Effect of light quality on the accumulation of photosynthetic pigments, proteins and mycosporine-like amino acids in the red alga Porphyra leucosticta (Bangiales, Rhodophyta).

Abstract

The effect of different light qualities (white, blue, green, yellow and red light) on photosynthesis, measured as chlorophyll fluorescence, and the accumulation of photosynthetic pigments, proteins and the UV-absorbing mycosporine-like amino acids (MAAs) was studied in the red alga Porphyra leucosticta. Blue light promoted the highest accumulation of nitrogen metabolism derived compounds i.e., MAAs, phycoerythrin and proteins in previously N-starved algae after seven days culture in ammonium enriched medium. Similar results were observed in the culture under white light. In contrast, the lowest photosynthetic capacity i.e., lowest electron transport rate and lowest photosynthetic efficiency as well as the growth rate were found under blue light, while higher values were found in red and white lights. Blue light favored the accumulation of the MAAs porphyra-334, palythine and asterina-330 in P. leucosticta. However, white, green, yellow and red lights favored the accumulation of shinorine. The increase of porphyra-334, palythine and asterina-330 occurred in blue light simultaneous to a decrease in shinorine. The accumulation of MAAs and other nitrogenous compounds in P. leucosticta under blue light could not be attributed to photosynthesis and the action of a non-photosynthetic blue light photoreceptor is suggested. A non-photosynthetic photoreceptor could be also involved in the MAAs interconversion pathways in P. leucosticta.

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Effect of light quality on the accumulation of
photosynthetic pigments, proteins and mycosporine-like amino
acids in the red alga Porphyra leucosticta (Bangiales, Rhodophyta)
Nathalie Korbee*, Fe ´lix L. Figueroa, Jose ´ Aguilera
Departamento de Ecologı ´a, Facultad de Ciencias, Universidad de Ma ´laga, Campus Universitario de Teatinos s/n, 29071-Ma ´laga, Spain
Received 4 November 2004; received in revised form 7 March 2005; accepted 7 March 2005
Available online 21 April 2005
Abstract
The effect of different light qualities (white, blue, green, yellow and red light) on photosynthesis, measured as chlorophyll fluo-
rescence, and the accumulation of photosynthetic pigments, proteins and the UV-absorbing mycosporine-like amino acids (MAAs)
was studied in the red alga Porphyra leucosticta. Blue light promoted the highest accumulation of nitrogen metabolism derived com-
pounds i.e., MAAs, phycoerythrin and proteins in previously N-starved algae after seven days culture in ammonium enriched med-
ium. Similar results were observed in the culture under white light. In contrast, the lowest photosynthetic capacity i.e., lowest
electron transport rate and lowest photosynthetic efficiency as well as the growth rate were found under blue light, while higher val-
ues were found in red and white lights. Blue light favored the accumulation of the MAAs porphyra-334, palythine and asterina-330
in P. leucosticta. However, white, green, yellow and red lights favored the accumulation of shinorine. The increase of porphyra-334,
palythine and asterina-330 occurred in blue light simultaneous to a decrease in shinorine. The accumulation of MAAs and other
nitrogenous compounds in P. leucosticta under blue light could not be attributed to photosynthesis and the action of a non-pho-
tosynthetic blue light photoreceptor is suggested. A non-photosynthetic photoreceptor could be also involved in the MAAs inter-
conversion pathways in P. leucosticta.
? 2005 Elsevier B.V. All rights reserved.
Keywords: Light quality; Mycosporine-like amino acid; Photoreceptor; Photosynthesis; Porphyra
1. Introduction
Light quality affects photosynthesis, growth, develop-
ment and morphogenesis in algae [1–6]. Physiological
and morphological processes are the main targets for
the acclimation of algae to the light field. In the red alga
Porphyra leucosticta, red light favored thallus expan-
sion, cell division and carbon accumulation meanwhile
blue light favored the accumulation of N compounds
i.e., photosynthetic pigment and protein with little
apparent thallus expansion [3,6]. In red light-adapted
thalli, most of the photosynthetic energy can be diverted
into new biomass due to high carbon investment effi-
ciency that minimized the demand for synthesis of new
photosynthetic components [5]. The white light effect is
the combination of both red and blue light effects [5].
In macroalgae, the accumulation of N compounds
under different light qualities was produced due to the
stimulation of N metabolism i.e., nitrate incorporation
and stimulation of nitrate reductase activity, under the
control of non-photosynthetic photoreceptors [4,7,8].
Mycosporine-like amino acids (MAAs) are UV
absorbing and nitrogenous compounds with low molec-
ular weight, high molar extinction coefficients and
absorption band in the UV with a maximum between
1011-1344/$ - see front matter ? 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2005.03.002
*Corresponding author. Tel.: +34 952 133337; fax: +34 952 132000.
E-mail address: nkorbee@uma.es (N. Korbee).
Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
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310 and 360 nm [9,10]. Recently, it has been shown that
both UV radiation and high inorganic nitrogen avail-
abilitystimulatetheaccumulation
P. leucosticta [11]. MAAs are passive sunscreens, prefer-
entially absorbing UV photons followed by a dissipation
of the absorbed radiation energy in the form of harmless
heat without generating photochemical reactions and
thereby protecting, at least partially, photosynthesis
and growth of phototropic organisms [12–14]. Besides
having a role in UV screening, several MAAs also show
antioxidant properties [15,16], which could act as com-
patible solutes [10,17], and other function as nitrogen
reserve could be inferred to these compounds from the
accumulation in presence of ammonium [11,18], as oc-
curred in the case of biliproteins [19].
Although the maximal absorption of MAAs is in the
UV region, other wavelengths such as blue light can
control the accumulation of MAAs in different algae
[20–27].
The aim of this work was to study the effect of differ-
ent light qualities (white, blue, green, yellow and red
light) for one week exposure under high nitrogen avail-
ability on photosynthesis and the accumulation of
photosynthetic pigments, proteins and MAAs in the
N-starved red alga P. leucosticta. Since MAAs are
nitrogenous compounds, a further objective was to
investigate if the accumulation of these compounds is
photoregulated in the same way as other nitrogenous
compounds i.e., photosynthetic pigments (chlorophyll
and phycobiliproteins) as had been reported in previous
studies [7,8].
ofMAAsin
2. Materials and methods
2.1. Plant material and culture conditions
Specimens of P. leucosticta Thuret in Le Jolis were
collected in April 2002 from the intertidal zone of La-
gos, Ma ´laga, Southern Spain (36?280N 4?10W). Discs
of approximately 1.2 cm in diameter were cut from thalli
of P. leucosticta and pre-acclimated in an illuminated
culture chamber at 16 ± 2 ?C in aerated cylinders filled
with artificial seawater (salinity of 33&, Instant Ocean
Sea Salt, Aquarium System, Sarrebourg, France) for
one week, without nitrogen (starved-conditions). During
this period algae were illuminated with fluorescent light
(Truelite, Duro-Test Corp., NJ, USA, 40 lmol m?2s?1)
with a photoperiod of 12 h light:12 h dark.
For light quality treatments, again at 16 ± 2 ?C, 90
discs per treatment were transferred to five aerated cyl-
inders with 300 lM NH4Cl and incubated for one week
with 45 lmol m?2s?1of white, blue, green, yellow and
red light and a photoperiod of 12:12 light:dark. Irradia-
tion with white light was provided with two Truelite
fluorescent lamps. Blue light was supplied with four Phi-
lips (20W fluorescent lamps) covered with two Plexiglas
blue filters (Ro ¨hm PG627 and PG602, Ro ¨hm GmbH,
Darmstadt, Germany). Irradiation with green light was
provided with a General Electric 20W and two Sylvania
fluorescent lamps covered with one Plexiglas Ro ¨hm
PG700 green filter. Irradiation with yellow light was
supplied with one Philips low pressure sodium lamp
SOX 135W attenuated with three neutral filters (gray
nets). Red light was obtained from four General Electric
20W red lamps filtered through one Plexiglas red filter
Ro ¨hm PG502. Spectral irradiance was determined by
means of a Licor 1800-UW spectroradiometer (LICOR,
Lincoln, NE, USA). The spectral characteristics are re-
ported in Fig. 1. In Table 1, effective irradiance for pho-
tosynthesis calculated according to the action spectra
reported in Porphyra umbilicalis [1] is shown.
Algal samples were taken after three and seven days
of incubation. Samples for pigment and protein analyses
were wrapped in aluminium foil, frozen in liquid nitro-
gen for several hours and then stored at ?20 ?C until
analysis were conducted. And samples for MAAs, C
and N analyses were blotted dry with paper tissue, and
then dried in silica gel in sealed plastic bags.
2.2. Photosynthetic activity as in vivo chlorophyll
fluorescence
In vivo chlorophyll fluorescence of photosystem II
(PSII) was determined with a portable pulse modulation
fluorometer (PAM 2000, Waltz GmbH, Effeltrich,
Germany). Determination of the maximal quantum
yield of fluorescence (Fv/Fm) was achieved according to
Korbee et al. [18]. Five replicates were taken for each
treatment.
The electron transport rate (ETR) was determined
using a Diving-PAM fluorometer (Waltz GmbH, Effel-
trich, Germany) according to Korbee et al. [18]. Five
replicates were taken for each treatment.
Fig. 1. Relative spectral irradiance of the lamps used: WL = white
light, BL = blue light, GL = green light, YL = yellow light, RL = red
light.
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2.3. Growth rate
The relative growth rate (R), expressed as % day?1,
was computed from the following expression [28]:
R ¼ ðlnat? lna0Þ=t;
where atis the area measured at time (t) in days and
a0is the area at the initial time. Ten replicates were
taken for each treatment. Disc area was determined
using an image analysis software (Visilog 5.2, Noesis,
France).
2.4. Photosynthetic pigments
Two discs of approximately 10–20 mg fresh weight
(FW) per sample were extracted in 1.5 ml N,N-dimethyl-
formamide for 24 h at 4 ?C in darkness. After centrifu-
gation at 5000g for 10 min (Labofuge 400R, Heraeus,
Kendro Laboratory Products, Langenselbold, Ger-
many), the supernatant was used for chlorophyll a
(Chl a) determination. Chl a concentration was calcu-
lated from the OD [29]. Triplicate samples were taken
from each treatment.
To determine phycobiliproteins (BP), phycoerythrin
(PE) and phycocyanin (PC), samples of 30–40 mg
(FW) of algal biomass were homogenized and extracted
in 2 ml of 0.1 M phosphate buffer (pH 6.5). The extracts
were centrifuged at 5000g for 10 min and the concentra-
tion of BP in the supernatant was determined from the
OD [30]. The supernatant was also used to determine
the content of soluble proteins (SP). And the pellet
was used to determine the content of structural proteins
(StP).Triplicatesamples
treatment.
weretaken from each
2.5. Soluble protein content
Soluble proteins were determined using a commercial
Protein Assay (BioRad), based on Bradford method
[31]. Protein content was determined spectrophotometri-
cally at 595 nm and concentrations were calculated by
means of standards made with bovine serum albumin
(SIGMA).
2.6. Structural protein content
Structural proteins were determined from the pellet
fraction obtained after centrifugation as above indi-
cated. Sodium hydroxide 1 M was added to the pellet,
the extraction occurred for 24 h at 4 ?C in darkness,
after this time HCl 1 N was added and finally the con-
tent of StP was determined as described above for SP.
2.7. Extraction, identification and quantification of MAAs
MAAs determination was assayed according to
Korbee-Peinado et al. [11]. Triplicate samples of dried
algal samples (10–20 mg DW) were extracted for 2 h in
centrifuge vials with 1 ml 20% (v/v) aqueous methanol
in a water bath at 45 ?C. Dried extracts were re-dis-
solved in 100% methanol and samples were analyzed
with an HPLC system (Waters 600). The mobile phase
was 2.5% aqueous methanol (v/v) plus 0.1% acetic acid
(v/v) in water, run isocratically at 0.5 ml min?1. Sample
volumes of 10 lL were injected into the C8chromato-
graphic column (5 lm pore size, 250 · 4 mm, Sphere-
clone?,Phenomenex,Aschaffenburg,
MAAs were detected online with a Waters Photodiode
Array Detector 996 at 330 nm.
Germany).
2.8. Internal carbon and nitrogen
Total intracellular carbon and nitrogen contents were
determined in triplicate dry samples at 1050 ?C by an
elemental analyzer CNHS LECO-932 (LECO Corp.,
MI, USA) couple with an IR detector.
2.9. Statistics
The results were analyzed by one-way ANOVA with
a significance level of p < 0.05. Previously, ANOVA
assumptions (homogeneity of variances and normal dis-
tribution) were examined. To determine differences
among treatments post hoc comparisons were made
with the Tukey test [32]. Computations were done with
SPSS for Windows, version 11.5.
3. Results
The photosynthetic activity was estimated as the
Fv/Fm and the ETR. Under blue light, thallus of
P. leucosticta showed an increase in the Fv/Fm, mean-
while there was a decrease in red light (Table 2). How-
ever, the maximal electron transport rate (ETRmax)
increased significantly (p < 0.05) after the incubation un-
der all light treatments, with the exception of blue light.
After the experimental period the greatest ETRmaxvalue
was found under red light. The slope of the ETR versus
irradiance curve (a) was greater after the incubation
Table 1
Irradiance and photosynthetic effective irradiance (PEI) expressed as
lmol m?2s?1in the different light qualities used
Irradiance (lmol m?2s?1)
Light quality
PEI (lmol m?2s?1)
White
Blue
Green
Yellow
Red
45
45
45
45
45
31
14
38
35
33
PEI was calculated according to the action spectra reported in
Porphyra umbilicalis [1].
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
73

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under white, green and yellow light than that in the ini-
tial time. No changes in a under blue and red light was
observed; however, a was greater under red than under
blue light (Table 2).
Although algal samples were taken and analyzed
after three and seven days, the pattern was the same
and therefore only data of day seven are presented.
Chl a content increased in all light treatments after the
incubation time. However, there were no significant dif-
ferences among the treatments (Table 3). PE and PC
content also increased in all treatments, except PE in
red light and PC in blue light (Table 3). The increase
of PE was greater than that of PC. Furthermore, the
content of PE after seven days was significantly
(p < 0.05) higher in white and blue than in red light,
but there were no significant differences among treat-
ments in PC content (Table 3). Soluble proteins also in-
creased in all light treatments, reaching the greatest
values under white and blue light and the lowest under
red light (Table 3). As other nitrogenous compounds,
blue light stimulated the accumulation of structural pro-
tein (Table 3).
The internal C content did not show any difference
amonglight treatments,
320 mg g?1DW. The internal N content increased in
all treatments after the incubation period (Table 3),
it wasapproximately
being higher under white, blue and green light than that
under red light. Due to the increase of N content and no
variation in C content, the relation C/N decreased in all
treatments (Table 3). The greatest C/N value was
reached in red light meanwhile the lowest value was ob-
served under white light (Table 3).
The relative growth rate was significantly (p < 0.05)
lower in blue light than that in white, red and green light
(Table 3).
The content of MAAs increased after the incubation
period under white, blue and green light, the highest
concentration was reached under blue light meanwhile
the lowest level was reached under yellow and red light
(Fig. 2). The composition of MAAs at the start of the
experimentswas 12.01 ± 0.39%
80.42 ± 1.50% porphyra-334, 3.94 ± 0.12% palythine
and 4.31 ± 0.17% asterina-330 (Fig. 3). During the incu-
bation period, the percentage of shinorine increased sig-
nificantly (p < 0.05) in all light treatments except in blue
light, in which a decrease was observed (Fig. 3(a)).
ofshinorine,
Table 3
Chlorophyll a (Chl a), phycoerythrin (PE), phycocyanin (PC), soluble (SP) and structural proteins (StP) and total intracellular nitrogen content (N),
expressed in mg g?1dried weight and carbon/nitrogen ratio (C/N) in Porphyra leucosticta at initial time and after seven days of incubation
Light
treatment
Chl a
PEPC SP StPNC/N
R (% day?1)
Initial 2.83 ± 0.44a
6.11 ± 0.30a
3.47 ± 0.67a
40.7 ± 3.8a
120.0 ± 4.8a
37.0 ± 2.0a
8.6 ± 0.2a
Seven-day
cultures
White
Blue
Green
Yellow
Red
6.83 ± 0.78b
5.80 ± 0.78b
7.06 ± 0.38b
6.37 ± 0.65b
5.60 ± 0.60b
11.80 ± 1.87b
11.83 ± 0.66b
10.26 ± 1.06b,c
9.84 ± 1.22b,c
7.82 ± 0.72a,c
6.31 ± 0.70b
4.91 ± 0.20a,b
5.81 ± 0.60b
5.54 ± 0.14b
5.68 ± 0.62b
78.1 ± 6.1b
77.4 ± 0.4b
75.2 ± 2.4b,c
72.4 ± 0.5b,c
65.2 ± 3.7c
112.1 ± 20.0a
151.6 ± 3.3b
108.0 ± 15.5a
115.1 ± 14.1a
98.2 ± 10.7a
62.7 ± 1.7b
56.9 ± 1.7b,c
56.1 ± 0.0b,c
54.0 ± 6.2c,d
47.3 ± 0.9d
5.6 ± 0.2b
6.0 ± 0.1b,c
6.3 ± 0.0c
6.4 ± 0.5c
7.1 ± 0.3d
6.5 ± 1.4a,b
3.3 ± 0.7c
7.3 ± 1.3b
4.9 ± 1.0a,c
6.0 ± 0.4a,b
Relative growth rate (R), expressed in % day?1, evaluated after seven days of incubation. Different letters indicate significant differences between
values (p < 0.05) for each parameter. Data given as means ± SD (n = 3 for Chl a, PE, PC, SP, StP, N and C/N; n = 10 for R).
Table 2
Optimal quantum yield (Fv/Fm), maximal ETR (ETRmax) and initial
slope (a) of the function ETR versus irradiance in Porphyra leucosticta
at initial time and after seven days of incubation
Treatment
Fv/Fm
ETRmax
a
Initial0.70 ± 0.03a
1.50 ± 0.29a
0.07 ± 0.02a,b
Seven-day
cultures
White
Blue
Green
Yellow
Red
0.70 ± 0.01a
0.72 ± 0.01b
0.69 ± 0.01a,d
0.68 ± 0.02d
0.65 ± 0.02c
4.43 ± 1.19b
2.82 ± 0.67a,c
3.37 ± 0.98b,c
3.24 ± 0.21b,c
7.60 ± 1.26d
0.13 ± 0.01c
0.05 ± 0.01a
0.13 ± 0.02c
0.13 ± 0.02c
0.09 ± 0.01b
Different letters indicate significant differences
(p < 0.05) for each parameter. Data given as means ± SD (n = 5).
between values
0
2
4
6
8
10
12
14
MAAs (mg·g-1DW)
WL BL GLYL RLInitial
Fig. 2. Concentration of MAAs, expressed in mg g?1dry weight, in
Porphyra leucosticta at initial time and after seven days of culture
under light treatments. Data are expressed as mean value ± SD (n = 3)
(WL = white light, BL = blue light, GL = green light, YL = yellow
light, RL = red light).
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Meanwhile, the percentages of palythine and asterina-
330 decreased compared to the initial values in all light
treatments except under blue-light treatment. Thus,
both palythine and asterina-330 accumulation was
clearly stimulated by blue light (Fig. 3(c) and (d)). There
was a decrease in the percentage of porphyra-334 in all
treatments, with the exception of blue light (Fig. 3(b)).
The concentration of each MAA was shown in Table 4.
4. Discussion
In P. leucosticta, it has been shown that red light fa-
vored thallus expansion, cell division, carbon accumula-
tion and a dramatic decrease of the protoplasmic area
and in the thickness of the cell walls, while blue light fa-
vored photosynthetic pigment, protein synthesis, numer-
ous well-formed phycobilisomes with little apparent
thallus expansion [3,6]. White light, as the result of the
combination of all light qualities, favors both C and N
metabolisms [3,6]. The accumulation of Chl a in
P. leucosticta after one week of incubation was similar
in all light treatments, as occurred in the same species
cultured under blue and red light [4]. The content of
PE was greater in white and blue than in red light, how-
ever, there were no differences among treatments in PC
content. Previously, it has been shown in P. leucosticta
an increase not only in PE but also in PC content [4].
Soluble and structural proteins were mainly stimulated
by blue light as it has been previously reported [3,4].
The UV-screen substances, MAAs, were also stimulated
by blue light as other nitrogenous compounds [4,33].
Lu ¨ning and Dring [1] demonstrated reduced photo-
synthetic activity in blue light compared with other spec-
tral bands in P. umbilicalis and other red algae
according to the action spectra for photosynthesis. In
P. leucosticta, lower photosynthetic rates have been
demonstrated under blue light relative to red light
WL BL GL YL RL
Shinorine (%)
0
5
10
15
20
25
30
0
2
4
6
0
20
40
60
80
100
0
2
4
6
Palythine (%)
Porphyra-334 (%)
Asterina-330 (%)
InitialWL BL GL YL RLInitial
(a)(b)
(c)(d)
Fig. 3. Composition of MAAs in Porphyra leucosticta at initial time and after seven days of culture under light treatments. Percentage of (a)
shinorine with respect to the total amount (%), (b) porphyra-334 (%), (c) palythine (%), (d) asterina-330 (%). Data are expressed as mean value ± SD
(n = 3) (WL = white light, BL = blue light, GL = green light, YL = yellow light, RL = red light).
Table 4
Concentration of shinorine, porphyra-334, palythine and asterina-330, expressed in mg g?1dried weight, in Porphyra leucosticta at initial time and
after seven days of incubation
TreatmentShinorine Porphyra-334 PalythineAsterina-330
Initial0.80 ± 0.16a
5.34 ± 0.83a
0.29 ± 0.02a
0.31 ± 0.04a
Seven-day culturesWhite
Blue
Green
Yellow
Red
2.67 ± 0.07b
0.57 ± 0.05a
2.39 ± 0.36b,c
1.96 ± 0.17c
1.87 ± 0.25c
7.27 ± 0.21b
8.88 ± 0.50c
6.54 ± 0.05a,b
5.75 ± 0.52a,b
5.69 ± 0.21a,b
0.22 ± 0.01a
0.60 ± 0.11b
0.19 ± 0.01a
0.14 ± 0.01a
0.16 ± 0.01a
0.24 ± 0.01a
0.64 ± 0.12b
0.21 ± 0.01a
0.15 ± 0.01a
0.18 ± 0.01a
Different letters indicate significant differences between values (p < 0.05) for each parameter. Data given as means ± SD (n = 3).
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