J. Plant Physiol. Vol. 146. pp.

527-534 (1995)

Pigment and Structural Changes in Chlorella zOfingiensis

upon Light and Nitrogen Stress

ETAN BAR, MOSHE RISE, MARINA VISHKAUTSAN, and SHOSHANA (MALIS) ARAD
The Institutes for Applied Research, Ben-Gurian University of the Negev, P.O.B. 653, Beer-Sheva 84105, Israel

Received October 14, 1994 . Accepted January 19, 1995

Summary

The green alga Chlorella zofingiensis was found to respond very rapidly to exposure to combined condi-
tions of high light intensity and nitrogen deficiency by accumulation of secondary carotenoids. Accumu-
lation of secondary carotenoids was detected as early as 60 min after induction to light stress
(300Ilmol'm-2's-1) in a nitrogen-free medium. Canthaxanthin and astaxanthin (free and monoesters)
were detected 2 - 3 h later, and after an additional 12 h an astaxanthin diester also appeared. The accumu-
lation of total secondary carotenoids was linear in relation to time. After 24 h the main secondary carot-
enoids were the monoester of astaxanthin (about 50% of total secondary carotenoids) and canthaxanthin
(20 - 25 %). During the first 8 h of stress the content of the primary carotenoids iJ-carotene and lutein in-
creased but subsequently the content of both chlorophyll and the primary carotenoids was reduced. The
reduction in the content of the photosynthetic pigments was followed by a degradation of the thylakoids
and a reduction of the potential rate of photosynthesis (a), but not of Pmax • After 3 days under light stress
the chloroplast was modified to a chromoplast-like organelle, full of secondary carotenoids and free of
thylakoid membranes. Twelve hours after the induction of stress, lipid bodies containing secondary ca-
rotenoids appeared around the chloroplast and accumulated at the periphery of the cell. The profile of
the secondary carotenoids in the lipid bodies was similar to that in the chromoplast, both containing
double the amount of astaxanthin diester and half the amount of free astaxanthin as compared with total
cell carotenoids. After 3 days under stress, a hydrophobic layer rich in secondary carotenoids formed in-
side the cell wall. Our results suggested that the lipid layer functions as a light filter to reduce irradiation
of the cell components, to prevent photooxidative damage and to reduce water losses.

Key words: Astaxanthin, canthaxanthin, Chlorella, chromoplast, photosynthesis, green algae, keto carot-
enoids, lipid body, primary carotenoid, secondary carotenoid.

Abbreviations: TEM = transmission electron microscope; HPLC = high performance liquid chroma-
tography; a = initial slope of photosynthesis.

Introduction 1979; Ben-Amotz et aI., 1982; An et aI., 1989; Arad et al.,

1993; Avalos et aI., 1993; Morelli et aI., 1993). The synthe-
Primary carotenoids constitute an integral part of the pho- sis of secondary carotenoids in green algae is induced by
tosynthetic apparatus in higher plants and algae, and func- stress conditions, such as high-intensity irradiation and ni-
tion as accessory pigments in the light-harvesting complex trogen deficiency (Donkin, 1976; Ben-Amotz et aI., 1982;
and as protective agents against photooxidative damage (Sie- Czygan, 1982; Ben-Amotz and Avron, 1989; Arad et aI.,
fermann-Harms, 1987; Lichtenthaler, 1987; 1993). The ac- 1993; Rise et aI., 1994).
cumulation of secondary carotenoids up to 10 % of the cell In the green microalgae Dunaliella bardawil and Tretepho-
dry weight is, however, a phenomenon unique to some spe- lia aurea, under light stress in combination with nutrition
a
cies of green algae, fungi and bacteria ohnson and Lewis, deficiency conditions that reduced the rate of cell division, iJ-

© 1995 by Gustav Fischer Verlag, Stuttgart

 528 ETAN BAR, MOSHE fuSE, MARINA VrSHKAUTSAN, and SHOSHANA (MALIS) ARAD

carotene, which usually appears as primary carotenoid, is ac- nitrogen free N-8 medium at an initial concentration of
cumulated and functions as a secondary carotenoid. No ke- 2 '10 6 cells' mL -I for further growth under the same conditions but
tocarotenoids were accumulated in those algae under these under a light intensity of 300 I1mol· m-z·s- I .
stress conditions (Czygan and Kalb, 1966; Ben-Amotz et al.,
1982; Ben-Amotz and A vron, 1989). In contrast, in other Extraction and HPLC analysis ofpigments
green microalgae, such as ChIarella emersonii, C. zojingiensis,
Cells from 5 mL of culture containing 2-4.10 6 cells' mL -I were
C. /usca, Coelastrum proboscideum, Scenedesmus and Haema·
collected by centrifugation (750 x g for 10 min), resuspended in
tococcus sp., the most common secondary carotenoids pro- DMSO: ethanol (2: 3 v/v) and heated at 70°C in the dark for
duced are ketocarotenoids. The main ketocarotenoids found 20 min (Rise et a!., 1994). The pigment extract was filtered through
in these species were canthaxanthin and astaxanthin (Good- 0045 11m filters and analyzed with a Varian 5000 LC HPLC system
win and Jamikorn, 1954; Droop, 1955; Donkin, 1976). Asta- with an L-4250 UVIVIS detector (Merck-Hitachi), a chromjet inte-
xanthin appeared largely in ester form (Renstrom et al., grator (Spectra Physics), and a 250 x 4 mm (id) Lichrospher 100 RP-
1981; Yong and Lee, 1991; Grung et al., 1992; Rise et al., 18,5 11m column (Merck) (Rise et a!., 1994). A stepwise elution pro-
1994). gram with two solvents, methanol: water (75: 25 v/v) and ethyl ace-
In Haematococcus and Dunaliella, secondary carotenoids tate, in a 30-min gradient at a flow rate of 0.6 mL' min -I was used
(Rise et a!., 1994). The principal absorbance detector was set at
have been found in lipid bodies (Ben-Amotz et al., 1982; 470 nm, and peaks were identified according to retention times and
Vechtel et al., 1992 a). In Haematococcus, the lipid bodies absorbance spectra (based on previous GC-MS and NMR identifica-
seem to be arranged at the cell periphery and to form a lipid tion (Rise et a!., 1994).
film containing carotenoids, possibly acting as a light filter.
It has also been suggested that in some species of green mi-
croalgae the secondary carotenoids serve as precursors for A nalysis of the photosynthetic potential
the synthesis of sporopollenin-like compounds in the algal Samples of cell suspension were diluted in duplicate to
cell wall (Atkinson et al., 1972; Burczyk, 1987; Derenne et 2·106cell·mL- 1 in a growth medium containing ImM NaHC0 3
al., 1992). This suggestion was based on four observations: i) for analysis with an oxygen electrode (Rank Brothers, Bottisham,
the appearance of sporopollenin upon stress, in parallel with UK). The oxygen electrode chamber was illuminated with a Hg
lamp at light intensities of 10-600 I1mol·s- l . m-Z.
the synthesis of secondary carotenoids, ii) the accumulation
of secondary carotenoids at the cell periphery, iii) inhibition
of carotenogenesis that was accompanied by the formation Electron microscopy
of sporopollenin-like compounds; and iv) in-vivo radiolabel- Cells were collected by centrifugation (600 x g for 5 min) and re-
i~g of secondary carotenoids that appear in the sporopolle- suspended in 3 % gluteraldehyde in 0.1 M phosphate buffer, pH 7.5,
mn. for 3 h. The cells were then washed three times with phosphate buf-
The accumulation of secondary carotenoids by C. emerso- fer, mixed into a 1.5 % agar solution at 40°C, and cooled rapidly in
nii and C. zojingiensis under a combination of high light in- ice. The cell-containing agar was cut into pieces, fixed in 1 % OS04
tensity and nitrogen deficiency has previously been observed for 1 h, and washed three times with phosphate buffer. The samples
and characterized by Arad et al. (1993) and by Rise et al. were dehydrated in ethanol followed by incubation in propylene
oxide. The samples were then embedded in Epon 812, sectioned
(1994). In C. zojingiensis, canthaxanthin, astaxanthin, an asta-
with an ultra-microtome to a thickness of 300 - 500 11m, and stained
xanthin monoester and an astaxanthin diester were found with saturated uranyl acetate solution and then with 0.3 % lead cit-
(Rise et al., 1994). Exposure of C. zojingiensis containing sec- rate. Specimens were examined with a Jeol 100CX transmission
ondary carotenoids to high light intensity caused removal of electron microscope (TEM).·
the ester groups from the astaxanthin; simultaneous chloro-
phyll degradation was not observed. De-esterification of the
Isolation oflipid bodies and chromoplasts
astaxanthin esters was suggested to be a mechanism for light
protection against photo oxidation. Cells from a 3-day-old culture, grown in a nitrogen-free medium
In order to further elucidate the functions of the secondary under a light intensity of 300 I1mol'm- z,s-t, were collected by cen-
carotenoids, we followed changes in the pigment profile and trifugation (750 x g for 10 min) and suspended in 5 mL of a buffer
made up of OAM sucrose, 100mM HEPES-NaOH, 10mM KCI,
structural modifications in C. zojingiensis subjected to high
1 mM MgCI 2 , and 1 mM EDTA at pH 7.5 (Murphy and Cummins,
light intensity and nitrogen deficiency, starting shortly after 1989). The cells were frozen in liquid air and crushed with a pestle
induction of stress conditions. and mortar in the presence of glass beads. Unbroken cells were re-
moved by centrifugation (750 x g for 20 min). The lipid bodies were
collected from the surface of the supernatant and washed twice with
Material and Methods the buffer. Chromoplasts were isolated according to Liedvogel et a!.
(1976) with the following modifications: the supernatant was cen-
Alga and growth conditions trifuged at 16,500 x g for 30 min to separate the chloroplasts (found
in the pellet) from the chromoplasts (occurring as a band in the su-
Chlorella zojingiensis (UTEX 32) was grown under constant fluo- pernatant). Since the culture was not synchronous, some of the cells
rescent light at an intensity of 50 I1mol· m-z's- I and at a controlled contained chloroplasts, which were removed with the pellet. The
temperature of 24 ± 2°C in conical tubes, 4cm in diameter, con- band of chromoplasts was transferred into 50 mL of HEPES buffer
taining 350mL of N-8 growth medium (Soeder et al., 1964) with amended with 0.1 M sucrose and centrifuged again. This step was re-
1 gr' L -I KN0 3, aerated with sterile air enriched with 0.2 % COz. peated twice. Pigments from the chromoplasts and the lipid bodies
For induction of synthesis of secondary carotenoids the cells were were extracted in DMSO: ethanol (2: 3 v/v) and analyzed by
collected by centrifugation (750 X g for 10 min) and resuspended in HPLC.
 Pigment and structural changes in Chlorella zojingiensls 529

:;l
A

"1
~

1 ~

.3 2

ec:
o
~ 02

: I
-S
l= "
<
j I
4

r
'0
I
20 30 .

...
<il
::1 B
'a::I 022 ~

.::-;
Gj '2~ 7
01. -<
I
"ii 016 .....;
I
,!:: I

ec: I
!
.J
01.

0
t-.
Fig. 1: HPLC chromatogram of pigments
extracted from C. zojingiensis grown under
~
-; "l
I
Gj 8
control conditions of low light intensity c:os .01 ~
<.J

(50 I1mol· m - 2. S-I) in nitrogen-containing

N-8 medium (A) and grown for 3 days un- -SQ
III
000 l I

der high light intensity (300 I1mol· m - 2 . S-I) ,.Q 0 .. ~

in nitrogen-free medium (B). 1 - lutein, < I !

2 - chlorophyll b, 3 - chlorophyll a, 4 - ()- "" 1 c

carotene, 5 - astaxanthin (free form), 6 - o~A

canthaxanthin, 7 - astaxanthin monoester I
(both peaks at retention times 28.32 and
28.98 min), 8 - astaxanthin diester.
. ,.:
RElEN110N nME (Min )
30

Statistics while no secondary carotenoids could be detected (Fig. 1).

In all experiments standard deviation (SD) was less than 5 % of Chlorophyll content remained constant up to the 8th h of
the mean. All results were statistically analysed for significance stress, when it began to fall, reaching less than 30 % of its ini-
(P < 0.05) by two-dimensional analysis of variance (Sokal and tial value after 24 h (Fig. 2). The concentrations of the pri-
Rohlf, 1981). mary carotenoids increased slowly, reaching a maximum af-
ter 8 -12 h and falling thereafter (Fig. 2). The ratio of {3-caro-
tene to lutein remained constant throughout the 24 h ({3-ca-
Results rotene/lutein 0.17 ± 0.013). Secondary carotenoids accumu-
lated over the 24 h period (Fig. 1, Fig. 2). After 4 h of stress,
Effect ofhigh light intensity and nitrogen deficiency on three secondary carotenoids were detected: canthaxanthin
pigment content during 24 h of stress (17 % of total secondary carotenoids), free astaxanthin
(30 %), and astaxanthin monoester (53 %) (Table 1). By 12 h a
The algal culture was subjected to a high light intensity of small amount of an astaxanthin diester was also detected,
300 ~mol· m -2. S -I and nitrogen deficiency. At zero time and reached up to 12 % of the total secondary carotenoids af-
only photosynthetic pigments, including chlorophyll and ter 24 h whereas the free astaxanthin decreased to less than
primary carotenoids ({3-carotene and lutein), were present 17 % (Table 1).
530 ETAN BAR, MOSHE RISE, MARINA VrSHKAUTSAN, and SHOSHANA (MALlS) ARAD

12 1.2
f; 6.0
i.e ;:;-
10 ..§ 1.0 .:..
5.0 i.e
'EOc ;;
;;" 0.8
..§
;;
..§ 8 Q
4.0
" ;;"
;; ."
8
'0 0.6
;;"
"" 3.0
e
6 "C
S '0
;; 0.4
e""
~

"~ 2.0
..."e 4 ~
."
~
0:
"S 0.2 1.0
~
e
~

2 "
<Il
;E
0.0 0.0
0 60 120 180 240 300
0
0 4 8 12 16 20 24 Time (min)

Time (h) Fig. 3: Effect of light stress and nitrogen deficiency on the content
of primary (PC) and secondary (SC) carotenoids during stress in C.
Fig. 2: Effect of high light intensity and nitrogen deficiency on zojingiensis. A logarithmic culture (50 mL of 2 '10 6 cells' mL -I) was
the content of primary (PC) and secondary (SC) carotenoids transferred to nitrogen-free medium in a 500 mL beaker. The
and chlorophyll (Chi) in C. zojingiensis. A logarithmic culture beaker was placed in a water bath to prevent heating and the culture
(2 '106 cells' mL -I) was transferred to nitrogen-free N-8 medium and was stirred using a magnetic stirrer. The culture was illuminated by
exposed to a light intensity of 300J.Lmol·m- 2 ·s- l • Samples were a light intensity of 800 J.Lmol· m -2. S -I and samples were taken for
taken after 0, 4, 8, 12 and 24 h under stress for pigment analysis. pigment analysis at 0, 60, 90, 150, 210 and 270 min after exposure to
light stress.

Table 1: Pigment content in a secondary carotenoid-free C. zojin·

gtensis culture exposed to high light intensity and nitrogen defi- 120
ciency. The alga culture (4 . 106 cells' mL -I) was transferred to ni-
trogen-free N-8 medium and exposed to a light intensity of 300
J.Lmol . m-2 . S-I. Samples were taken after 0, 4, 8, 12 and 24 h of ex- 100
posure to the stress conditions for analysis of pigments (/-1g' mL -I).
Three experiments were done, each in duplicates with no signifi-
cant differences. ND - not detectable (less than 0.5 J.Lg' ml-I).
0;
C
. 80

Carotenoids Time (h) ...Q

<J

Q 60
0 4 8 12 24 t<

Canthaxanthin ND 0.04 0.2 0.43 1.95 40

Astaxanthin (free) ND 0.07 0.2 0.57 1.57
Astaxanthin monoester ND 0.13 0.31 0.90 4.78
Astaxanthin diesters ND ND ND 0.06 1.14 20
0 4 8 12 16 20 24
Time (h)
When the algae were illuminated at a higher intensity of
Fig.4: Effect of light intensity of 300/-1mol'm- 2 's- 1 and nitrogen
800 Ilmol· m -2. S-I secondary carotenoids could already be deficiency on the photosynthetic potential in C. zojingiensis. A loga-
detected by 60 min (Fig. 3). Accumulation of secondary ca- rithmic culture (2 '106 cells' mL -I) was transferred to nitrogen-free
rotenoids was linear with time, and after 4 h their content N-8 medium and exposed to a light intensity of 300 J.Lmol·m- 2·s- l .
was four to five times higher than that at 300 Ilmol· m- 2. S-I. Samples in triplicates were taken after 0, 4, 8, 12 and 24 h under
Primary carotenoids accumulated very rapidly during the stress for analysis of oxygen evolution under various light inten-
first hour, but thereafter their accumulation rate ceased. The sities up to 600/-1mol·m- 2·s- l . Control Pm", was 3.4J.Lmole
maximal amount of primary carotenoids was obtained after 02·s- l ·min - l .
150min under a light intensity of 800Ilmol'm-2's-1 vs.
8-12 hat 300 Ilmol· m- 2 's- l •
trol level, while Pmax was reduced to 50% of the control
level.
Effect ofhigh light intensity and nitrogen deficiency on
In order to verify the protective role played by secondary
photosynthesis
carotenoids in photosynthesis, the effect of a very high light
The photosynthetic potential was measured in a culture of intensity (1500 Ilmol· m -2. S-I) on photosynthetic potential
C. zofingiensis growing under a light intensity of in a pre-induced culture (containing secondary carotenoids)
300 Ilmol· m- 2 's- 1in a nitrogen-free medium for 24h (Fig. 4). was compared with that in a non-induced culture (Fig. 5).
The initial slope a dropped before reduction in Pmax was The initial value of Pmax in the secondary carotenoid-contain-
observed. After 24h a had decreased to 25-30% of its con- ing culture was less than half of that in the secondary carote-
 Pigment and structural changes in Chiarella za/ingiensis 531

4.--------------------------------, Table 2: Profile of secondary carotenoids in total cell extract, lipid

bodies and chromoplasts. A secondary carotenoid-free culture of C.
za/ingiensis was exposed to high light and nitrogen deficiency and
analyzed after 3 days under stress. Concentration of each secondary
carotenoid was expressed as a percentage of the total secondary ca-
rotenoids. The experiment was repeated independently three
D
times, and single samples were analyzed each time. No significant
differences were found between the experiments as found by 2D
D D D analysis of variance.
Carotenoids Lipid bodies Chromoplast Whole cell
Canthaxanthin 25.7 25.9 26.1
Astaxanthin (free) 4.9 6.1 10.4*
Astaxanthin monoester 49.8 47.2 52.2
30 60 90 120 150 Astaxanthin diestes 19.6 20.8 11.3"
Time (min) * Significantly different (P < 0.05).

Fig. 5: Effect of extremely high light intensity of (1500 !Lmol· m -2. S-I)
on Pmax in a culture of C. za/ingiensis containing secondary carotenoids
after 24h under high light intensity of (300 !Lmol·m- 2 ·s- 1) and nitro- were detected. However, chlorophyll, /3-carotene, and lutein
gen deficiency (0) and in a culture devoid of secondary carotenoids were found in the total cell extract, probably from incom-
(0). pletely transformed chloroplasts that were removed from
the chromoplast fraction (see Materials and Methods). No
significant differences were found between the profile of
noid-free culture. During the time of stress Pmax in the cul- secondary carotenoids of the lipid bodies and that of the
ture containing secondary carotenoids did not change, chromoplasts (Table 2): in both, the astaxanthin diester
whereas in the culture devoid of secondary carotenoids Pm• x constituted about 20 % of the total amount of secondary
dropped within 20 min to about 15 % of its initial level, and carotenoids (vs. about 11 % in the total cell extract). The
after 60 min no oxygen evolution could be detected. relative amount of free astaxanthin in the total secondary
carotenoids in the lipid bodies and in the chromoplasts was
Effect ofhigh light intensity and nitrogen deficiency on cell about half in the total cell extract. No significant differences
structure were found between the relative amounts of total astaxanthin
and canthaxanthin in the lipid bodies, chromoplasts and to-
TEM was used to follow structural changes in the algal cell tal cell extract. In all fractions astaxanthin amounted to 74.1
during stress (high light intensity of 300 !lmol· m -2. S-1 and ± 2 % of total secondary carotenoids and canthaxanthin to
nitrogen deficiency). The most significant changes were 25.9 ± 2 %. In an older culture (~ 7 days) no differences
found in the chloroplast: after 4 h exposure to stress, degra- were found in the profile of secondary carotenoids among
dation of thylakoids was observed and after 12 to 24 h the the total cell extract, lipid bodies and chromoplasts, and no
grana structure of thylakoids had almost disappeared and the chlorophyll or primary carotenoids were detected (results
content of thylakoid membranes was reduced (Fig. 6 a, b). not shown).
After 3 days (Fig. 6 c) the chloroplast had completely lost its
typical structure and took the form of an orange chromo-
plast-like organelle (as observed by light microscope) with-
out an inner membrane. Discussion
Two additional significant changes in the cell were ob-
served in response to stress. In specimens examined after 12 Carotenoids playa highly effective role in protecting pho-
and 24 h, lipid bodies were seen to accumulate outside the tosynthetic pigments, enzymes, membranes and nucleic
chloroplast close to the cell membrane (Fig. 6 a, b). In the acids against photooxidative damage (Lichtenthaler, 1987;
3-day specimen (Fig. 6 c), a lipid layer containing secondary Siefermann-Harms, 1987). During photosynthesis, oxygen
carotenoids was observed under the cell wall. A very thick radicals are formed even under relatively low light inten-
cell wall, which was found to be stable in concentrated sulfu- sities. Under high irradiation the photosynthetic apparatus
ric acid (95 %), indicated the presence of an additional com- does not sufficiently utilize light energy, and the excess
ponent in the cell wall, such as sporopollenin-like molecules. energy leads to the formation of highly active oxygen mole-
cules. In this case the primary carotenoids cannot scavenge
the radicals sufficiently, and additional mechanisms there-
Pigment analysis of the lipid bodies and the chromoplasts fore are required for eliminating radicals or for reducing the
Lipid bodies and chromoplasts were isolated from a culture illumination reaching the cell components under such condi-
grown for 3 days under a light intensity of 300 !lmol· m -2. S-1 tions. In green microalgae such as Chlorella and Haematococ-
in a nitrogen-free medium. Pigments were analyzed in the cus, secondary carotenoids that accumulate in response to
lipid bodies, the chromoplasts and a cell extract (Table 2). stress conditions may serve as agents protective against the
Chlorophyll was not found in the chromoplasts or the lipid effects of photooxidation (Rise et al., 1994; Vechtel et al.,
bodies, and only very small amounts of primary carotenoids 1992 b).
532 ETAN BAR, MOSHE RISE, MARINA VISHKAUTSAN, and SHOSHANA (MALIS) AKAD

In our study, secondary carotenoids that were not present thetic apparatus against irradiation damage. Significant sup-
in algae grown under regular conditions (Fig. 1) appeared port for the premise that secondary carotenoids provide pro-
very shortly after exposure to combined stress conditions of tection against light stress comes from the fact that in cells
high light intensity and nitrogen deficiency (Fig. 3), which is containing secondary carotenoids and exposed to high irra-
evidence for a rapid mechanism for protecting the photosyn- diation, equivalent to the intensity of sunlight, a sharply re-

~ .~ .
." ~ .. ""
• V' ,J
'

'- ~:'J ' ': 6 ~

12
6a
Fig. 6: Transmission electron micrographs of C. zojingiensis subjected to a high light intensity of 300 limol· m -2. S-1 and nitrogen defi-
ciency. 6 a - Cell structure in speciemens subjected to stress for 0, 4, 12 and 24 h, 6 b - Sample taken after 24 h of stress at a higher magnifi-
cation, 6 c - Sample taken after 72 h of stress. S-starch granules, L - lipid bodies, m - mitochondria, g - grana, arrows - thylakoids. Bars
are 2 lim.
 Pigment and structural changes in Chlorella zoJingiensis 533

duced photosynthetic potential in cells lacking secondary ca- reduction in photosynthetic pigments. Since photosynthesis
rotenoids but not in cells in which secondary carotenoids is almost inactive during the period that secondary carot-
had accumulated was found (Fig. 5). Indeed, in parallel to the enoids continue to accumulate, it seems that they protect
accumulation of secondary carotenoids, the chloroplast lost photosynthesis only at the early stages of the stress. The ac-
its function due to the degradation of the thylakoids and the cumulation of high concentrations of secondary carotenoids
during the later stages of stress suggests that they play an ad-
ditional role in helping the cell to survive during a long pe-
riod of stress by preventing irradiation damage to cell com-
ponents and not only protecting photosynthesis against pho-
tooxidation but also preventing irradiance damage to other
cell components.
We suggest that the response to stress is made up of three
stages. During the first stage (0 - 8 h, depending on light in-
tensity) both primary and secondary carotenoids protect the
\ photosynthetic apparatus against photooxidation. During
:~, the second stage (4 -12 h), when the content of primary ca-
:.,..~
rotenoids is reduced and secondary carotenoids begin to ac-
cumulate, the secondary carotenoids replace the primary ca-
rotenoids in protecting photosynthesis. During the third
stage (12 h and more) a lipid layer containing secondary ca-
rotenoids surrounds the cell to form a hydrophobic light fil-
ter, as previously suggested by Santos and Mesquita (1984)
and by Hagen et al. (1994). The secondary carotenoids may
also function as a substrate for the synthesis of sporopollenin
in the algal cell wall, as suggested by Derenne et al. (1992),
Atkinson et al. (1972) and Burczyk (1987). The thick and
stable cell wall found in the 3-day-old culture (Fig. 6 c) pos-
sibly contains a sporopollenin-like substance.
The transportation of the secondary carotenoids from the
chloroplast to the cell periphery is possibly accomplished by
lipid bodies. Formation of a lipid layer enriched with carot-
enoids was observed clearly in the 24-h and 72-h specimens
(Fig. 6). This lipid layer seems to function as a sunshade filter
to reduce irradiation of the cell components and to prevent
formation of oxygen radicals. It may also function as an ef-
fective hydrophobic layer that reduces water losses upon de-
hydration or salinization of the water body (occurring upon
evaporation).

Acknowledgements

The authors wish to thank Dr. S. Guttman from the Biologial

Services, Electron Microscopy Department, at the Wiezmann In-
stitute of Sciences, Israel for technical assistance in the microscopy
study and to Ms I. Mureinik and Ms D. Imbar for editing the manu-
script.
The work was partially supported by the Ben-Gurion Foundation
of the Ministry of Science and Arts of Israel.

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