Tree Physiology 21, 549–554
© 2001 Heron Publishing—Victoria, Canada
Light- and cold-stress effects on the greening process in epicotyls and
young stems of red oak (Quercus rubra) seedlings
A. SKRIBANEK1 and B. BÖDDI2,3
1
Doctoral School, Eötvös Loránd University, Pázmány Péter S. 1., Budapest, H-1117, Hungary
2
Department of Plant Anatomy, Eötvös Loránd University, Puskin Street 11-13, Budapest, H-1088, Hungary
3
Author to whom correspondence should be addressed
Received July 26, 2000
Summary Protochlorophyllide (Pchlide) and protochlorophyll (Pchl) were found in epicotyls of 14-day-old dark-germinated seedlings and in 100-day-old dark-grown stems of red
oak (Quercus rubra L.). Fluorescence spectroscopy measurements of epicotyls at 77 K showed that the majority of Pchlide
and Pchl is present as a shorter wavelength-emitting monomer
with a fluorescence emission maximum at 629–631 nm. A
small amount of a monomeric form emitting at 635–636 nm
was also present. Minor amounts of Pchlide were aggregated
into larger complexes with fluorescence emission maxima at
640, 644–646 and 652–654 nm, as seen in etiolated leaves.
Flash illumination transformed the 652–654-nm-emitting
form to chlorophyllide, but not those forms with emission
maxima at 629–631, 635–636 and 644–646 nm. These shorter
wavelength-emitting forms were transformed to chlorophyllide by continuous illumination, but the process took several hours. Epicotyls and young stems were light sensitive,
with exposure to full daylight causing strong pigment bleaching and tissue destruction. Complete greening took place only
at low irradiances. Light sensitivity was greater at 4 °C than at
room temperature. We conclude that the monomeric arrangement of the pigments accounted for the light and temperature
sensitivity of the greening process in epicotyls and stems.
Keywords: fluorescence, protochlorophyllide.
Introduction
Chlorophyll biosynthesis in the chlorenchymatous tissues of
stems and stem-related organs (i.e., epicotyls and hypocotyls)
differs from that of leaves (Skribanek et al. 2000). If stems are
grown in the dark, they form etioplasts with only a few inner
membranes that are less organized than the inner membranes
of leaf etioplasts. Instead of the regular prolamellar bodies
(PLB) found in leaf etioplasts, stem-type etioplasts have more
abundant prothylakoids (Böddi et al. 1996). The protochlorophyllide (Pchlide) concentration of stems is usually only
5–10% that of leaves (Böddi et al 1994, Skribanek et al. 2000)
and is mainly in the monomeric state (Böddi et al. 1998). The
majority of Pchlide has 77 K emission bands at 629–631 and
635–636 nm, with only a small proportion of the pigment aggregated into forms emitting at 644–646 and 652–654 nm.
These longer wavelength-emitting forms or their complexes,
which are abundant in etiolated leaves, are highly photoactive
and play a role in the greening process of etiolated leaves
(Sundqvist and Dahlin 1997). As a consequence of the specific
arrangement of Pchlide, the greening of dark-grown stems differs from that of leaves. Flash illumination converts almost all
Pchlide in pea (Pisum sativum L.) leaves on a millisecond
time-scale, but it causes only minor changes in the Pchlide or
protochlorophyll (Pchl) of pea epicotyls (Böddi et al. 1996). In
response to flash illumination, only the longer wavelength,
644–646- and 652–654-nm-emitting Pchlide forms are converted to chlorophyllide (Chlide), whereas two monomeric
forms, which emit at 629–631 and 635–636 nm, remain unchanged. The monomeric forms can be transformed with continuous low irradiance in the course of several hours; however,
exposure to full daylight causes pigment bleaching and tissue
damage in stems, epicotyls and hypocotyls (Skribanek et al.
2000). Greening of leaves is much slower at 5 °C than at room
temperature (Krol et al. 1987) and in pea epicotls, no greening
occurs at 0 °C (Böddi et al. 1996).
Oak is usually propagated from seed. Acorns require
well-defined conditions to germinate, including cold treatment (below 10 °C) for about 1 month to ensure normal differentiation of the embryo, otherwise only the radicle emerges
(Wareing and Phillips 1978). Acorns are usually sown at a
depth of 4–8 cm (Morris and Perring 1974). Within this soil
layer, epicotyls develop up to several cm in length. The outer
cell layers of the epicotyl primary cortex are green on emergence from the soil surface, and contain normally developed,
photosynthetically active chloroplasts (Metcalfe and Chalk
1950). After several weeks, however, these tissues become
lignified and their photosynthetic activity gradually decreases.
Chloroplasts have also been detected around young vessels in
deeper tissues of stems up to 2 years old (Esau 1969). Plastid
initials and their seasonal transformation into plastids with
PLB-like structures were also observed in cortical cells of living bark of apple (Malus domestica Mill.) trees in mid-winter
(January–March) (Sagisaka 1993).
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SKRIBANEK AND BÖDDI
This study was undertaken to compare non-woody pea epicotyls and the lignifying epicotyls and stems of oak seedlings
with respect to the native arrangement of Pchlide, the greening
process, and their responses to light and cold stress. Oak seedlings were germinated and grown in the dark and illuminated
with white light at difference irradiances and temperatures.
Pigment concentrations of epicotyls and stems were determined, and 77 K fluorescence emission spectra were studied at
different stages during the greening process.
Materials and methods
Plant material
Acorns of red oak (Quercus rubra L.) were stored at 6 °C for
4 weeks, then germinated in petri dishes on wet filter papers at
25 °C for 2 weeks. The germinating acorns were transferred to
stainless steel wire-nets and grown hydroponically in tap water and total darkness at 25 °C for up to 100 days. The water
was changed every second day. All handling and sample collections were done in dim green light, which had previously
been shown not to cause phototransformation of Pchlide in etiolated leaves (data not shown).
Illumination
Some seedlings were illuminated with a 2 ms light flash of
200 µmol m –2 s –1 at room temperature. Other seedlings were
illuminated continuously with a tungsten lamp at irradiances
adjusted with neutral filters to 0, 5, 15, 250 or 625 µmol m –2
s –1.
dissolved in 80% acetone and the Pchl-containing residue was
dissolved in petroleum ether. Dilution series were prepared
and the fluorescence emission in the 629–631 nm waveband
was measured at room temperature with excitation at 430 nm.
Fluorescence spectroscopy
Fluorescence spectra were measured with a FluoroMax 2
Jobin Yvon-Spex spectrofluorometer (Jobin Yvon Ltd., Edison, NJ). To measure 77 K spectra of epicotyl or stem pieces, a
liquid nitrogen accessory was used. During the measurements,
samples were immersed in liquid nitrogen. The emission spectra were measured with excitation at 440 or 460 nm. The integration time was 0.2 s, and the data frequency was 0.5 nm. The
excitation and emission slits were 2 nm. Three spectra were
measured and averaged automatically for each sample. Five
samples were collected and measured per treatment. The spectra shown in the figures are means of these five measurements
(i.e., 15 spectra). All spectra were corrected for the wavelength-dependent sensitivity changes of the fluorometer. Calculation of the means, 5-point linear smoothing, derivative
calculations, baseline corrections and Gaussian resolutions
were done with the software SPSERV - V.3.14 (C. Bagyinka,
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary). Gaussian resolution was done in the wave number function of the spectra and
the results then reversed to the wavelength function (Böddi et
al. 1992). The error of resolution was less than 0.5% (the
sum-residual-square value was less than 5 × 10 –4 ).
Cold stress
Results
Some seedlings were transferred to a 4 °C cold-room for
60 min and then illuminated at various radiant flux densities.
Both dark-grown epicotyls and young stems contained
Pchlide and Pchl. Mean total pigment concentration of the
14-day-old epicotyls on a fresh mass basis was 1.2 µg g –1.
There was no significant gradient in pigment distribution
along the 15-cm epicotyls. Overall, pigment concentrations in
the 40-cm-long young stems on a fresh mass basis were higher
(2.7 µg g –1) than pigment concentrations in the epicotyls. The
mean molar concentrations of Pchlide and Pchl in stems were
3 × 10 –9 and 1 × 10 –9 mol g –1 fresh mass, respectively. The
gradient in pigment concentration along the stems was highest
in the tip segments and gradually decreased basipetally. The
oldest segments contained only 50% of the pigment concentration of the tip segment. The molar ratio of Pchlide to Pchl
was 3:1 in both epicotyls and stems, and the ratio did not
change with seedling age.
The 77 K fluorescence emission spectrum of epicotyls was
dominated by a 629–631-nm band, with a low intensity shoulder around 652–654 nm when the excitation was at 440 nm.
The fourth derivative of this spectrum (not shown) had main
positive bands at 629, 640 and 652 nm. The dominating central
and side-peaks of the derivative of the 629–631-nm band,
however, did not allow further analysis. Therefore, a combination of Gaussian resolution, spectrum modification and derivative analysis was carried out as described by Böddi et al.
(1992). If the bands at 629–631 and 652–654 nm were sub-
Pigment extraction, separation and determination
Epicotyls and young stems were cut into small pieces, homogenized, and the pigments extracted with 80% acetone containing 1 N NH 4OH. The pigment solution was passed through a
sintered glass filter (Pyrex England 5044). To separate Pchlide
from Pchl and other phytolized pigments, 10 cm3 of petroleum
ether (pro analysi boiling point 40–70) followed by 5 cm3 of
distilled water were added to 10 cm3 of pigment extract in 80%
acetone and the mixture shaken. A few mg of NaCl was added
to separate the phases, which were collected and measured.
The petroleum ether phase contained the phytolized pigments,
and Pchlide accumulated in the aqueous phase. Fluorometric
analysis showed (see below) that the petroleum ether phase
contained only traces of Pchlide or Pchl.
Pigment concentrations were determined based on calibration curves created by a combination of absorption and fluorescence spectroscopy as follows. The concentrations of
Pchlide and Pchl in the diethyl ether solutions were determined spectrophotometrically with a Shimadzu UV-2101PC
spectrophotometer (Shimadzu Corporation, Kyoto, Japan),
based on the extinction coefficient reported by Houssier and
Sauer (1969). The solvent was evaporated and Pchlide was
TREE PHYSIOLOGY VOLUME 21, 2001
LIGHT- AND COLD-STRESS EFFECTS ON OAK SEEDLINGS
tracted, the modified spectrum had a clear band at
645–646 nm and a shoulder around 635–636 nm. If the excitation wavelength was shifted from 440 to 460 nm, the emission
maximum appeared at 652 nm and a shoulder was found at
640 nm (Figure 1). Similar spectra were registered for
100-day-old dark-grown stems; however, the amplitude of the
652–654-nm band was higher than for epicotyls. In addition,
the intensities of the emission bands were about twice as high
for dark-grown stems as for epicotyls, because of the higher
total pigment concentration in stems (Figure 1). As an example, Figure 2 shows the spectrum of an epicotyl measured with
excitation at 440 nm resolved into Gaussian components. All
spectra measured for the non-illuminated samples were resolved into the same four components; however, the band positions varied within 1.5 nm, and the half-bandwidth values
varied within 2 nm.
Flash illumination at room temperature caused phototransformation of the 652–654-nm-emitting Pchlide form only. In
spectra measured with excitation at 440 nm, the band at
652–654 nm disappeared and fluorescence increased above
680 nm, indicating Chlide formation. A 7-fold decrease in signal was found as a result of pigment bleaching caused by the
flash illumination (Figure 3, curve B). In the spectrum measured with excitation at 460 nm, the maximum was at
639–640 nm (Figure 3, curve C). Curve resolution showed
that this band comprised two peaks with maxima at
635–636 and 645–646 nm (data not shown). Regeneration of
the Pchlide forms was observed when plants that had received
flash illumination were kept in the dark at room temperature
for 7 days. With excitation at 440 nm, the amplitude of the fluorescence signal doubled, and a pronounced regeneration of
the 652–654-nm band occurred (Figure 3, curve D). The regenerated, 652–654-nm-emitting Pchlide form was photoactive, and transformed into the 680–688-nm-emitting Chlide
form after flash illumination (data not shown).
Figure 1. The 77 K fluorescence emission spectra of a 14-day-old,
dark-germinated epicotyl and a 100-day-old dark-grown stem of red
oak (Quercus rubra). Symbols: solid line = epicotyl and excitation at
440 nm; intermediate length dashes = epicotyl and excitation at
460 nm; short dashes = stem and excitation at 440 nm; and long
dashes = stem and excitation at 460 nm.
551
Figure 2. Gauss components of the 77 K fluorescence emission spectrum of a 14-day-old, dark-germinated red oak (Quercus rubra)
epicotyl. Excitation wavelength was 440 nm. Solid line = experimental spectrum, dashed lines = Gaussian components and the error of the
fit. The numbers indicate the positions of the Gaussian components
and the half-bandwidth values (nm) are given in parentheses.
A slow phototransformation of the 629–631-, 635–636and 644–646-nm-emitting Pchlide forms was observed when
the stems were illuminated with continuous light at room temperature. At high irradiance (625 µmol m –2 s –1), however,
bleaching occurred. After 60 min of illumination at high
irradiance, no fluorescence spectra could be measured indicating the breakdown of all Pchlide or Pchl and Chlide or chlorophyll (Chl) molecules. In the first minutes of illumination at
low irradiance, the longer wavelength-emitting forms were
transformed to Chlide, whereas transformation of the shorter
wavelength-emitting forms occurred over several hours. Even
after 6 h of illumination at 250 mmol m –2 s –1 or lower
irradiance at room temperature, a band at around 630 nm was
Figure 3. The 77 K fluorescence emission spectra of a dark-germinated young stem of red oak (Quercus rubra). A: Before flash illumination, excitation at 440 nm; B: after flash illumination, excitation at
440 nm; C: after flash illumination, excitation at 460 nm; D: after
1 week dark-regeneration, excitation at 440 nm. The spectra are normalized at their maxima. The normalization factors were: A: 3.8 ×
10 6; B: 5.3 × 10 5; C: 2.1 × 10 5; and D: 1.3 × 10 6.
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552
SKRIBANEK AND BÖDDI
observed (Figure 4). During the first 6 h of the greening process with an irradiance of 0.5, 15 or 250 µmol m –2 s –1, the
spectra were characterized by bands around 630 and 680 nm.
Illumination at 15 µmol m –2 s –1 at room temperature resulted
in a consistent change in the ratio of these bands during the
first 6 h of greening (Figure 4). Further illumination resulted in
the appearance of emission bands above 680 nm, indicating
the formation of Chl–protein complexes of the photosynthetic
apparatus (data not shown). Ratios of the amplitude of the
bands around 680 nm (designated 68x) and 629–630 nm (designated 63y), (i.e., 68x/63y) corresponding to the Chlide and
Pchlide forms, respectively, calculated from spectra registered
after different durations of illumination, seemed to be robust
indicators of the greening process. We note, however, that
bleaching of pigments may also alter this ratio. At 250 µmol
m –2 s –1 illumination, the 68x/63y band ratio showed an increase within the first 2 h; however, with longer illumination,
the ratio decreased (Figure 5, filled columns) with a marked
decrease in total fluorescence of the stems. At lower irradiances, the greening process was flux density dependent. A continuous increase in the 68x/63y ratio was observed during the
first 6 h of illumination at 15 µmol m –2 s –1 (Figure 5, open columns). Much slower accumulation of Chlide and Chl was
found when the stems were illuminated at 0.5 µmol m –2 s –1
light. Large amounts of unconverted Pchl or Pchlide were
found even after 6 h of illumination at 0.5 µmol m –2 s –1 (Figure 5, shaded columns).
The greening process was delayed and light sensitivity of
the stems increased when illumination was carried out at 4 °C.
At this temperature, illumination at 250 µmol m –2 s –1 caused
total bleaching of the stems within 60 min. At 4 °C, even illumination at 15 µmol m –2 s –1 caused bleaching and this was reflected in a decrease in the 68x/63y ratio after 120 min of
illumination (Figure 6, open columns) and after 24 h of illumination at 15 µmol m –2 s –1 the stems were fully bleached (data
not shown). Because epicotyls contain little woody tissue,
bleaching caused wilting as a result of turgor loss. No bleach-
Figure 4. The 77 K fluorescence emission spectra of a dark-germinated young stem of red oak (Quercus rubra) illuminated at 15 µmol
m –2 s –1 for different periods at room temperature. The excitation
wavelength was 440 nm. The spectra are normalized at their maxima.
Figure 5. Ratios of the amplitudes of the 77 K fluorescence emission
bands at 680–682 nm (68x) and 629–631 (63y) nm, (corresponding to
chlorophyllide and protochlorophyllide forms, respectively) in spectra of dark-germinated young stems of red oak (Quercus rubra) continuously illuminated at 20 °C for different time periods. Shaded
columns = irradiation at 0.5 µmol m –2 s –1; open columns = irradiation
at 15 µmol m –2 s –1; and filled columns = irradiation at 250 µmol m –2
s –1.
ing, but slow phototransformation was observed when the
stem was illuminated at 0.5 µmol m –2 s –1 at 4 °C. After 6 h of
illumination at 0.5 µmol m –2 s –1, the 68x/63y ratio was still under 2 (Figure 6, shaded columns). After 48 h of illumination,
the 629–631-nm band disappeared and the fluorescence bands
of the forming Chl–protein complexes appeared.
Discussion
Although the pigment concentration, the greening process and
the light sensitivity of dark-grown oak (Quercus robur L.)
leaves have been studied in detail (Axelsson et al. 1979, 1981),
Figure 6. Ratios of the amplitudes of the 77 K fluorescence emission
bands at 680–682 nm (68x) and 629–631 (63y) nm, (corresponding to
chlorophyllide and protochlorophyllide forms, respectively) in spectra of dark-germinated young stems of red oak (Quercus rubra) continuously illuminated at 4 °C for different time periods. Shaded
columns = irradiation at 0.5 µmol m –2 s –1; and open columns = irradiation at 15 µmol m –2 s –1.
TREE PHYSIOLOGY VOLUME 21, 2001
LIGHT- AND COLD-STRESS EFFECTS ON OAK SEEDLINGS
there are no published data on these parameters for epicotyls
of dark-germinated oak seedlings or for young stems of
dark-grown oak plants. Pigment concentrations in both epicotyls and young stems were lower than in etiolated oak leaves.
The total Pchlide and Pchl concentration on a fresh mass basis
of etiolated oak leaves is 6 µg g –1 (Axelsson et al. 1981),
whereas this value was only 1.2 µg g –1 in epicotyls and 2.7 µg
g –1 in young, dark-grown stems. Similarly low values were
found in pea epicotyls (Böddi et al. 1994).
The native arrangement of the pigments in oak epicotyls and
stems was similar to that in pea epicotyls. The shorter wavelength, 629–631-nm-emission band predominated, but longer
wavelength-emitting forms with emission maxima above
650 nm were present in small amounts. The 629–631-nmemitting Pchl or Pchlide forms only dominated in 2–3-day-old
etiolated leaves (Klein and Schiff 1972, Schoefs and Franck
1993), where the development of proplastids into etioplastids
was still in the initial stages. Thus, oak epicotyls and young
stems differ from oak leaves in retaining protoplasts or undeveloped etioplasts, even after 100 days of growth in the dark.
Besides the predominant, shorter wavelength-emitting
Pchlide forms, young leaves always have a narrow emission
band at 657 nm (Böddi et al. 1992, Schoefs and Franck 1993)
and a band at 644 nm (Böddi et al. 1992). In spectra of oak epicotyls and stems, longer wavelength bands with low amplitudes were found at 635–636, 644–646 and 652–654 nm. The
635–636-nm-emission band may correspond to the 635–636nm band found in pea epicotyls (Böddi et al. 1994). The
644–646-nm band was less pronounced in the spectra of pea;
it was detected only in fluorescence spectra measured at 10 K
(Böddi et al. 1998). The 644–646-nm band overlapped with
the 635–636-nm band in the spectra of flash-illuminated oak
stem resulting in a maximum at 639 nm. Thus, the form emitting at 644–646 nm is not flash photoactive in oak epicotyl and
stem. In contrast, in etioplast inner membranes isolated from
leaves, the 644-nm-emitting Pchlide form is flash photoactive
(Böddi et al. 1991). Thus, despite their spectral similarities,
the Pchlide forms in epicotyls and stems have different characteristics from those in leaves. Their appearance is related to the
relatively high phytolized pigment concentration in oak epicotyls and stems, but the physiological role of this pigment is
uncertain. We attempted to study the phototransformation of
this pigment by separating the pigments after flash illumination, but only minute amounts were transformed in response to
flash illumination. On illumination with continuous light, the
transformation was so slow that parallel phytolization could
occur. Fluorescence spectroscopy cannot distinguish between
the native Pchl or Pchlide forms, because the presence of the
phytol chain has only a minute effect on the π-electron system
of the porphyrin ring in solution (Sironval et al. 1965). In vivo,
the complicated molecular environment screens the effect of
the phytol chain on the electron system of the porphyrin ring
even more. The flash photoactivity of Pchl pigments is controversial. In vitro measurements showed that NADPH:protochlorophyllide oxidoreductase (POR) is inactive when esterified pigment is added as substrate (Griffiths 1991). However,
the shorter wavelength-emitting forms may regenerate (El
553
Hamouri et al. 1981) the longer wavelength-emitting forms
and thus take part indirectly in a slow greening process (Cohen
and Rebeiz 1981). The longer wavelength- emitting Pchlide
forms are integral components of the PLBs in leaves (Böddi et
al. 1989). The pea epicotyl, which has similarly low amounts
of the 652–655-nm-emitting Pchlide form as oak stems and
epicotyls, has small, irregular PLBs and most of the etioplasts
contain only prothylakoid membranes (Böddi et al. 1994).
These spectral similarities and the similarly modest reactions
in response to flash illumination (Böddi et al. 1996) suggest
that chlorenchyma cells of oak epicotyls and young stems
have etioplasts containing mainly prothylakoid membranes in
which the majority of the Pchlide is in the monomeric state
(Böddi et al. 1998). The spectral difference between the
flash-photoactive forms of leaves and non-leaf organs
(657 and 652–654 nm, respectively) and differences in their
regeneration processes (fast regeneration in proplastids of
2-day-old leaves (Schoefs and Franck 1993) and slow regeneration in epicotyls and stems) also reflect differences in the arrangements of these complexes and in the regulation of their
formation. The dominating monomer structure does not allow
rapid (time scale of seconds) regeneration processes to take
place, unlike the aggregates of PLBs present in dark- grown
leaf etioplasts (Böddi and Franck 1997). In these aggregates,
Chlide is released and new Pchlide molecules replace them in
the active sites of POR after illumination. Also, NADP + is rapidly reduced in preventing photooxidation of the leaf pigments
(Franck et al. 1999). In the monomeric structure, however, the
replacement of the pigment and the reduction of NADP + are
dependent on diffusion processes, which are slow and strongly
temperature dependent. As a result of these characteristics of
epicotyl etioplasts, transformation of the shorter wavelengthemitting Pchlide forms was inhibited at 0 °C in pea epicotyl
(Böddi et al. 1994).
Because a large proportion of the pigments in epicotyls and
stems occurs as monomers, a photoprotective Chlide cycle
(Franck et al. 1995) is less probable in these tissues than in
leaves. In the absence of the Chlide cycle, the monomer pigments (both Pchlide and Chlide) are unprotected from strong
light and so the early Chlide forms are less stable in epicotyls
and stem than in leaves of oak (Axelsson et al. 1981) and other
plants (Sundqvist and Dahlin 1997). In epicotyls and stems,
the excitation energy causes photooxidation rather than photoreduction, resulting in bleaching of the pigments. The products of photooxidation may initiate processes leading to the
destruction of cell membranes, resulting in turgor loss and
epicotyl destruction. The latter processes were not found in
young stems, probably because lignification provides protection against photodamage. A decrease in temperature results
in rigidity of the cell membranes, which may increase light
sensitivity.
In nature, acorns germinating on the soil surface are exposed to both light and cold stress, resulting in significant destruction of germinating seedlings. The stress response that we
have characterized provides a physiological explanation for
improved seedling establishment when acorns are sown several cm below ground (Morris and Perring 1974).
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SKRIBANEK AND BÖDDI
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