Journal
of
Photochemistry
Photobiology
B:Biology
and
www.elsevier.hi/ locate/jphotobiol
ELSEVIER
J. Photochem.Photobiol.B: Biol. 50 (1999) 149-158
Effects of changes of irradiance on the pigment composition of
Gracilaria tenuistipitata var. liui Zhang et Xia
E. Carnicas *, C. Jim6nez, F.X. Niell
Department of Ecology, Faculty of Sciences, Universityof Mdlaga, E-29071 Malaga, Spain
Received 2 February 1999; accepted 5 July 1999
Abstract
In plants, excess irradiation can damage the photosynthetic apparatus, although some protective mechanisms exist. The excess energy can
be dissipated as thermal energy, and pigments (i.e., carotenoids) also play an important role in protecting the photosynthetic apparatus by
epoxidating reactions. Chromatographic analysis of pigment extracts of Gracilaria tenuistipitata shows that zeaxanthin is the major carotenoid
in this alga, accounting for up to 82% of total carotenoids. Short-term (55 h) and long-term (10 days) response of the pigments shows that
Chl a, 13-carotene and zeaxanthin degradation after light increase follows negative exponential trends, while the response of biliproteins is
almost linear. Decreasing the irradiance results in a clear saturating response of the synthesis of Chl a and 13-carotene after one to two days.
Biliprotein synthesis displays a double linear trend, the first one lasting for four days in the cases of both R-phycoerythrin (RPE) and Rphycocyanin (RPC). The response of zeaxanthin is always faster than that of Chl a or biliproteins to changes of irradiance. Our results might
indicate the presence of two pools of zeaxanthin in this alga, with different acclimation responses to the changes in the photon flux density.
© 1999 Elsevier Science S.A. All rights reserved.
Keywords: Gracilaria tenuistipitata; Xantophyllcycle; Zeaxanthin; Pigmentcontent; HPLC
1. Introduc~on
Light is the essential energy source for photosynthesis.
Photosynthetic organisms are capable of operating a lightabsorbing pigment system over a wide range of photon flux
densities (PFDs). However, excess solar energy can lead to
photodestruction of the photosynthetic apparatus [ 1-3].
Irradiance levels are very high in some latitudes, reaching
values around 2500 p~mol m -2 s -1 in the tropics. Typical
sun algae become photosynthetically light-saturated at photon fluence rates around 500 p,mol m - 2 s - t and shade algae
at 60-150 pLmol m - 2 s - ~ [4]. Algae, like higher plants, have
developed mechanisms to adapt to excess sunlight, Macrophytes, in contrast to phytoplankton which are free to move
vertically, are restricted to their site of growth and survive by
developing internal mechanisms that increase the dissipation
of excitation energy through non-photosynthetic metabolism.
Therefore, components of the photosynthetic system of the
chloroplast involved in light absorption, electron transport
and carbon fixation undergo acclimation to the level of irradiance [5,6]. When an excess of light reaches the photosyn* Corresponding author. Tel.: + 34-952-131844; Fax: + 34-952-132000;
E-mail: mecarnicas@uma.es
thetic apparatus, an increased proportion of absorbed quanta
is dissipated as thermal energy [7].
The irradiance level also has a pronounced effect on algal
pigment composition. The chlorophylls do most of the light
harvesting, whereas carotenoids play two important roles, one
as light-harvesting pigments and another in protecting the
photosynthetic apparatus against excess irradiance through
epoxidating reactions [ 8-10]. Two main xanthophyll cycles
have been described, acting as photoprotective mechanisms
of the photosynthetic apparatus. The first one, called the 'violaxanthin cycle', has been reported in higher plants, green
algae and in some brown algae. Under excess light, violaxanthin de-epoxidates through the intermediate antheraxanthin
to form zeaxanthin [ 11 ]. Conversely, the action of an epoxidase transforms zeaxanthin to violaxanthin, via anteraxanthin. The 'diadinoxanthin cycle' is found in diatoms [ 12]
and it involves similar reactions. Under high irradiance, diadinoxanthin de-epoxidates to diatoxanthin. In both cycles,
the reverse epoxidating reactions occur under low irradiance.
A third xanthophyll cycle, which involves the xanthophylls
lutein and taraxanthin (5,6 diepoxilutein), has been described
in Dunaliella tertiolecta [ 13]. Up to the present, no xanthophyU cycles have been described in red algae. In addition to
this photoprotective function, in some marine organisms
1011-1344/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PIISIO1 1-1344(99)00086-X
150
E. Carnicas et al. / J. Photochem. Photobiol. B: Biol. 50 (1999) 149-158
carotenoids also contribute significantly to light harvesting
[141.
Changes in pigment composition and in the different components of the xanthophyll cycle under luminous stress have
been widely studied in higher plants [ 15-18 ] and green algae
[8,19]. In contrast, the effects of irradiance on the concentration of Chl a and phycobiliproteins in red algae have been
studied by several authors [20--231, but less work has been
done concerning the xanthophyll composition and the change
in carotenoid concentration under different irradiances [ 9].
In this study we analyse the pigment composition of a
Rhodophyta, Gracilaria tenuistipitata, when thalli are submitted to drastic changes of the irradiance level. To perform
the experiments, G. tenuistipitata was kept in a controlled
system in which light was the only variable that was modified.
The pigment response dynamic to light intensity is presumed
to be vary with time, e.g., it has been demonstrated to be very
fluctuating in the first hours in other red algae [21,23]. So,
the acclimation of G. tenuistipitata to a luminous change was
studied in short-term (55 h) and long-term ( 11 days) experiments. A detailed analysis of carotenoid composition was
made in order to establish the xanthophyll composition of
this rhodophyta, as well as the possible photoprotective
mechanisms when the plants were exposed to excess light.
60
A
0
350
-~
400
i
450
t
500
550
Wavelength
t
600
r
650
i
700
i
750
(nm)
Fig. 1. Spectrumof the halogenlamp used as a light sourcein the two sets
of experiments.
and once a day for 10 days after the starting day in the longterm acclimation experiments.
In the second set of experiments, the light treatment was
the reverse. The algae were kept at a relatively high irradiance
(500 ixmol m -2 s- l) for one month and then were exposed
to 40 ixmol m -2 s -1 for 55 h or for i0 days, following the
same sampling strategy as in the experiment above. Experiments were repeated several times; samples were taken in
triplicate.
2.3. Pigment analysis
2. Materials and methods
2.1. Organism and culture conditions
G. tenuistipitata Zhang et Xia (1983) [24] was obtained
from the Institute of Plant Physiology of the University of
Uppsala (Sweden). The algae were cultivated in 41 cylinders
at a temperature of 25°C and a plant density of 3 g 1-1. The
culture medium consisted of filtered natural sea water (Whatman GF/F), enriched with Provasoli's solution [25]. The
final concentrations of nitrate and phosphate were 820 and
30 IxM, respectively. It was renewed every three days and the
cultures were bubbled with air at a rate of 4.4 1 min -1 [2628 ]. White light was continuously provided by halogen lamps
(Mazda MAIH/400 W) (see spectrum in Fig. 1).
2.2. Experimental design
Two sets of experiments were performed for the description of the acclimation of the photosynthetic pigments of G.
tenuistipitata to short-term (55 h) and long-term ( 10 days)
changes of the PFD. In the first one, the algae were grown at
a PFD of 40 ixmol m-2 s - 1 for one month to acclimate to
low irradiance. After this period, the plants were suddenly
transferred to a higher PFD (500 Ixmol m -2 s - l) for 55 h or
10 days and the pigment composition ofG. tenuistipitata was
analysed. Samples were taken three times a day during the
first three days after the change of irradiance in the 55 h
experiments (starting time, 4, 7, 24, 28, 31, 48, 52 and 55 h)
The pigment composition of G. tenuistipitata was determined using two different techniques: spectrophotometry to
obtain a fast although coarse result and high-performance
liquid chromatography (HPLC) to obtain more accurate
results. Whole plants were randomly taken from the cultures
and water was removed to avoid a progressive reduction in
plant density during the experiments.
The extraction of hydrosoluble pigments (R-phycoerythrin, RPE, and R-phycocyanin, RPC) was performed by
grinding the plants (50 mg fresh weight (FW)) in phosphate
buffer (0.1 M, pH 6.5) at 4°C. After 12 h, the extracts were
centrifuged for 15 min at 19 000g at 4°C (Heraeus 17-SB)
and the supernatant was used to determine the phycobiliprotein concentration, according to Ref. [29]. In order to determine the concentration of liposoluble pigments, samples of
around 100 mg FW were extracted in 4 ml dimethylformamide (DMF) and kept overnight at 4°C. This method avoids
grinding the plants and has previously been used efficiently
in algae [30] and higher plants [31]. Pigments were then
transferred to a more volatile solvent by adding 2 ml of
diethylether and 2 ml of bidistilled water to the extracts. A
short centrifugation pulse of 1 min at 1000g was given after
sample mixing and the diethylether phase containing the pigments was transferred to a 2 ml glass container and kept at
- 2 0 ° C until HPLC analysis (less than one week). If the
DMF/water fraction was still coloured, the process was
repeated until it became totally transparent. Before injection
in the chromatograph, the diethylether was evaporated under
a nitrogen flow and pigments were finally resuspended in
20 Ixl of terbutylmethylether.
E. Carnicas et al. / J. Photochem. Photobiol. B: Biol. 50 (1999) 149-158
A Hewlett-Packard HP-1090 system equipped with a
reverse-phase VYDAC 201TP54 C-18 column (25 cm × 4.6
mm, 5 Ixm particle size) was used. Elution was performed in
a two-solvent gradient system. The solvents were a mixture
of acetonitrile (75%), methanol (15%) and tetrahydrofuran
(10%) (solvent A) and 100% bidistilled water (solvent B).
The gradient system was as follows: initial 80% A + 20% B;
0-30 min linear gradient to 100% A; 30-40 min isocratic
100% A; 40-50 min linear gradient to 80% A + 2 0 % B; 5060 min isocratic 80% A + 20% B for column stabilization
before next injection, at a flow of 1.0 ml m i n - 1. A programmable photodiode array detector was used for pigment detection, at 445 and 412 nm. Identification of chromatographic
peaks was carried out by injection of commercial standards
(Sigma, St Louis); zeaxanthin was kindly provided by
Dr Olimpio Montero (Instituto de Ciencias Marinas de
Andaluc{a, Puerto Real, C{tdiz) and by comparison with
several spectral and chromatographic characteristics reported
in the literature [32].
As the relationship between fresh and dry weight may vary
at different irradiances, pigment content was expressed on a
dry weight basis.
2.4. Statistics
Experiments were repeated several times, and samples for
HPLC were analysed in triplicate in each experiment. Results
presented here correspond to one representative experiment
for each of the treatments. Two different statistical tests were
employed. A Student's t test was used to establish significant
differences between initial and final pigment concentrations.
An ANOVA was performed to determine the significance of
the changes in the pigment concentration of G. tenuistipitata
during the experiments [33].
3. Results
G. tenuistipitata, like all the Rhodophyta, contains Chi a,
and carotenoids and phycobiliproteins (RPE and RPC) as
major accessory pigments. Fig. 2 shows the chromatograms
of plants acclimated to PFDs of 40 and 500 I~mol m - 2 s - 1.
Four main liposoluble pigments were identified: Chl a, 13carotene ([3-CT), zeaxanthin (Z) and lutein (L), but neither
violaxanthin nor anteraxanthin. The percentage zeaxanthin
concentration (on a total carotenoid basis) represented 76.7
and 75.7% at 40 and 500 ixmol m -2 s - 1 respectively, and it
did not vary significantly among the plants acclimated to low
and high irradiance.
In the first set of experiments, the change in the pigment
composition of G. tenuistipitata was followed after an
increase of the PFD from 40 up to 500 ixmol m - 2 s - 1. When
plants that were acclimated to 40 Ixmol m -2 s-~ were
exposed to a higher irradiance for 55 h, the concentration of
all pigments decreased markedly (Fig. 3). Chl a concentration decreased from an initial value of 5.26 mg g - 1dry weight
151
(DW) to a final value of 2.94 mg g - 1 DW (Fig. 3 (A) ); this
decrease fitted a negative logarithmic function (r 2= 0.92).
[3-Carotene decreased in a similar way, from 0.12 to 0.05 mg
g - 1DW (Fig. 3 ( B ) ) (r 2 = 0.94 ). The long-term acclimation
of G. tenuistipitata after the increase in irradiance was also
studied (Fig. 4). When the algae were exposed to 500 ~mol
m - 2 S- 1 for 10 days, the concentration of Chl a decreased
from 5.71 to 2.63 mg g - 1 DW and the logarithmic trend of
the decay was maintained during the whole period (Fig.
4 ( A ) ) . The results obtained for 13-carotene were different
(Fig. 4 ( B ) ) . As described above, its concentration decreased
during the first 55 h but it increased sharply to the initial value
during the third day. Then it decayed continuously during the
rest of the experiment, reaching a final value of 0.047 mg g - 1
DW.
Two main xanthophyUs were identified in G. tenuistipitata,
zeaxanthin and lutein (Fig. 2). Zeaxanthin was found to be
the major carotenoid in G. tenuistipitata, with concentrations
four to six times higher than that of lutein and three to five
times higher than 13-carotene. The changes of the two xanthophylls were markedly different from the logarithmic
trends described for Chl a and 13-carotene (Fig. 3(C), ( D ) ) .
Zeaxanthin concentration (Fig. 3 ( C ) ) decreased sharply
during the first 7 h of exposure to 500 Ixmol m - 2 s - ~. During
this time, its concentration dropped to half of the initial value
(0.45 to 0.22 mg g - 1 D W ) . Then, it fluctuated between 0.22
and 0.30 mg g - ~DW. The concentration of zeaxanthin over
10 days behaved in a similar way to that of [3-carotene (Fig.
4(C). It decreased during the first two days from an initial
value of 0.62 to 0.53 mg g - 1 D W , increasing sharply on day
three to a value of 0.68 mg g-~ DW and finally decaying
during the rest of the experiment, reaching a final value of
0.45 mg g - 1 DW.
The change in lutein concentration (Fig. 3 ( D ) ) was very
irregular during the first 55 h the plants were exposed to high
irradiance. Its concentration decreased sharply in 7 h, from
an initial value of 0.08 mg g - 1 DW to a value of 0.042 mg
g-- 1 D W , and it continued decreasing to a minimum of 0.035
mg g-1 DW after 24 h. Then it rose to 0.07 mg g-1 DW,
with a final decay to the end of the experiments (0.05 mg
g - ~ DW). Changes of the lutein content over 10 days were
also significant (Fig. 4(D) ), with an initiai decay from 0.08
to 0.043 mg g - 1 DW during the first three days, followed by
daily fluctuations ranging between 0.03 and 0.05 mg g-1
DW.
G. tenuistipitata contains both RPE and RPC as accessory
light-harvesting pigments. The RPE concentration was 2.52.6 times higher than that of RPC. Both pigments decreased
when plants acclimated to 40 txmol m - 2 s - 1 were exposed
to 500 ixmol m - 2 s - 1. The RPE concentration decreased from
an initial value of 22.05 to 13.96 mg g-~ DW at the end
of the experiment (55 h) (Fig. 3 ( E ) ) . The experimental
results were also fitted to a negative logarithmic curve
(r2=0.92). In contrast, the RPC concentration decreased
from 8.84 to 5.89 mg g - 1 DW following a linear behaviour
(Fig. 3 ( F ) ) . Changes in the concentration of phycobilipro-
152
E. Carnicas et al. / J. Photochem. Photobiol. B: Biol. 50 (1999) 149-158
]SO0-
A
4
3"
1100 -
v
TN-
,,R
3
n.i,
-IN
I
S
I
I
J0
I
1S
Z0
1
2$
1
30
I
35
i
~0
I
4S
I
SO
I
SS
I
60
Time (min)
l|oo*
B
IN-
~D
So0.
..B.
Z00.
3
n.i.
-100
1
i
|
r
I
I
I
f
I
I
I
I
I
5
10
J$
20
2S
30
3S
qO
4S
SO
5S
60
Time (min)
Fig. 2. HPLC chromatograms of G. tenuistipitata cultured at two irradiances, 40 ixmol m 2 s - 1 (A) and 500 ~mol m -2 s - t (B). The following pigments
were identified: lutein (peak 1 ), zeaxanthin (peak 2), Chl a (peaks 3, 3' and 3") and 13-carotene (peak 4), n.J. not identified.
E. Carnicas et al. / J. Photochem. Photobiol. B: Biol. 50 (1999) 149-158
153
0.13~ 0.11-
5
~ 0.08m
u
~ 0.06J .
2 8
10
20
3'0
4'0
5'0
Time (hours)
0"71
i
0.04
6O
o
1'o
2~0
3'0
4~0
5'0
0'0
5~0
8'0
Time (hours)
0.09"
C
~ 0.07.
~
O. 5
.,~
,u o . o s -
0.4
i
8.3
~
0.03-
o.2
0.1
~
0
10
20
30
40
Time (hours)
50
35-
1'0
2'0 3~0 4]0
Time (hours)
lit
30-
~ 25'7~o
~u 20-
10-
5
0.01
60
F
4-
1'0
2'0
3'0
4'0
5'0
6'0
Time (hourS)
2
0
i
10
i
20
i
30
i
40
i
50
6'0
Time (hours)
Fig. 3. Changes in pigment concentrations (rag g - ~ DW) of G. tenuistipitata when plants acclimated to low irradiance (40 I~mol m -2 s - l ) were exposed for
55 h to 500 is.rnol m - 2 s - ]. The extracts were analysed using HPLC and mean values with their standard deviation are shown (n = 34).
teins were also studied over 10 days after increasing the
irradiance level. RPE decreased from an initial value of 32.67
mg g - ] DW to a final value of 7.92 mg g - ~ DW, while the
RPC concentration decayed from 11.3 to 2.79 mg g - 1 DW
(Fig. 4(E), ( F ) ) . Both responses followed clear negative
logarithmic patterns.
In the second set of experiments, cultures of G. tenuistipitata that were acclimated to high irradiance (500 Ixmol m - 2
s -~) were transferred to a lower one (40 p~mol m -2 s - ] )
during the same time periods as above, 55 h and 10 days. As
expected, the concentrations of all pigments increased during
the experiments. The concentration of Chl a (Fig. 5 ( A ) )
rose from 1.62 to 2.87 mg g - ~ DW after 55 h of exposure.
When the experiments were extended over 10 days, the concentration of Chl a continued to increase, reaching a final
concentration of 3.62 mg g-1 DW. Changes in 13-carotene
concentration are shown in Fig. 5 (B). The initial concentration (0.034 mg g-~ DW) increased to a value of 0.072 mg
g - ] DW after 55 h. In contrast to Chl a, the I~-carotene
concentration did not significantly vary from day three to the
end of the experiment. Both Chl a and 13-carotene concentrations increased following a logarithmic pattern over the 10
days (r 2 = 0.72 and r 2 = 0.75, respectively).
In plants acclimated to high irradiance, the concentration
of zeaxanthin was around six to eight times higher than that
of lutein and five to six times higher than that of 13-carotene.
The change in zeaxanthin and lutein concentrations is shown
in Fig. 5(C, D). When plants acclimated to high irradiance
were exposed to a lower photon flux density for 55 h, the
zeaxanthin content increased from an initial value of 0.21 mg
g - ] DW to a final value of 0.35 mg g - 1DW with a maximum
of 0.45 mg g - ] DW after 24 h. The lutein concentration
increased in a similar way, from 0.035 to 0.049 mg g - 1DW,
showing a peak of 0.055 mg g - ~ DW, also after 24 h. This
behaviour totally changed when exposure to 40 Ixmol m - a
s - t was extended for 10 days. The concentration of both
xanthophylls fluctuated widely and the final zeaxanthin content was not significantly different from the initial value
(0.27 mg g-1 DW), while lutein increased to a final value
of 0.054 mg g - 1 DW.
The change in the concentration of RPE and RPC is shown
in Fig. 5(E, F). The concentration of both pigments continuously increased after the shift, although they did it in a
different way from the rest of the pigments. The amount of
RPE (Fig. 5 ( E ) ) rose from an initial value of 5.41 mg g - i
DW to a value of 10.5 mg g - 1 DW in 55 h, while the con-
E. Carnicas et al. / J. Photochem. Photobiol. B: Biol. 50 (1999) 149-158
154
0.14-
A
B
~ 0.12~
a
0.1-
~ 0.08-
~
;--'L'.--,a~..~_l
~ 0.060.04
Time (days)
Time (days)
0.7-
0.09-
0.60.07-
0.5-
~ 0.2
0.40.3-
~ o.o3.
0.20.1
0.01
0
Time (days)
~
~
~'
i'o
~
d
d
lb
Time (days)
35-
14.
E
30-
12-
25- \
~
10-
~
6-
2015105
4
6I
Time (days)
8'
1;
2
Time (days)
Fig. 4. Changes in the concentrations ( m g g - 1 D W ) of pigments when plants grown at 40 ~ m o l m -2 s - 1 were exposed to 500 Ixmol m -2 s - ] for 10 days.
Mean values with their standard deviations are shown (n = 22).
centration of RPC increased from 2.1 to 3.2 mg g-~ DW
(Fig. 5 ( F ) ) . In both cases, the upward trend was linear, in
contrast to the logarithmic trend of the liposoluble pigments.
The ratio between these two pigments was similar to that
obtained in the experiment described above (2.5-2.8).
Longer exposure to low irradiance ( 10 days) induced a further linear increase of the concentration of phycobiliproteins,
but at a lower rate.
Table 1 shows the changes in the proportions of pigments
(final concentration/initial concentration) at different times
when the incident PFD was changed from 500 to 40 ixmol
m -2 s - ~ and vice versa over 10 days. The ratio between the
final and the initial concentrations of zeaxanthin and Chl a
increased to 1.62 and 1.41, respectively, in 4 h, while that of
RPE and RPC increased only to 1.22 and 1.19, respectively.
When the experiments were extended to 10 days, it was found
that Chl a was the pigment that underwent the widest range
of variation, either when increasing or decreasing the irradiance. The ratio Chl a f / C h l a i rose to 3.22 when the irradiance
was decreased, and dropped to 0.24 when it was increased.
On the contrary, the variation of the zeaxanthin concentration
was narrow, increasing to 1.25 after 10 days in low PFD and
decreasing only to 0.72 after the same period in high PFD. It
is also seen that while the phycobiliprotein and Chl a content
increased gradually over the 10 days when the light level was
reduced from 500 to 40 lxmol m - 2 s - l, the concentration of
zeaxanthin increased sharply during the first hours and then
decayed to the end of the experiments. A similar behaviour
occurred when the irradiance was increased: the zeaxanthin
concentration dropped sharply during the first 4 h, while the
Chl a and phycobiliprotein concentrations decreased
gradually.
When algae were acclimated to low irradiance, the ratio
between the phycobiliproteins ( R P E + R P C ) and Chl a
(Table 2) was higher (7.7) than when the algae were grown
at high irradiance (4.77). The ratio ( R P E + R P C ) / C h l a
significantly decreased when the irradiance was increased
from 40 to 500 ~mol m - 2 s-~, after an initial increase on
day one. On the contrary, when the irradiance was decreased
from 500 to 40 ~mol m -2 s -1, the ratio (RPE + RPC)/Chl
a increased; nevertheless, an initial drop of this ratio was
detected on the first day.
155
E. Carnicas et al. /J. Photochera. Photobiol. B: Biol. 50 (1999) 149-158
A
4.5-
0.11-
B
43.53-
el
0.0,.
................
,u
~ 0.05-
2.52-
0.03
1.5
8
Time (days)
1'0
Time (days)
0.7-
C
0.09-
D
0.6~ 0.07' ~ 0.5-
•~ 0.05-
0.4-
o.a~0.03~0.20.1
~
~
~;
1'o
0.01
Time (days)
Time (days)
18-
E •
7-
16~ 14~
~
12-
5-
~ 10~
03-
6
j
4
F
~
~
Time (days)
Time (days)
~
1'o
Fig. 5. Increase of the pigment concentrations (mg g- 1 DW) after a decrease of the photon flux density of growth of G. tenuistipitata from 500 to 40 p,mol
m- 2 s- t for 10 days. Samples were taken three times a day during the first three days ( days 0 to 2 ) and then once a day up to the 10th day ( n = 34).
Table 1
Final concentration/initial concentration ratios of the main pigments of G.
tenuistipitata, Chl a, zeaxanthin and the phycobiliproteins RPE and RPC,
when the algae are exposed to a change of the irradiance level for 10 days
Time
(days)
Chl af/Chl a i
RPEf/RPE i
RPCf/RPC i
Decrease of irradiance from 500 to 40 ltmol m -2 s- t
0
1.0
1.0
1.0
0.17
1.41
1.22
1.19
1
1.84
1.48
1.25
2
1.97
1.84
1.7
2.3
1.77
1.94
1.54
4
2.43
2.11
1.66
7
2.77
2.27
2.12
Increase of irradiance from 40 to 500 p,mol m- 2 S--I
0
1.0
1.0
1.0
0.17
0.71
0.75
0.88
1
0.62
0.71
0.77
2
0.51
0.62
0.68
2.3
0.61
0.63
0.66
4
0.46
0.49
0.62
7
0.31
0.32
0.5
10
0.24
0.24
0.46
Zf/Z_q
1.0
1.62
1.9
1.86
1.62
1.49
1.06
1.0
0.61
0.65
0.7
0.63
0.84
0.76
0.72
4. D i s c u s s i o n
T h e p i g m e n t c o m p o s i t i o n o f red algae shows s o m e differences related to other plants; the light-harvesting antennae
consist o f large extrinsic protein c o m p l e x e s , the phycobilisomes, w h i c h are attached to the thylakoid m e m b r a n e s . In
addition to this, only the p r e s e n c e o f chlorophyll a has b e e n
clearly demonstrated as a chlorophyllic p i g m e n t [ 32]. All o f
the c h l o r o p h y l l a and m o s t carotenoids are attached to a f e w
specific proteins. 13-Carotene is active in p h o t o s y s t e m I
( P S I ) , and probably in P S I I too [ 3 4 ] , and the presence o f a
variable n u m b e r o f other carotenoids has b e e n described in
red algae ( m a i n l y lutein, zeaxanthin, 13-cryptoxanthin and
anteraxanthin), although their contribution to photosynthesis
is not yet clear [ 3 5 ] .
The c h r o m a t o g r a p h i c analysis o f p i g m e n t extracts f r o m G.
t e n u i s t i p i t a t a shows a particular p i g m e n t c o m p o s i t i o n in
w h i c h zeaxanthin and c h l o r o p h y l l a, along with R P E and
R P C , are the m o s t abundant p i g m e n t s at both l o w and high
irradiance. In fact, zeaxanthin concentrations are m u c h h i g h e r
than those o f lutein or 13-carotene ( a p p r o x i m a t e d l y 75% o f
156
E. Carnicas et al. / J. Photochem. Photobiol. B: Biol. 50 (1999) 149-158
Table 2
Change in the accessory pigments (RPE and RPC)/Chl a ratios and in the percentage of zeaxanthin (on a zeaxanthin + lutein + [3-carotene basis) after a shift
of irradiance. Standard deviations in brackets
Time (days)
0
1
2
4
7
10
500 to 40 tzmol m -2 s - i
40 to 500 i~mol m -2 s -I
(RPE + RPC)/Chl a
%Z
7.77
8.04
6.98
5.84
4.75
4.07
76.7
77.9
80.4
81.7
82.4
82.1
(1.48)
(0.7)
(0.19)
(0.3)
(0.05)
(0.16)
total carotenoids). Thus, no components of the 'xanthophyll
cycle' have been detected and zeaxanthin is the main carotenoid of G. tenuistipitata and not lutein or [3-carotene, as
described in higher plants and other algae [ 32].
Several studies in higher plants point out the photoprotective role of the xanthophyll zeaxanthin, which is related to
detoxification processes when plants are exposed to excess
irradiation [3,11 ]. This photoprotective role of zeaxanthin
via the 'xanthophyll cycle' has also been reported in macroand microalgae [8-10]. Under photoinhibiting irradiance,
violaxanthin is converted to zeaxanthin via anteraxanthin,
and zeaxanthin dissipates part of the absorbed energy via
non-photochemical quenching of the light-harvesting complexes. In G. tenuistipitata zeaxanthin is the major carotenoid,
but neither anteraxanthin nor violaxanthin has been found,
indicating that the violaxanthin-anteraxanthin-zeaxanthin
cycle is not operative in this Rhodophyta. Lutein has been
found, but its concentration is very small compared with that
of zeaxanthin.
The first pigment to respond to a change of PFD is zeaxanthin. Its concentration increases or decreases proportionately more than the other pigments when light is reduced or
increased, respectively, in a short period of time (4 h) (Table
1 ). The change of zeaxanthin concentration follows a similar
pattern to the other liposoluble pigments in the short-term
experiments, but when they were prolonged to 10 days, the
results were different. When the irradiance level was lowered
(500 to 40 ixmol m - 2 s - l ) the zeaxanthin concentration
increased first and then decreased up to the end of the experiments, reaching similar values to the initial ones. In contrast,
when plants were shifted to a higher irradiance, the zeaxanthin concentration decreased sharply in 24 h, then increased
and finally reached values slightly lower than the initial ones.
These two responses lead us to consider the possible presence
of two pools of zeaxanthin, one acting as an accessory lightharvesting pigment and the other acting as a photoprotective
pool. In groups that lack the xanthophyll cycle, e.g., cyanobacteria, the putative photoprotective function of zeaxanthin
is not convincing [ 36] and zeaxanthin may still act as a lightharvesting pigment, transferring energy from its higherenergy excited state to chlorophyll a [37]. When the light
irradiance is reduced, the zeaxanthin concentration increases
first, indicating an adaptation of the photosynthetic apparatus
(7%)
(7%)
(3.8%)
(3.8%)
(4%)
(3.9%)
( R P E + RPC)/Chl a
%Z
4.77
3.88
4.23
6.72
5.89
6.42
75.7
76.3
74.6
71.7
62.8
67.8
(0.28)
(0.24)
(0.26)
(0.49)
(0.84)
(0.05)
(2.5%)
(9.6%)
(3.8%)
(13%)
(9%)
(18.2%)
to low light. Then its concentration decreases, probably due
to destruction of the photoprotective pool. In contrast, when
light is increased, the zeaxanthin concentration falls rapidly
as a result of an acclimative response of the light-harvesting
pool. The sharp increase on day three may be due to an
increase of the photoprotective pool as an acclimation to high
irradiance. According to these results, the acclimative
response of the light-harvesting zeaxanthin pool is faster than
the response of the photoprotective pool. In spite of these
changes, the amount of zeaxanthin did not vary much during
the two sets of experiments performed (Table 2), indicating
that the main changes of its concentration took place in a
short period of time and that zeaxanthin would undergo a
subsequent equilibrium. The probability of a harvesting role
for zeaxanthin is very small and it is not generally accepted
in the literature, although we think that it cannot be totally
excluded in G. tenuistipitata, as zeaxanthin is the major carotenoid. However, further research on the photochemistry of
zeaxanthin in this alga should be done before assuring the
two proposed roles.
After a decrease of PFD, the concentrations of all pigments
increase in a logarithmic way except for the phycobiliproteins, which increase linearly. Chlorophyll a and carotenoids
are closely related to the reaction centre and physically bound
to the thylakoid membranes. The rise in the concentration of
these pigments is limited by the free space in the membranes
and, therefore, the kinetics is a saturation curve. In contrast,
phycobilisomes are structures that are asociated with PSII
and located on the thylakoid membranes. The phycobiliprotein localization does not depend on the strict spatial availability in the thylakoid membranes, as they form prominent
structures, being the possible cause for these linear responses.
The first response to a decrease of the irradiance is a rise
in the concentration of pigments [ 20,21,38 ]. But this increase
is not equal for all the pigments: accessory pigments increase
faster than Chl a [39,40]. The Z/Chl a ratio over 10 days is
presented in Fig. 6. When G. tenuistipitata was grown at high
PFD (500 txmol m -2 s -1) and shifted to a lower one (40
Ixmol m -2 s - 1) for 10 days, the ratio Z/Chl a increased first
and then decayed and reached a similar value to the initial
one in two days. When the experiment was prolonged for 10
days, the ratio Z/Chl a continued to decay up to the end of
the experiment. On the contrary, when the irradiance level
E. Carnicas et al. / J. Photochem. Photobiol. B: BioL 50 (1999) 149-158
•
fl
Decrease of irradiance from 500 to 40 p.moles m "2 s "1
Increase of irradiance from 40 to 500 ~moles m "2 s "1
0.25,I
0.20.150.1-
o os-
Time
(days)
Fig. 6. Variation of the zeaxanthin/Chl a ratio following either an increase
or a decrease of the photon flux density.
was increased from 40 to 500 ixmol m -2 s-1, the Z/Chl a
ratio decayed first and then increased progressively during
the first two days. When the exposure to high PFD was
extended, the Z/Chl a ratio showed a clear upward trend.
Two hypotheses have been proposed to explain the changes
of pigment proportions when plants are exposed to decreasing
irradiance. One of them has been described in terrestrial
plants, Chlorophyceae and Rhodophyceae, suggests a higher
increase of accessory pigment proportions through changes
in the accessory pigments/Chl a ratio [41-43 ]. As it stimulates the synthesis of the most efficient pigments, this strategy
results in an energy saving economy for plants. The second
hypothesis proposes an increase of all of the pigments, and it
has been observed in diatoms and Phaeophyceae [44], some
Cyanobacteria and in a Rhodophyta, Bostrychia binderii
[45]. This strategy implies a higher energy consumption. In
the case of G. tenuistipitata, the concentration of all pigments
increased when light was lowered and vice versa. The accessory pigments ( R P E + R P C ) / C h l a ratio remained around
3.8-4.7 in the first two days and then started to increase. The
acclimative strategy of G. tenuistipitata is probably the second one described above in a short period of time, with an
increase of all the pigments. However, when algae were kept
for 10 days at low irradiance this ratio increased, indicating
that the phycobiliproteins were the last pigments to respond
to a decrease of irradiance. Zeaxanthin could be an exception
to this behaviour due to the possible presence of two pools
of this xanthophyll with different acclimation response to
changes in the PFD.
Acknowledgements
This work was supported by project AMB-96-0782 of the
CICYT. E.C. was supported by a fellowship from the Spanish
Minister of Education and Culture.
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