Journal of Experimental Botany, Vol. 47, No. 294, pp. 49-60, January 1996
Journal of
Experimental
Botany
Ageing of Euglena chloroplasts in vitro
I. Variations in pigment pattern and in morphology
Simonetta Pancaldi, Angelo Bonora, Giuseppe Dall'Olio, Alessandro Bruni and Maria P. Fasulo1
Department of Biology, Section of Botany, University of Ferrara, Corso Porta Mare 2,1-44100 Ferrara, Italy
Received 3 July 1995; Accepted 13 September 1995
Abstract
Isolated chloroplasts of Euglena gracilis Klebs were
kept for 10 d in complete darkness at 4 C in a maintenance buffer (pH 7.5) without shaking. During incubation, the qualitative and quantitative changes in the
pattern of photosynthetic pigments were evaluated by
the combined use of spectrophotometry in the visible
range of whole chloroplasts and their acetone extracts,
of in vivo spectrofluorimetry and of reversed-phase
HPLC. Microscopic and submicroscopic modifications
were also followed by UV and transmission electron
microscopy.
The main findings were as follows: (1) a fast decay
of all photosynthetic pigments, chiefly chlorophylls,
not accompanied by evident signs of alteration of the
thylakoid system during the first 5 d; (2) a higher
stability of PSII compared to PSI and of antenna
complexes compared to the relative reaction centres
during the first 24-48 h; (3) a low accumulation of
phaeoderivative compounds in spite of the marked
decrease of chlorophyll content; (4) a lack of dephytylated compounds; (5) a quicker decay of the intensity
of fluorescence emission with respect to the decreasing chlorophyll a content; and (6) a fast degradation of
xanthophylls and //-carotene with the consequent lack
of defence from the ageing oxidative stresses. This
accounts for the rapid loss of pigments, although the
lack of other antioxidant defence mechanisms is not
excluded.
The characterization of some of the steps involved
in plastid degradation may render this experimental
model viable for further studies on plastid senescence,
a multifactorial process still awaiting definite answers.
Key words: Euglena gracilis, ageing, isolated chloroplasts,
morphological changes, pigment degradation.
Introduction
Chloroplasts are the most studied cellular organelles
during plant cell senescence. During this process, they
show qualitative and quantitative changes of pigment and
macromolecular composition, of molecular structure and
organization of thylakoid membranes, of primary photochemical reactions, and of the activity of Calvin enzymes
(Woolhouse, 1984). Alterations in the absorption
and emission characteristics have also been described
(Panigrai and Biswal, 1979). Most reports on this subject
regard higher plant chloroplasts both in vivo and in vitro
(Choe and Thimann, 1975; Biswal and Biswal, 1988;
Hashimoto et al, 1989; Ginsburg and Matile, 1993;
Ginsburg et al, 1994; Smart, 1994; Shioi et al., 1995),
while the literature dealing with the organelles in algae is
still scarce (Werthmuller and Senger, 1971; Kulandaivelu
and Senger, 1976; Engel et al, 1991). However, in spite
of the numerous studies, many aspects of the process
remain unclear. In particular, no unified picture of the
differential rates of degradation of pigment pattern is
available. Considering that in vitro experiments may provide a useful tool in studies on the senescence of Euglena
gracilis (Mares et al., 1993), the morphophysiological
changes that occur in isolated chloroplasts during ageing
have been followed, an aspect still controversal in the
algae (Ehara et al., 1975; Schoch et al., 1981; Scheer and
Parthier, 1982; Suzuki et al., 1987). Since a limitation in
these studies is imposed by the quick degradation of the
organelles, the experiments were made on isolated chloroplasts maintained axenically for 10 d in darkness at 4^C
' To whom correspondence should be addressed. Fax: +39 532 208561.
Abbreviations: car. = carotenoids; Chi = chlorophyll, Chlide = chk>rophyllide; EDTA = ethy1enediamine tetraacetic acid; HEPES = A/-(2-hydroxyethyl)
piperazine-W-(2-ethanesulphonic acid); LHC = light-harvesting chlorophyll-protein; Phaeo = phaeophytin; Pphaeo = pyrophaeophytin; PSI and
PSII = photosystem I and photosystem II; RC = reaction centre; rp-HPLC = reversed-phase high-performance liquid chromatography; R, = retention time.
© Oxford University Press 1996
50
Pancaldi et al.
without shaking. In fact, in these environmental conditions the rate of plastid degradation slows down (Panigrai
and Biswal, 1979; Walker et al., 1987), so that the study
could be continued for a longer time. In this paper, the
qualitative and quantitative variations of the photosynthetic pigments occurring throughout the experiment are
described. The evaluations were performed by combining
spectrophotometry in the visible range of whole chloroplasts and their acetone extracts, in vivo spectrofiuorimetry and reversed-phase HPLC. Microscopic and submicroscopic modifications were also followed by UV and
transmission electron microscopy. Attention was focused
on the catabolites that are detectable in the visible range
of the spectrum with regard to pigment degradation and,
with regard to Chls in particular, among the products of
the two reaction types of degradation I and II (Brown
et al., 1991; Shioi et al., 1995), only type I was considered,
i.e. the compounds still having an intact porphyrin macrocycle (Chlide, Phaeo, Pphaeo, phaeophorbides, and pyrophaeophorbides). Type II degradation, involving the
oxidative cleavage of the tetrapyrrole macrocycle and the
subsequent degradation to smaller carbon/nitrogen fragments (Ginsburg and Matile, 1993; Ginsburg et al., 1994;
Hortensteiner et al., 1995; Vicentini et al., 1995), will be
the object of further studies.
Materials and methods
Algal culture
Wild-type cells of Eugkna gracilis Klebs, strain Z, were
cultivated under constant shaking in Hutner-Provasoli's liquid
medium (Wolken, 1961) in a thermostatically controlled
incubator at 24±1°C under ordinary air (0.04% (v/v) CO 2 )
with daily light-dark periods of 16 h light (3.5 W m" 2 ) and 8 h
dark. For the experiments, the cells were collected at the early
stationary phase when cell density reached about 6 mg Chi I" 1 .
Cell disruption
The procedure reported by Suzuki el al. (1987) was applied
with some modifications as follows. The cells, filtered through
a layer of cotton cloth, were centrifuged at 1500 g for 2 min.
The pellet was rinsed with 50 mM K-phosphate solution
(pH7.0) containing 0.33 M sorbitol (K-P buffer). 10ml of
cellular suspension in K-P buffer were treated with 40 mg of
trypsin (Sigma Chemical Co, St Louis, MO, USA; type II) at
room temperature with shaking. The trypsin used was not
highly purified since it is reported that a certain amount of
chymotrypsin may be needed for the digestion of the pellicle of
Euglena (Ortiz et al., 1980). The course of digestion was
followed by light microscopy, and sufficient weakening of the
pellicle was usually achieved in 1 h. Soybean trypsin inhibitor
was also added to a concentration of 1 mg m l " ' . All subsequent
procedures were carried out on ice under a dim green safe light.
The suspension, diluted with K-P buffer, was centrifuged at 160
g for 3 min and the pellet was rinsed and resuspended in 20 mM
HEPES-NaOH buffer (pH 7.5) containing 0.33 M sorbitol and
0.5 mM Na 2 -EDTA (H-S buffer).
To achieve disruption, an original method has been devised
employing a pressure pump HPLC (Varian Model 5020). The
pump was placed between an H-S buffer reservoir and an empty
stainless steel chromatographic column (Merck Hibar,
12x1 cm) ending with a two-way Valco efflux-valve. The
column was filled with 3 ml of cell suspension and subjected to
a constant pressure of about 90 atm, and the valve was
regulated in order to obtain an efflux rate of 5 ml min" 1 .
Chloroplast isolation
The pressate was diluted with H-S buffer and then centrifuged
twice at 300 g for 3 min each to sediment unbroken cells and
cell debris. The supernatant fluid was centrifuged at 1500g for
3 min and the pellet was resuspended in H-S buffer. These steps
were repeated twice and finally the pellet was resuspended in
the buffer. The suspension was subsequently loaded on a stepgradient of Percoll (Sigma) in an H-S buffer which consisted
from the bottom upwards of four layers of 60%, 40%, 20%,
and 10% (v/v) solutions. The intact chloroplast fraction was
obtained at the 20%-40% interface after centrifugation at 400
g for 10 min. This fraction was washed twice with H-S buffer
plus 5 mM MgCl2 (maintenance buffer) by centrifugations at
1200 ^ for 3 min to remove any residual Percoll. The concentrate
suspension of the chloroplasts was stored at 4°C in the dark
without shaking. For all the analyses, aliquots of the suspension
were taken immediately after the preparation (0 time) and after
1, 2, 5, and 10 d. To minimize bacterial contamination, sterile
media and glassware were used throughout the experiments.
The samples, examined microscopically, were found to be free
of bacterial and fungal contamination during the entire 10 d
incubation period. The morphological intactness of chloroplasts
was established with a Zeiss Photomicroscope II equipped with
a phase contrast apparatus, while functional integrity was
assessed by the Hill reaction before and after osmotic shock
(Lilley et al., 1975) (four experiments). Ferricyanide-dependent
O 2 evolution was determined with a Clark-type oxygen electrode
(Yellow Springs Instrument Co., Yellow Springs, Ohio).
Chlorophylls were determined by the method of Lichtenthaler
(1987) (four experiments).
Visible absorption spectra
Absorption spectra were measured by an UV-VIS spectrophotometer Perkin-Elmer Model 554. The operating conditions were
as follows: band pass 1 nm, scansion speed 120 nm min"',
recorder speed 20nmcm~'. The analyses were carried out on
intact chloroplasts in maintenance buffer, on plastidic extracts
in absolute acetone and on standard pigments in a range of
solvents as reported below (see 'HPLC determinations'). For
the preparation of plastidic extracts, aliquots of the suspension
were pelleted and extracted with absolute acetone and filtered
on Millipore FH (0.4 ^m).
To accentuate small shoulders and asymmetries, which
otherwise may be overlooked, second-derivative analyses of
absorption spectra were performed by a built-in microcomputer
(band pass 2 nm; speed 60 nm min" 1 ). The determinations
were made in the 360-760 nm range, at room temperature. The
amount of chloroplasts used for the measurement was adjusted
to give approximately the same absorbance at 760 nm.
HPLC determinations
Preparation, identification and quantification of standard pigments: The cell suspension was pelleted by centrifugation at
1500 g for 3 min and the supernatant fluid was removed. The
pellet was extracted with methanol:acetone:n-hexane ( 3 : 1 : 1 ;
by vol.) and the solvent mixture was removed by centrifugation.
The extraction procedure was repeated until the pellet appeared
greyish-white. The crude extracts were combined and purified
Degradation of Euglena chloroplasts in vitro
by passage through an octadecyl silica cartridge (C18 Sep-Pak,
Waters Associates) and eluted with a few millilitres of ethyl
acetate. The solvent mixture was removed on a rotary
evaporator at 30°C, and the residue was dissolved in an
appropriate volume of acetone and filtered through a 0.4 ^m
pore size Millipore FH filter and injected into the chromatographic system.
Chi a (Rt 27.7 min), Chi b (Rt 26.0 min), neoxanthin («, 12.6
min), diadinoxanthin (Rt 16.4 min), diatoxanthin (Rt 19.8 min),
and ^-carotene (Rx 34.2 min) were separated by semi-preparative
rp-HPLC (see 'rp-HPLC equipment and procedure'), each peak
being collected after passing through the detector flow cell. The
isolated compounds were examined in terms of their purity by
further chromatography and fractionated again, if necessary.
Chi a allomer (Rt 27.0 min) was obtained by action of light, air
and LiCl for 2 h at room temperature (Berger et al., 1990). Chi
a epimer (Rl 28.4 min) was prepared by dissolving Chi a in
pyridine and leaving it to react in the dark at room temperature
(Zapata et al., 1987). For pigment quantification, Chi a allomer
and Chi a epimer were estimated together with Chi a. Phaeos
a and b (7?, 30 min and 28.9 min) were obtained by treating the
respective chlorophyll solutions with 0.1 N HC1 for a few min
at room temperature (Bacon and Holden, 1967). Chlides a and
b (Rt 5.5 min and 3.2 min) were prepared by enzymic
de-esterification of the respective Chls following the method
proposed by Jones et al. (1972). Phaeophorbides a and b {Rl
11.8 min and 9.9 min) were then obtained from their respective
Chlides by acidification (Bacon and Holden, 1967). Pphaeos a
and b (Rt 32.3 min and 30.3 min) were obtained by keeping
pyridine solutions of the respective Phaeos in evacuated sealed
vials for 24 h at 100°C (Pennington et al., 1964).
For the identification of the individual peaks, the standard
pigments obtained were compared by co-elution and by analysis
of spectral properties with published data (Wright and Shearer,
1984; Minguez-Mosquera et al., 1991; Wright et al., 1991).
Furthermore, the presence of epoxide groups was confirmed in
neoxanthin and diadinoxanthin fractions by the specific reaction
with ethanolic HC1 (Davies, 1976).
For quantification, the standards were dissolved in a known
volume of appropriate solvent and their visible light absorption
spectra were recorded; the X^^ and specific absorptions used
for the quantitative determinations were those reported by
Vernon (1960), Pennington et al. (1964), Davies (1976), and
Jackson (1976).
Finally, definite amounts of each standard acetone solution,
found photometrically, were injected into the chromatographic
column and plotted against the resulting peak area. Six to eight
determinations were made for each pigment, resulting in a
linear calibration graph (the standard deviation was less than
1.5%). The samples were stored at — 20°C and all the operations
involving the handling of pigment solutions were carried out
under a dim green safe light to avoid photodegradation of the
compounds.
HPLC equipment and procedure: The liquid chromatograph
consisted of a Varian 5020 apparatus, a Rheodyne 7126 injector
with a 100 ^1 injection loop for analytical and a 2 ml injection
loop for semi-preparative separations, and of a Varian UV-100
visible-UV variable wavelength detector set at 425 nm. This
wavelength represents a good compromise for the detection of
cars and Chls together with the phaeoderivatives which can not
be evidenced at the greater wavelengths (440-455 nm) usually
employed (Minguez-Mosquera et al., 1992). Chromatograms
were recorded on a Merck-Hitachi D-2000 chromato-integrator.
The separations were performed on a prepacked column MerckHibar-Lichrosorb Rp 18(7 ^m, 10 x 250 mm) with a precolumn
51
Lichrocart-Lichrosorb Rpl8 (5 Jim, 4 x 4 mm). Pigment peaks
were collected with a Gilson 201 fraction collector. The mobile
phase, methanol-water (8:2; v/v) (solvent A) and ethyl acetate
(solvent B), was pumped at flow-rate of 4 ml min" 1 as a step
gradient constituted as follows: 0% B (0-3 min), 0% B versus
35% B (3-16 min), and 35% B versus 65% B (16-24 min). One
minute after the elution of the final pigment (|S-carotene) the
solvent composition was returned to the initial conditions over
a 5 min gradient, after which a further time span of 5 min was
allowed to equilibrate the system before injecting the next
sample.
Measurement of the fluorescence characteristics
The room temperature fluorescence excitation spectra of
chloroplast suspensions were recorded by a Perkin Elmer MPF
fluorimeter. Excitation spectra were taken in the range of
390 nm to 510 nm for fluorescence at 688 nm. The fluorescence
emission, in the range of 630 nm to 750 nm, was recorded with
a 438 nm excitation wavelength. Exciting and measuring slits
were 6 nm. The spectra were not corrected for the spectral
sensitivity of the instrument. The chloroplast suspensions were
equivalent to 2 ^ g m l " 1 of Chi in the maintenance buffer.
Morphological observations
The examinations were performed on chloroplasts incubated at
0 time and at 1,2, 5, and 10 d.
UV light microscopy: A photomicroscope Zeiss model Axiophot
equipped with a reflected fluorescence condenser was used for
the fluorescence microscopy study. The light source was a
pressure mercury vapour lamp HBO 50 W with a filter set for
UV excitation: BP 436/10, FT 460, LP 470. High speed Ilford
film HP5 400 ASA was employed for photography.
The fluorescence intensity of Chi a was measured with a
MPM Microscope Photometer equipped with a MSP 21
Microscope System Processor installed on the microscope. The
analysis was performed on 20 single chloroplasts for each
experimental time (three determinations).
Electron microscopy: The chloroplasts were fixed with an equal
volume of 3% glutaraldehyde in 50 mM potassium phosphate
(pH 7.5) and 2 mM MgCl2 for 45 min at 4°C, sequentially
washed with 50 mM HEPES buffer (pH 8) containing decreasing
sorbitol concentrations (0.33, 0.16, 0.00 M) and incubated in
2% OsO 4 in phosphate buffer (pH 7.5) for 2 h at room
temperature. After being rinsed with phosphate buffer, chloroplasts were dehydrated in a graded ethanol series (12.5, 25, 40,
60, 75, 85, 95, and 100%), followed by three cycles in absolute
acetone, without embedding in agar (Yuan et al., 1991). The
materials were then embedded in Epon-Araldite. Embedding
and staining procedures were performed as previously described
(Fasulo et al., 1983, 1991). Observations were made with a
Zeiss EM 109N electron microscope.
Results and discussion
Chloroplast preparation
The use of an HPLC pump for cell disruption after
trypsin digestion made it possible to apply a pressure
which was not harmful for the integrity of the chloroplasts
themselves (Kitaoka et al., 1989). The percentage of
chloroplasts that, with the phase-contrast light micro-
52
Pancaldi et al.
scope, appeared pale yellow-green and refractile and then
intact (Walker et al., 1987), was about 88%. Moreover,
the ferricyanide-dependent oxygen evolution was 26 ± 4
^mol O 2 m g " ' Chi h " 1 and 156 Mmol O 2 m g ~ ' Chi h " 1
before and after osmotic shock, respectively. This suggests
that about 83% of the chloroplasts retained an intact
envelope.
The amount of purified chloroplasts estimated by
Chi concentration from a 1 1 culture was 300 ±80 /xg Chi
(average of the four independent experiments).
Visible light absorption spectra
The second-derivative absorption spectra of isolated
chloroplasts at 0 time and after 10 d (Fig. 1) showed
different bands corresponding to those reported in the
literature for other green algae and higher plants
(Anderson et al., 1978; Murata and Satoh, 1986;
Govindjee and Satoh, 1986; Daley, 1990). The spectral
profiles showed six main absorption peaks: 680, 670, 542,
490, 436, and 412 nm. The peak at 680 nm (Chi a) is
mainly attributable to mixed signals from both the LHCs
themselves and whole PSI and PSII. The peak at 670 nm
(Chi a) is principally ascribable to components of PSII
plus LHC signals. The peaks at 490 nm and 436 nm are
attributable to cars and Chi a, respectively. Finally, based
on the spectral characteristics of Phaeo a, Pphaeo a and
phaeophorbide a in a polar organic solvent that showed
bands at 534 nm and 409 nm (Schoch et al., 1981), the
peaks at 542 nm and 412 nm could originate from Chi a
phaeoderivative compounds. The approximately 10 nm
shift towards longer wavelengths, in the peak positions
of the absorption spectra of isolated chloroplasts with
respect to the standard pigment maximum peaks (Fig. 2)
obviously depends on the two different in vivo and in
vitro systems.
With regard to the quantitative variations of pigments
during chloroplast ageing, the graphics in Fig. 1 show a
decrease of all the pigments with the exception of the
Phaeo a-like compounds. In particular, the decrease of
Chi a involved to a greater extent the peak at 680 nm
compared to that at 670 nm. Since the signals at 680 nm
and 670 nm are approximately assigned to PSI and PSII
components, respectively, it seems reasonable to postulate that, during chloroplast senescence, a preferential
degradation of the PSI with respect to PSII occurs. This
fact was confirmed by fluorimetric analysis (see below).
Spectroscopic determinations of the acetone extracts of
isolated chloroplasts showed only quantitative, but not
qualitative, variations (data not shown). Due to the
similar spectral characteristics of the various photosynthetic pigments, qualitative variations were evaluated
only by HPLC procedure.
HPLC analysis
Qualitative distribution of pigments: The elution profiles
of pigment extracts from chloroplasts at 0 time and after
a 10 d incubation are reported in Fig. 3. Rt increased
according to the apolarity values of the pigments: neoxan-
o
430
O
to
.a
j_
o
m
.a
<e
CD
O
C
ai
410
360
Wavelength (nm)
667
760
Fig. 1. Second-derivative absorption spectra of chloroplasts isolated
from E. gracilis stored in maintenance buffer al 4 C. in the dark,
without shaking. The spectra refer to freshly isolated (-) and to 10 d
incubated (—) chloroplasts. (760 nm = 0 absorbance).
360
Wavelength (nm)
760
Fig. 2. Second-derivative absorption spectra of standard pigments in
absolute acetone. (A) Chi a: (B) phaeophytin a (760 nm = 0 absorbance).
Degradation of Euglena chloroplasts in vitro
53
Dd
N
~f"
LJL
_A_J
10
Minutes
25
30
35
a/pa(:10)
i
Fig. 3. Elution profiles of pigments extracted from E. gracilis freshly
isolated (top profile) and incubated for 10 d (bottom profile)
chloroplasts stored as in Fig. 1. The pigments were separated by
rp-HPLC and recorded by monitoring the absorption at 425 nm. The
absorption spectra of the main chromatographic fractions were
performed in the elution solvent. N = neoxanthin, Dd = diadinoxanthin;
D = diatoxanthin; b = ChJ b; a l t M 3 H =Chl a allomer; a = Chl o; pa =
phaeophytin a; /3 = /3-carotene.
thin, diadinoxanthin, diatoxanthin, Chi b, Chi a allomer,
Chi a, Chi a epimer, Phaeo a, and ^-carotene. The extracts
were devoid of Chlide, Phaeo b, Pphaeos, phaeophorbides
and pyrophaeophorbides. With respect to 0 time, in 10 d
incubated chloroplasts a generalized decrease of all the
fractions was noted except for Phaeo, whose level remained unchanged. Diatoxanthin was completely absent.
Quantitative distribution of pigments: The kinetics of loss
of the various photosynthetic pigments during a 10 d
incubation period is drawn in Fig. 4. The behaviour of
the total Chls/total cars molar ratio shows a relative
stability of cars during a 4-5 d dark incubation. Cars are
integral components of the photosystems, and particularly
the xanthophylls which represent about 75% of total cars
(Fig. 4), are necessary for the assembly of the LHCs
0 1
i
i
10
Fig. 4. Pigment contents and their molar ratios in E. gracilis isolated
chloroplasts stored as in Fig. 1. Each value, determined by rp-HPLC,
is expressed in nmol/ml of chloroplast suspension for pigment content
(
), and in nmol/nmol for molar ratios of the pigments (
). Each
point represents the mean of four experiments. a = Chl a; b (x 10)= 10
times amplified Chi b values; a/b = molar ratio between Chi a and Chi
b; pa (x 10) = 10 times amplified phaeophytin a values; a/pa (. 10)= 10
times diminished molar ratio between Chi a and phaeophytin a values;
x = xanthophylls; j8 = (9-carotene; x//3 = molar ratio between xanthophylls and /3-carotene; a + b/car = molar ratio between total Chi, and
total car,.
(Brandt and Wilhelm, 1990). Thus, it is tempting to
suggest that the lower breakdown of the cars was correlated to the maintenance of the antenna rather than core
complexes. This is also deduced from the trend of the
molar ratios of Chi a/Chl b and xanthophylls//3-carotene.
Indeed, during dark incubation the Chi a content exhibited a forward and remarkable decrease, which was about
16% after 24 h and 78% at the end of the experiment. In
contrast, the decrease of Chi b reached values of 16%
54
Pancaldi et al.
only at the 2nd day and then exceeded that of Chi a
(85% after 10 d) (Fig. 5). Consequently, the Chi a/Chl b
molar ratio decreased appreciably within the first 2 d and
then increased gradually (Fig. 4). The content of j3carotene showed a concentration gradient similar to that
of Chi a during the first days with a decrease of 21%
after 24 h and of 82% after 10 d. The diminution of
xanthophylls, represented essentially by diadinoxanthin,
paralleled that of Chi b with values of 28% and 77% after
2 and 10 d, respectively. Thus, during the first 24-48 h,
the increase of the xanthophylls/)3-carotene molar ratio
paralleled the decrease of the Chi a/Chl b ratio.
Considering that in Euglena (a) among carotenoids, /3carotene is the major component of the core complexes
as in the other green algae (Cunningham and Schiff,
1986), (b) the larger part of the ChJ b and xanthophyll
molecules is associated with the LHCs (Cunningham and
Schiff, 1986), (c) a decrease in the value of Chi a/Chl b
and an enhancement of xanthophylls//3-carotene ratios
are very likely related to the accumulation of the LHCs
with respect to the core complexes (Anderson, 1986;
Busheva et al, 1991), the behaviour of the molar ratios
ChJ a/Chl b and xanthophylls//3-carotene during ageing
further suggests an enhanced stability of the antenna
complexes with respect to their respective cores during
the first 24 h. This may be interpreted as an attempt of
adaptation to the light-dark transition similar to the
(i
%
80
en
*
•
MOO
*
•
••
1.1.1
I.I.I
LJJL1
40—
on
oo—l
0
2
10
Days
Fig. 5. Same as in Fig. 4. Percentage variations in pigment composition
of chloroplasts isolated from E. gracilis at 0, 2 and 10 d incubations.
a = Chl a; b = Chl b; c = carotenoids; and d = Chl derivatives.
long-term adaptation occurring in algae and higher
plants kept under light-limiting conditions (Anderson and
Barrett, 1986).
In spite of the rapid disappearance of Chi from ageing
chloroplasts, HPLC analysis revealed the presence of only
a low concentration of Phaeo a that remained unchanged
during the entire experiment (Fig. 4). In particular, the
molar ratio Chi a/Phaeo a decreased from about 40:1 to
10:1 after 10 d while the percentage of the Phaeo related
to the total pigments increased from 1.7% to 6.1%
(Fig. 5). The absence of Phaeo b may be due to the
conversion of Chi b to Chi a as reported recently by Ito
et al. (1993) and by Hortensteiner et al. (1995).
The constant low presence of Phaeo a could derive
from an inadequate Mg-dechelatase activity due to the
absence of the cytosolic cell compartment. Indeed, in the
in vivo chloroplasts of Euglena, phaeoderivative compounds normally accumulate during senescence (Schoch
et al., 1981; Bonora et al., unpublished). However, the
possibility remains that in this system Phaeo may be
destroyed as soon as it is produced. The fact that in
isolated chloroplasts Chls diminished without accumulation of their usual catabolites detectable in the visible
range of the spectrum indicates the occurrence of the
oxygenolytic cleavage of the macrocyclic ring system and
of the subsequent degradation to smaller carbon/nitrogen
fragments unabsorbing in the visible range (Hendry et al.,
1987; Ginsburg and Matile, 1993; Smart, 1994). This
seems to be likely, considering that the involved oxidative
enzymes are, reportedly, constitutive of the gerontoplasts
(Ginsburg et al., 1994).
As regards cars, neoxanthin diminished quickly and a
substantial decrease of jS-carotene occurred. A similar
pattern of degradation was found in some higher plants
both in vivo and in vitro during senescence under stress
conditions (Young et al., 1989, 1991; Young and Britton,
1990a, b). Surprisingly, in aged chloroplasts among the
car catabolites detectable by HPLC no fraction detected
at 0 time was found and no new chromatographic peaks
were noted. At present, this behaviour is inexplicable.
Indeed, unlike the breakdown of ChJs in which progresses
have been made by Matile's group (Shioi et al., 1995,
and references therein), the fate of cars during senescence
is not well understood. As viewed in Fig. 3, the destruction
of diadinoxanthin was not accompanied by the accumulation of its de-epoxi-derivative diatoxanthin whose formation is a light-dependent process (Krinsky, 1968). Since,
in Euglena, diadinoxanthin and diatoxanthin, similar to
their acetylene-functional analogues antheraxanthin and
zeaxanthin in higher plants and other green algae, in
addition to a light-harvesting function, also play a role
in the epoxide cycle (Siefermann-Harms, 1977; Parry and
Horgan, 1991), the lack of diatoxanthin in the aged
chloroplasts may determine an inadequate defence from
oxidative damages. In fact, ageing involves the formation
Degradation of Euglena chloroplasts in vitro
of reactive oxygen species capable of damaging thylakoid
lipoproteins (Peterman and Siedow, 1985). That cars,
such as |9-carotene, are able to protect cellular structures
from oxidative damage by inactivating electronically
excited molecules (i.e. singlet molecular oxygen ('O2))
has been clearly proved in vitro (Di Mascio et al., 1990).
The process is termed 'quenching' and occurs not only
by photoexcitation but also in darkness via chemiexcitation (Di Mascio et al., 1989). However, the lack of other
'O 2 quenching systems such as tocopherols, thiols, and
ascorbate (Di Mascio et al., 1990), can not be excluded.
Furthermore, it is possible that the activities of superoxide
dismutase and peroxidase, two enzymes that scavenge
active oxygen species (Larson, 1988; Fryer, 1992), are
compromised. These enzymes are important in chloroplast
degradation since they are localized in the organelle and
their activities decrease during senescence (Biswal and
Biswal, 1988).
55
1-
u
u
c
CD
HI
o
Fluorescence characteristics
Excitation spectra: The changes of the room temperature
fluorescence excitation spectra of chloroplasts at 0 time
and after 10 d are shown in Fig. 6 (insert A). The control
displayed a typical tracing, for fluorescence at 688 nm
(Fggg), with a distinct peak at 438 nm (F438) and a broad
peak at 475 nm (F475) corresponding to Chi a and cars
plus Chi b, respectively (Kramer et al., 1981; Lebedev
et al., 1985). The data correspond essentially to those
reported for Euglena by Brody (1968) and are also
comparable with those of the organelles of other green
algae and higher plants (Govindjee and Satoh, 1986).
During the 10 d incubation, the spectrum did not show
any significant qualitative change, whereas it presented
relevant quantitative variations. More precisely, the behaviour of the F438/F475 ratio indicates that the 475 nm
signal declined mostly during the first 5 d, after a lag
phase of 24 h (Fig. 6).
It is established that the excitation spectrum of fluorescence provides information not only on the composition
of the pigment systems, but also on the efficiency of the
excitation energy transfer (Govindjee and Satoh, 1986).
Consequently, in our aged chloroplasts the light energy
absorbed by the LHCs is probably less effectively passed
on to the RC complexes (Dhal and Biswal, 1990; Ivanov
and Kicheva, 1993).
0
1 2
Days
10
Fig. 6. Fluorescence intensity and Chi a content variations in E. gracilis
isolated chloroplasts stored as in Fig. 1. Fluorescence emission maximum
F
688 ( * - * ) and Chi a content determined by rp-HPLC (O-O); the
values are reported in percentage. Fluorescence intensity ratio
( • - • ) and fluorescence intensity ratio F438/F473 ( • - • ) . Insert A:
excitation spectra (510-390 nm) of freshly isolated (top profile) and 10
d incubated (bottom profile) chloroplasts. Fluorescence emission
monitored at 688 nm. Insert B: emission spectra (750-630 nm) of
freshly isolated (top profile) and 10 d incubated (bottom profile)
chloroplasts. Fluorescence excitation monitored at 438 nm.
daily to LHCI (Goedheer, 1972; Mullet et al., 1980).
During incubation, chloroplasts exhibited a progressive
quenching of the fluorescence at 688 nm that accompanied
the remarkable decrease of Chi a content evaluated by
HPLC analysis (Fig. 6). However, the variations were
not strictly interrelated. In fact, while the reduction of
the emission after a 2 d incubation was about 55%, the
diminution of Chi a content did not exceed 25%. On the
other hand, the level of Chi may not, by itself, be a
sufficiently sensitive parameter to probe the functional
Emission spectra: The room temperature emission spec- status of photosynthetic pigments in thylakoids and the
trum (for fluorescence excitation monitored at 438 nm)
degree of their possible disorganization. In contrast, this
of freshly isolated chloroplasts (Fig. 6 (insert B)) showed
information may be acquired by evaluating the variations
a distinct peak at 688 nm that is known to be originated
of thefluorescencecharacteristics of Chi a (Biswal et al.,
from PSII, principally from RC1I (Satoh, 1980; Murata
1989). From Fig. 6 it is apparent that the emission
and Satoh, 1986). Furthermore, the evident asymmetry
intensity ratio Fggg/F-^j, corresponding to the ratio of
of the tracing also suggests the presence of a component
PSII/PSI fluorescence intensity emission (Mullet et al.,
emitting at about 735 nm (F 735 ), attributed to PSI, essen1980; Satoh, 1980; Dahl and Biswal, 1990), increased
56
Pancaldi et al.
quickly during the first 24 h and then remained almost
constant. Since the above-named ratio provides a reliable
indication for the distribution of excitation light energy
between PSII and PSI (Murata and Satoh, 1986), it is
very likely that ageing affects PSII and PSI to different
degrees,with a greater involvement of PSI. This confirms
that PSII is more stable than PSI as already observed by
in vivo spectroscopy and that this stability occurs particularly during the first 24 h.
During the first 5 d of dark incubation, no appreciable
loss of structure was observed despite the remarkable
decrease in pigment content. At most, there was some
separation into individual discs, but this was not an
extensive process (Plate IB, C). Subsequently, the pattern
of the thylakoids became more and more disorganized
and after 10 d the inner membrane system was also
entirely damaged (Plate ID). The maintenance of a
normal internal structure for a long period of time,
notwithstanding the large drop in photosynthetic pigMorphological observations
ments, is in agreement with the observations of BenFluorescence microscopy: The results obtained from fluor- Shaul et al. (1965) in dark-adapted non-dividing cells of
Euglena. This confirms that the pigments are not always
imetric analysis were generally confirmed by the microa
decisive factor in the thylakoidal structure (Biswal et al.,
fluorimetric analysis and by the UV light microscopy
1989)
and that during senescence there is a sequential
observations (Fig. 7). As regards the latter, at 0 time
degeneration
of the chloroplast in which Chi degradation
chloroplasts appeared as brightly red fluorescent entities
occurs
earlier
than the disintegration of the thylakoid
having the normal aspect repeatedly described for these
membrane
system
(Biswal et al., 1983).
organelles in whole cells of Euglena (Fasulo et al., 1979,
1983). Chloroplast aspect and level of fluorescence
remained almost unchanged during the first 24 h. Later,
Conclusions
the plastidic contours became less marked and the autoOn the basis of these results it may be concluded that
fluorescence gradually declined.
isolated chloroplasts of Euglena, maintained for 10 d in
complete
darkness at low temperature without shaking,
Transmission electron microscopy: At 0 time, the isolated
undergo
several
qualitative and quantitative variations in
chloroplasts essentially showed the typical structure
the
pattern
of
pigments
that only latterly were accompandescribed for the normal fully developed organelles of
ied
by
signs
of
morphological
degradation. The main
Euglena (Schiff and Schwartzbach, 1982; Fasulo et al.,
aspects observed can be summarized as follows:
1983; Vannini et al., 1985) (Plate 1A). They were elongated in shape and had a stroma crossed by numerous
(1) remarkable decay of all the photosynthetic pigments,
lamellae each formed by two or three appressed
chiefly Chls;
thylakoids.
(2) quicker degradation of PSI than PSII during the first
24-48 h as inferred from the spectroscopic analysis
100
in the visible plusfluorescencedata;
(3) higher stability of the LHC complexes in comparison
to the relative RCs as inferred from the behaviours
of Chi a/Ch\ b and xanthophylls//?-carotene ratios;
(4) apparent maintenance of thylakoid organization
during the first days of incubation in spite of the
substantial decrease in pigment content and fluorescence intensity;
(5) absence of Chlides and of the other dephytylated
compounds;
(6) lack of correspondence between Chi decrease and
accumulation of phaeopigments;
(7) rapid degradation of carotenoids with the consequent
reduced defence from ageing oxidative stresses.
10
Fig. 7. Fluorescence intensity changes in E. gracilis isolated chloroplasts
stored as in Fig. 1. Percentage decay of the fluorescence emission
monitored microfluorimetrically on single chloroplast. The values are
expressed as the means and standard deviations of three separate
experiments of 20 determinations each (left). Fluorescent microscopy
aspects (right), x 833; x 1000; x 1000.
The absence of dephytylated compounds is not surprising.
In fact, in the in tow cells of Euglena these Chi catabolites
are not found (Schoch et al., 1981; Bonora et al., unpublished). This signifies that the alga is not competent with
regard to chlorophyllase in the first step of the catalytic
pathway and this is different from other lower and higher
plants in which the first step of Chi degradation involves
a dephytylation (Smart, 1994). This peculiarity of Euglena
Degradation of Euglena chloroplasts in vitr:
57
Plate 1. Electron microscopy aspects of E. gracilis isolated chloroplasts stored as in Fig 1. (A) Image of a freshly isolated chloroplast, x 27 500;
(B) partially disorganized pattern of the thylakoids in a 7-d-old chloroplast, x 29 200; (C) detail showing a lamellar system still organized in a
4-d-old chloroplast, x 57 500 and (D) a 10-d-old chloroplast exhibiting a completely disorganized membraneous system, x 17 300.
deserves further attention and helps to confirm the variability of a process which remains a biological enigma.
The presence of Phaeo a, even at a low concentration,
confirms the existence of a Mg-dechelatase activity.
Indeed, in Euglena, Phaeo is known to be the first Chi
catabolite (Schoch et al., 1981). However, the complete
absence of Pphaeo, which is the main derivative of Phaeo
in the in toto cells kept in darkness (Schoch et al., 1981),
suggests that, in the isolated chloroplasts, the relative
degradative enzyme is lacking or inactive perhaps because
it is encoded in the nucleus. Further studies will improve
the understanding of this metabolic aspect.
The lack of correspondence between ChJ decrease and
Phaeo pigment accumulation is ascribable to the oxidative
cleavage of the porphyrin ring, resulting in the formation
of small fragments not absorbing in the visible range.
This is likely since colourless compounds from Chi catabolism may be both intermediary and primary products
of porphyrin cleavage. They are eventually employed for
the assessment of Chi breakdown in isolated chloroplasts
and thylakoids (Schellenberg et al., 1990, 1993; Ginsburg
and Matile, 1993; Hfirtensteiner et al., 1995). The characterization of these compounds still requires investigation.
In the isolated chloroplasts, no strict relationship
between the rates of degradation of pigments, thylakoid
system and PSI and PSII was observed. This finding was
also reported in Euglena cells during dark-adaptation by
other workers (Ophir et al., 1975; Scheer and Parthier,
1982) and indicates that, under such conditions, certain
components of the photosynthetic apparatus are more
stable than others. This selective degradation supports
the hypothesis of Guiamet et al. (1991) that senescence
is a set of parallel, co-ordinated events which are not
necessarily interconnected, rather than a single chain of
changes.
The sequence of the events occurring in our isolated
chloroplasts during ageing may be difficult to relate to
that found by others since data are often contradictory
due to different system specificity and to the lack of
uniformity in the experimental conditions employed.
Furthermore, our data reflect the obvious limitations
caused by the model employed. Chloroplast senescence is
a multifactorial process which depends above all on
nuclear-cytosolic gene expression (Biswal and Biswal,
1988; Smart, 1994) even if there are arguments in favour
of the contribution of plastid gene expression to the
process (Guiamet et al., 1991).
However, this model, even though artificial, could
furnish an efficient and reproducible system for clarifying
some aspects of chloroplast senescence, a process which
is still largely unsolved. Few studies have characterized
the nature and location of degradative enzymes for
chloroplast degradation and plastid-specific proteases,
their specificity, time-dependent activation, and de novo
synthesis are still largely unknown (Shioi et al., 1995).
Acknowledgements
The authors are grateful to Professor Rino Cella, Department
of Biology, Section of Botany, University of Ferrara, Italy, for
his critical reading of the manuscript and to Dr Teresa Indelli,
Department of Chemistry, University of Ferrara, Italy, for
58
Pancaldi et al.
kindly providing the Perkin-Elmer MPF fluorimeter and for
careful technical assistance.
This work was supported by grants from Consiglio Nazionale
delle Ricerche (CNR) and Ministero dell'Universita e della
Ricerca Scientifica e Tecnologica (MURST) of Italy.
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