Copyright C Physiologia Plantarum 2002
ISSN 0031-9317
PHYSIOLOGIA PLANTARUM 114: 296–302. 2002
Printed in Denmark – all rights reserved
Lignification and lignin heterogeneity for various age classes of bamboo
(Phyllostachys pubescens) stems
Jinxing Lina,b,*, Xinqiang Hea, Yuxi Hua, Tingyun Kuanga and R. Ceulemansb
a
Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Department of Biology, University of Antwerpen, Wilrijk B-2610, Belgium
*Corresponding author, e-mail: linjx/ns.ibcas.ac.cn
b
Received 27 November 2000; revised 6 April 2001
The lignification process and lignin heterogeneity of fibre, vessel and parenchyma cell walls for various age classes of bamboo stems of Phyllostachys pubescens Mazel were investigated. It was shown that protoxylem vessels lignified in the
early stage of vascular bundle differentiation, metaxylem vessel and fibre walls initiated lignification from the middle lamella and cell corners after the completion of vascular bundle
differentiation. Most of the parenchyma cell walls lignified
after the stem reached its full height, while a few parenchyma
cells remained non-lignified even in the mature culm. The cell
walls of fibres and most parenchyma cells thickened further
during the stem growth to form polylamellate structure and
the lignification process of these cells may last even up to 7
years. The fibre walls were rich in guaiacyl lignin in the early
stage of lignification, and lignin rich in syringyl units were
deposited in the later stage. Vessel walls mainly contained
guaiacyl lignin, while both guaiacyl and syringyl lignin were
present in the fibre and parenchyma cell walls.
Introduction
Bamboos are perennial grasses, which occur mainly in
tropical, subtropical regions but with some taxa also in
the temperate areas of China, Japan, Chile and the
USA. They are fast-growing plants and their culms can
grow to their full height of 3–30 m within a few months
due to the expansion of individual internodes already
present in the buds (Metcalfe 1960, Liese 1998). With
the dwindling timber resources in the world over the past
several decades, woody monocotyledons, arborescent
bamboos in particular, have received considerable attention (Liese 1987). To better understand the growth
characteristics and physical properties of these bamboos, vast investigations have already been conducted.
However, most of these studies focused on the general
mode of growth and anatomical structure of the culms
(Lee and Chin 1960, Alvin and Murphy 1988, Murphy
and Alvin 1992, 1997). Few investigations dealt with the
evaluation of the lignification and lignin heterogeneity
for various age classes. In studies on tissue lignification
during growth, Itoh and Shimaji (1981) showed that lig-
nification increased progressively in both the fibres and
the ground tissue parenchyma during the first year of
growth. Itoh (1990) further concluded that lignification
was completed at the end of the first growing season
with no further increase of the lignification occurring in
the later stages. Nevertheless, such conclusions can be
challenged because both studies were largely based on
histochemical analysis.
The purpose of the present study is to examine the
variability in the cell wall lignification process with a
particular focus on lignin deposition within the vascular
bundles and seeking a general pattern of lignification
related to growth and maturation of the culm. A microscopic evaluation of the cell wall thickening and lignification processes in bamboo stems by histochemical
staining, as well as the autofluorescence traits of cell
walls by fluorescence microscopy before and after treatments with ammonia and H2O2/HAC, were performed.
In addition, the heterogeneous formation and distribution of lignin in different tissues were investigated by
Abbreviations – Ph-Phloem; Mv-Metaxylem, vessel; Pv-Protoxylem, vessel; Pa-Parenchyma, cell;
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microspectrophotometry coupled with the Wiesner or
Mäule reaction.
Materials and methods
Plant samples
Immature bamboo (Phyllostachys pubescens Mazel)
shoots of 0.5, 1.0, 2.0, 4.0, and 6.0 m in height were
harvested on April 25 1999 in the Bamboo Garden
(32æ2ƒ N, 118æ7ƒ E) of Nanjing Forestry University, Jiangsu province, China. Small blocks of about 1 mm in
thickness were cut from the middle part of the internodes at the top, middle and basal portions of the bamboo stems, fixed in 50% ethanol, and then preserved in
70% alcohol. For comparison, 1-, 3- and 7-year-old
culms were harvested on September 15, 1999 in the Anji
Bamboo Garden (30æ6ƒ N, 119æ6ƒ E), Zhejiang Province,
China. Sampled internodes were numbered from the
basal internode of the culms, then small blocks were cut
from the middle part of the internodes, fixed in 50%
alcohol and stored in 70% alcohol.
Sectioning and microscopy
Transverse sections of 8–20 mm in thickness were cut
with a cryomicrotome. Some sections were observed directly under the polarized microscope. Others were used
for observation of autofluorescence of cell walls by
fluorescence microscopy with UV-excitation (filter:
UG1, DM400, L410, L420) before and after chemical
treatment. For chemical treatment, the sections were extracted with 0.1 M ammonia for 5 min (Harris and
Hartley 1976), 30% hydrogen peroxide and 97% glacial
acetic acid (H2O2/HAC), 1 : 1 (v/v) at 100æC for 30 min,
respectively (Willemse and Emons 1991). Treatment with
10% glycerin served as the control. Sections were then
subjected to microspectrophometry except those observed directly under the light microscope after histochemical staining.
Histochemical staining and microspectrophotometry
Two histochemical tests with bright-field coloured reagents were used to observe polysaccharides in the fresh
sections. Acid polysaccharides were stained with 0.02%
ruthenium red for 5 min (Jensen 1962). For staining
crystalline polysaccharides, sections were treated for 5
min with 1% aqueous Congo red in water, washed and
mounted in water (Vallet et al. 1996).
Lignin was stained with Wiesner or Mäule reagent, respectively. Some sections were treated for 5 min with 2%
phloroglucinol in 95% ethanol and mounted in 6 M HCl to
demonstrate the cinamaldehyde groups present in lignin.
To detect the syringyl moieties in lignin, sections were
treated for 5 min with KMnO4, followed by washing in
water prior to the transfer of sections to 8 M HCl until the
sections were partially decoloured, and mounted in ammonia (Meshitsuka and Nakano 1979, Vallet et al. 1996).
Physiol. Plant. 114, 2002
Absorption spectra from 400 nm to 700 nm were measured
with a microspectrophotometer (Leitz MPV II, spot size:
1.5 mm; band width: 10 nm) in the sections after reaction
with Wiesner or Mäule reagents. For the average value, 10
measurements for each tissue were performed in one section within 10 min because the colour may gradually have
faded away after reaction.
Results
Histochemical staining of the cell walls
The tissues throughout the culm are entirely primary
and formed by the activity of the apical meristem and,
for a certain period of time, by the intercalary nodal
meristem (Liese 1985). The internodes were mainly composed of epidermis, cortex, ground parenchyma and vascular bundles.
The vascular bundles at the shoot apex were considered to be in the initial stage of differentiation, since
only protoxylem vessel and phloem cells appeared in this
stage (Itoh and Shimajii 1981, He et al. 1999). Metaxylem cells and fibres were not fully developed, as shown
in Fig. 1. All the tissues first developed showed blue
fluorescence after UV irradiation (Fig. 2). However, after
treatment with H2O2/HAC, only protoxylem vessels kept
their fluorescence, while other tissues lost their autofluorescence (Fig. 3). With the treatment with ammonia,
only protoxylem vessels remained blue in colour, while
others changed into green because of the existence of
ferulic acid (Harris and Hartley 1976). It was further
noted that only protoxylem vessels reacted positively to
the Wiesner reaction at this time. All tissues except protoxylem and phloem cells were stained faintly red with
Ruthenium red. Protoxylem and phloem cells showed
strong birefringence under polarized microscopy.
At different internodes other than the shoot apex, all
tissues showed blue fluorescence after irradiation of UV
light (Fig. 4). The phloem changed its fluorescence from
blue to green after ammonia treatment (Fig. 5), whereas
the vessel walls of fibre, protoxylem and metaxylem
showed blue fluorescence even after H2O2/HAC treatment (Fig. 6). Protoxylem vessels and fibres stained in
deep red, while metaxylem vessels stained faintly red in
the Wiesner reaction (Fig. 7). Fibres, the middle lamella
of fibres in particular, were stained deep red while the
vessel appeared brown after the Mäule reaction (Fig. 8).
Parenchyma cells and part of the metaxylem vessels near
the protoxylem vessels showed faintly red after staining
with Ruthenium red. Phloem and parenchyma cells
stained deep red with Congo red, while protoxylem, metaxylem vessels and fibres stained yellow. After staining
with safranin and fast green, protoxylem vessels, the
middle lamella and cell corner of the metaxylem vessels
and some fibres turned reddish, while most other cell
walls turned blue. All tissues had a strong birefringence.
In the 1-year-old culm, all tissues showed blue fluorescence after irradiation with UV light, the phloem
changed to green after ammonia treatment (Fig. 9),
297
Fig. 1. Safranine and fast green staining, the vascular bundle was in the early stage of differentiation, protoxylem vessel and phloem had
appeared, while metaxylem vessel and fibres were not formed. Magnification 380 x
Fig. 2. All tissues at the shoot apex showing blue autofluorescence after UV irradiation. Magnification 380 x
Fig. 3. Showing fluorescence remained in the protoxylem vessel but lost in other tissues after H2O2/HAC treatment. Magnification 380 x
Fig. 4. All tissues at the basal internode of shoot showed blue autofluorescence after UV irradiation. Magnification 190 x
Fig. 5. Phloem changed its colour into green, while other tissues remain blue fluorescence after treatment of ammonia. Magnification 190 x
Fig. 6. Showing fluorescence remained in the cell walls of protoxylem vessles, metaxylem vessles and fibres after H2O2/HAC treatment.
Magnification 190 x
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Physiol. Plant. 114, 2002
Fig. 7. The cell walls of protoxylem vessels, metaxylem vessels and fibres stained red in Wiesner reation. Magnification 190 x
Fig. 8. The cell walls of fibres were stained deep red while the vessels appeared brown at the basal internode of shoot in Mäule reaction.
Magnification 380 x
Fig. 9. All the tissues of the 1-year-old culm showed blue autofluorescence after UV irradiation. Magnification 190 x
Fig. 10. Phlom cells lost their fluorescence while other tissues kept their fluorescence after after H2O2/HAC treatment. Magnification 190 x
Fig. 11. The cell walls of fibres were stained deep red, parenchyma cells were stained light red in 1-year-old culm in the Wiesner reaction.
Magnification 160 x
Fig. 12. Safranine and fast green staining, most parenchyma cell walls were lignified, but there were some parenchyma cells that remained
thin and non-lignified wall (arrowhead) in 1-year-old culm. Magnification 250 x
Fig. 13. Polarized microscope, fibre and parenchyma cell wall exhibited a typical polylamallate structure in 7-year-old culm. Magnification 400 x
Physiol. Plant. 114, 2002
299
Fig. 14. Visible light absorption spectra of the fibre (I), vessel (II) and parenchyma walls (III) in bamboo stem after Wiesner reaction (A):
In the bamboo shoot, fibre walls exhibited a relatively high absorption in comparison with the weak absorption in the vessel walls and no
absorption in the parenchyma cell walls around 560 nm. (B): In the 7-year-old culm, fibre, vessel and parenchyma walls all exhibited an
absorption peak around 560 nm with a slight deviation.
while all the tissues except the phloem showed blue
fluorescence after H2O2/HAC treatment (Fig. 10), and
they had positive reactions with Wiesner (Fig. 11) and
Mäule reagents. Nevertheless, Ruthenium red was no
longer active in the tissues. Counter-staining with safranin and fast green has demonstrated that most of the
parenchyma cell walls, except a few of them, were
thickened and lignified (Fig. 12).
In the 3-year-old and 7-year-old culms, the staining
traits were mostly similar to those of the 1-year-old
culms, but the parenchyma cell walls were much more
thickened and lignified compared with tissues in the 1year-old culm. Fibre and parenchyma cell walls exhibited the typical polylamallate structure (Fig. 13).
Visible light absorption spectra
Visible light absorption spectra varied remarkably with
culm age. Figure 14A and 14B represented the spectra
Table 1. Absorbance [log(I0/I)] at 560 nm in tissues of the bamboo
shoot and mature culms after the Wiesner reaction. Values are the
means of 10 measurements for each tissue.
Discussion
Age of bamboo stems
Tissues
Bamboo
shoot
3-yr-old
5-yr-old
7-yr-old
Fibre
Vessel
Parenchyma
0.37
0.21
0.02
0.46
0.28
0.10
0.49
0.30
0.11
0.51
0.33
0.13
Table 2. Absorbance [log(I0/I)] at 560 nm in tissues of basal internode of the shoot and mature culms after the Mäule reaction.
Values are the means of 10 measurements for each tissue.
Age of bamboo stems
Tissues
Bamboo
shoot
3-yr-old
5-yr-old
7-yr-old
Fibre
Vessel
Parenchyma cell
0.98
0.23
0.09
1.17
0.24
0.57
1.21
0.24
0.59
1.28
0.24
0.61
300
of fibre, vessel and parenchyma cell walls at the basal
internode of the shoot and the 7-year-old culm with a
positive Wiesner reaction, respectively. In the bamboo
shoot, fibre walls exhibited a relatively high absorption
in comparison with the weak absorption in the vessel
walls and no absorption in the parenchyma cell walls
around 560 nm. In the 7-year-old culm, fibre, vessel and
parenchyma walls all exhibited an absorption peak
around 560 nm, with a slight deviation. The absorption
spectra at 560 nm of these tissues in different culm ages
are summarized in Table 1.
Figure 15 A and 15B illustrated the absorption spectra
of the fibre, vessel and parenchyma cell walls at the basal
internode of the shoot and 7-year-old culm with the
Mäule reaction, respectively. It was noted that no peak
was visible in the absorption curve of all tissues in the
bamboo shoot and shoulder-rises around 530 nm in the
absorption curve of the fibre and parenchyma cells of
the 7-year-old culm were observed. However, the spectra
of vessel walls remained more or less similar to that in
the bamboo shoot. The absorbance at 560 nm of these
tissues in different ages is summarized in Table 2 .
The present study displayed the colour reactions reflecting the differences in the staining properties among
vascular tissues, and even in the separate wall layers of
individual cells. Visible light microspectrophotometry
also revealed some differences in the absorption maximum and the absorbance among tissues. These findings
can be interpreted as indicating variable ratios of
guaiacyl and syringul units among the cell types at different ages of bamboo growth (He and Terashima 1991,
Yoshizawa et al. 1993).
The autofluorescence in the cell walls has already been
successfully adopted to determine the content and nature of lignin in gymnosperms and dicotyledons (Willemse and Emons 1991, Yoshizawa et al. 1993). For
monocotyledons, however, the autofluorescence in the
Physiol. Plant. 114, 2002
Fig. 15. Visible light absorption spectra of the fibre (I), vessel (II) and parenchyma walls (III) in bamboo stem after Mäule reaction.(A): In
the bamboo shoot, no peak in the absorption curve of all tissues.(B): In the seven-year-old culm, shoulder-rises around 530 nm in the
absorption curve of the fibre and parenchyma cells, the spectra of vessel walls remained more or less similar to that in the bamboo shoot.
lignified cell walls can be interfered because of the presence of phenolic acids esterified to hemicelluloses of the
non-lignified cell walls, which may also excite autofluorescence. To discriminate between phenolic acid bound
to hemicelluloses and phenolics in the lignin molecule,
treatment with ammonia has proved to be successful,
causing ferulic acid to green fluoresence, and lignin to
blue fluorescence (Harris and Hartley 1976). Furthermore, the H2O2/HAC treatment can remove hemicellulose and phenolic acid, but lignin and cellulose remained
intact (He et al. 1999). In the present study, the autofluorescence of young tissues after being treated with
ammonia changed from blue to green in most of the
tissues except in the protoxylem vessels. The colour
change revealed that the ferulic acid was widely distributed in the young tissues (Harris and Hartley 1976). The
young tissues in the shoot apex exhibited a positive reaction to Ruthenium red, indicating that the tissues were
in a state of polysaccharide deposition (Jensen 1962).
The presence of autofluorescence in protoxylem vessels
after treatment of H2O2/HAC at the shoot apex may be
interpreted as an earlier onset of lignification of the protoxylem vessels than that of the metaxylem vessels and
fibres of the differentiating vascular bundles. As the lignification advanced, most of the tissues showed no distinct colour changes after ammonia treatment in mature
tissues, suggesting that there was a decrease of ferulic
acid (Harris and Hartley 1976, Fujii et al. 1993).
Both the Mäule reaction and Wiesner reaction are
used to detect the presence of lignin (Higuchi 1957).
However, they represent different staining properties
that occur in tissue types and internodes of various bamboo ages. For example, protoxylem vessels strongly reacted with Wiesner reagents while all the tissues showed
no colour reaction with the Mäule reagents in the young
stage of differentiation. In the mature culms, protoxylem
vessels, metaxylem vessels and fibres had highly intensive
Physiol. Plant. 114, 2002
reactions with Wiesner reagents in contrast to the weak
reaction in the parenchyma cells. With the Mäule reagents, fibres together with parenchyma cells were heavily stained, but vessels reacted weakly in the mature
stage. The difference in colour reactions appeared to be
associated with the ratio of monomeric units of lignin
in the cell walls. The colour differences in response to
the reactions were associated with the ratio of monomeric units of lignin in the cell walls (Meshitsuka and
Nakano 1979).
Microspectrophotometric determination of the
samples reacted with Mäule or Wiesner reagents can reveal specific variations in the monomers of dimethoxylated syringyl compared with the corresponding
monomethoxylated guaiacyl monomeric unit (Yoshizawa et al. 1993). In conifers, there was only one absorption maximum around 390 nm, while there were two
maximum peaks for dicotyledons, one at 380–400 nm,
another at 470–530 nm (Higuchi 1957). In the present
study, the absorption curves detected for bamboo were
similar to those of dicotyledons. The absorption peak
around 385 nm was caused by the guaiacyl propane
component, whereas the other peak around 508 nm was
caused by the syringyl propane component of the lignin
of dicotyledons and bamboos (Fujii et al. 1991, Yoshizawa et al. 1993). As shown in Fig. 15(A), all tissues at the
shoot showed no absorption after the Mäule reaction,
while fibre and parenchyma cell walls of the 1-year-old
culm exhibited a shoulder-rise in the absorption curve
around 530 nm in Fig. 15(B). Furthermore, as shown in
Fig. 14 A and 14B, the fibre and vessel walls at the shoot
showed a weak absorption and the parenchyma cell walls
showed a very weak absorption, respectively, in comparison with the high peak absorption that occurred in fibre,
vessel and parenchyma cell walls of the 7-year-old culms
around 560 nm after the Wiesner reaction. These results
again demonstrated that vessel walls lignified at the first
301
stage, incorporate a lower syringyl/guaiacyl ratio lignin,
while the content of the syringyl unit increased particularly in the fibre and parenchyma walls as the lignification progressed (Shimada et al. 1970, Itoh 1990).
It is of interest to note that lignification can be continued after the cessation of culm growth. As shown in
Table 1, the spectral absorption in fibres increased from
0.46 at 3-years-old to 0.51 at 7-years-old. The absorbance in the parenchyma cells increased from 0.10 at 3years-old to 0.13 at 7-years-old, though the absorbance
in the vessels remained invariable. In addition, there was
an increase of the fibre wall thickness by 1–8 lamellae
from the shoot to 7-year-old culms. This finding is in
contrast to a more recent report, which stated that lignification of all the cell components completed within one
growth season in the old culm (Itoh 1990). The present
findings support the fact that the radial and tangential
bending strength of the bamboo culms estimated by
physical tests may last up to the 6-year-old culms. The
findings also support the experience of the management
of bamboo forest that culms are generally best harvested
at the age of 3–4 years (Liese 1985, Wei et al. 1997).
Acknowledgements – This study was financially supported by the
research grant of Chinese Academy of Sciences and State Key Basic
Research and Development Plan (G1998010100). JXL acknowledged support from the special research fund of the University of
Antwerpen (UIA). The authors acknowledge Mrs. Yuehui Lin and
Mrs. Lizhi Zhang for their technical assistance, as well as I. Laureysens and L. Pronk for comments on an earlier draft of this manuscript.
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