- Email: [email protected]
Effect of light quality on the accumulation of photosynthetic pigments, proteins and mycosporine-like amino acids in the red alga Porphyra leucosticta (Bangiales, Rhodophyta)
Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78 www.elsevier.com/locate/jphotobiol
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 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. 2005 Elsevier B.V. All rights reserved. Keywords: Light quality; Mycosporine-like amino acid; Photoreceptor; Photosynthesis; Porphyra
1. Introduction Light quality affects photosynthesis, growth, development 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 expansion, 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 *
Corresponding author. Tel.: +34 952 133337; fax: +34 952 132000. E-mail address: [email protected] (N. Korbee).
1011-1344/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.03.002
thalli, most of the photosynthetic energy can be diverted into new biomass due to high carbon investment efficiency 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 molecular weight, high molar extinction coefficients and absorption band in the UV with a maximum between
72
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
310 and 360 nm [9,10]. Recently, it has been shown that both UV radiation and high inorganic nitrogen availability stimulate the accumulation of MAAs in P. leucosticta [11]. MAAs are passive sunscreens, preferentially 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 compatible 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 occurred 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 different light qualities (white, blue, green, yellow and red light) for one week exposure under high nitrogen availability 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].
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 Lagos, Ma´laga, Southern Spain (3628 0 N 41 0 W). 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 m2 s1) 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 cylinders with 300 lM NH4Cl and incubated for one week with 45 lmol m2 s1 of white, blue, green, yellow and red light and a photoperiod of 12:12 light:dark. Irradiation 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 reported in Fig. 1. In Table 1, effective irradiance for photosynthesis 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 nitrogen 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, Effeltrich, 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.
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78 Table 1 Irradiance and photosynthetic effective irradiance (PEI) expressed as lmol m2 s1 in the different light qualities used Light quality
Irradiance (lmol m2 s1)
PEI (lmol m2 s1)
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].
2.3. Growth rate The relative growth rate (R), expressed as % day1, was computed from the following expression [28]: R ¼ ðln at ln a0 Þ=t; where at is the area measured at time (t) in days and a0 is 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).
73
2.6. Structural protein content Structural proteins were determined from the pellet fraction obtained after centrifugation as above indicated. 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 content 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-dissolved 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 min1. Sample volumes of 10 lL were injected into the C8 chromatographic column (5 lm pore size, 250 · 4 mm, Sphereclone, Phenomenex, Aschaffenburg, Germany). MAAs were detected online with a Waters Photodiode Array Detector 996 at 330 nm.
2.4. Photosynthetic pigments 2.8. Internal carbon and nitrogen Two discs of approximately 10–20 mg fresh weight (FW) per sample were extracted in 1.5 ml N,N-dimethylformamide for 24 h at 4 C in darkness. After centrifugation at 5000g for 10 min (Labofuge 400R, Heraeus, Kendro Laboratory Products, Langenselbold, Germany), the supernatant was used for chlorophyll a (Chl a) determination. Chl a concentration was calculated 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 concentration 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). Triplicate samples were taken from each treatment. 2.5. Soluble protein content Soluble proteins were determined using a commercial Protein Assay (BioRad), based on Bradford method [31]. Protein content was determined spectrophotometrically at 595 nm and concentrations were calculated by means of standards made with bovine serum albumin (SIGMA).
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 distribution) 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, meanwhile there was a decrease in red light (Table 2). However, the maximal electron transport rate (ETRmax) increased significantly (p < 0.05) after the incubation under all light treatments, with the exception of blue light. After the experimental period the greatest ETRmax value was found under red light. The slope of the ETR versus irradiance curve (a) was greater after the incubation
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
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 Initial Seven-day cultures
Fv/Fm 0.70 ± 0.03
White Blue Green Yellow Red
ETRmax a
0.70 ± 0.01a 0.72 ± 0.01b 0.69 ± 0.01a,d 0.68 ± 0.02d 0.65 ± 0.02c
a a
1.50 ± 0.29
0.07 ± 0.02a,b
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 between values (p < 0.05) for each parameter. Data given as means ± SD (n = 5).
under white, green and yellow light than that in the initial 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 differences 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 treatments in PC content (Table 3). Soluble proteins also increased 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 protein (Table 3). The internal C content did not show any difference among light treatments, it was approximately 320 mg g1 DW. The internal N content increased in all treatments after the incubation period (Table 3),
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 observed 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 experiments was 12.01 ± 0.39% of shinorine, 80.42 ± 1.50% porphyra-334, 3.94 ± 0.12% palythine and 4.31 ± 0.17% asterina-330 (Fig. 3). During the incubation period, the percentage of shinorine increased significantly (p < 0.05) in all light treatments except in blue light, in which a decrease was observed (Fig. 3(a)).
14 12
MAAs (mg·g-1 DW)
74
10 8 6 4 2 0 Initial
WL
BL
GL
YL
RL
1
Fig. 2. Concentration of MAAs, expressed in mg g dry 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).
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 g1 dried weight and carbon/nitrogen ratio (C/N) in Porphyra leucosticta at initial time and after seven days of incubation Light treatment Initial Seven-day cultures
White Blue Green Yellow Red
Chl a
PE
PC
SP
StP
2.83 ± 0.44a
6.11 ± 0.30a
3.47 ± 0.67a
40.7 ± 3.8a
b
b
b
b
6.83 ± 0.78 5.80 ± 0.78b 7.06 ± 0.38b 6.37 ± 0.65b 5.60 ± 0.60b
11.80 ± 1.87 11.83 ± 0.66b 10.26 ± 1.06b,c 9.84 ± 1.22b,c 7.82 ± 0.72a,c
6.31 ± 0.70 4.91 ± 0.20a,b 5.81 ± 0.60b 5.54 ± 0.14b 5.68 ± 0.62b
78.1 ± 6.1 77.4 ± 0.4b 75.2 ± 2.4b,c 72.4 ± 0.5b,c 65.2 ± 3.7c
N
120.0 ± 4.8a
C/N
37.0 ± 2.0a a
112.1 ± 20.0 151.6 ± 3.3b 108.0 ± 15.5a 115.1 ± 14.1a 98.2 ± 10.7a
b
62.7 ± 1.7 56.9 ± 1.7b,c 56.1 ± 0.0b,c 54.0 ± 6.2c,d 47.3 ± 0.9d
R (% day1)
8.6 ± 0.2a 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 % day1, 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).
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
(a)
Porphyra-334 (%)
Shinorine (%)
30 25 20 15 10 5
100 (b) 80 60 40 20 0
(c)
Asterina-330 (%)
Palythine (%)
0 6 4 2 0
Initial WL BL GL YL RL
75
(d)
6 4 2 0
Initial WL BL GL YL RL
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 g1 dried weight, in Porphyra leucosticta at initial time and after seven days of incubation Treatment Initial Seven-day cultures
White Blue Green Yellow Red
Shinorine
Porphyra-334
Palythine
Asterina-330
0.80 ± 0.16a
5.34 ± 0.83a
0.29 ± 0.02a
0.31 ± 0.04a
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).
Meanwhile, the percentages of palythine and asterina330 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 favored thallus expansion, cell division, carbon accumulation and a dramatic decrease of the protoplasmic area and in the thickness of the cell walls, while blue light favored photosynthetic pigment, protein synthesis, numerous 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, however, 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 photosynthetic activity in blue light compared with other spectral 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
76
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
[3,5]. Our results are consistent with these studies; the lowest ETRmax and the lowest photosynthetic efficiency (a) had been shown in blue light. The lower values of ETRmax and a under blue light has been interpreted by a higher package effect [5] and higher amount of PE pool not attached to phycobilisomes, as observed in this species previously by Tsekos et al. [6]. In contrast, Fv/Fm was greater under blue than under red light. So, low ETRmax in parallel to high Fv/Fm values under blue light could be related to an imbalance between PSII and PSI, with a simultaneous high demand for electron sinks i.e., Mehler reaction or the electron demand for the inorganic nitrogen assimilation, which is stimulated by blue light [3,5]. Growth rate under blue light was significantly lower than in white, red and green lights in P. leucosticta, as it has been previously shown in this species [3,5]. The lower growth rate under blue light could be related to the lowest ETRmax and the lowest photosynthetic efficiency (a) observed under these culture conditions, as described above. Other authors ascribed the lower growth rate in blue light to two different processes in P. leucosticta; first, to a low efficiency in photosynthetic performance mainly produced at level of the water splitting complex of the PSII and secondly to a lower carbon investment efficiency, leading to a deficient accumulation of photosynthetic and osmotic-related products [5]. Previous studies showed that in the Bangiales the accumulation of MAAs was not affected by light quality [34,35]. These results have been explained by the fact that these species contain very high constitutive level of MAAs (8–10 mg g1 DW), close to the maximal potential concentration [18,34,36,37]. In contrast, in our study the initial level of MAAs was reduced to 6.7 ± 1.1 mg g1 DW, due to the previous incubation under N-starvation conditions. When algae were transferred to a medium with high ammonium concentration, the synthesis of MAAs was stimulated by blue or white light. Similar stimulation, by blue light after the addition of inorganic nitrogen, has been reported for phycobiliproteins and chlorophylls [7,8]. The accumulation of nitrogenous compounds under blue light was related to the stimulation of nitrate incorporation, and the stimulation of nitrate reductase and glutamine synthetase activities [8]. This is the second report regarding the variation of MAAs in Porphyra sp. due to light quality [11]. It has been well established that algae respond to nutrient limitation by increasing uptake capacity and/or efficiency for the specific nutrient [38]. The response of MAAs to light quality and nitrogen enrichment (after starvation) is according to the fast incorporation of nitrogen into organic compounds i.e., PE and protein, as it has been previously reported [3]. Wavelength dependence for MAA synthesis in algae has been published [15,25,39]. The total UV absorbance of the microalgae Alexand-
rium excavatum increased when cells where grown in blue light, but not to the same degree as white light [20]. The intracellular concentration of MAAs in the microalgae Gloeocapsa sp. achieved were directly related to the intensity of the UV radiation (maximum at 320 nm) received by the cells; however, a small but significant increase was attained with UV radiation at 365 nm, and neither blue, green nor red light increased MAAs concentration in this species [21]. Antarctic diatoms synthesized MAAs preferentially between 370 and 460 nm [22]. Our results indicate that blue light favored the accumulation of palythine and asterina-330 in P. leucosticta. However, white, green, yellow and red lights favored the accumulation of shinorine. The induction of MAAs by red, green, blue and white light was examined in Chondrus crispus collected from Helgoland [25]. Palythine was the most abundant MAA in this species [24]. Similar results as in P. leucosticta were obtained, palythine was synthesized in thalli under blue light to the same extent observed in control samples in white light, in contrast, no MAAs were accumulated under green and red light [25]. The differences observed under different light qualities in the accumulation of MAAs in P. leucosticta seem to be mediated by a non-photosynthetic photoreceptor. Some authors suggest the presence of photoreceptors in the synthesis of MAAs; a reduced pterin (UV-B photoreceptor) was suggested in Chlorogloeopsis PCC 6912 [40]. On the other hand, flavins have been discussed in the induction of MAAs in C. crispus, they demonstrated that blue light and UV radiation interact to boost the synthesis of shinorine [25], which is in line with the photoregulation of flavin synthesis [41,42]. In addition, recently a monochromatic action spectrum for shinorine in C. crispus indicated that the photoreceptors mediating shinorine photoinduction might be an unidentified UVA-type photoreceptor with absorption peaks at 320, 340 and 400 nm [27]. The fact that the increase of palythine and asterina-330 occurred in blue light simultaneous to a decrease in shinorine in P. leucosticta suggests that blue light photoreceptor could be involved in the MAA conversion pathway. Carreto et al. [43] illustrated the structural relationship among several MAAs commonly detected in the marine environment. We propose the conversion from shinorine to palythine and asterina-330 in P. leucosticta under blue light. In summary, the greatest accumulation of MAAs, PE and proteins in blue light cannot be attributed to photosynthesis i.e., photosynthetic rate is low under blue light. But rather, the accumulation of nitrogenous compounds seems to be controlled by a non-photosynthetic blue light photoreceptor, as it has been shown for the control of photosynthetic pigment synthesis in red macroalgae [7]. The identification of this photoreceptor and the transduction pathway must be still elucidated.
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
5. Abbreviations a photosynthetic efficiency a area BP phycobiliprotein Chl chlorophyll DW dried weight ETR electron transport rate ETRmax maximal electron transport rate FW fresh weight Fv/Fm maximal quantum yield MAAs mycosporine-like amino acids OD optical density PAM pulse amplitude modulated fluorescence PC phycocyanin PE phycoerythrin R relative growth rate SP soluble protein StP structural protein UV ultraviolet radiation
Acknowledgements This study was supported by a grant from de Ministry of Education and Science (Spain) to N.K.P. The CICYT project AGL 2001-1888-C03-02 (MCYT) and the Research group RNM-295 (Junta de Andalucı´a). We thank Dr. A. Cabello for improving the English grammar, and two anonymous reviewers who provided helpful comments and suggestions on the manuscript.
References [1] K. Lu¨ning, M.J. Dring, Action spectra and spectral quantum yield of photosynthesis in marine macroalgae with thin and thick thalli, Mar. Biol. 87 (1985) 119–129. [2] M.J. Dring, Photocontrol of development in algae, Ann. Rev. Plant Physiol. Mol. Biol. 39 (1988) 157–174. [3] F.L. Figueroa, J. Aguilera, F.X. Niell, Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (Bangiales, Rhodophyta), Eur. J. Phycol. 30 (1995) 11–18. [4] F.L. Figueroa, J. Aguilera, C. Jime´nez, J.J. Vergara, M.D. Robles, F.X. Niell, Growth, pigment synthesis and nitrogen assimilation in the red alga Porphyra sp. (Bangiales, Rhodophyta) under blue and red light, Sci. Mar. 59 (1995) 9–20. [5] J. Aguilera, F.J.L. Gordillo, U. Karsten, F.L. Figueroa, F.X. Niell, Light quality effect on photosynthesis and efficiency of carbon assimilation in the red alga Porphyra leucosticta, J. Plant Physiol. 157 (2000) 86–92. [6] I. Tsekos, F.X. Niell, J. Aguilera, F.L. Figueroa, S.G. Delivopoulos, Ultrastructure of the vegetative gametophytic cells of Porphyra leucosticta (Rhodophyta) grown in red, blue and green light, Phycol. Res. 50 (2002) 251–264. [7] W. Ru¨diger, F. Lo´pez-Figueroa, Photoreceptors in algae, Photochem. Photobiol. 55 (1992) 949–954.
77
[8] F.L. Figueroa, Effects of light quality on nitrate reductase and glutamine synthetase activities in the red alga Porphyra leucosticta Thur. in Le Jol. and other macroalgae, Sci. Mar. 60 (1996) 163–170. [9] C.S. Cockell, J. Knowland, Ultraviolet radiation screening compounds, Biol. Rev. 74 (1999) 311–345. [10] J.M. Shick, W.C. Dunlap, Mycosporine-like amino acids and related gadusols: biosynthesis, accumulation and UV-protective functions in aquatic organisms, Ann. Rev. Physiol. 64 (2002) 223–262. [11] N. Korbee-Peinado, R.T. Abdala-Dı´az, F.L. Figueroa, E.W. Helbling, Ammonium and UV radiation stimulate the accumulation of mycosporine-like amino acids in Porphyra columbina (Rhodophyta) from Patagonia, Argentina, J. Phycol. 40 (2004) 248–259. [12] F.R. Conde, M.S. Churio, C.M. Previtali, The photoprotector mechanism of mycosporine-like amino acids. Excited-state properties and photostability of porphyra-334 in aqueous solution, J. Photochem. Photobiol. B: Biol. 56 (2000) 139–144. [13] F.R. Conde, M.O. Carignan, M.S. Churio, J.I. Carreto, In vitro cis–trans photoisomerization of palythene and usujirene. Implications on the in vivo transformation of mycosporine-like amino acids, Photochem. Photobiol. 77 (2003) 146–150. [14] J.M. Shick, W.C. Dunlap, G.R. Buettner, in: S. Yoshikawa, S. Toyokuni, Y. Yamamoto, Y. Naito (Eds.), Free Radicals in Chemistry, Biology and Medicine, OICA Int, London, 2000, pp. 215–228. [15] W.C. Dunlap, J.M. Shick, UV radiation absorbing mycosporinelike amino acids in coral reef organisms: a biochemical and environmental perspective, J. Phycol. 34 (1998) 418–430. [16] H.J. Suh, H.W. Lee, J. Jung, Mycosporine-glycine protects biological systems against photodynamic damage by quenching singlet oxygen with a high efficiency, Photochem. Photobiol. 78 (2003) 109–113. [17] A. Oren, Mycosporine-like amino acids as osmotic solutes in a community of halophilic cyanobacteria, Geomicrobiol. J. 14 (1997) 231–240. [18] N. Korbee, P. Huovinen, F.L. Figueroa, J. Aguilera, U. Karsten, Availability of ammonium influences photosynthesis and the accumulation of mycosporine-like amino acids in two Porphyra species (Bangiales, Rhodophyta), Mar. Biol. 146 (2005) 645–654. [19] L. Talarico, G. Maranzana, Light and adaptative responses in red macroalgae: an overview, J. Photochem. Photobiol. B: Biol. 56 (2000) 1–11. [20] J.I. Carreto, V.A. Lutz, S.G. De Marco, M.O. Carignan, in: E. Graneli, L. Edler, B. Sundstrom, D.M. Anderson (Eds.), Toxic Marine Phytoplankton, Elsevier, Amsterdam, 1990, pp. 275–279. [21] F. Garcı´a-Pichel, C.E. Wingard, R.W. Castenholz, Evidence regarding the UV sunscreen role of a mycosporine-like compound in the cyanobacterium Gloecapsa sp., Appl. Environ. Microbiol. 59 (1993) 170–176. [22] L. Riegger, D. Robinson, Photoinduction of UV-absorbing compounds in Antarctic diatoms and Phaeocystis antarctica, Mar. Ecol. Prog. Ser. 160 (1997) 13–25. [23] U. Karsten, T. Sawall, D. Hanelt, K. Bischof, F.L. Figueroa, A. Flores-Moya, C. Wiencke, An inventory of UV-absorbing mycosporine-like amino acids in macroalgae from polar to warm-temperate regions, Bot. Mar. 41 (1998) 443–453. [24] L.A. Franklin, I. Yakovleva, U. Karsten, K. Lu¨ning, Synthesis of mycosporine-like amino acids in Chondrus crispus (Florideophyceae) and the consequences for sensitivity to ultraviolet B radiation, J. Phycol. 35 (1999) 682–693. [25] L.A. Franklin, G. Kra¨bs, R. Kuhlenkamp, Blue light and UVA radiation control the synthesis of mycosporine-like amino acids in Chondrus crispus (Florideophyceae), J. Phycol. 37 (2001) 257–270. [26] K. Hoyer, U. Karsten, T. Sawall, C. Wiencke, Photoprotective substances in Antarctic macroalgae and their variation with
78
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35]
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78 respect to depth distribution, different tissues and developmental stages, Mar. Ecol. Prog. Ser. 211 (2001) 117–129. G. Kra¨bs, M. Watanabe, C. Wiencke, A monochromatic action spectrum for the photoinduction of the UV-absorbing mycosporine-like amino acid shinorine in the red alga Chondrus crispus, Photochem. Photobiol. 79 (2004) 515–519. J.M. Kain, Seasonal growth and photoinhibition in Plocamium cartilagineum (Rhodophyta) of the Isle of Man, Phycologia 26 (1987) 88–99. W. Inskeep, P.R. Bloom, Extinction coefficients of chlorophyll a and b in N,N-dimethylformamide and 80% acetone, Plant Physiol. 77 (1985) 483–485. S. Beer, A. Eshel, Determining phycoerythrin and phycocyanin concentrations in aqueous crude extracts of red algae, Aust. J. Mar. Freshw. Res. 36 (1985) 785–792. M.M. Bradford, A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. R.R. Sokal, F.J. Rohlf, Biometry, third ed., W.H. Freeman and Company, New York, 1995. F.L. Figueroa, Photoregulation of nitrogen metabolism ad protein accumulation in the accumulation in the red alga Corallina elongata Ellis et Soland, Z. Naturforsch. 48 (1993) 788–794. A. Gro¨niger, C. Hallier, D.P. Ha¨der, Influence of UV radiation and visible light on Porphyra umbilicalis: photoinhibition and MAA concentration, J. Appl. Phycol. 11 (1999) 437–445. U. Karsten, J.A. West, Living in the intertidal zone-seasonal effects on heterosides and sun-screen compounds in the red alga
[36]
[37]
[38] [39]
[40]
[41]
[42]
[43]
Bangia atropurpurea (Bangiales), J. Exp. Mar. Biol. Ecol. 254 (2000) 221–234. A. Gro¨niger, R.P. Sinha, M. Klisch, D.P. Ha¨der, Photoprotective compounds in cyanobacteria, phytoplankton and macroalgae-a database, J. Photochem. Photobiol. B: Biol. 58 (2000) 115–122. K. Hoyer, U. Karsten, C. Wiencke, Induction of sunscreen compounds in Antarctic macroalgae by different radiation conditions, Mar. Biol. 141 (2002) 619–627. J. Beardall, E. Young, S. Roberts, Approaches for determining phytoplankton nutrient limitation, Aquat. Sci. 63 (2001) 44–69. R.P. Sinha, M. Klisch, A. Gro¨niger, D.P. Ha¨der, Ultravioletabsorbing/screening substances in cyanobacteria, phytoplankton and macroalgae, J. Photochem. Photobiol. B: Biol. 47 (1998) 83– 94. A. Portwich, F. Garcı´a-Pichel, A novel prokaryotic UVB photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912, Photochem. Photobiol. 71 (2000) 493–499. J.M. Christie, G.J. Jenkins, Distinct UV-B and UV-A/blue light transduction pathways induce chalcone synthase gene expression in Arabidopsis cells, Plant Cell 8 (1996) 1555–1567. G. Fuglevand, J.A. Jackson, G.J. Jenkins, UV-B, UV-A and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis, Plant Cell 8 (1996) 2347–2357. J.I. Carreto, M.O. Carignan, N.G. Montoya, A high-resolution reverse-phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms, Mar. Biol. 146 (2005) 237–252.
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 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. 2005 Elsevier B.V. All rights reserved. Keywords: Light quality; Mycosporine-like amino acid; Photoreceptor; Photosynthesis; Porphyra
1. Introduction Light quality affects photosynthesis, growth, development 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 expansion, 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 *
Corresponding author. Tel.: +34 952 133337; fax: +34 952 132000. E-mail address: [email protected] (N. Korbee).
1011-1344/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.03.002
thalli, most of the photosynthetic energy can be diverted into new biomass due to high carbon investment efficiency 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 molecular weight, high molar extinction coefficients and absorption band in the UV with a maximum between
72
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
310 and 360 nm [9,10]. Recently, it has been shown that both UV radiation and high inorganic nitrogen availability stimulate the accumulation of MAAs in P. leucosticta [11]. MAAs are passive sunscreens, preferentially 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 compatible 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 occurred 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 different light qualities (white, blue, green, yellow and red light) for one week exposure under high nitrogen availability 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].
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 Lagos, Ma´laga, Southern Spain (3628 0 N 41 0 W). 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 m2 s1) 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 cylinders with 300 lM NH4Cl and incubated for one week with 45 lmol m2 s1 of white, blue, green, yellow and red light and a photoperiod of 12:12 light:dark. Irradiation 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 reported in Fig. 1. In Table 1, effective irradiance for photosynthesis 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 nitrogen 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, Effeltrich, 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.
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78 Table 1 Irradiance and photosynthetic effective irradiance (PEI) expressed as lmol m2 s1 in the different light qualities used Light quality
Irradiance (lmol m2 s1)
PEI (lmol m2 s1)
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].
2.3. Growth rate The relative growth rate (R), expressed as % day1, was computed from the following expression [28]: R ¼ ðln at ln a0 Þ=t; where at is the area measured at time (t) in days and a0 is 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).
73
2.6. Structural protein content Structural proteins were determined from the pellet fraction obtained after centrifugation as above indicated. 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 content 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-dissolved 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 min1. Sample volumes of 10 lL were injected into the C8 chromatographic column (5 lm pore size, 250 · 4 mm, Sphereclone, Phenomenex, Aschaffenburg, Germany). MAAs were detected online with a Waters Photodiode Array Detector 996 at 330 nm.
2.4. Photosynthetic pigments 2.8. Internal carbon and nitrogen Two discs of approximately 10–20 mg fresh weight (FW) per sample were extracted in 1.5 ml N,N-dimethylformamide for 24 h at 4 C in darkness. After centrifugation at 5000g for 10 min (Labofuge 400R, Heraeus, Kendro Laboratory Products, Langenselbold, Germany), the supernatant was used for chlorophyll a (Chl a) determination. Chl a concentration was calculated 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 concentration 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). Triplicate samples were taken from each treatment. 2.5. Soluble protein content Soluble proteins were determined using a commercial Protein Assay (BioRad), based on Bradford method [31]. Protein content was determined spectrophotometrically at 595 nm and concentrations were calculated by means of standards made with bovine serum albumin (SIGMA).
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 distribution) 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, meanwhile there was a decrease in red light (Table 2). However, the maximal electron transport rate (ETRmax) increased significantly (p < 0.05) after the incubation under all light treatments, with the exception of blue light. After the experimental period the greatest ETRmax value was found under red light. The slope of the ETR versus irradiance curve (a) was greater after the incubation
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
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 Initial Seven-day cultures
Fv/Fm 0.70 ± 0.03
White Blue Green Yellow Red
ETRmax a
0.70 ± 0.01a 0.72 ± 0.01b 0.69 ± 0.01a,d 0.68 ± 0.02d 0.65 ± 0.02c
a a
1.50 ± 0.29
0.07 ± 0.02a,b
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 between values (p < 0.05) for each parameter. Data given as means ± SD (n = 5).
under white, green and yellow light than that in the initial 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 differences 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 treatments in PC content (Table 3). Soluble proteins also increased 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 protein (Table 3). The internal C content did not show any difference among light treatments, it was approximately 320 mg g1 DW. The internal N content increased in all treatments after the incubation period (Table 3),
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 observed 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 experiments was 12.01 ± 0.39% of shinorine, 80.42 ± 1.50% porphyra-334, 3.94 ± 0.12% palythine and 4.31 ± 0.17% asterina-330 (Fig. 3). During the incubation period, the percentage of shinorine increased significantly (p < 0.05) in all light treatments except in blue light, in which a decrease was observed (Fig. 3(a)).
14 12
MAAs (mg·g-1 DW)
74
10 8 6 4 2 0 Initial
WL
BL
GL
YL
RL
1
Fig. 2. Concentration of MAAs, expressed in mg g dry 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).
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 g1 dried weight and carbon/nitrogen ratio (C/N) in Porphyra leucosticta at initial time and after seven days of incubation Light treatment Initial Seven-day cultures
White Blue Green Yellow Red
Chl a
PE
PC
SP
StP
2.83 ± 0.44a
6.11 ± 0.30a
3.47 ± 0.67a
40.7 ± 3.8a
b
b
b
b
6.83 ± 0.78 5.80 ± 0.78b 7.06 ± 0.38b 6.37 ± 0.65b 5.60 ± 0.60b
11.80 ± 1.87 11.83 ± 0.66b 10.26 ± 1.06b,c 9.84 ± 1.22b,c 7.82 ± 0.72a,c
6.31 ± 0.70 4.91 ± 0.20a,b 5.81 ± 0.60b 5.54 ± 0.14b 5.68 ± 0.62b
78.1 ± 6.1 77.4 ± 0.4b 75.2 ± 2.4b,c 72.4 ± 0.5b,c 65.2 ± 3.7c
N
120.0 ± 4.8a
C/N
37.0 ± 2.0a a
112.1 ± 20.0 151.6 ± 3.3b 108.0 ± 15.5a 115.1 ± 14.1a 98.2 ± 10.7a
b
62.7 ± 1.7 56.9 ± 1.7b,c 56.1 ± 0.0b,c 54.0 ± 6.2c,d 47.3 ± 0.9d
R (% day1)
8.6 ± 0.2a 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 % day1, 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).
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
(a)
Porphyra-334 (%)
Shinorine (%)
30 25 20 15 10 5
100 (b) 80 60 40 20 0
(c)
Asterina-330 (%)
Palythine (%)
0 6 4 2 0
Initial WL BL GL YL RL
75
(d)
6 4 2 0
Initial WL BL GL YL RL
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 g1 dried weight, in Porphyra leucosticta at initial time and after seven days of incubation Treatment Initial Seven-day cultures
White Blue Green Yellow Red
Shinorine
Porphyra-334
Palythine
Asterina-330
0.80 ± 0.16a
5.34 ± 0.83a
0.29 ± 0.02a
0.31 ± 0.04a
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).
Meanwhile, the percentages of palythine and asterina330 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 favored thallus expansion, cell division, carbon accumulation and a dramatic decrease of the protoplasmic area and in the thickness of the cell walls, while blue light favored photosynthetic pigment, protein synthesis, numerous 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, however, 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 photosynthetic activity in blue light compared with other spectral 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
76
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
[3,5]. Our results are consistent with these studies; the lowest ETRmax and the lowest photosynthetic efficiency (a) had been shown in blue light. The lower values of ETRmax and a under blue light has been interpreted by a higher package effect [5] and higher amount of PE pool not attached to phycobilisomes, as observed in this species previously by Tsekos et al. [6]. In contrast, Fv/Fm was greater under blue than under red light. So, low ETRmax in parallel to high Fv/Fm values under blue light could be related to an imbalance between PSII and PSI, with a simultaneous high demand for electron sinks i.e., Mehler reaction or the electron demand for the inorganic nitrogen assimilation, which is stimulated by blue light [3,5]. Growth rate under blue light was significantly lower than in white, red and green lights in P. leucosticta, as it has been previously shown in this species [3,5]. The lower growth rate under blue light could be related to the lowest ETRmax and the lowest photosynthetic efficiency (a) observed under these culture conditions, as described above. Other authors ascribed the lower growth rate in blue light to two different processes in P. leucosticta; first, to a low efficiency in photosynthetic performance mainly produced at level of the water splitting complex of the PSII and secondly to a lower carbon investment efficiency, leading to a deficient accumulation of photosynthetic and osmotic-related products [5]. Previous studies showed that in the Bangiales the accumulation of MAAs was not affected by light quality [34,35]. These results have been explained by the fact that these species contain very high constitutive level of MAAs (8–10 mg g1 DW), close to the maximal potential concentration [18,34,36,37]. In contrast, in our study the initial level of MAAs was reduced to 6.7 ± 1.1 mg g1 DW, due to the previous incubation under N-starvation conditions. When algae were transferred to a medium with high ammonium concentration, the synthesis of MAAs was stimulated by blue or white light. Similar stimulation, by blue light after the addition of inorganic nitrogen, has been reported for phycobiliproteins and chlorophylls [7,8]. The accumulation of nitrogenous compounds under blue light was related to the stimulation of nitrate incorporation, and the stimulation of nitrate reductase and glutamine synthetase activities [8]. This is the second report regarding the variation of MAAs in Porphyra sp. due to light quality [11]. It has been well established that algae respond to nutrient limitation by increasing uptake capacity and/or efficiency for the specific nutrient [38]. The response of MAAs to light quality and nitrogen enrichment (after starvation) is according to the fast incorporation of nitrogen into organic compounds i.e., PE and protein, as it has been previously reported [3]. Wavelength dependence for MAA synthesis in algae has been published [15,25,39]. The total UV absorbance of the microalgae Alexand-
rium excavatum increased when cells where grown in blue light, but not to the same degree as white light [20]. The intracellular concentration of MAAs in the microalgae Gloeocapsa sp. achieved were directly related to the intensity of the UV radiation (maximum at 320 nm) received by the cells; however, a small but significant increase was attained with UV radiation at 365 nm, and neither blue, green nor red light increased MAAs concentration in this species [21]. Antarctic diatoms synthesized MAAs preferentially between 370 and 460 nm [22]. Our results indicate that blue light favored the accumulation of palythine and asterina-330 in P. leucosticta. However, white, green, yellow and red lights favored the accumulation of shinorine. The induction of MAAs by red, green, blue and white light was examined in Chondrus crispus collected from Helgoland [25]. Palythine was the most abundant MAA in this species [24]. Similar results as in P. leucosticta were obtained, palythine was synthesized in thalli under blue light to the same extent observed in control samples in white light, in contrast, no MAAs were accumulated under green and red light [25]. The differences observed under different light qualities in the accumulation of MAAs in P. leucosticta seem to be mediated by a non-photosynthetic photoreceptor. Some authors suggest the presence of photoreceptors in the synthesis of MAAs; a reduced pterin (UV-B photoreceptor) was suggested in Chlorogloeopsis PCC 6912 [40]. On the other hand, flavins have been discussed in the induction of MAAs in C. crispus, they demonstrated that blue light and UV radiation interact to boost the synthesis of shinorine [25], which is in line with the photoregulation of flavin synthesis [41,42]. In addition, recently a monochromatic action spectrum for shinorine in C. crispus indicated that the photoreceptors mediating shinorine photoinduction might be an unidentified UVA-type photoreceptor with absorption peaks at 320, 340 and 400 nm [27]. The fact that the increase of palythine and asterina-330 occurred in blue light simultaneous to a decrease in shinorine in P. leucosticta suggests that blue light photoreceptor could be involved in the MAA conversion pathway. Carreto et al. [43] illustrated the structural relationship among several MAAs commonly detected in the marine environment. We propose the conversion from shinorine to palythine and asterina-330 in P. leucosticta under blue light. In summary, the greatest accumulation of MAAs, PE and proteins in blue light cannot be attributed to photosynthesis i.e., photosynthetic rate is low under blue light. But rather, the accumulation of nitrogenous compounds seems to be controlled by a non-photosynthetic blue light photoreceptor, as it has been shown for the control of photosynthetic pigment synthesis in red macroalgae [7]. The identification of this photoreceptor and the transduction pathway must be still elucidated.
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78
5. Abbreviations a photosynthetic efficiency a area BP phycobiliprotein Chl chlorophyll DW dried weight ETR electron transport rate ETRmax maximal electron transport rate FW fresh weight Fv/Fm maximal quantum yield MAAs mycosporine-like amino acids OD optical density PAM pulse amplitude modulated fluorescence PC phycocyanin PE phycoerythrin R relative growth rate SP soluble protein StP structural protein UV ultraviolet radiation
Acknowledgements This study was supported by a grant from de Ministry of Education and Science (Spain) to N.K.P. The CICYT project AGL 2001-1888-C03-02 (MCYT) and the Research group RNM-295 (Junta de Andalucı´a). We thank Dr. A. Cabello for improving the English grammar, and two anonymous reviewers who provided helpful comments and suggestions on the manuscript.
References [1] K. Lu¨ning, M.J. Dring, Action spectra and spectral quantum yield of photosynthesis in marine macroalgae with thin and thick thalli, Mar. Biol. 87 (1985) 119–129. [2] M.J. Dring, Photocontrol of development in algae, Ann. Rev. Plant Physiol. Mol. Biol. 39 (1988) 157–174. [3] F.L. Figueroa, J. Aguilera, F.X. Niell, Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (Bangiales, Rhodophyta), Eur. J. Phycol. 30 (1995) 11–18. [4] F.L. Figueroa, J. Aguilera, C. Jime´nez, J.J. Vergara, M.D. Robles, F.X. Niell, Growth, pigment synthesis and nitrogen assimilation in the red alga Porphyra sp. (Bangiales, Rhodophyta) under blue and red light, Sci. Mar. 59 (1995) 9–20. [5] J. Aguilera, F.J.L. Gordillo, U. Karsten, F.L. Figueroa, F.X. Niell, Light quality effect on photosynthesis and efficiency of carbon assimilation in the red alga Porphyra leucosticta, J. Plant Physiol. 157 (2000) 86–92. [6] I. Tsekos, F.X. Niell, J. Aguilera, F.L. Figueroa, S.G. Delivopoulos, Ultrastructure of the vegetative gametophytic cells of Porphyra leucosticta (Rhodophyta) grown in red, blue and green light, Phycol. Res. 50 (2002) 251–264. [7] W. Ru¨diger, F. Lo´pez-Figueroa, Photoreceptors in algae, Photochem. Photobiol. 55 (1992) 949–954.
77
[8] F.L. Figueroa, Effects of light quality on nitrate reductase and glutamine synthetase activities in the red alga Porphyra leucosticta Thur. in Le Jol. and other macroalgae, Sci. Mar. 60 (1996) 163–170. [9] C.S. Cockell, J. Knowland, Ultraviolet radiation screening compounds, Biol. Rev. 74 (1999) 311–345. [10] J.M. Shick, W.C. Dunlap, Mycosporine-like amino acids and related gadusols: biosynthesis, accumulation and UV-protective functions in aquatic organisms, Ann. Rev. Physiol. 64 (2002) 223–262. [11] N. Korbee-Peinado, R.T. Abdala-Dı´az, F.L. Figueroa, E.W. Helbling, Ammonium and UV radiation stimulate the accumulation of mycosporine-like amino acids in Porphyra columbina (Rhodophyta) from Patagonia, Argentina, J. Phycol. 40 (2004) 248–259. [12] F.R. Conde, M.S. Churio, C.M. Previtali, The photoprotector mechanism of mycosporine-like amino acids. Excited-state properties and photostability of porphyra-334 in aqueous solution, J. Photochem. Photobiol. B: Biol. 56 (2000) 139–144. [13] F.R. Conde, M.O. Carignan, M.S. Churio, J.I. Carreto, In vitro cis–trans photoisomerization of palythene and usujirene. Implications on the in vivo transformation of mycosporine-like amino acids, Photochem. Photobiol. 77 (2003) 146–150. [14] J.M. Shick, W.C. Dunlap, G.R. Buettner, in: S. Yoshikawa, S. Toyokuni, Y. Yamamoto, Y. Naito (Eds.), Free Radicals in Chemistry, Biology and Medicine, OICA Int, London, 2000, pp. 215–228. [15] W.C. Dunlap, J.M. Shick, UV radiation absorbing mycosporinelike amino acids in coral reef organisms: a biochemical and environmental perspective, J. Phycol. 34 (1998) 418–430. [16] H.J. Suh, H.W. Lee, J. Jung, Mycosporine-glycine protects biological systems against photodynamic damage by quenching singlet oxygen with a high efficiency, Photochem. Photobiol. 78 (2003) 109–113. [17] A. Oren, Mycosporine-like amino acids as osmotic solutes in a community of halophilic cyanobacteria, Geomicrobiol. J. 14 (1997) 231–240. [18] N. Korbee, P. Huovinen, F.L. Figueroa, J. Aguilera, U. Karsten, Availability of ammonium influences photosynthesis and the accumulation of mycosporine-like amino acids in two Porphyra species (Bangiales, Rhodophyta), Mar. Biol. 146 (2005) 645–654. [19] L. Talarico, G. Maranzana, Light and adaptative responses in red macroalgae: an overview, J. Photochem. Photobiol. B: Biol. 56 (2000) 1–11. [20] J.I. Carreto, V.A. Lutz, S.G. De Marco, M.O. Carignan, in: E. Graneli, L. Edler, B. Sundstrom, D.M. Anderson (Eds.), Toxic Marine Phytoplankton, Elsevier, Amsterdam, 1990, pp. 275–279. [21] F. Garcı´a-Pichel, C.E. Wingard, R.W. Castenholz, Evidence regarding the UV sunscreen role of a mycosporine-like compound in the cyanobacterium Gloecapsa sp., Appl. Environ. Microbiol. 59 (1993) 170–176. [22] L. Riegger, D. Robinson, Photoinduction of UV-absorbing compounds in Antarctic diatoms and Phaeocystis antarctica, Mar. Ecol. Prog. Ser. 160 (1997) 13–25. [23] U. Karsten, T. Sawall, D. Hanelt, K. Bischof, F.L. Figueroa, A. Flores-Moya, C. Wiencke, An inventory of UV-absorbing mycosporine-like amino acids in macroalgae from polar to warm-temperate regions, Bot. Mar. 41 (1998) 443–453. [24] L.A. Franklin, I. Yakovleva, U. Karsten, K. Lu¨ning, Synthesis of mycosporine-like amino acids in Chondrus crispus (Florideophyceae) and the consequences for sensitivity to ultraviolet B radiation, J. Phycol. 35 (1999) 682–693. [25] L.A. Franklin, G. Kra¨bs, R. Kuhlenkamp, Blue light and UVA radiation control the synthesis of mycosporine-like amino acids in Chondrus crispus (Florideophyceae), J. Phycol. 37 (2001) 257–270. [26] K. Hoyer, U. Karsten, T. Sawall, C. Wiencke, Photoprotective substances in Antarctic macroalgae and their variation with
78
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35]
N. Korbee et al. / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 71–78 respect to depth distribution, different tissues and developmental stages, Mar. Ecol. Prog. Ser. 211 (2001) 117–129. G. Kra¨bs, M. Watanabe, C. Wiencke, A monochromatic action spectrum for the photoinduction of the UV-absorbing mycosporine-like amino acid shinorine in the red alga Chondrus crispus, Photochem. Photobiol. 79 (2004) 515–519. J.M. Kain, Seasonal growth and photoinhibition in Plocamium cartilagineum (Rhodophyta) of the Isle of Man, Phycologia 26 (1987) 88–99. W. Inskeep, P.R. Bloom, Extinction coefficients of chlorophyll a and b in N,N-dimethylformamide and 80% acetone, Plant Physiol. 77 (1985) 483–485. S. Beer, A. Eshel, Determining phycoerythrin and phycocyanin concentrations in aqueous crude extracts of red algae, Aust. J. Mar. Freshw. Res. 36 (1985) 785–792. M.M. Bradford, A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. R.R. Sokal, F.J. Rohlf, Biometry, third ed., W.H. Freeman and Company, New York, 1995. F.L. Figueroa, Photoregulation of nitrogen metabolism ad protein accumulation in the accumulation in the red alga Corallina elongata Ellis et Soland, Z. Naturforsch. 48 (1993) 788–794. A. Gro¨niger, C. Hallier, D.P. Ha¨der, Influence of UV radiation and visible light on Porphyra umbilicalis: photoinhibition and MAA concentration, J. Appl. Phycol. 11 (1999) 437–445. U. Karsten, J.A. West, Living in the intertidal zone-seasonal effects on heterosides and sun-screen compounds in the red alga
[36]
[37]
[38] [39]
[40]
[41]
[42]
[43]
Bangia atropurpurea (Bangiales), J. Exp. Mar. Biol. Ecol. 254 (2000) 221–234. A. Gro¨niger, R.P. Sinha, M. Klisch, D.P. Ha¨der, Photoprotective compounds in cyanobacteria, phytoplankton and macroalgae-a database, J. Photochem. Photobiol. B: Biol. 58 (2000) 115–122. K. Hoyer, U. Karsten, C. Wiencke, Induction of sunscreen compounds in Antarctic macroalgae by different radiation conditions, Mar. Biol. 141 (2002) 619–627. J. Beardall, E. Young, S. Roberts, Approaches for determining phytoplankton nutrient limitation, Aquat. Sci. 63 (2001) 44–69. R.P. Sinha, M. Klisch, A. Gro¨niger, D.P. Ha¨der, Ultravioletabsorbing/screening substances in cyanobacteria, phytoplankton and macroalgae, J. Photochem. Photobiol. B: Biol. 47 (1998) 83– 94. A. Portwich, F. Garcı´a-Pichel, A novel prokaryotic UVB photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912, Photochem. Photobiol. 71 (2000) 493–499. J.M. Christie, G.J. Jenkins, Distinct UV-B and UV-A/blue light transduction pathways induce chalcone synthase gene expression in Arabidopsis cells, Plant Cell 8 (1996) 1555–1567. G. Fuglevand, J.A. Jackson, G.J. Jenkins, UV-B, UV-A and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis, Plant Cell 8 (1996) 2347–2357. J.I. Carreto, M.O. Carignan, N.G. Montoya, A high-resolution reverse-phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms, Mar. Biol. 146 (2005) 237–252.