lm (a–c),

50 lm (d–g), 10 lm (h, i)

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Fig. 2 Immunogold localization of xylans by LM10 antibody in

fibers. a, b Cambium cells and fibers in radial expansion zone. Note

no labeling in the cell wall composed of intercellular (IL) and primary

cell wall (PW) layers. c–g Fibers during secondary cell wall

formation. Labeling was first detected at the cell corner of the S1

layer (c arrows). During maturation, the outer layer showed stronger

labeling than the inner layer (d–g). Some strong labeling was also

detected in the innermost layer (d–g arrowheads). Note strong

labeling on the less dense area of the middle lamella cell corner

(MLcc) (d, e). h Mature fibers. Note stronger labeling in the outer

secondary cell wall than the inner wall and some strong labeling in the

MLcc. Bar 500 nm

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Vessel cell walls

Like fibers, xylan labeling was not detected in the

IL ? PW developmental stage of vessels in the radial

expansion zone (Fig. 4a). Xylan labeling was detected in

the vessel cell wall during S1 formation (Figs. 4c, 5b) at a

later developmental stage than that for the fiber cell wall

(Figs. 4b, 5a). The formation of the secondary cell wall in

vessels also began later than in fibers (Figs. 4b, 5a). As in

fibers, LM10 showed an heterogeneous labeling pattern in

vessel cell walls. During the early stage of S1 formation,

LM10 showed stronger labeling in the outer cell wall than

the inner layer, and then labeling increased gradually from

the outer cell wall layer during the secondary cell wall

formation of vessels (Fig. 4c–e). In contrast, LM11 showed

uniform labeling in the vessel cell wall in a similar manner

to fibers as described above (Fig. 5d–e). In mature vessels,

unlike fibers, a uniform distribution of xylan labeling was


fibers. a–e Fibers during secondary cell wall formation. Occurrence of

labeling was detected from the cell corner of the S1 layer (a) as shown

with LM10 (Fig. 2c). However, LM11 showed a much more uniform

labeling across the cell wall than LM10 (Fig. 2d–g). Note some strong

labeling in the middle lamella cell corner (MLcc) (c–e). f Mature

fibers. Note uniform distribution of labeling across the whole

secondary cell wall. Bar 500 nm

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detected in the vessel cell wall by both LM10 and LM11

(Figs. 4f, 5f). However, LM10 showed much stronger

labeling in vessels than fibers, especially the inner S2 layer

(Fig. 4f). In contrast, LM11 revealed a similar labeling

density between the two cells (Fig. 5f).

Ray cell walls

The LM10 and LM11 antibodies showed almost the same

patterns of xylan distribution in the ray cell wall, except for

some differences in labeling density. Xylan labeling was


vessels. a Vessel (V) and fibers (F) in radial expansion zone. Note the

absence of labeling in the cell wall composed of intercellular (IL) and

primary cell wall (PW) layers. b The vessel and fibers at the stage of

S1 formation in fibers. Note no labeling of the vessel cell wall, but

some strong labeling in the fiber cell walls. c–e Vessel and fibers

during secondary cell wall formation. Labeling in the vessel began

during S1 formation (b) and showed similar distributional character-

istics as fibers during the early stages of secondary cell wall

formation, with stronger labeling in the outer than inner layer (d).

Vessels showed a more even distribution than fibers in the late stage

of secondary cell wall formation (e). f Mature vessel and fibers. Note

the much stronger and more uniform labeling in the vessel cell wall

than fiber cell walls. Bar 500 nm

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first detected in the ray cell wall during S2 formation in

fibers (Figs. 6a–d, 7a–c), particularly at the beginning of

secondary cell wall formation in ray cells, which was often

observed after the first ray cross wall was formed by

cambium cells (Figs. 6e, 7c). This initial occurrence of

labeling in ray cells was much later than that in fibers and

vessels (Figs. 6b, c, 7a, b). During secondary cell wall

formation of ray cells, xylan labeling was observed in the

whole cell wall from the outer to innermost layers as shown

in fibers and vessels by LM11, regardless of antibody type

(Figs. 6d, f, 7d). In the mature stage of fibers, a uniform

distribution of xylan labeling was observed around the

whole ray cell wall regardless of antibody type, with

similar labeling intensity to the outer S2 layer of fibers by

LM10 and the whole secondary cell wall by LM11

(Figs. 6g, 7e).


vessels. As with LM10 (Fig. 4b, c), labeling was detected in the

vessel cell wall (V) during S1 formation (b), which was later than in

fibers (F, a). During secondary cell wall formation, uniform labeling

was detected in the cell wall regardless of the developmental stages of

vessels (c–e). f Mature vessel and fibers. Note uniform labeling in

vessels and similar labeling density between the vessel and fiber cell

walls. Bar 500 nm

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Fig. 6 Immunogold localization of xylans by LM10 antibody in ray

cells. a The ray cell (R) and fibers (F) in radial expansion zones. No

labeling was detected in ray cells and fibers composed of intercellular

(IL) and primary cell wall (PW) layers. Labeling was detected in the

ray cell wall during early S2 formation in fibers (d), which was much

later than in fibers (b) and vessels (V, c). In particular, the initial

occurrence of labeling in ray cells was often observed after the

beginning of secondary cell wall (SW) formation in ray cells (e),

which was detected after the first ray cross wall from cambium.

During secondary cell wall formation, ray cells showed even

distribution of labeling in the whole cell wall (f). g Ray cell at the

mature stage of fibers. Note uniform and similar labeling density to

the outer cell wall of fibers of the ray cell wall. Bar 500 nm

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Pits

Three pit types were mainly observed in the secondary

xylem of aspen, including fiber– (between fibers), vessel–

(between vessels) and ray–vessel pits (between ray cells

and vessels). No specific labeling patterns were detected in

pit borders compared to other parts of the cell wall of

fibers, vessels and ray cells, regardless of antibody type. Pit

membranes were not labeled by LM10. Specific xylan

labeling patterns were limited to pit membranes by LM11

during pit development.

From the early stage of fiber and vessel development,

xylan labeling was detected in pit membranes of fiber and

vessel pits (Figs. 8a, b, 9a–c). In particular, xylan labeling

increased in pit membranes during maturation of vessel pits

(Fig. 9a–c). However, xylan labeling was not detected in

pit membranes of mature fibers and vessels regardless of

antibody type (Figs. 8c, d, 9d, e). During maturation of

Fig. 7 Immunogold localization of xylans by LM11 antibody in ray

cells. Like LM10 (Fig. 6d–f), labeling was first detected in the ray

cell wall during S2 formation in fibers, especially after the beginning

of secondary cell wall (SW) formation (c), which was much later than

that in fibers (a) and vessels (b), and showed an even distribution in

the whole cell wall during maturation (d). The ray cell at the mature

stage of fibers (e) showed uniform and similar labeling density to the

secondary cell wall of fibers. Bar 500 nm

1324 Planta (2012) 235:1315–1330

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ray–vessel pits, xylan labeling was successively detected in

the vessel side membrane (Fig. 10a), the ray side mem-

brane (Fig. 10b), and then both sides of the membrane

(Fig. 10c). As in fiber and vessel pits, no xylan labeling

was detected in both sides of the pit membrane at the

mature stage of vessels with either antibody (Fig. 10d, e).

However, some strong labeling was detected in the pro-

tective layer (PL, Chafe 1974) by LM11 (Fig. 10d).

Discussion

The secondary xylem of hardwoods is composed of several

different cell types. Each cell type performs different

functional roles in secondary xylem formation with differ-

ent anatomical and chemical properties observed between

cell types. However, the functional correlations between

cell types and chemical properties of each cell type are not

fully understood. In particular, the specific cellular distri-

bution of hemicelluloses in the cell wall is poorly under-

stood. In this work, we clearly demonstrate different

distributional characteristics of xylans among cell types.

Since the masking effect of pectins in xylan localization

was reported in tobacco (Herve et al. 2009), the present

work focused mainly on xylan localization in secondary cell

walls (except for pit membranes) because the primary cell

walls in woody plants also contain significant amounts of

pectins. Even for the secondary cell walls, we do not

completely exclude the possibilities of some masking effect

in xylan labeling by several factors, particularly by its

interaction with lignin or other polysaccharides.

Temporal and spatial distribution of xylans

in the aspen xylem

Our results demonstrate that xylan deposition in the sec-

ondary cell wall of fibers began from the cell corner of the

S1 layer after initiation of S1 formation. This observation is

spatially consistent with general lignin deposition events in

wood cell walls (reviewed by Donaldson 2001). However,

our results showed that xylan deposition and secondary cell

wall formation began earlier in fibers than in vessels; in

contrast to Terashima et al. (1986) who reported that lignin

deposition in poplar was first observed in the vessel cell

Fig. 8 Immunogold localization of xylans by LM10 (d) and LM11

(a–c) antibodies in pits between fibers (fiber pits). Some labeling was

detected in pit membranes by LM11 during differentiating stages of

fibers (a, b arrows), but was not detected in pit membranes of mature

fibers regardless of antibody type (c, d). Bar 500 nm

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Fig. 9 Immunogold localization of xylans by LM10 (e) and LM11

(a–d) antibodies in pits between vessels (vessel pits). Some labeling

was detected in pit membranes in the early stage of vessel secondary

cell wall formation by LM11 (a, arrows), after which labeling

increased gradually during vessel secondary cell wall formation

(b, c). At the mature stage of vessels, no labeling was observed in pit

membranes regardless of antibody type (d, e). Bar 500 nm

1326 Planta (2012) 235:1315–1330

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Fig. 10 Immunogold localization of xylans by LM10 (e) and LM11

(a–d) antibodies in pits between vessels and ray cells (ray–vessel

pits). At early stages of the pit formation, strong labeling was detected

in the membranes by LM11, but was mostly limited on the vessel

(V) side membrane (a). During pit maturation, labeling was mostly

detected on the ray (R) side membrane (b), and then detected on both

sides of the membrane (c). At vessel maturity, labeling was not

detected in pit membranes regardless of antibody type (d, e), but some

strong labeling was detected in the protective layer (PL) by LM11 (d).

Bar 500 nm

Planta (2012) 235:1315–1330 1327

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wall and later in the fiber cell wall using autoradiography

of precursors of lignin biosynthesis. This result indicates

that xylan deposited in the cell corner of the S1 layer may

not be associated to a role as a lignin primer for further

lignification of the secondary cell wall (Atalla 2005;

Terashima et al. 2004, 2009). At present, we assume that

the beginning of xylan deposition from the cell corner of

the S1 layer in fibers may represent one of the initial steps

in general secondary cell wall deposition processes in

fibers (Grunwald et al. 2002), rather than relationship with

initiation of lignification.

Interestingly, LM10 and LM11 antibodies showed dif-

ferent labeling patterns in fiber cell walls during secondary

cell wall development. LM10 showed strong labeling in the

outer cell wall layer, weak labeling in the inner layer and

strong labeling again in the innermost layer in differenti-

ating cell walls, suggesting that low substituted xylans

(lsAcGXs) may be mostly deposited through the intussus-

ceptional deposition mode, i.e., lsAcGXs penetrate the

preexisting cell walls without binding with cellulose

microfibrils (CMFs) and deposit from the outer part of cell

walls (Awano et al. 1998). In contrast, LM11 labeling was

always evenly distributed across the whole developing

secondary cell wall from the outer to the inner cell wall,

suggesting that highly substituted xylans (hsAcGXs) may

prefer to bind with newly synthesized CMFs in differen-

tiating fiber cell wall without penetration of preexisting cell

walls (appositional deposition mode, Awano et al. 1998).

From the specificity of LM11, we can expect that both

lsAcGXs and hsAcGXs may be simultaneously deposited

in the developing poplar fiber cell walls. Furthermore, in

mature fibers, LM10 showed stronger xylan localization in

the outer secondary cell wall than inner layer, while LM11

showed uniform xylan labeling in the whole secondary cell

wall, indicating heterogeneous composition of xylans in the

fiber cell wall.

Although not as prominent in vessels due to their thin

cell walls, differentiating vessels showed similar patterns

of xylan labeling as fibers with the two antibodies, i.e.,

heterogeneous labeling by LM10 and uniform labeling by

LM11 during vessel development. However, in the mature

stage, vessels showed almost uniform xylan labeling with

both LM10 and LM11 antibodies. In particular, LM10

showed a much more uniform and stronger xylan labeling

in the vessel than fiber cell wall, especially the inner S2

layer. Yoshinaga et al. (1993) also reported that vessels

contain more xylans than fibers in oak xylem by neutral

sugar analysis of various tissue fractions. Vessels are

generally rich in G lignin (reviewed by Donaldson 2001)

and it has been suggested that xylans have a close rela-

tionship with G lignin in vessel cell walls and play

important roles in the water conducting function of vessels

(Yoshinaga et al. 1993). However, it was not possible to

imply that vessels contain more xylans than fibers in the

present work because LM11 showed almost identical

intensity of xylan labeling of the two cell types. At present,

we assume that the vessel cell wall may be composed

primarily of a higher proportion of lsAcGXs than the fiber

cell wall.

In ray cells, xylan labeling occurred at the S2 formation

stage in fibers which was much later than that in fibers and

vessels. As in fibers and vessels, xylan labeling in ray cells

was also initially detected after the beginning of secondary

cell wall formation in ray cells. These results suggest that

differences in the initial occurrence of xylan labeling in the

cell wall among fibers, vessels and ray cells may be basi-

cally caused by temporal differences in secondary cell wall

formation among cells. Unlike fibers and vessels, ray cells

showed almost identical xylan labeling patterns between

LM10 and LM11 in differentiating and mature stages,

suggesting that ray cells may be composed chemically of a

more homogeneous distribution of xylans than fibers and

vessels.

Although only a limited number of pits were observed in

the cell wall, our results showed clearly that pit membranes

contain hsAcGXs xylans, specifically labeled by LM11, in

differentiating secondary xylem cells. Interestingly,

hsAcGXs were not detected in pit membranes at mature

stages, indicating gradual degradation (or disappearance)

of xylans during pit maturation. Such changes in hemi-

cellulose distribution in pit membranes were also observed

in the secondary xylem of softwood, but in this case with

galactoglucomannans (Kim et al. 2011; see below). At

present, the reason for gradual disappearance of xylans

from pit membranes is unknown. We can only assume that

water conductance between cells or enzymes capable of

degradation of xylans may cause the gradual degradation of

xylans from pit membranes. Interestingly, ray–vessel pits

showed specific spatial sequences of xylan labeling in

developing pit membranes; the vessel side membrane, the

ray side membrane, both vessel and ray side membranes,

and finally the absence of labeling in membranes but

present in the PL. Although we cannot explain the bio-

logical function of xylans in ray–vessel pit membranes, it is

assumed that xylan deposition in pit membranes may have

a role as a reinforcing agent to maintain pit membrane

structure during secondary cell wall formation of ray cells

and vessels because the active translocation and internal

turgor pressure between ray cells and vessels during sec-

ondary cell wall formation can easily damage pit mem-

brane structures composed mainly of pectins and cellulose.

In case of mature cells, xylans may contribute to the

enforcement of PL structures since the PL is unlignified

(Murakami et al. 1999) and needed to maintain pit structure

or living protoplasts in ray cells even after vessel formation

is completed (Barnett et al. 1993).

1328 Planta (2012) 235:1315–1330

123

Comparison of xylan labeling between hardwoods

and softwoods

Hardwood xylem is mainly composed of vessels, fibers and

parenchyma cells, whereas softwood xylem is composed of

tracheids and parenchyma cells. Here, we compare the

xylan labeling patterns between cells in hardwood and

softwood xylems to extend our understanding on the var-

iation of xylan distribution among cell types in relation to

their functions during wood formation. The information of

xylan labeling in softwood xylem (Japanese cedar, Cryp-

tomeria japonica) derived in a similar way as the present

work is used for comparison (Kim et al. 2010, 2011). For

convenience, the basic structural difference of xylans

between softwoods (AGXs) and hardwoods (AcGXs) are

not considered. We consider primarily the degree of sub-

stitution linked to the backbone of hardwood and softwood

xylans.

Fibers versus tracheids

Both fibers (Figs. 1, 2, 3) and tracheids (Kim et al. 2010)

showed the initial presence of xylan labeling in the corner

of the S1 layer after the initiation of S1 formation and in

general similar labeling patterns in the secondary cell wall

by both LM10 and LM11 even though they have different

lignin compositions i.e., S lignin is rich in fibers while G

lignin is rich in tracheids. These results suggest that xylan

labeling patterns in combination with xylan substitutions in

the secondary cell wall may not be closely related to the

lignin types of fibers and tracheids. Interestingly, the

boundary between S1 and S2 layers (S1/S2 region) showed

some different characteristics in xylan labeling between

fibers and tracheids. In tracheids, the S1/S2 regions showed

lower xylan labeling than other parts of the secondary cell

wall at early stages of S2 formation by LM10 and LM11

(Kim et al. 2010). Even in mature tracheids, LM10 showed

weak xylan labeling in the S1/S2 regions (Kim et al. 2010).

However, fibers showed strong xylan labeling in the S1/S2

region like other parts of the cell wall during whole sec-

ondary cell wall formation with both LM10 and LM11

(Figs. 2d–h, 3c–f). These results suggest that the S1/S2

regions are composed of different xylans in fibers and

tracheids, mainly low substituted xylans (AcGXs) in fibers

and highly substituted xylans (AGXs) in tracheids. In

addition, strong xylan labeling was always detected in

MLcc regions of tracheids after the beginning of xylan

deposition in these regions (Kim et al. 2010), while fibers

(Figs. 2d–h, 3c–f) showed various types of xylan labeling

patterns depending on spatial differences in location on

transverse view, including complete absence of xylan

labeling.

Rays

Xylan was detected in ray cell walls after the beginning of S2

formation in both tracheids (Kim et al. 2011) and fibers

(Figs. 1d, e, 6d, 7c). The initial occurrence of xylan labeling

in ray cells was much later than that observed in either

tracheids or fibers. These results suggest that the occurrence

of xylans is temporally similar in ray cells between Japanese

cedar and aspen even though they have different ultrastruc-

tural and chemical characteristics of the ray cell walls.

Pits

Xylan labeling was not detected in pit membranes of cedar

xylem during pit formation, including bordered (between

tracheids) and cross-field pits (between tracheids and ray

cells) (Kim et al. 2011), while some strong xylan labeling

was detected in pit membranes of aspen xylem at the

developing stage of pit formation (Figs. 8, 9, 10). These

results indicate that the chemical composition of pit mem-

branes differs between cedar and aspen even though the

presence of galactoglucomannans in developing pit mem-

branes as shown in cedar is not clearly understood in aspen.

From the gradual disappearance of galactoglucomannans

(Kim et al. 2011) or xylans (Figs. 8, 9, 10) from pit mem-

branes of cedar and aspen respectively, we can also assume

that similar enzymatic or physical processes are involved in

the formation of pit membranes in cedar and aspen.

In conclusion, the present work indicates that there are

temporal and spatial variations in xylan deposition between

different cell types in aspen xylem. The observations also

confirm that the chemical structure of xylans deposited in

the cell wall differ depending on the developmental stage

and cell wall layer. The work also indicates that xylans in

hardwoods may differ from those in softwoods not only in

concentration and structure, but also localization properties

in the same functional cells. Together our results suggest

that variations in xylan distribution among cells may be an

important factor regulating the ultrastructure of cells and be

associated with their different functions during wood for-

mation. Finally, our basic information of xylan distribution

in aspen should help in the interpretation of genetically

modified populus trees.

Acknowledgments The authors gratefully acknowledge funding

provided by the Formas FuncFiber Center of Excellence (http://www.

funcfiber.se).

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