Tree Physiology 26, 201–210
© 2005 Heron Publishing—Victoria, Canada
Lignification in cell cultures of Pinus radiata: activities of enzymes and
lignin topochemistry
RALF MÖLLER,1,2 GERALD KOCH,3 BERNADETTE NANAYAKKARA4 and UWE SCHMITT3
1
New Zealand Forest Research Institute Ltd., Private Bag 3020, Rotorua, New Zealand
2
Corresponding author ([email protected])
3
Federal Research Centre for Forestry and Forest Products, Leuschnerstr. 91, 21031 Hamburg, Germany
4
The University of Waikato, Private Bag 3105, Hamilton, New Zealand
Received January 28, 2005; accepted May 20, 2005; published online November 8, 2005
Summary Enzymatic and topochemical aspects of lignification were studied in a Pinus radiata D. Don cell culture system
that was induced to differentiate tracheary elements and sclereids with lignified secondary cell walls. The activities of the
lignin-related enzymes phenylalanine ammonia lyase (PAL;
EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD; EC
1.1.1.195) increased concomitantly with cell differentiation,
indicating that the increase in enzyme activity was related to
lignification of the cell walls and was not induced by stress.
This result also indicates that PAL and CAD are suitable markers for tracheary element differentiation in coniferous gymnosperms. To further characterize lignification in this cell culture
system, cellular UV-microspectrophotometry and thioacidolysis were employed. Typical UV-absorption spectra of lignin
were obtained from the secondary cell walls of the tracheary elements and sclereids and from the compound middle lamella
connecting differentiated cells, and the presence of lignin was
confirmed by thioacidolysis. Certain aspects of lignin topochemistry in the cell walls of the tracheary elements were similar to cell walls of P. radiata wood, such as the high lignin
concentration in the compound middle lamella connecting adjacent cells and the lower lignin concentration in the secondary
cell walls. Therefore, the P. radiata cell culture system appears
to be well suited to study the formation of lignified secondary
cell walls in coniferous gymnosperms.
Keywords: callus culture, cell differentiation, cinnamyl alcohol dehydrogenase, phenylalanine ammonia lyase, sclereid,
secondary cell wall, tracheary element, tracheid, UV-microspectrophotometry.
Introduction
Lignin is a complex phenolic polymer formed by radical coupling reactions of mainly three hydroxycinnamyl alcohols or
monolignols: p-coumaryl (4-hydroxy-cinnamyl), coniferyl
(3-methoxy-4-hydroxy-cinnamyl) and sinapyl (3,5-dimethoxy-4-hydroxy-cinnamyl) alcohol, which are synthesized via
the phenylpropanoid pathway (Terashima 2000, Anterola and
Lewis 2002, Boerjan et al. 2003). After cellulose, it is the most
abundant biopolymer on earth and is one of the major components of wood. In coniferous gymnosperms, it is synthesized in
tracheary elements, phloem fibers, sclereids and xylem parenchyma cells and represents an integral component of the primary and secondary walls of these cells. Stress-induced lignin
biosynthesis also takes place in parenchyma cells near sites of
wounding or pathogen attack and lignin has been detected in
the primary cell walls and the lumen of these cells (Hawkins
and Boudet 2003).
Biochemical and enzymatic studies of lignin biosynthesis
have often been investigated in plant cell cultures. For this purpose, cell cultures have several advantages over entire plants
because: first, they are available in great quantities independent of season; second, large amounts of cells, cell walls or conditioned medium can be isolated from cell cultures for chemical analysis; and third, enzymes can be extracted from cell
cultures in sufficient quantities for biochemical characterization. However, lignification in most cell cultures of coniferous
gymnosperms has been induced by stress factors, such as treatment with UV-light and fungal elicitors, or by culturing in media with changed growth regulators or high concentrations of
sucrose (Brunow et al. 1990, Campbell and Ellis 1992,
Eberhardt et al. 1993, Messner and Boll 1993, Nose et al.
1995, Hotter 1997, Anterola et al. 2002, Stasolla et al. 2003).
Lignin was secreted into the cell culture medium and in some
cases also incorporated into the thickened walls of cells present in the elicited cell cultures (Campbell and Ellis 1992,
Eberhardt et al. 1993, Nose et al. 1995, Stasolla et al. 2003).
Lignification induced in these cell culture systems, however,
was unaccompanied by the formation of secondary cell walls.
Moreover, it was found that the secreted “extracellular lignin”
had a more condensed structure compared with lignin in secondary xylem from the same species (Brunow et al. 1993).
Möller et al. (2003) reported on a Pinus radiata D. Don
(radiata pine) cell culture system that had been induced to differentiate cells with lignified secondary cell walls. To investigate the suitability of this cell culture system for studying the
formation of secondary cell walls, in particular their lignifica-
202
MÖLLER, KOCH, NANAYAKKARA AND SCHMITT
tion, we monitored the activities of the first enzyme in the
phenylpropanoid pathway, phenylalanine ammonia lyase
(PAL; EC 4.3.1.5) and of the last enzyme involved in the
biosynthesis of the monolignols, cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195). In cell cultures of isolated mesophyll cells of Zinnia elegans L. that differentiated tracheary
elements, PAL and CAD activities increased concomitantly
with cell differentiation (Fukuda and Komamine 1982, Lin
and Northcote 1990, Sato et al. 1997), suggesting that PAL and
CAD are useful markers for the differentiation of tracheary elements in cell cultures of angiosperms (Northcote 1995).
However, this has not been investigated in cell cultures of the
evolutionarily distant coniferous gymnosperms.
Another important aspect of lignification is the distribution
of lignin in cell walls. It is unknown whether the lignin distribution in the cell walls of tracheary elements and sclereids differentiated in vitro is similar to that in the cell walls of tracheids differentiated in vivo. We used scanning UV-microspectrophotometry, which is a sensitive, high-resolution microspectroscopic method (Chabannes et al. 2001, Koch and
Kleist 2001), to investigate lignin topochemistry in the lignified secondary walls of cells induced to differentiate in callus
cultures of P. radiata. With this method, lignin distribution in
ultrathin sections of samples can be analyzed by measuring the
UV-absorption at a fixed wavelength in numerous areas of a
section with a resolution of 0.25 × 0.25 µm 2 (Koch and Kleist
2001, Koch et al. 2003). These UV-scanning images reveal detailed information about lignin distribution in plant cell walls
and can be analyzed by a variety of new graphical and statistical methods. Ultraviolet-absorption spectra can also be obtained, which are useful for characterizing phenolic cell wall
components and secreted phenolic compounds (Koch and
Kleist 2001, Koch et al. 2003).
We present evidence that lignin formation was not stress-induced and that lignin topochemistry was similar to that in
tracheid cell walls of P. radiata wood. These results demonstrate that the P. radiata cell culture system is useful for studying the lignification of cells with secondary walls in coniferous
gymnosperms.
dium, transferred to nylon mesh discs and then subcultured as
described above on embryo development medium (Walter and
Grace 2000) supplemented with activated charcoal (charcoal
activated Guaranteed Reagent for analysis; Merck, Darmstadt,
Germany) (2 g l – 1) and solidified with 3 g l – 1 Gelrite, but without phytohormones.
Materials and methods
UV-microspectrophotometry
In vitro culture
Callus cultures were initiated from xylem strips of P. radiata
as described previously (Möller at al. 2003). Xylem strips
(3 cm long × 0.8 cm wide) were obtained from shoots of
3-year-old P. radiata trees, transferred to solidified P6-SHvmedium (Hotter 1997), and incubated at 24 °C in continuous
low light (5 µmol m – 2 s –1) to induce callus formation. Calli
were suspended in liquid P6-SHv medium and the suspension
transferred to nylon mesh discs (mesh size 20 × 30 µm). The
excess fluid was absorbed and the discs bearing the callus cells
transferred to fresh medium. The cell line was subcultured in
the same way every 14 days (control cultures).
To induce cell differentiation, callus from xylem strips (1 g
fresh mass (FM) in 4 ml) was suspended in liquid P6-SHv-me-
Cell counting and histochemistry
The presence of tracheary elements and sclereids in callus cultures was monitored by randomly picking pieces of callus, suspending them in water, homogenizing them with a cell
disrupter (FastPrep FP 120; Savant Instruments, Farmingdale,
NY), treating them with phloroglucinol-HCl (Krishnamurthy
1999) and examining them by bright-field and polarized-light
microscopy. The number of tracheary elements and sclereids
was determined as a percentage of the total number of cells.
The mean number of differentiated cells was determined and
the standard deviation calculated.
Callus tissue was also histochemically examined for the
presence of catechins and condensed tannins (pro-anthocyanidins) using the vanillin test (Sarkar and Howarth 1976). Callus cells were transferred to a centrifuge tube and treated for
5 min in a solution of vanillin (10% w/v) in ethanol, followed
by 1 volume of concentrated HCl. Samples were treated in the
same way with a control solution of ethanol and concentrated
HCl (1:1 v/v). A red color reaction with the vanillin solution
indicates the presence of flavanols and a red color reaction in
the control solution indicates the presence of anthocyanins
(Sarkar and Howarth 1976).
Confocal laser scanning microscopy
Callus cells were stained with an aqueous solution of basic
fuchsin (0.001% w/v) for 10 min, mounted in glycerol and examined with a confocal laser scanning microscope (CLSM)
(Model TCS NT; Leica, Wetzlar, Germany). Samples were illuminated with an excitation wavelength of 568 nm and the
emission passing through a long-pass filter (LP 600) was recorded.
Ultraviolet-microspectrophotometry (UMSP) was carried out
as described by Koch and Kleist (2001). Briefly, P. radiata callus cells and wood samples from the stem of a 20-year-old
P. radiata tree were dehydrated in an acetone series and embedded in Spurr’s epoxy resin as described by Singh (1996).
Sections (1 µm thick) were cut with a diamond knife, fixed to
quartz glass slides and immersed in non-UV-absorbing glycerine. The sections were covered with quartz cover slips and analyzed with a ZEISS UMSP 80 microspectrophotometer (Carl
Zeiss AG, Oberkochen, Germany) equipped with a scanning
stage. Before measurements, an area devoid of plant material
was selected to adjust the instrument. The reference value was
defined as 100% transmission. Image profiles at a constant
wavelength of 280 nm (absorbance maximum of softwood
lignin) with a geometrical resolution of 0.25 × 0.25 µm 2 were
TREE PHYSIOLOGY VOLUME 26, 2006
LIGNIFICATION IN PINUS RADIATA CELL CULTURES
acquired using the scan program APAMOS (Carl Zeiss AG,
Oberkochen, Germany). This program controlled the scanning
stage and allowed the measurement of a field consisting of up
to 65,000 data points. Absorption spectra, ranging from 240 to
400 nm, were obtained for spot sizes of 1 µm 2 with the
program LAMWIN (Carl Zeiss AG, Oberkochen, Germany).
The UV-micrographs were taken at a wavelength of 280 nm
with a ZEISS microspectrophotometer UMSP I. Because the
plane of the section may affect absorption measurements, particularly in thick-walled sclereids, only those cells that showed
well defined inner and outer wall boundaries were selected for
UV-absorption measurements.
Extraction of soluble components
Callus tissue (about 50 mg FM) from control and induced callus cultures was homogenized with a cell disrupter (FastPrep)
in 1.5 ml of aqueous acetone (50% v/v). The homogenate was
extracted on a tube shaker for 30 min at 24 °C, centrifuged
(14,926 g, 5 min), the extract removed and the pellet resuspended in aqueous acetone (50% v/v). This procedure was repeated three times and the pooled extracts were stored at 4 °C.
Determination of the total amount of soluble phenolic
components by the Folin-Ciocalteu method
The soluble extract (50 µl) was transferred to a centrifuge tube,
475 µl of 0.25 N Folin-Ciocalteu reagent (Sigma Chemical, St.
Louis, MO) added and the solution mixed immediately. After
3 min, 475 µl of 1 M Na 2CO 3 was added, the solution mixed,
incubated for 1 h and the absorbance measured at 750 nm. The
total amount of soluble phenolic components was quantified
against a standard curve for (+)-catechin.
203
let washed by centrifugation twice with water (1 ml). The
TGAL was dissolved in NaOH (1 M, 2 ml) and the absorbance
measured at 280 nm. The content of TGAL in the cell walls of
the callus cultures was calculated based on an extinction coefficient of 13.4 l g – 1 cm – 1, determined from a linear calibration
curve obtained for milled-wood lignin of P. radiata.
Thioacidolysis
Callus cells were homogenized in 20 mM MOPS-KOH buffer
(pH 6.8) containing 20 mM sodium metabisulfite. The homogenate was filtered on a nylon filter and the filtrate was washed
with 20 ml buffer and 20 ml of deionized water. The filtrate
was collected, extracted with methanol for 20 min and collected by centrifugation. The resulting pellet was extracted in a
methanol:chloroform mixture (1:1 v/v) for 18 h at 24 °C, collected by centrifugation and washed with methanol. The pellet
was resuspended in a mixture of toluene:ethanol (2:1 v/v) and
the homogenate stirred for 24 h at 24 °C. The extracted cell
walls were washed twice with acetone, vacuum dried and
stored over silica gel in a desiccator. Thioacidolysis was carried out as described by Rolando et al. (1992), except reactions
were performed at 110 °C for 2 h (Pasco and Suckling 1994).
The products were analyzed by gas chromatography-mass
spectrometry (GC/MS) and the mass spectra compared with
those published in the literature (Rolando et al. 1992). Further,
peak identity was confirmed by comparison with products
formed on thioacidolysis of authentic samples (p-hydroxyphenylglycerol-β-guaiacyl ether and guaiacylglycerol-βguaiacyl ether).
Preparation of extracts and assay of CAD and PAL
Thioglycolic acid lignin assay
The insoluble residues from the acetone (50% v/v) extractions
were homogenized in methanol:chloroform:H 2O (2:1:0.8 v/v)
with a cell disrupter (FastPrep), incubated for 5 min, centrifuged (900 g, 3 min) and the supernatant discarded; this process was repeated once. The same procedure was repeated
with aqueous ethanol (70% v/v) and then with acetone (100%)
and the pellet was dried overnight at 70 °C. One ml of 1 M
NaOH was added and the sample extracted on a tube rocker
overnight at 24 °C. The sample was acidified with 2 M HCl,
centrifuged (10,000 g, 3 min) and the supernatant discarded.
The pellet was washed with water (1 ml), the supernatant discarded and HCl (2 M, 900 µl) and thioglycolic acid (100 µl)
were added. The mixture was incubated at 100 °C for 4 h,
cooled on ice, centrifuged (2900 g, 3 min) and the supernatant
discarded. The pellet was washed with water (1 ml), resuspended in NaOH (1 M, 1 ml) on a tube rocker overnight at
24 °C, centrifuged and the supernatant containing the thioglycolic acid lignin (TGAL) recovered. The pellet was washed
twice with water (0.5 ml) and the supernatants pooled. Concentrated HCl (100 µl) was added and the sample incubated for
3 h at 4 °C to precipitate the TGAL. The sample was centrifuged (26,000 g, 5 min), the supernatant discarded and the pel-
Protein extraction was carried out at 4 °C. Callus cells were
homogenized with a cell disrupter (FastPrep) in Tris-HCl
buffer (100 mM, pH 8.8) containing 10 mM dithiothreitol
(DTT), 25 mg ml –1 polyvinylpolypyrrolidone (PVPP) and
0.5% (w/v) polyethylene glycol (PEG) 8000. The extract was
centrifuged (14,926 g, 2 min) and the total protein in extracts
was quantified using the Protein Assay Dye Reagent Concentrate (Bio-Rad, CA) (Bradford 1976). Standard curves were
constructed with bovine serum albumin (BSA) (Sigma Chemical). Extracts containing 20 µg total protein were assayed
spectrophotometrically for CAD (at 30 °C) by the method of
Wyrambik and Grisebach (1975). The assay solution (1 ml)
contained 2 mM coniferyl alcohol and 2 mM NADP in TrisHCl buffer (100 mM, pH 8.8) and production of cinnamyl aldehyde measured as the change in absorbance at 400 nm. We
used a modification of the spectrophotometric method described by Edwards and Kessmann (1992) to assay PAL activity. Protein extract (100 µl) was incubated (37 °C) with 2.9 ml
of the assay solution, which contained 12.1 mM L-phenylalanine in Tris-HCl buffer (50 mM, pH 8.5). Production of
cinnamic acid was measured as the change in absorbance at
290 nm over a 2-h period. Control samples were incubated
with an assay solution containing D-phenylalanine instead of
L-phenylalanine.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
204
MÖLLER, KOCH, NANAYAKKARA AND SCHMITT
Results
Cell differentiation
Callus cultures grown on a maintenance medium did not contain cells with secondary cell walls. However, five days after
transfer of these cultures to induction medium, the first tracheary elements and sclereids were detected. After 18 days of
culture when the experiments were terminated, the number of
differentiated cells in the induced callus cultures was 18 ± 5%.
No tracheary elements or sclereids differentiated in the control
cultures kept on maintenance medium over the same period.
Enzyme activities in control cultures and induced cultures
Activities of PAL and CAD in callus cultures of P. radiata increased transiently during the first three days after transfer of
the cells to new maintenance or induction medium (Figures 1A and 1B). In induced callus cultures, enzymatic activities increased further after 5 days of culture. The PAL activity
in induced cultures reached 68 ± 6 pkat mg –1 protein after six
days of culture and then remained stable. This is a sixfold increase in PAL activity compared with the control cultures. The
CAD activity in induced cultures increased continually and
peaked at 1207 ± 63 pkat mg –1 protein after 18 days of culture
when the experiment was completed. This is a threefold increase in CAD activity compared with the control cultures.
Contents of soluble phenolic components and lignin analysis
The amount of soluble phenolic components in acetone extracts and the amount of TGAL in cell walls from control cultures remained low and showed little change over the 18 days
of culture. However, in induced callus cultures, the amount of
soluble phenolic components in acetone extracts and the
amount of TGAL in cell walls increased concomitantly with
the increase in PAL and CAD activities (Figures 2A and 2B).
After 18 days, the concentration of TGAL was 18 ± 2%. The
presence of lignin was further confirmed by thioacidolysis, a
technique extensively used for the analysis of lignin in wood
(Rolando et al. 1992) (Figure 3). Results showed that cell walls
isolated from the induced cultures released 3349 µmol g –1
lignin units from uncondensed guaiacyl β-ethers and 137 µmol
g –1 lignin units from uncondensed p-hydroxyphenyl β-ethers.
In comparison, wood samples from the stem of 20-year-old
P. radiata trees released 1233 µmol g –1 lignin units from
uncondensed guaiacyl β-ethers and 35 µmol g –1 lignin units
from uncondensed p-hydroxyphenyl β-ethers.
UV-absorption spectra of cell walls and extracellular
droplets
Representative UV-absorption spectra of sections of lignified
cell walls from tracheary elements and sclereids differentiated
in induced callus cultures are shown in Figure 4. The spectra of
the secondary cell walls and of the middle-lamella/primarywall (compound middle lamella) regions from tracheary elements and sclereids showed the characteristic UV-absorption
spectrum of lignin with a minimum near 260 nm and a maximum near 280 nm. Two types of UV-absorption spectra were
Figure 1. Induction of enzymes associated with lignification in
P. radiata callus cultures during differentiation of tracheary elements
and sclereids. Enzymatic activities were monitored at 24-h intervals
during the first 6 days of culture and at 72-h intervals thereafter. (A)
Time course of phenylalanine ammonia lyase (PAL) activity in extracts of control and induced P. radiata callus cultures. (B) Time
course of cinnamyl alcohol dehydrogenase (CAD) activity in extracts
of control and induced P. radiata callus cultures. Black and white bars
represent enzymatic activities in control and induced callus cultures,
respectively. Arrows indicate when the first tracheary elements and
sclereids were detected.
observed for droplets that were attached to the outer surface of
the walls of callus cells. For most of the droplets the spectrum
was characteristic of lignin. For other droplets, the spectrum
showed a maximum near 284 nm but lacked a minimum near
260 nm (Figure 4).
Lignin topochemistry
The tracheary elements differentiated in induced callus cultures had either reticulate secondary cell wall patterns or pitted
secondary cell wall patterns with bordered pits (Figure 5). In
cross sections examined by UV-microspectrophotometry at a
wavelength of 280 nm, the secondary wall thickenings were
readily identified (Figure 6A). From the contrast of the UVmicrographs it was deduced that the lignin concentration in the
secondary cell wall thickenings was lower than in the compound middle lamella region connecting the differentiated
TREE PHYSIOLOGY VOLUME 26, 2006
LIGNIFICATION IN PINUS RADIATA CELL CULTURES
205
Figure 3. A GC/MS (selected ion monitoring) chromatogram of
monomeric degradation products after thioacidolysis of a cell wall
preparation from induced P. radiata callus cultures. Peaks 1 and 2 represent lignin degradation products released from uncondensed
p-hydroxyphenyl β-aryl ethers, and Peaks 3 to 5 represent lignin degradation products released from uncondensed guaiacyl β-aryl ethers.
Abbreviation: IS = internal standard (hexacosane).
centration was present in the cell corners and in the compound
middle lamella region (A280nm 0.48 to 1). The lignin concentration of the secondary cell wall was lower (A280nm 0.22 to 0.35).
Histochemical analysis of phenolic components
Figure 2. (A) Concentration of soluble phenolics determined as
(+)-catechin equivalents in control and induced P. radiata callus cultures. (B) Lignin concentration quantified as thioglycolic acid lignin
(TGAL) in control and induced P. radiata callus cultures. Callus cultures were monitored at 72-h intervals. Black and white bars represent
values for control and induced callus cultures, respectively. Arrows
indicate when the first tracheary elements and sclereids were detected.
cells. The UV-scanning images of sections confirmed this
qualitative analysis. The lignin concentration in cell walls was
highest in the compound middle lamella region (A280nm 0.49 to
0.95) and lower in the secondary cell wall (A280nm 0.22 to 0.35)
(Figure 6B). Transverse sections of sclereids revealed a layered cell wall structure and pit channels (Figure 7A). The secondary cell walls of the sclereids often had higher lignin
concentrations (A280nm 0.37 to 0.90) compared with the cell
walls of the tracheary elements (Figure 7B). However, lignin
distribution was variable with areas of high and low lignin
concentration within the wall of an individual cell. Frequently,
cells with phenolic substances attached in the form of droplets
to the outer surface of the cells were observed. The droplets
were found attached to cells with or without secondary cell
walls. These droplets stained pink after treatment with phloroglucinol-HCl (data not shown). They strongly absorbed UVradiation at 280 nm as indicated by the dark contrast in the
UV-micrographs (Figure 8A) and the high absorption values
measured by UV-microspectrophotometry (A280nm 0.5 to 1.13)
(Figure 8B). In comparison, the lignin topochemistry of
P. radiata wood is shown in Figure 9. The highest lignin con-
Control cultures stained strongly red after treatment with the
vanillin solution, indicating the presence of flavanols and
dihydrochalcones or condensed tannins, or both (Sarkar and
Howarth 1976). A weak color reaction was detected with the
ethanolic control solution, indicating that anthocyanins were
present in only small amounts. The phloroglucinol-HCl reaction for lignin was negative. Induced callus cultures stained
only weakly red in the vanillin solution and very weakly in the
ethanolic control solution; however, a strong red color reaction
Figure 4. Representative UV absorbance spectra of a secondary wall
of a tracheary element (䉭) and of a secondary wall of a sclereid (䊊)
differentiated in P. radiata callus cultures; the compound middle lamella region between differentiated cells (䉱); and excreted phenolic
substances that were produced by P. radiata callus cells and which adhered as droplets to the outer surface of the cells (䊏, 䊐).
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
206
MÖLLER, KOCH, NANAYAKKARA AND SCHMITT
Figure 5. Confocal micrograph
of tracheary elements differentiated in P. radiata callus cultures. Tracheary elements
showing reticulate or pitted
secondary cell-wall patterns.
The pits, which had borders,
had different sizes and shapes
(white arrows).
was detected with phloroglucinol-HCl, indicating the presence of lignin (data not shown).
Discussion
The activities of PAL and CAD were investigated in relation to
the differentiation of tracheary elements and sclereids in the
P. radiata cell cultures. Phenylalanine ammonia lyase is in-
volved in the biosynthesis of phenylpropanoids that can be
incorporated into cell wall polymers such as lignin, or phenolic compounds such as flavonoids or stilbenes (Boerjan et al.
2003). We detected low PAL activity in extracts from unlignified P. radiata callus cultures. Our histochemical results
showed that flavonoids were present in these control cultures
and that PAL activity is required for their biosynthesis. Similar
PAL activities have been detected previously in unlignified
Figure 6. (A) A UV-micrograph of tracheary elements
differentiated in P. radiata callus cultures. Note the secondary wall thickenings and the
darker appearing compound
middle lamella region. A
dense fibrillar network was observed between some callus
cells (arrows). The marked
area corresponds to the area
that was scanned at a wavelength of 280 nm. (B) The
scanned area had a size of
48.5 × 49 µm and the scanning image represents 38,415 UV-absorption measurements at a resolution of 0.25 µm 2. The highest absorption values
were measured in the compound middle lamella region (A280nm 0.42 to 0.48). Absorption values are color coded, ranging from the lowest absorption of 0 (white) to the highest absorption of 1 (black).
TREE PHYSIOLOGY VOLUME 26, 2006
LIGNIFICATION IN PINUS RADIATA CELL CULTURES
207
Figure 7. (A) A UV-micrograph of a sclereid differentiated in a P. radiata callus
culture. Note the layered cell
wall structure and the pit channels (arrows). (B) A UV-scanning image (280 nm) of the
sclereid shown in Figure 7A.
The scanned area was 53.0 ×
63.0 µm and the scanning image represents 53,889 UV-absorption measurements at a
resolution of 0.25 µm 2. Absorption values (A280nm) in
the secondary cell wall ranged from 0.37 to 0.90. Absorption values were color coded, ranging from the lowest absorption (value = 0, white) to the
highest absorption (value = 1, black).
Figure 8. (A) A UV-micrograph of callus cells in a
P. radiata callus culture secreting phenolic substances on induction medium. The droplets
were either attached to the
outer cell walls or in the close
vicinity of the cells (arrows).
Phenolics were also found in
the vacuoles of the cells. (B) A
UV-scanning image (280 nm)
of the marked area in Figure
8A. The scanned area was 52.5
× 56.75 µm and the scanning
image represents 48,108 UV-absorption measurements at a resolution of 0.25 µm2. Absorption values were color coded, ranging from the lowest
absorption (value = 0, white) to the highest absorption (value = 1, black).
Figure 9. A UV-scanning image of a transverse section of
P. radiata wood scanned at a
wavelength of 280 nm. The
scanned area was 56.25 ×
47.25 µm and the scanning image represents 43,392 UV-absorption measurements at a
resolution of 0.25 µm 2. Highest absorption values were detected in the cell corners
(A280nm 0.8 to 0.93). The compound middle lamella regions
(A280nm 0.48 to 0.54) had a
higher lignin concentration
than the secondary cell walls
(A280nm 0.29 to 0.35).
Absorption values were color coded, ranging from the lowest absorption (value = 0, white) to the highest absorption (value = 1, black).
callus and suspension cultures of various coniferous gymnosperms; however, it was not reported whether flavonoids were
present (Messner et al. 1991, Campbell and Ellis 1992, Hotter
1997). The transient increase in PAL activity within one day
after transfer of the callus cells to fresh maintenance or induction medium was probably caused by wounding of the cells
when they were suspended and then spread onto the nylon
mesh. A similar transient increase in PAL activity has been ob-
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
208
MÖLLER, KOCH, NANAYAKKARA AND SCHMITT
served in suspension and callus cultures of Picea abies L.
(Norway spruce) (Messner et al. 1991).
The second increase in PAL activity in induced callus cultures that differentiated tracheary elements and sclereids was
due to lignification of the walls of these cells and was not a
stress response. Similar observations have been made in Z. elegans mesophyll suspension cultures that differentiated tracheary elements with lignified cell walls. In these cultures, PAL
activity reached maximum values when lignin was deposited
(Lin and Northcote 1990). In contrast, cell cultures of conifers
that were elicited by fungal elicitors showed only a transient
increase in PAL activity on elicitation, although “extracellular
lignin” was secreted into the cell culture medium (Messner et
al. 1991, Campbell and Ellis 1992, Messner and Boll 1993).
The secretion of the “extracellular lignin” in these studies can
be regarded as a stress response.
Cinnamyl alcohol dehydrogenase is responsible for the reduction of hydroxycinnamyl aldehydes to hydroxycinnamyl
alcohols (monolignols), which are lignin precursors (Boerjan
et al. 2003). We detected CAD activity in the undifferentiated,
unlignified control callus cultures that did not stain after treatment with phloroglucinol-HCl. Similar CAD activities have
been reported for suspension cultures of P. radiata and Pinus
banksiana Lamb. (Jack pine) (Campbell and Ellis 1992, Hotter
1997) and for megagametophytes of Pinus taeda L. (loblolly
pine), which also have unlignified cell walls (MacKay et al.
1995). Hydroxycinnamyl alcohols are also necessary for the
biosynthesis of lignans such as dehydrodiconiferyl alcohol
(Orr and Lynn 1992) and we conclude that this may account
for the CAD activity in the control cultures.
The strong increase in CAD activity in induced callus cultures after 5 days of subculture was due to the lignification of
the cell walls of tracheary elements and sclereids. A similar increase in CAD activity has been observed in Z. elegans mesophyll suspension cultures that differentiated tracheary elements with lignified secondary cell walls (Sato et al. 1997).
We conclude that the activities of PAL and CAD are useful
markers for in vitro studies of differentiation of cells with lignified secondary cell walls in gymnosperms; however, these
enzymes are also involved in the biosynthesis of phenolic
compounds other than lignin and can be transiently stimulated
by treatment with elicitors.
The lignin synthesized in induced callus cultures was analyzed chemically and by cellular UMSP. Monomers specific
for lignin were released by thioacidolysis and the absorption
spectra obtained from cell walls of the tracheary elements and
sclereids were found to be typical for lignin, with a minimum
at about 260 nm and a maximum near 280 nm (Goldschmid
1970). The thioacidolysis results indicated that the lignin present in the induced callus cultures contained similar proportions of p-hydroxyphenyl and guaiacyl units as the lignin in
P. radiata wood. In contrast, Brunow et al. (1993) found that
the “extracellular lignin” secreted by spruce suspension cultures contained a higher proportion of p-hydroxyphenyl to
guaiacyl units compared with milled wood lignin from spruce.
These differences indicate that lignification in P. radiata cell
cultures is not stress-induced, but related to secondary cell
wall formation.
Compared with a lignin content of 24 to 28% for P. radiata
wood (Uprichard 1991), the lignin content of induced callus
cultures after 18 days appears to be relatively high (18%),
given that only 18 ± 5% of the cells had lignified secondary
cell walls. However, a similar lignin content for induced callus
cultures of P. radiata was reported earlier (Möller et al. 2003).
One reason for this comparatively high content is that the
lignin concentration in the cell walls of the sclereids, which accounted for 5% of the differentiated cells, was much higher
than that of the tracheids of normal P. radiata wood as determined by UV-microscopy. It is also possible that lignin was
present in the primary cell walls of parenchymatous cells in
the induced callus cultures. Furthermore, the thioglycolic acid
lignin may have contained proteins, in which case the method
we used would have slightly overestimated the lignin concentration (Chen et al. 2000).
Our UMSP results show that the lignin topochemistry of lignified cell walls of tracheary elements in induced callus cultures and in xylem tissue were similar. The compound middle
lamella and the cell corners had the highest lignin concentration, whereas the secondary cell walls had a lower lignin concentration. The lignin concentrations in cell walls of in vitro
tracheary elements and in cell walls of xylem tracheids from
P. radiata were slightly lower than those measured by Koch
and Kleist (2001) for cell walls of tracheids from secondary
xylem of P. abies, who reported absorbance values of log
Abs 280nm 0.61–0.87 for cell corners and compound middle
lamella and values of log Abs 280nm 0.35–0.54 for the S2 layers
of the secondary cell wall. Fergus et al. (1969) reported that
the mean lignin concentration in the compound middle lamella
of tracheids from Picea mariana Mill. (black spruce) was
about twice that in the secondary wall. In contrast, the lignin
concentration in the cell walls of sclereids was variable and
generally higher than in the cell walls of tracheary elements.
Areas of higher and lower lignin concentration were observed
in the secondary cell walls of the sclereids; however, we did
not observe lignin distribution matching the pattern of the layered secondary cell walls of these cells. It may be that the resolution of the method used was not high enough to show this
pattern.
Our histochemical and microspectrophotometric results
showed the secretion of phenolic substances by callus cells in
the induced callus cultures. These droplets, which were either
attached to cell walls or present in intercellular spaces of the
callus cultures, stained red after treatment with phloroglucinol-HCl, indicating that they contained aromatic aldehydes,
which are present in lignin (Pomar et al. 2002). Because the
UV-absorption spectra of most extracellular droplets were
similar to those of lignin, we conclude that the cultures secreted lignin-like substances similar to those described earlier
(Brunow et al. 1990, Nose et al. 1995, Anterola et al. 2002).
However, we also found that the UV-absorption spectra of
some droplets were similar to those of phenolic extractives,
such as tannins (Koch and Kleist 2001, Koch et al. 2003). The
spectra were characterized by higher absorbance values and
the absorbance maxima showed a bathochromic shift to a
TREE PHYSIOLOGY VOLUME 26, 2006
LIGNIFICATION IN PINUS RADIATA CELL CULTURES
wavelength of 284 nm. This spectral behavior can be explained by the presence of conjugated double bonds. The
higher degree of conjugation stabilizes π–π* transitions, resulting in absorbance bands shifted to a higher wavelength
(Goldschmid 1970). It is also possible that these secreted droplets contained a mixture of different compounds such as
lignin-like substances and proteins. Lange et al. (1995) found
that the extracellular stress-induced lignin secreted by elicited
spruce suspension cultures contained 32% protein and 14%
carbohydrates.
In conclusion, we have shown that PAL and CAD are useful
markers for tracheary element differentiation in cell cultures
of coniferous gymnosperms. We also found that the lignin
topochemistry in tracheary elements differentiated in P. radiata callus cultures was similar to that in tracheids of P. radiata
wood. It has been shown previously that cell cultures of coniferous gymnosperms provide a valuable model system for
studying different aspects of lignification (Brunow et al. 1990,
Eberhardt et al. 1993, Nose et al. 1995, Anterola et al. 2002,
Stasolla et al. 2003). The P. radiata cell culture system, however, is particularly useful because of the differentiation of
cells with lignified secondary cell walls. Because the P. radiata cell culture system can be genetically transformed before
induction of secondary cell-wall formation and lignification
(Möller et al. 2003), we believe that it will be useful for studying the effects of altered gene expression on the biosynthesis
of phenylpropanoids and the process of lignification.
Acknowledgments
We thank Gertie Carroll, Karen Hignett and Tomoko Pearson for technical assistance with tissue culture, Tanja Potsch for technical support, and Christina Waitkus and John Smith for photographic assistance. Many thanks also to Dr. Ian D. Suckling for his advice on
thioacidolysis and Assoc. Prof. Philip J. Harris and Drs. Adya Singh,
Christian Walter and Armin Wagner for critical reading of the manuscript. R.M. acknowledges financial support through a Royal Society
of New Zealand ISAT Linkages Fund Travel Grant.
References
Anterola, A.M., J.-H. Jeon and N.G. Lewis. 2002. Transcriptional
control of monolignol biosynthesis in Pinus taeda. J. Biol. Chem.
277:18,272–18,280.
Anterola, A.M. and N.G. Lewis. 2002. Trends in lignin modification:
a comprehensive analysis of the effects of genetic manipulations/
mutations on lignification and vascular integrity. Phytochemistry
61:221–294.
Boerjan, W., J. Ralph and M. Baucher. 2003. Lignin biosynthesis.
Annu. Rev. Plant. Biol. 54:519–546.
Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 72:248–254.
Brunow, G., R.M. Ede, L.K. Simola and J. Lemmetyinen. 1990.
Lignins released from Picea abies suspension cultures: true native
spruce lignins? Phytochemistry 29:2535–2538.
Brunow, G., I. Kilpeläinen, C. Lapierre, K. Lundquist, L.K. Simola
and J. Lemmetyinen. 1993. The chemical structure of extracellular
lignin released by cultures of Picea abies. Phytochemistry 32:
845–850.
209
Campbell, M.M. and B.E. Ellis. 1992. Fungal elicitor-mediated response in pine cell cultures I. Induction of phenylpropanoid metabolism. Planta 186:409–417.
Chabannes, M., K. Ruel, A. Yoshinaga, B. Chabbert, A. Jauneau,
J.-B. Joseleau and A.M. Boudet. 2001. In situ analysis of lignins in
transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular
and subcellular level. Plant J. 28:271–282.
Chen, M., A.J. Sommer and J.W. McClure. 2000. Fourier transform-IR determination of protein contamination in thioglycolic
acid lignin from radish seedlings, and improved methods for extractive-free cell wall preparation. Phytochem. Anal. 11:153–159.
Eberhardt, T.L., M.A. Bernards, H. Lanfang, B.D. Laurence,
J.B. Wooten and N.G. Lewis. 1993. Lignification in cell suspension
cultures of Pinus taeda. J. Biol. Chem. 268:21,088–21,096.
Edwards, R.A. and H. Kessmann. 1992. Isoflavonoid phytoalexins:
their biosynthetic enzymes. In Molecular Plant Pathology: A Practical Approach. Eds. S.J. Gurr, M.J. McPherson and D.J. Bowles.
Oxford University Press, Oxford, pp 54–55.
Fergus, B.J., A.R. Procter, J.A.N. Scott and D.A.I. Goring. 1969. The
distribution of lignin in sprucewood as determined by ultraviolet
microscopy. Wood Sci. Technol. 3:117–138.
Fukuda, H. and A. Komamine. 1982. Lignin synthesis and its related
enzymes as markers of tracheary-element differentiation in single
cells isolated from the mesophyll of Zinnia elegans. Planta 155:
423–430.
Goldschmid, O. 1970. Ultraviolet spectra. In Lignins: Occurrence,
Formation, Structure and Reactions. Eds. K.V. Sarkanen and
C.H. Ludwig. Wiley-Interscience, New York, pp 241–266.
Hawkins, S. and A. Boudet. 2003. “Defence lignin” and hydroxycinnamyl alcohol dehydrogenase activities in wounded Eucalyptus
gunnii. For. Pathol. 33:91–104.
Hotter, G.S. 1997. Elicitor-induced oxidative burst and phenylpropanoid metabolism in Pinus radiata cell suspension cultures. Aust.
J. Plant Physiol. 24:797–804.
Koch, G. and G. Kleist. 2001. Application of scanning UV microspectrophotometry to localise lignins and phenolic extractives in
plant cell walls. Holzforschung 55:563–567.
Koch, G., J. Puls and J. Bauch. 2003. Topochemical characterization
of phenolic extractives in discoloured beechwood (Fagus sylvatica L.). Holzforschung 57:339–345.
Krishnamurthy, K.V. 1999. Methods in cell wall cytochemistry. CRC
Press, Boca Raton, FL, 318 p.
Lange, B.M., C. Lapierre and H. Sandermann, Jr. 1995. Elicitor-induced spruce stress lignin. Plant Physiol. 108:1277–1287.
Lin, Q. and D.H. Northcote. 1990. Expression of phenylalanine ammonia-lyase gene during tracheary-element differentiation from
cultured mesophyll cells of Zinnia elegans L. Planta 182:591–598.
MacKay, J.J., W. Liu, R. Whetten, R.R. Sederoff and D.M. O’Malley.
1995. Genetic analysis of cinnamyl alcohol dehydrogenase in
loblolly pine: single gene inheritance, molecular characterization
and evolution. Mol. Gen. Genet. 247:537–545.
Messner, B. and M. Boll. 1993. Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material
in a cell suspension culture of spruce (Picea abies). Plant Cell Tissue Organ Cult. 34:261–269.
Messner, B., M. Boll and J. Berndt. 1991. L-Phenylalanine ammonia-lyase in suspension cultures of spruce (Picea abies). Plant Cell
Tiss. Org. Cult. 27:267–274.
Möller, R., A.G. McDonald, C. Walter and P.J. Harris. 2003. Cell differentiation, secondary cell-wall formation and transformation of
callus tissue of Pinus radiata D. Don. Planta 217:736–747.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
210
MÖLLER, KOCH, NANAYAKKARA AND SCHMITT
Northcote, D.H. 1995. Aspects of vascular differentiation in plants:
parameters that may be used to monitor the process. Int. J. Plant
Sci. 156:245–256.
Nose, M., M.A. Bernards, M. Furlan, J. Zajicek, T.L. Eberhardt and
N.G. Lewis. 1995. Towards the specification of consecutive steps
in macromolecular lignin assembly. Phytochemistry 39:71–79.
Orr, J.D. and D.G. Lynn. 1992. Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell
growth. Plant Physiol. 98:343–352.
Pasco, M.F. and I.D. Suckling. 1994. Lignin removal during Kraft
pulping. An investigation by thioacidolysis. Holzforschung 48:
504–508.
Pomar, F., F. Merino and A. Ros Barcelo. 2002. O-4-linked coniferyl
and sinapyl aldehydes in lignifying cell walls are the main targets
of Wiesner (phloroglucinol-HCl) reaction. Protoplasma 220:
17–28.
Rolando, C., B. Monties and C. Lapierre. 1992. Thioacidolysis. In
Methods in Lignin Chemistry. Eds. S.Y. Lin and C.W. Dence.
Springer-Verlag, Berlin, pp 334–349.
Sarkar, S.K. and R.E. Howarth. 1976. Specificity of the vanillin test
for flavanols. J. Agric. Food Chem. 24:317–320.
Sato, Y., T. Watanabe, A. Komamine, T. Hibino, D. Shibata, M. Sugiyama and H. Fukuda. 1997. Changes in the activity and mRNA of
cinnamyl alcohol dehydrogenase during tracheary element differentiation in Zinnia. Plant Physiol. 113:425–430.
Singh, A.P. 1996. Ultrastructural features of compression wood cells
in relation to bacterial decay in Pinus radiata. In Recent Advances
in Wood Anatomy. Eds. L.A. Donaldson, A.P. Singh, B.G. Butterfield and L.J. Whitehouse. New Zealand Forest Research Institute
Ltd., Rotorua, pp 400–407.
Stasolla, C., J. Scott, U. Egertsdotter, J.F. Kadla, D.M. O’Malley,
R.R. Sederoff and L. van Zyl. 2003. Analysis of lignin produced by
cinnamyl alcohol dehydrogenase-deficient Pinus taeda cultured
cells. Plant Physiol. Biochem. 41:439–445.
Terashima, N. 2000. Formation and ultrastructure of lignified plant
cell walls. In New Horizons in Wood Anatomy. Ed. Y.S. Kim.
Chonnam National University Press, Kwangju, Korea, pp
169–180.
Uprichard, J.M. 1991. Chemistry of wood and bark. In Properties and
Uses of New Zealand Radiata Pine. Vol. 1. Wood Properties. Eds.
J.A. Kininmoth and L.J. Whitehouse. Ministry of Forestry and Forest Research Institute, Rotorua, New Zealand, pp 4-1-4/45.
Walter, C. and L.J. Grace. 2000. Genetic engineering of conifers for
plantation forestry: Pinus radiata transformation. In Molecular Biology of Woody Plants. Eds. S.M. Jain and S.C. Minocha. Kluwer
Academic Publishers, Dordrecht, The Netherlands, pp 79–104.
Wyrambik, D. and H. Grisebach. 1975. Purification and properties of
isoenzymes of cinnamyl-alcohol dehydrogenase from soybeancell-suspension cultures. Eur. J. Biochem. 59:9–15.
TREE PHYSIOLOGY VOLUME 26, 2006