Plant Science 176 (2009) 413–419

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Anatomy, ultrastructure and lignin distribution of stone cells in two Pyrus species
Shutian Tao a, Shahrokh Khanizadeh b, Hua Zhang a, Shaoling Zhang a,*
a
Pear Engineering Research Centre, College of Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing, Jiangsu Province 210095, PR China
b
Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, Saint Jean sur Richelieu, Quebec J3B 3E6, Canada

A R T I C L E I N F O A B S T R A C T

Article history: The statement ‘‘seeing is believing’’ expresses the importance of microscopy in basic and applied
Received 26 September 2008 research. In this study, anatomy, ultrastructure and lignin distribution in stone cells from the fruit of two
Received in revised form 16 December 2008 Pyrus species (Pyrus bretschneideri cv. ‘Jingaisu’ and Pyrus pyrifolia cv. ‘Kousui’) were examined by using
Accepted 16 December 2008
light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy
Available online 31 December 2008
(TEM) as well as autofluorescence analysis. To our knowledge, this is the first time this combined method
has been used to analyze the stone cells in pear fruit. Sections stained with phloroglucinol–HCl revealed
Keywords:
the presence of lignin in stone cells, and showed that stone cells were distributed in a mosaic pattern in
Stone cells
Ultrastructure
the flesh, with larger stone cells concentrated around the core and smaller ones in the pericarp. There
Lignin distribution were no obvious differences in stone cell structure between the two varieties, but stone cell size and
Autofluorescence content was much greater in ‘Jingaisu’ than in ‘Kousui’. Further, lignin accounted for 29.8% of stone cell
SEM composition in ‘Jingaisu’, a significantly higher proportion than in ‘Kousui’ (24.6%); this result was
TEM confirmed by autofluorescence analysis. More detailed information on lignin distribution across the cell
wall was obtained by TEM combined with the KMnO4 staining technique. In TEM images, cell walls of
both pear varieties were typically divided into four layers: compound middle lamella (CML), secondary
wall 1 (S1), outer secondary wall 2 (S2L) and secondary wall 2 (S2), with different staining intensities for
different lignin concentrations in those regions.
ß 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Lignin is a polymer derived from the dehydrogenative poly-
merization of three different monolignols—p-coumaryl alcohol,
conferyl alcohol and sinapyl alcohol. Lignin is widely distributed in
plant cell walls and confers strength, rigidity and impermeability. In
some plants with extreme needs for both structural support and
water transport, e.g. terrestrial trees, lignin makes up 15–36% of the
dry weight of the wood. Lignin is therefore one of the world’s most
abundant natural polymers, along with cellulose. In addition, it plays
crucial roles in plant defense against biotic and abiotic stresses [1–4].
The presence of lignin in high concentrations in cell walls is regarded
as a positive benefit, for example in the fiberboard industry, and
lignification of plant tissues also has a well-recognized impact on
foods, particularly on forage digestibility and bioavailability [5].
However, there is a further unusual instance of lignification in
foods—an increase in the lignin content of some fruit, specifically in
* Corresponding author at: Pear Engineering Research Centre, College of
Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing, Jiangsu Province
210095, PR China. Tel.: +86 25 84396580; fax: +86 25 84395266.
E-mail addresses: shutian_tao@hotmail.com (S. Tao), khanizadehs@agr.gc.ca
(S. Khanizadeh), zhanghu6337@sina.com (H. Zhang), nnzsl@njau.edu.cn (S. Zhang).
the form of stone cells [6], which occurs during normal development
and/or ripening after harvest [7].
Until now, considerable effort has been devoted to investigating
the process of lignin formation and lignin distribution in cell walls,
and various techniques have been developed in this process. For
example, the distribution of lignin can be determined by
interference microscopy and confocal laser scanning microscopy
(CLSM). Furthermore, fluorescence microscopy has been used to
visualize the lignin distribution in wood cell walls by autofluor-
escence [8,9]. The technique of transmission electron microscopy
coupled with energy dispersive X-ray analysis (TEM-EDXA) was
successfully used to quantify the lignin distribution in mercurized
wood tissue [10]. The staining of ultrathin sections with potassium
permanganate (KMnO4) in order to determine the lignin distribu-
tion in woody cell walls experienced somewhat of a renaissance in
the 1980s and 1990s. In this case, the lignin molecule is oxidised by
KMnO4. The permanganate anion is reduced to manganese dioxide,
which then precipitates, indicating the site of reaction. Singh et al.
[11], in particular, used staining intensity as an indicator of lignin
distribution in the S2 layer of Pinus radiata tracheids.
Pyrus bretschneideri and Pyrus pyrifolia, which originated in
China, are widely cultivated in Asia, and they are characterized by
the existence of stone cells in the fruit pulp. Several factors
influence the formation of stone cells, including cultural practices,

0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2008.12.011
414 S. Tao et al. / Plant Science 176 (2009) 413–419
post-harvest handling [12–17], but the most important factor is
genetic variability, different species even different varieties show
variation in stone cell content with various size [18–21]. Some
times, stone cells are not a major constituent of the edible portion,
however in some varieties, such as ‘Jingaisu’ (Pyrus bretschneideri),
which accounts for 32% of the total area under cultivation in China,
they impart a very gritty texture. Due to the crucial impact of stone
cells to pear fruit texture, some studies have been performed on
these undesirable cells. However, to the best of our knowledge, the
research was carried out mainly based on the anatomical
characteristics and content or size analysis in the fruit [13,22–
25]. The chemical nature of stone cells was described by Sterling
[26] and Ranadive and Haard’s reports [27], which showed the
formation of stone cells resulted from cell lignification and the
deposition of lignin in the cell wall. We still have a very incomplete
understanding of stone cells, especially their ultrastructure and
lignin distribution. As a part of our research program aimed at
improving pear quality by reducing stone cell content, the
techniques used in wood research were employed in the present
study to investigate the stone cells and lignin distribution in two
different pear varieties, ‘Jingaisu’ (Pyrus bretschneideri) and
‘Kousui’ (Pyrus pyrifolia), characterized by gritty and smooth
textures, respectively. The observations can partly overcome the
limitations in view of stone cells in pear fruit.
2. Materials and methods
2.1. Plant materials
Fresh pear (Pyrus) fruit harvested from ‘Jingaisu’ (Pyrus
bretschneideri) and ‘Kousui’ (Pyrus pyrifolia) trees grown on the
experimental farm of Nanjing Agricultural University were used as
samples.
2.2. Stone cell content in flesh
Each fruit was peeled, cored and diced into cubes. A 100-g sample
of pear flesh was homogenized with distilled water in a blender for
10 min. The homogenate was then diluted with distilled water. The
suspension was incubated at room temperature for 30 min and the
supernatant phase was decanted. Finally, the sediment was
suspended in 0.5N HCl for 30 min, decanted and washed with
distilled water. This operation was repeated several times until the
stone cells were almost free of extraneous cell debris [15].
2.3. Lignin determination
The method was carried out as described by Syros et al. [28]
with some modifications. Stone cells were pestled in 95% ethanol,
then the sediment was washed with 95% ethanol and ethanol:-
hexane (1:2, v/v) three times, respectively, and dried. Dried
sediments were digested in 2 ml of 25% (v/v) acetyl bromide in
acetic acid and incubated for 30 min at 70 8C. The reaction was
terminated by adding 0.9 ml of 2N NaOH with an extra 5 ml of
acetic acid and 0.1 ml of 7.5 M hydroxylamine hydrochloride. The
volume was corrected to 10 ml with acetic acid and the absorbance
at A280 was measured. The amount of lignin was calculated from a
linear calibration curve with commercial alkali lignin (Sigma–
Aldrich, USA).
2.4. Specimen preparation for light microscopy (LM) and
autofluorescence analysis
3
The fruit tissues were cut into approximately 2 mm cubes and
fixed in FAA solution (formalin:glacial acetic acid:90% etha-
nol = 5:5:90, v/v). The tissues were then processed as follows:
sequentially dehydrated at room temperature in 70%, 85%, 95% and
100% ethanol (30 min each step); vitrified with a gradient from
100% ethanol to 100% xylene; infiltrated and embedded in paraffin;
sections (10 mm) obtained from a microtome (Leica RM2015,
Germany) were subsequently mounted on microscopic slides.
Sections were then rehydrated and allowed to dry. The Wiesner
reaction was performed by pouring a few drops of 1% phlor-
oglucinol ethanol solution on the section, adding one drop of 35%
HCl, and then covering the section with a cover slip.
Some additional samples without staining were directly
examined for autofluorescence with a confocal laser scanning
microscope (Leica DM IRB2) using laser (405 nm) as the excitation
wavelength. The exposure was strictly kept identical to insure the
comparability between samples.
Hand-cut sections of fresh pear fruit tissues were mounted in
water and directly observed using a fluorescence microscope (Zeiss
Axioskop 40) and images were captured with Canon PowerShot
A640.
2.5. Electron microscopy (EM)
For transmission electron microscopy (TEM) observation, flesh
cubes (<2 mm3) were post-fixed for 2 h in 2% osmic acid,
dehydrated in a gradient ethanol series and embedded in Spurr’s
epoxy resin. Sections approximately 100-nm thick were cut with a
diamond knife on an ultramicrotome (Power Tome-XL, USA), post-
stained with 1% KMnO4 (prepared in 0.1% sodium citrate buffer),
and examined with a Hitachi H7650 transmission electron
microscope at 80 kV [11].
For scanning electron microscopy (SEM) observation, blocks of
1–2 mm3 prepared from fruit tissue with razor blades were coated
with a 10-nm thick layer of gold using a sputter coater after fixing,
cleaning and drying. Finally, the sections were examined on a
Hitachi S-3000N scanning electron microscope under high vacuum
[29].
3. Results and discussion
3.1. Histochemical test for stone cells
Staining with phloroglucinol–HCl (Wiesner reagent) clearly
shows highly lignified stone cells. As shown in Fig. 1, in both
varieties, stone cell clusters were unevenly scattered in the
parenchyma, concentrating around the core and the pericarp;
however, the stone cell clusters around the core were much
bigger than those in other regions. Size distribution of the stone
cell clusters from different parts of the fruit was shown in Fig. 2;
Stone cell clusters around the core of ‘Jingaisu’, which were
larger than 0.05 mm2, accounted for 49.21% of the total amount
compared with 26.67% in ‘Kousui’; in the outer region of
‘Jingaisu’ pulp (edible part), stone cell clusters of that size
accounted for 23.17% compared with only 14.14% in ‘Kousui’
(Fig. 3). Furthermore, measurement of the stone cell content
showed that ‘Jingaisu’ fruit contained many more stone cells
than ‘Kousui’ (P < 0.05, Table 1), which meant the distribution
density of stone cells in ‘Jingaisu’ was significantly higher than
that in ‘Kousui’. The information presented above indicates that
Table 1
Stone cell content, lignin concentration and fluorescence energy of stone cells in the
two pear varieties.
Variety Stone cell Lignin Fluorescence
content (% FW) concentration (%) energy
‘Jingaisu’ 0.379  0.019 29.83  1.93 36456.26  3470.81
‘Kousui’ 0.071  0.004 24.55  0.53 31015.77  9181.34
 S. Tao et al. / Plant Science 176 (2009) 413–419 415

Fig. 1. Sections stained with phloroglucinol–HCl; the lignin is present mainly in stone cells as a red color. (A and C) ‘Jingaisu’; (B and D) ‘Kousui’. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of the article.)

the different texture found in these two varieties mainly result
from the different amount of stone cell aggregates and their
different size. This finding is supported by other scientists’
studies [22,30–33].
3.2. The presence of lignin in stone cells
To examine lignin deposition in the flesh, we prepared hand-
cut (Fig. 1A and B) and paraffin sections (Fig. 1C and D) from the
fruit tissues and stained them with lignin-staining dye
phloroglucinol–HCl (Wiesner reagent) to show the lignin. The
use of phloroglucinol–HCl staining is intended to highlight those
cell walls that have lignin deposition; however the non-lignified
cells are not clearly shown [34]. As shown in Fig. 1, examination
of the sections after Wiesner reaction showed that the stone
cells were stained deep red, whereas the parenchyma cells were
barely stained. This showed that the lignin was specifically
deposited in stone cells in pear fruit during normal growth and
development; and lignin content accounted for 29.83% and
24.55%, respectively of ‘Jingaisu’ and ‘Kousui’ dried stone cells
(Table 1), which was much higher than the proportion of 18%
measured in ‘Yuzuhada’ (Pyrus serotina) [27] and might be due
to the difference between species. However, the stone cell
distribution in the pulp exhibited a mosaic pattern, with

Fig. 2. Histogram representing the size distribution of the stone cell clusters in different parts of the fruit; the size is defined as area (mm2) measured by Image-Pro Plus
Version 6.0. (A) The core region of ‘Jingaisu’; (B) the outer region of ‘Jingaisu’; (C) the core region of ‘Kousui’; (D) the outer region of ‘Kousui’.
416 S. Tao et al. / Plant Science 176 (2009) 413–419

scattering in the parenchyma. This indicates that all parenchyma

Fig. 3. The percentage of stone cell clusters larger than 0.05 mm2 in different regions
of the pear pulp. (A) The core region of ‘Jingaisu’; (B) the outer region of ‘Jingaisu’;
(C) the core region of ‘Kousui’; (D) the outer region of ‘Kousui’.
cells in the pulp have the potential to become lignified and
subsequently to become stone cells. The mosaic pattern raises
the possibility that a genetic control mechanism is involved as
that in Arabidopsis [34,35], the lignin deposition and formation
of stone cells has their genetic background [18,19]. For example,
different varieties show the variation on the content and size of
stone cells [20,21,36]. Besides, peroxidase which is believed to
catalyse the final step of lignin biosynthsis is localized in the cell
wall regions where lignin deposition occurs [37,38], the positive
relationship between peroxidase activities and stone cell
content in pear fruit was also established in our previous work
[14,23]. Furthermore, it is reported that the down regulation of
an anionic peroxidase can alters both lignin content and
composition in hybrid aspen [39].

Fig. 4. Fluorescence images of stone cells. (A, C, and E) ‘Jingaisu’; (B, D, and F) ‘Kousui’; Side bars in C, D, E, and F represent the energy of the autofluorescence.
 S. Tao et al. / Plant Science 176 (2009) 413–419
Fig. 5. SEM micrographs showing the ultrastructure of stone cells. (A) ‘Jingaisu’; (B) ‘Kousui’; (C) higher magnification indicating the pits in cell wall; (D) higher magnification
indicating the lamellar structure of secondary cell wall.
3.3. Autofluorescence analysis
The application of autofluorescence [40,41] permits an assess-
ment of the gross localization of lignin in lignified tissues. It is
believed that autofluorescence is primarily due to lignin based on
its general appearance. Cellulose is also known to be autofluor-
escent, but it is generally much dimmer than lignin in cases where
comparisons have been made [8,42]. From the fluorescence
micrographs, almost all blue signals were presented in stone cells
in both pear varieties (Fig. 4A and B); however, some ramose
stripes were observed in the cell wall synchronously, which
indicates a lack of lignin deposition in these regions; and these
structures are postulated to be protoplast branches connected to
the pits, which ensure the interchange of water and nutritive
liquids between cells during lignifications [43]. Further examina-
tion of the lignin autofluorescence was conducted with laser
excitation by CLSM and analyzed with Leica Confocal Software
(Leica Microsystems Heidelberg GmbH). As shown in Fig. 4C–F, in
agreement with the results obtained by fluorescence microscopy,
sections under laser excitation showed greater autofluorescence in
the cell walls and the entire stone cells compared to surrounding
parenchyma cells, indicating strong lignification in the stone cells.
It is known that lignification is initiated at the outermost regions
and then works its way back towards the plasma membrane, after
which the cell dies [44]. In the confocal images, higher fluorescence
energy was mainly observed in the cell wall of fruitlets (Fig. 4C and
D); however, the fluorescence was observed in the entire stone
cells in mature samples (Fig. 4E and F), indicating that the process
of lignification begins in the outer layers of the cell wall and then
proceeds towards the secondary wall.
Also, the relative amounts of lignin in stone cells as determined
from the energy of the autofluorescence can be directly visualized
with CLSM and analyzed by Leica Confocal Software (Leica
Microsystems Heidelberg GmbH). In this study, the fluorescence
energy of stone cells in ‘Kousui’ was recorded as 31015.77 
9181.34, while the energy was higher in ‘Jingaisu’ with a value of
417
36456.26  3470.81. In comparison, lignin content in dried stone
cells as determined by the acetyl bromide method was higher in
‘Jingaisu’ than in ‘Kousui’ (Table 1). There is reasonable agreement
between the two techniques with respect to the relative lignification
levels of the two pear varieties. However, the acetyl bromide method
revealed a significantly higher lignin content in ‘Jingaisu’ than in
‘Kousui’ (P < 0.05), while autofluorescence did not. There are several
possible reasons for this discrepancy. Autofluorescence of lignin may
vary with changes in chemical composition or may not vary linearly
with lignin concentration, at least not in pear fruit. Variability in
lignin composition between different plants or even between tissues
within the same plant [45] is also known to interfere with other
methods for determining lignin [46].
3.4. SEM and TEM analysis coupled with potassium permanganate
staining
In pear pulp, stone cells were again observed in the form of
aggregates surrounded by parenchyma cells (Fig. 5A and B).
Lignified cells were anatomically characterized by the presence of
simple pits (Fig. 5C). Their function is to ensure the interchange of
water and nutritive liquids between cells. Because pits are natural
irregularities in the cell wall and vary in dimension (Fig. 5C), pit
regions are preferred areas of enzymatic hydrolysis which causes a
loose texture [10]. Besides, in the micrograph, stone cells revealed
a lamellar structure (Fig. 5C), indicating that the deposition of
lignin in the cell wall was layered, subsequently forming the
distinctly ring structure seen in both SEM and TEM micrographs
(Figs. 5D and 6).
Since the early 1950s, conventional transmission electron
microscopy has been used to study various aspects of wood cell
wall structure, and it has proven to be a very effective tool in wood
research. From the 1990s on, the KMnO4 technique was used in
order to learn more about the course of lignification during cell
wall differentiation [47]. The lignin-staining ability of KMnO4
makes it a very suitable technique for TEM [10]. In this case, the
418 S. Tao et al. / Plant Science 176 (2009) 413–419
Fig. 6. TEM micrographs of ultrathin sections taken at 80 kV. The specimens were stained with 10% KMnO4. (A) ‘Jingaisu’; (B) ‘Kousui’. CC, cell corner; CML, compound middle
lamella; S1, secondary wall 1; S2, secondary wall 2; S2L, outer S2 layer.
lignin molecule is oxidised by KMnO4. The permanganate anion is
reduced to manganese dioxide which then precipitates as electron-
opaque sediments indicating the site of reaction.
It is well known that the concentration of lignin varies across
wood cells, and the compound middle lamella (CML) generally is
more highly lignified than other cell wall layers. In the present
study, various cell wall layers could be identified clearly in TEM
micrographs because of the electron-opaque sediments in those
regions (Fig. 6). The CML and cell corner (CC) showed darker
staining compared to the adjacent layer secondary wall 1 (S1),
indicating the different levels of lignification. This observation is
partly consistent with other determinations of lignin concentra-
tion by interference microscopy, fluorescence images in radiata
pine [8] and KMnO4 staining in spruce [10] and in Caragana
korshinskii [29]. However, the total lignin content in the CML was
relatively low for its size (approximately 150–200 nm width). This
observation is supported by the study done by Westermark et al. in
spruce [48]. They measured a relatively low lignin content (55–
58%) in the true middle lamella by mercerization with the SEM-
EDX technique. Furthermore, a distinct outer S2 layer (S2L), which
was characteristic of severe compression wood, appeared in the
sections in this study, approximately 200–300 nm wide, and
samples from both varieties showed an increased lignin concen-
tration in S2L. This region seems to be lignified to approximately
the same extent as the CML region. This observation is supported
by the analysis conducted by Singh and Donaldson [49] in radiata
pine compression wood with CLSM and subsequently confirmed
with TEM. Examination of the sections at 80 kV revealed that less
lignified secondary wall 2 (S2) accounted for a very great
proportion of the entire cell wall; the lignin distribution in S2
appeared to be more uniform and to have a fibrillar texture due to
Fig. 7. Intensity of KMnO4 staining as a line scan across the cell wall layers
conducted with Image-Pro Plus Version 6.0. CML, compound middle lamella; S1,
secondary wall 1; S2, secondary wall 2; S2L, outer S2 layer.
the presence of alternating dark and light lines (Fig. 6). The dark
lines are believed to be deposits of lignin in the spaces between
cellulose microfibrils, which are oriented in different directions in
the CML, S1, and S2 [50]. In addition, the relative lignin content in
different regions was shown in Fig. 7. Although the lignin
concentration in the entire S2 layer is relatively low, the volume
ratio is the largest. On the other hand, S2 contains the highest
lignin content compared to the other layers.
4. Conclusion
The deposition of abundant large stone cell clusters in pears,
especially in Asian pear fruit flesh, plays a key role in fresh fruit
texture and in post-harvest processing [51]. In this study,
observations made from hand-cut sections dyed with phloroglu-
cinol–HCl showed that the higher distribution density, bigger size
and content of stone cells containing lignin results in the gritty
texture characterizing ‘Jingaisu’ (Pyrus bretschneideri) in contrast
with ‘Kousui’ (Pyrus pyrifolia). The higher lignification level of the
stone cells in ‘Jingaisu’ was subsequently confirmed by CLSM and
by the acetyl bromide method. KMnO4 combined with the TEM
technique was employed to investigate the more detailed
information on lignin distribution in the cell wall. The results
indicate that the lignin concentration and distribution pattern
varies across the cell wall. The stone cells show a highly lignified
outer S2 layer (S2L), which is also a characteristic of severe
compression wood. The usefulness of combining different tech-
niques in studying lignin distribution and the formation of stone
cells is also demonstrated. It is hoped that information presented
here can help to advance the view of stone cells in pear fruit and
help to improve the fruit quality by reducing the lignifying process.
Acknowledgement
The authors would like to acknowledge the support that
received from Specialized Research Fund for the Doctoral Program
of Higher Education (SRFDP) (B200523).
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