Download Changes in cellular structures and enzymatic

Tree Physiology 20, 467–475
© 2000 Heron Publishing—Victoria, Canada
Changes in cellular structures and enzymatic activities during
browning of Scots pine callus derived from mature buds
HANNA LAUKKANEN, LEA RAUTIAINEN, ERJA TAULAVUORI and ANJA HOHTOLA
Department of Biology/Botany, University of Oulu, PO Box 3000, FIN-90401 Oulu, Finland
Received March 29, 1999
Summary Visible browning is a typical feature of callus cultures derived from shoot tips of mature Scots pine (Pinus
sylvestris L.). Because the ability of callus to regenerate is low,
we determined the effect of browning on growth and changes
in cellular structure during culture. Striking alterations in cellular structure were detected by LM (light microscopy), EM
(electron microscopy) and SEM (scanning electron microscopy). Accumulation of phenolic substances was shown by
histochemical staining. Staining for $-glucosidase activity of
soluble proteins that had been subjected to polyacrylamide gel
electrophoresis indicated lignification of cells. The measured
growth rate of callus was low compared with a hypothetical
growth curve. Peroxidase activity increased rapidly soon after
the start of the culture period, but especially between the second and third weeks of culture. At this time, the degradation of
cell membranes and browning began coincident with the loss
of chlorophyll. We conclude that browning is associated with
cell disorganization and eventual cell death, making tissue culture of mature pine especially difficult.
Keywords: extracellular matrix, growth, $-glucosidase,
phenolics, Pinus sylvestris, peroxidase, recalcitrance, tannin.
ameliorative effect on browning of Scots pine callus derived
from mature pine buds.
Peroxidases have many roles in plant growth and development. Campa (1991) reported that peroxidases were associated with chlorophyll degradation and lipid peroxidation in
senescent plant tissues. The presence of phenol compounds
accelerates this degradation (Kato and Shimizu 1985). Elevated peroxidase activity is often associated with the production of polyphenols, e.g., lignins and tannins, which may also
act as antioxidants against oxidative stress (Sakagami et al.
1995). Although Scots pine callus showed oxidative activity
when catechol was used as a substrate for polyphenol oxidase
(Laukkanen et al. 1999), addition of catalase revealed that the
oxidation was mainly (60–70%) caused by peroxidases.
Catechol oxidation activities did not correlate with the onset
or progress of browning.
Because of the recalcitrance of mature Scots pine in tissue
culture, we investigated changes in growth, cytoplasmic and
extracellular structures during browning to assess the effect of
browning on the regeneration ability of tissue cultures of mature pine. The roles of peroxidase, lipid peroxidation, pigment
degradation, lignification and onset of browning were also investigated.
Introduction
Browning or blackening is frequently observed in tissue cultures of woody plants. Browning is caused by the oxidation of
phenols after cellular disorganization. Disorganization of cellular components is often a result of the degradation of membranes by the toxic forms of oxygen (Lee and Whitaker 1995).
Oxidation of phenols may occur nonenzymatically, or may be
catalyzed by phenol oxidases or peroxidases (Webb 1966,
Rhodes and Wooltorton 1978, Vaughn and Duke 1984, Ke and
Saltveit 1988).
The brown polymers of phenols have been reported to hamper tissue culture of many high-tannin plants, such as Sorghum
(Cai et al. 1987), Quercus (Tóth et al. 1994) and Pinus
(Laukkanen et al. 1997) species. Attempts have been made to
prevent the browning of many food plants, especially fruits
and vegetables, by various chemicals, to enhance preservation
(Vámos-Vigyázó 1995). Hohtola (1988) tested a range of concentrations of various oxidase inhibitors and antioxidants in
the growth medium and found that they had no conclusive
Materials and methods
Plant material and culture conditions
Twigs were collected in April and November from 15- to
20-year-old Scots pine (Pinus sylvestris L.) trees growing in
natural stands in Oulu, northern Finland (65o N; 25o30¢ E). The
buds were surface-sterilized in 5–6% calcium hypochlorite
for 20 min and rinsed three times in sterile distilled water, after
which the scales were removed aseptically. The bud tips
(2–3 mm), including the apical meristem, were placed on MS
medium (Murashige and Skoog 1962) modified as described
by Hohtola (1988), except that the only inorganic nitrogen
source was ammonium nitrate (2 mM), the concentration of
sucrose was 2%, and arginine and glucose were not used. The
growth regulators 2,4-D (2,4-dichlorophenoxy acetic acid,
4.5 m M), BA (N 6-benzyladenine, 1.7 m M) and kinetin
(1.8 m M) were used. The pH was adjusted to 5.7. The medium
was solidified with 0.6% Phytagel (Sigma, St. Louis, MO). All
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LAUKKANEN, RAUTIAINEN, TAULAVUORI AND HOHTOLA
tissues were cultured in test tubes for the first 2 weeks and then
transferred to fresh medium in test tubes, and finally transferred to petri dishes after 4 weeks. The cultures were maintained in a 16-h photoperiod (white fluorescent Osram 18 W
tubes (Germany), at 1.6 W m –2, 400–700 nm) at 24 ± 2 oC.
Suspension cultures were initiated by transferring 2-week-old
callus to liquid growth medium including the same salts as the
solid growth medium and agitated on an orbital shaker
(130 rpm).
On the days of measurement, 1 ml of suspension culture was
taken for dry weight estimation. It was filtered through a
preweighed filter paper and washed with deionized water. The
filter with the culture was dried overnight at 100 oC before
weighing.
Color and pigment estimation
Color changes in callus were followed visually and spectrophotometrically during culture. The color changes were classified into the following categories: green, yellow-green,
yellow-brown, brown and dark brown. Samples (0.5 g) were
collected at the beginning of the culture period and at weekly
intervals throughout the 7 weeks of culture and homogenized
in liquid nitrogen. Pure acetone was used to extract green and
yellow pigments from the samples. The extracts were centrifuged (12 000 g, 5 min) and the supernatants were measured
spectrophotometrically at 435 (chlorophylls) and 665 (carotenoids) nm (Goodwin 1976).
Preparation of samples for LM, EM and SEM
During callus culture, small pieces of tissue (3–5 mm) were
collected weekly and fixed in 10:5:85 (v/v) FAA (formalin:acetic acid:94% ethanol). The fixed tissues were dehydrated in an ethanol series, infiltrated with 1:1 (v/v)
Historesin:95% EtOH and embedded in Historesin
(Reichert-Jung, Heidelberg, Germany). Sections were cut
with an LKB Historange microtome at 4 m m thickness from 10
blocks. Fresh pieces or LM (light microscopy) sections of callus were stained with 0.05% toluidine blue (Feder and O’Brien
1968), 10% acidic vanillin (Mueller-Harvey et al. 1987), 2%
acidic phloroglucinol (Harris et al. 1994) and 0.01% acridine
orange (Oparka and Read 1994), or a solution for phenolic
compounds containing 2% K3Fe(CN)6 and 2% FeCl3 in water
(100 ml) plus 10 crystals of KMnO4 (Mueller-Harvey et al.
1987). The colors of the cell walls after toluidine blue staining
indicate the viability of the cells. The cell wall is reddish purple in a viable cell and blue to greenish blue in a dead cell without cytoplasm. Polyphenolic material gives a green coloration
and lignified matter a blue coloration (Feder and O’Brien
1968, Harris et al. 1994). Vanillin–HCl characteristically produces a bright cherry red product in the presence of flavan-3-ol
monomers (catechin) and polymers.
Unstained sections and fresh suspension cells were examined with a fluorescence microscope with UV excitation
(Nikon Optiphot 2 with a 100-W HG lamp and an automatic
Nikon Microflex UFX 35 DX camera, filter combination Ex
365/10, DM 400, BA 400).
Samples for EM (electron microscopy) were prepared according to Kupila-Ahvenniemi and Hohtola (1979), except
that pieces of callus tissue were fixed overnight in cold 3%
glutaraldehyde in sodium phosphate buffer (pH 7.0), rinsed
three times in sodium phosphate buffer, post-fixed in cold 1%
osmium tetroxide for 3 h, rinsed three times in sodium phosphate and embedded in Ladd’s epon. For scanning electron
microscopy (SEM), the samples were fixed in FAA, dehydrated by ethanol series, dried with a critical point dryer, covered with gold, and viewed in a JEOL JEM 100B scanning
electron microscope.
Peroxidase assay
Vegetative buds (0.3 g) were transferred to an ice-cold beaker
after quick removal of the scales. Enzymatic activities were
measured in buds (Day 0) and in callus during culture
(14, 28 and 42 days after the initiation of culture). For the assay of peroxidase (POD), buds and callus (0.3–0.5 g) were
frozen in liquid N2 and ground to a fine powder with a mortar
and pestle. The powder was homogenized (45 s, on ice) in 3 ml
of extraction buffer, with an electric grinder (Heidolph Elektro
KG, Kelheim, Germany). The extraction buffer for POD was
0.167 M potassium phosphate (pH 7.8). After homogenization, 2.5 ml of the extraction buffer containing insoluble PVPP
(polyvinyl polypyrrolidone, final concentration 2.5% w/v,
soaked for 24 h in buffer) was added to the sample and the
mixture allowed to stand for 40 min in the dark with occasional stirring. The mixture was centrifuged at 25,000 g
(25 min, 4 oC) and filtered. One hundred m l of the filtrate was
added to 100 ml of 0.167 M potassium phosphate buffer
(pH 5.5) containing 10 mM guaiacol and 20 mM H2O2. The
mixture was incubated at room temperature and the change in
absorbance at 470 nm monitored for 5 min. Soluble protein
content was measured as described by Bradford (1976).
Lipid peroxidation analyses
Lipid peroxidation was measured in 2- to 7-week-old callus
(0.5 g) by determining malondialdehyde (MDA, a product of
lipid peroxidation) content by the thiobarbituric acid (TBA)
reaction according to Dhindsa et al. (1981).
Protein analyses
Soluble proteins were extracted from 0.5-g samples that were
ground in liquid nitrogen and homogenized with a mortar and
pestle in 2 ml of extraction buffer (50 mM Tris-HCl, pH 8.6,
20 mM KCl and 10 mM MgCl2) containing 1.5% (w/v) of insoluble PVPP. The homogenate was centrifuged at 13 000 g
for 15 min. The supernatant was subjected to one-dimensional
SDS-PAGE (ExcelGel ™ SDS), as recommended in the
Pharmacia (Upsala, Sweden) instruction manual. After electrophoresis, the proteins were visualized by silver staining
(Bio-Rad, silver staining kit). The molecular mass standards
were from Amersham gradient 8-18 (Pharmacia).
Soluble proteins obtained from 1 g samples of callus were
separated by non-denaturing PAGE and stained for $-glucosidase activity as described by Dharmawardhana et al. (1995)
by incubating with 0.2 mM MUG (4-methylumbelliferyl
$-glucoside). Electrophoresis was performed at 4 ° C according to the Laemmli (1970) procedure but without SDS and
2-mercaptoethanol.
TREE PHYSIOLOGY VOLUME 20, 2000
BROWNING OF PINE CALLUS
For glycoprotein analysis, 1.0 g samples of callus were
ground in liquid nitrogen and homogenized with a mortar and
pestle in sterile water, after which 2.5 volumes of ethanol were
added and the mixture allowed to stand overnight at 4 oC. The
ethanol-insoluble residue was precipitated by centrifugation at
15 000 g for 10 min and resuspended in 100 ml of buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM
MgCl2 and 1 mM MnCl2. Glycoproteins were purified according to Gavish et al. (1992). The purified glycoproteins were
analyzed by one-dimensional SDS-PAGE (Pharmacia) and
stained with Coomassie Brilliant Blue G-250 (Neuhoff et al.
1988).
Results
Color changes of tissues
The rate of browning varied among the different callus tissues,
but in most cases the callus changed from green to yellow-green after three days of culture and thereafter changed
gradually through yellow-brown to dark brown. The first sign
of brown color usually appeared as small yellow-brown spots
on the surface of callus after 2 weeks of culture, or after 1 week
on the cut surface of the explant. Several dark brown calli were
found among the 4-week-old callus samples, and browning
was often complete after 8 weeks of culture, though color variations among similar callus masses and among areas of the
same callus were evident. The color of suspension cultures derived from callus remained green for 2 weeks, but thereafter
the cultures gradually turned to yellow-brown and brown.
Concentrations of green and yellow pigments
The amounts of green (chlorophylls) and yellow pigments (carotenoids) decreased during culture, but the reduction in
chlorophylls usually started after 2 weeks of culture, whereas a
decrease in carotenoids was already evident after 1 week (Table 1). The yellow pigments disappeared more slowly than the
green pigments, and this was manifest as a color change from
green to yellow-green in young callus. The water content of
the explants increased after 1 week (Laukkanen et al. 1997),
which partly explains the difference in pigment concentrations
469
between the 1-week-old culture and the buds. The peeling of
buds probably also affected the amounts of pigments detected.
Changes in peroxidase activity and lipid peroxidation during
culture
Peroxidase activity increased during culture. The activity in
6-week-old callus was 5-fold higher than in buds (Table 1).
The rate of increase slowed after 4 weeks of culture. Lipid peroxidation was maximal after 2 weeks of culture, but the process decreased gradually during aging of the callus.
Growth of callus and suspension cultures
Figures 1A and 1B show the growth of callus and cell suspensions during culture. The fresh weight of callus increased in
parallel with the increase in dry weight (cf. Lindfors et al.
1990). The increase in callus production was 2.6-fold from the
first to the second week, whereas from the sixth to the seventh
week it was only 1.1-fold. The standard deviations of the
growth curve increased during culture, reflecting differences
in growth among individual calli. The relative growth rate of
the callus cultures declined over time, so that there was no exponential growth phase (Figure 1A) (King et al. 1972). The
growth curve of the suspension culture showed a large decline
in dry weight after 2 weeks of culture (Figure 1B), coinciding
with the onset of browning.
Alterations in cellular structure during browning
The vanillin–HCl test for tannin gave a positive but variable
pink-red reaction in a fresh, crushed callus sample. The red
color was usually weak in green tissues and bright cherry red
in dark brown tissues. Fresh brown callus reacted with
phloroglucinol–HCl and acridine orange, indicating lignification of the cells. The cell walls of brown callus also showed a
positive blue color after staining with a solution for phenolic
compounds (results not shown).
The proportion of greenish blue cells increased during the
culture, and approximately 50% of the cells were without cytoplasm after 4 weeks of culture (Figure 2A). Fibrillar material
(phenolics) was observed in vacuoles of fixed (Figure 2A) and
fresh callus cells (Figure 2C) and between the cells (Figure
2D). The young green callus tissue expressed blue
Table 1. Changes in the amounts (± SD) of chlorophylls, carotenoid, peroxidase (POD) activity and lipid peroxidation during callus culture. Standard deviations < 10 –1 are not shown. There were three (n) samples for pigment analysis (except Week 1, n = 1); POD = activity of guaiacol
peroxidase, n = 3–11; and MDA = malondialdehyde, a product of lipid peroxidation, n = 2–3.
Culture time
(Weeks)
Chlorophylls
(A435)
Carotenoids
(A665)
POD
(A min –1 mg prot –1)
0
1
2
3
4
5
6
7
2.05
1.58
1.22
1.15
0.80
0.63
0.42
0.36
2.68
1.01
1.07
0.84
0.43
0.34
0.25
0.21
58.7 ± 5.6
121.4 ± 19.8
180.5 ± 50.7
263.3 ± 39.2
307.2 ± 98.4
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Peroxidation of lipids
(m M MDA gFW –1)
27.2 ±
32.4 ±
16.7 ±
21.3 ±
19.5 ±
21.8 ±
6.4
2.5
6.5
1.5
1.7
5.4
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LAUKKANEN, RAUTIAINEN, TAULAVUORI AND HOHTOLA
The cells with thin walls were rich in starch, whereas the yellow-brown cells often lacked cytoplasm.
Under the light microscope, sections of old callus usually
showed bright white fluorescence in the intercellular spaces
(Figure 2F). The cytoplasm of dark brown pine callus was
completely disorganized and rich in tannin (Figure 3F, 4A
and 4B) or devoid of cytoplasm. Osmiophilic material was
seen in the cytoplasm (Figure 3F) and in the intercellular
spaces in dying brown callus (Figure 4B). The number of
microvesicles (Figure 4B) and other membraneous structures,
such as multivesicular bodies (Figure 4C), increased during
culture. The number of fibrillar structures increased during
culture, and the brown tissues often had very dense fibrillar areas (Figure 4D).
Polypeptide analyses
Figure 1. (A) Growth curve based on fresh mass (FW, mg) determinations of calli (n = 10) initiated from bud apices. Symbols: 䊉 = measured growth curve; 䊏 = exponential growth curve. (B) Dry weight
determinations of suspension cultures (n = 3) derived from pine callus. Symbols: 䊉 = dry mass (mg), measured six times during the culture period (18 days).
autofluorescence on UV excitation, whereas the cells just before browning emitted bright white fluorescence (Figure 2B).
Unstained fresh suspension cells that were yellow-brown under the light microscope exhibited white fluorescence on UV
excitation (Figure 2E).
In the SEM image of callus, membrane-like structures were
seen (Figure 3A), resembling those found in the superficial
cells of Coffea arabica L. callus (Nakamura and Maeda 1990).
Well-organized organelles, such as mitochondria and
chloroplasts (Figure 3B), were visible in the cytoplasm of
green callus. Other plastids without grana, such as amyloplasts, were also present, as shown in Figure 3C. The amount
of starch increased during culture, as did the osmiophilic material (tannins) in vacuoles (Figure 3E). Myelin-like structures
were found within the cytoplasm and vacuoles of young callus
that had apparently formed as a result of ER activity or through
tonoplast folding (Figure 3D). The fresh unstained cells of
completely dark brown callus had thick cell walls that emitted
a yellow-brown fluorescence typical of phenol substances on
UV excitation, or thinnish cell walls with white fluorescence.
The concentration of a polypeptide with a molecular mass of
24 kDa (Figure 5) was high after 2 weeks of culture. In a previous study, Laukkanen et al. (1997) reported that the concentration of a 24 kDa protein increased from the beginning of
culture and was highest after 12 to 14 days of culture.
Polypeptides of 28, 14 and 10 kDa were abundant, whereas the
24 kDa glycoprotein was seen as a faint band. This
polypeptide can be detected better by silver staining of purified glycoproteins (Laukkanen et al. 1997).
When soluble proteins were subjected to PAGE and then
stained for $-glucosidase, a single fluorescent band of approximately 60 kDa was observed in UV (Figure 5). This fluorescent band had a similar molecular mass to the
coniferin-specific $-glucosidase of lodgepole pine reported by
Dharmawardhana et al. (1995).
Discussion
Scots pine, like several other pine species, is recalcitrant in tissue culture. To determine if this is associated with tissue
browning, we studied the relationship between browning and
growth, cellular structure and enzyme activity during in vitro
culture of callus derived from mature pine buds.
The dry mass growth curve of cell suspension cultures
showed that deterioration of cells began after about 2 weeks in
culture. Similarly, the first visible signs of browning in callus
were usually seen after 2 weeks in culture when peroxidase
(POD) reached its maximum activity and the growth rate
started to decrease. At the same time, the amount of chlorophyll started to decline and lipid peroxidation reached its maximum. Earlier work on pine callus has shown that PODs, and
to some extent polyphenol oxidases (PPOs), are able to oxidize catechol producing dark brown quinones (Laukkanen et
al. 1999). However, we found that after 2 weeks of culture,
POD activity with catechol as substrate was considerably
lower (approximately 11,000´ ) than POD activity with guaiacol as substrate. Furthermore, POD activity with catechol as
substrate did not correlate with the onset of browning. Our results are in agreement with the finding that some peroxidases
function as phenol oxidases by oxidizing hydroxycinnamic
derivatives and flavans (see references in Siegel 1993 and
Richard-Forget and Gauillard 1997).
TREE PHYSIOLOGY VOLUME 20, 2000
BROWNING OF PINE CALLUS
471
Figure 2. (A) Callus tissue after
4 weeks of culture. The reddish
blue color indicates viable cells
and the blue to greenish blue color
indicates degraded cells without
cytoplasm (arrow); 175´ . (B)
Autofluorescent material (arrow)
inside young (10-day) callus; 85´ .
(C) Fibrillar structures (arrow) of
fresh callus cells, CW = cell wall;
345´ . (D) Fibrillar structures (arrow) between callus cells (UV fluorescence); 85´ . (E) Unstained
fresh cells from suspension culture. The cell (left) with a yellow-brown tone under a light
microscope showed bright white
autofluorescence on UV excitation, whereas the normal-looking
cell (right) showed blue; 175´ . (F)
Fluorescence of a microscope section of brown callus (42 days).
Note the white autofluorescence in
the intercellular spaces; 175´ .
Scots pine callus is able to produce ethylene during the first
2 or 3 weeks; thereafter the amount of ethylene decreases in
parallel with browning (Lindfors et al. 1990). Ethylene is
known to induce de novo synthesis of peroxidase and
phenylalanine ammonia-lyase (PAL), an enzyme involved in
lignification and phenylpropanoid synthesis of cells (Abeles et
al. 1988, Hyodo and Fujinami 1989). An increase in PAL activity during the first 2 or 3 weeks of culture has been detected
in pine callus (Laukkanen et al. 1997).
The enzyme activity staining for $-glucosidase in Scots
pine callus revealed a single fluorescent band of 60 kDa indicating lignification of cells. Lignification was confirmed by
histochemical staining of cells. The 60-kDa fluorescent band
has a similar molecular mass to the purified coniferin
$-glucosidase isolated from lignifying xylem of lodgepole
pine by Dharmawardhana et al. (1995). Coniferin
$-glucosidase is a homodimer of 28 kDa subunits, although a
subunit of 24 kDa was also seen on SDS-PAGE after purification. Similarly, we observed polypeptides of 28 and 24 kDa
when a soluble protein extract from Scots pine callus was subjected to SDS-PAGE. The 24 kDa band may represent subunits of methyltransferase, an enzyme associated with lignin
synthesis (Pakusch et al. 1991). The production of lignin compounds in callus of pine species may be a result of stress reac-
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LAUKKANEN, RAUTIAINEN, TAULAVUORI AND HOHTOLA
Figure 3. Bars = 5 m m. (A) A
tions because several stresses are known to induce the
synthesis of phenylpropanoids (cf. Dixon and Palva 1995,
Lange et al. 1995).
The amount of (+)catechin, a precursor of tannin, was
10-fold higher and that of condensed tannins approximately
3-fold higher in 4-week-old callus of Scots pine compared
with buds (Laukkanen et al. 1997). These compounds contributed to the formation of the red color in callus that was stained
with vanillin–HCl. Because Scots pine callus produces large
amounts of ethylene (Lindfors et al. 1990, Laukkanen and
Sarjala 1997), we suggest that the production of ethylene,
rather than the browning process, resulted in the accumulation
of (+)catechin, as in the russet spotting of iceberg lettuce,
Lactuca sativa L. (Ke and Saltveit 1988).
The toxicity of large tannin deposits has been observed in
other tissues in addition to Scots pine callus. For example,
large tannin deposits were reported to be toxic in brown tissues of slash pine (Hall et al. 1972, Baur and Walkinshaw
1974). The results from slash pine suggested that the
foam-like tannin is significantly more soluble and causes
more pronounced cellular degradation than the dense spherule
type. The foam-like tannin was also observed in our Scots pine
TREE PHYSIOLOGY VOLUME 20, 2000
BROWNING OF PINE CALLUS
473
Figure 4. Bars = 5 m m. (A) The
fine structure of dark brown callus.
Note the large starch granules (arrows) and the dark tannin deposits
(T) in the absence of cytoplasm.
(B) Dark osmiophilic material (arrow) in the intercellular space of
brown callus. Note the numerous
small vesicles (small arrow) in the
cytoplasm (C). (C) Multivesicular
bodies inside the cell wall (CW) of
brown callus. (D) A dense fibrillar
network between the cell walls
(CW) of brown callus.
callus culture.
Our EM studies indicated that accumulation of large
amounts of tannin and starch in cells precedes the browning
Figure 5. Polyacrylamide gel electrophoresis of soluble proteins extracted from 3-week-old callus. Lane A: SDS-PAGE of soluble proteins stained with silver. Arrow indicates a 24 kDa polypeptide. Lane
B: Purified glycoproteins stained with Coomassie blue. Arrows indicate polypeptides of 28, 14 and 10 kDa. Lane C: Enzyme activity
staining for $-glucosidase after native PAGE. Arrow indicates an
approximately 60 kDa polypeptide.
and deterioration of tissues. The results are in accordance with
the idea that the accumulation of tannins in the cytoplasm
leads to cell death (Baur and Walkinshaw 1974, Barnett 1978,
Hall et al. 1992). We observed cellular alterations similar to
those observed during aging of callus derived from hypocotyl
segments of Pinus radiata D. Don (Barnett 1978), where cell
aging was accompanied by increases in the amount of
osmiophilic material, probably tannins, in the vacuoles, and in
the size of the starch grains. The final stage of tannin accumulation in callus cells appears to be its incorporation into the cytoplasm with accompanying degeneration of the cytoplasm
and its organelles. At this stage, tannin may also be observed
in the intercellular spaces.
The increase in lipid peroxidation during browning suggests that browning of Scots pine callus and cell suspensions
markedly reduced their regeneration capacity. Hall et al.
(1972) observed a relationship between cytoplasmic disorganization and oxygen consumption in slash pine callus derived
from embryos. Oxygen consumption was lower in brown and
dark brown calli compared with green, yellow-green, yellow
or yellow-brown calli.
Although proembryos have been found in suspension cultures derived from buds, no further differentiation into somatic embryos has been observed (Hohtola 1995). There is
evidence that autofluorescence of the cell wall in UV light, together with the presence of a large central nucleus (Blervacq et
TREE PHYSIOLOGY VOLUME 19, 1999
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LAUKKANEN, RAUTIAINEN, TAULAVUORI AND HOHTOLA
al. 1995) and extracellular fibrillar structures of proembryos
(Rohr et al. 1989, Dubois et al. 1992, Jasik et al. 1995, Šamaj et
al. 1995) indicate embryogenic potential in tissue cultures.
However, because autofluorescence can also be a sign of incipient lignin synthesis under stress, we conclude that the
autofluorescence and fibrillar structures found in browning
Scots pine callus reflect high oxidative stress in the cells rather
than the potential development of somatic embryos.
In conclusion, microscopic studies revealed that, during
browning, large changes occurred in the cellular structure of
callus derived from mature Scots pine buds. Accumulation of
tannins in the cell walls, intercellular spaces and cytoplasm
was observed in brown tissues. Disorganization or absence of
cytoplasm and lignified cell walls were typical of dark brown
callus. This type of browning is a consequence of high oxidative stress and causes serious disorganization in the cytoplasm
and eventual cell death, thereby reducing the regeneration capacity of callus and suspension cultures. High peroxidase activity precedes and stimulates the deterioration of tissues
leading to rapid browning.
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