Differential behaviour of two cyanobacterium species to UV radiation. Artificial UV radiation induces phycoerythrin synthesis

Journal

of

Photochemistry and Photobiology ELSEVIER

Journal of Photochemistryand PhotobiologyB: Biology 44 (1998) 175-183

B:Biology

Differential behaviour of two cyanobacterium species to UV radiation. Artificial UV radiation induces phycoerythrin synthesis Rdmulo Ar~ioz a, Miranda Shelton b, Michael Leben a, Donat-P. Hiider a,, ~'lnstitut fiir Botanik und Pharmazeutische Biologie, Lehrstuhl Botanik 1, Friedrich-Alexander-Universiti~t, StaudtstraJ3e 5. D-91058 Erlangen, Germany b University of Portsmouth, School of Biological Sciences, King Heno' 1st Street, Portsmouth, PO12DY. UK

Received 6 April 1998; accepted 5 June 1998

Abstract

Altitude is an important factor contributing to the local UV-B climate. In the European Alps solar UV-B increases ~ 21% 1000 m -~. A Nostoc muscorum (UTEX 389) originating from Scotland and a Nostoc sp. isolated from a highland lake (Yanaqocha) located 3980 m above

sea level (Cusco, Peril) have been used in a study where the tolerance to UV radiation (UVR) stress of both species was determined. Following irradiation doses of 15 kJ UV (UV-A plus UV-B, equivalent to ~ 6 h exposure to unfiltered solar light at noon for a standard midlatitude region with normal ozone concentration), the viability o f N o s t o c sp. is 30% compared to 3% for Nostoc muscorum. UV-B induces the reduction of the number of phycobilisomes per cell, phycobilisome disassembly and/or degradation as well as phycobilisome uncoupling. Following UV exposure, phycoerythrin (PE) fluorescence emission increases dramatically in both species, indicating accumulation of PE in the phycobilisome rods. The detected increase in PE due to UVR is confirmed using a monoclonal antibody anti-PE. © 1998 Elsevier Science S.A. All rights reserved. Keywords." Cyanobacteria: Photobleaching;Phycobilisomes;UV-B

1. I n t r o d u c t i o n

The discovery of a large spring-time loss of lower-stratospheric ozone over Antarctica initiated the reassessment of ultraviolet radiation ( U V R ) effects on living organisms because of the effective filter capacity of ozone for shortwavelength UV-B (280-315 n m ) [ 1,2]. In the recent past, the extension of stratospheric ozone loss to other areas of the planet including the Arctic and mid latitudes has been observed [ 3-5 ]. In response to this critical situation, several international ozone-protection agreements were reached in order to restrict a n d / o r phase out the production and use of chlorofluorocarbons ( C F C s ) , halons and methyl chloroform known to degrade ozone [6]. Photosynthetic activity and light-stress avoidance mechanisms such as orientation and motility are readily impaired by artificial UV and unfiltered solar radiation in many phytoplankton organisms [ 7 - 9 ] . UVR-induced detrimental effects may reduce the biomass productivity of marine phytoplankton estimated to incorporate 90 to 100 Gt of atmospheric carbon into organic material annually (for review, see [ 10] ). *Corresponding author. Tel.: +49-9131-858216; Fax: +49-9131858215; E-mail: [email protected]

UVR reaching the Earth's surface is subject to natural fluctuations caused by wavelength-dependent atmospheric transmission, solar elevation and scattering by air molecules and clouds, to name the most important ones [ 11,12]. Geographical differences in the UV-B climate are however more significant than the corresponding differences in the total solar radiation climate [13]. The altitude effect ( A E ) , defined as the increase in solar radiation with altitude in per cent per 1000 m, is due to the.lower atmospheric pressure, less scattering because of the smaller aerosol content and the reflection of UVR by natural coverage ( A l b e d o ) at higher altitudes [ 14]. A E mean values measured since 1981 in the Alps (Jungfraujoch, Switzerland) are ~ 10% 1000 m 1 for U V R and ~ 2 1 % 1000 m ~ for solar UV-B [15]. Global irradiance for U V R measured in January and February 1991 in the Chilean Andes ( A t a c a m a ) increased 8-10% 1000 m - 1 [161. Terrestrial plants are able to adapt to UVR stress by accumulating UV-B screening pigments in the epidermal layer to reduce UV-B-induced damage to the photosynthetic apparatus or other molecular targets [ 17,18]. In non-photosynthetic microorganisms D N A is the main target for UV-B; it absorbs ~ 50% of the incident UV-B radiation. In contrast, photosynthetic cyanobacteria have been shown to accumulate

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176

R. Arc'loz et al. / Journal of PhotochemistD' and Photobiology B: Biology 44 (1998) 175-183

UV-A/UV-B screening pigments to attenuate UVR damage. Although the photoprotection is not complete, the absorbance in the UV range is between 30 and 70% of the incoming radiation [ 19-21]. Light-harvesting protein complexes and chlorophyll are thought to absorb more than 99% of the incoming UV-B in photosynthetic organisms [22]. In the present work the differential UV-B tolerance of a Nostoc sp. isolated from a high-altitude Andean lake (3980 m above sea level) and aNostoc muscorum originating from Scotland was studied in association with the possible role of phycobilisomes in UV-B photoprotection.

spread on agar in BG11 medium and cultured for two weeks. Colonies were counted manually afterwards. These experiments were repeated three times.

2. Materials and methods

2.5. Biochemical analysis

2.1. Strains and culture conditions

Phycobilisomes were isolated as described previously [ 24]. The cells were resuspended in phycobilisome isolation buffer (0.75 M potassium phosphate buffer, 1 mM EDTA, 0.5 mM PMSF at pH 7), pre-homogenized in a homogenizer (HO4 Biihler, Tiibingen, Germany), three bursts of 5 s at 15 000 rpm, and then passed through a French pressure cell at 1030 bar. The homogenate was incubated for 15 min at room temperature with constant stirring in the presence of 2% (vol./vol.) Triton X-100. After centrifugation at 30 000 g for 15 min, aliquots of 1.5 ml of the supernatant were layered over a discontinuous sucrose density gradient from 0.5 to 1.5 M sucrose in 0.75 M potassium phosphate, pH 7 and centrifuged for 12 h at 100 000 g. Free phycobiliproteins and intact phycobilisomes were recovered from the interface between 0.5 and 0.75 M sucrose and from the 1 M sucrose layer, respectively, by suction with a Pasteur pipette. The protein samples were precipitated with (NH4)2SO4, resuspended in TE buffer ( 10 mM Tris-HC1, 1 mM EDTA, pH 8) and dialysed extensively against the same buffer. The protein concentration was determined according to Bradford [ 25 ] prior to SDS-PAGE on a 10-18% S DS polyacrylamide linear gradient gel [ 26]. The gels were silver-stained following the procedure described by Morrisey [27] and scanned densitometrically with an Ultroscan XL (LKB, Sweden). Phycoerythrin (PE) was purified in a two-step chromatographic procedure as described elsewhere [ 28 ]. After dialysis of phycobilisomes against 1 mM EDTA in deionized water to disrupt the phycobilisome structure, phycobiliproteins were separated by gel filtration chromatography (Superdex 200) in an FPLC system (Pharmacia, Uppsala, Sweden) using 50 mM potassium phosphate, 500 mM NaC1 at pH 7.0 as eluent buffer. The partially purified PE fraction was loaded onto an anion exchange column (Resource, Pharmacia) using 20 mM Tris-HC1, pH 8 as start buffer and 20 mM TrisHC1, 250 mM NaC1 as eluent buffer. The protein fractions were precipitated with (NH4)2SO4 and resuspended in TE buffer, dialysed against the same buffer and stored at - 20°C. Equal protein samples in 100 Ixl TE buffer were blotted by vacuum onto a Fluorotrans membrane, 0.2 p,m pore size (Pall Filtron, USA) using a dot-blot transfer unit (Hoefer, USA). Immunodetection of PE was carried out using a monoclonal

Nostoc sp. was isolated from Yanaqocha, an Andean lake located in the Urubamba valley (Cusco, Peril, 13 ° 2' S, 72 ° W) at 3980 m above sea level. It is a filamentous cyanobacterium that presents motile hormogonia lacking heterocysts. Mature filaments are sessile and present terminal heterocysts. It was classified into the second subgroup, Section 4 of cyanobacteria according to Ref. [ 23 ]. The second cyanobacterium species was Nostoc muscorum (UTEX 389) originating from Scotland. Both strains were cultivated in BG11 medium at 18°C, at a white-light irradiance of 6 W m-2. 2.2. UV irradiation

For spectroscopic analysis, Nostoc cells were immobilized in quartz cuvettes containing 0.3% agar (wt./vol.) in BG11 prior to irradiation with a broad-band UV transilluminator (280-400 nm) (peak at 312 nm, Bachofer, Reutlingen, Germany). A total UV irradiance of ~ 2 0 W m -2 (10 W m -2 UV-B and 10 W m 2 UV-A) was measured at 13.5 cm distance with a double-monochromator spectral radiometer (Optronic 754, Orlando, FL, USA); cellulose acetate foils were used to filter the remaining UV-C. Since photosynthetic active radiation (PAR) is also known to photobleach phycobiliproteins at higher irradiances, all exposures were limited to artificial UV radiation. So no attempt was made to simulate solar radiation. To analyse the in vivo effects of artificial UVR at the phycobilisome level, Nostoc cells of both strains were suspended in BG11 medium in Petri dishes and irradiated under the conditions indicated above. All the experiments were repeated three times. 2.3. Clonogenic assay

Cell samples with an ODsoo of 0.1 (approximately 1 × 10 4 cells ml - l ) were submitted to UVR treatment. Aliquots of 1 ml cell suspension were removed after 0.25, 0.5, 1, 2, 4, 6, 20 and 30 h exposure to artificial UVR (reduced to 4 W m 2 by the insertion of a UV-transmitting neutral density filter) and diluted 10 times. 1 ml samples from these dilutions were

2.4. Spectroscopic analysis

Absorption spectra of immobilized cells and isolated phycobilisomes were measured in quartz cuvettes with an optical path length of 10 mm in a DU 70 spectrophotometer (Beckman, Palo Alto, CA, USA). Fluorescence emission spectra were recorded using a spectrofluorimeter (RF 5000, Shimadzu, Japan). The cell density was adjusted to 0.10Dsoo.

R. Ardoz et al. /Journal of Photochemist O' and Photobiology B: Biology 44 (1998) 175-183

antibody ( IgG2B type) anti-PE produced in mouse cells (Lot 078F4816, Sigma, USA). The working dilution was 1:1250. A secondary antibody anti-mouse IgG (whole molecule) produced in goat conjugated with alkaline phosphatase was used to detect the primary antibody using a dilution of 1:2500. The blots were developed using BCIP/NBT tablets (Sigma, USA). Anti-PE antibody was tested for sensitivity against purified PE from both N o s t o c strains as well as for crossreactions. Small amounts of protein were blotted onto the membrane filters to enhance the sensitivity and the difference in intensity among the samples (5 Ixg protein in the case of the homogenate and the first fraction, and 2.5 Ixg protein in the case of phycobilisome samples).

100

~

177

~

\ ~Nostocsp.

=...,,=

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10

-i

Nostoc muscorum ~

1

3. Results

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1

i

,111111]

,

10

i

"~/ ~""-

i1,1,11

,

100

,1111,

1000

UV-B dose [ka m "z]

3.1. C l o n o g e n i c c a p a c i t y

The survival curves presented in Fig. 1 indicate t h a t N o s t o c sp. isolate Yanaqocha is able to repair UV-B-induced damage more efficiently than N. m u s c o r u m . After an initial period during which no statistical difference ( p > 0 . 0 1 ) between both species was observed, the number of colonies decreased drastically in the case ofN. m u s c o r u m in comparison to N o s toc sp. Following an irradiation dose of 30 kJ m - 2 (equiva_ lent to ~ 6 h exposure to unfiltered solar radiation at noon for a standard mid-latitude region with a normal ozone concentration of 300 Dobson units [29] ) the viability of N. sp. was 30% compared to 3% for N. m u s c o r u m . 3.2. S p e c t r o s c o p i c a n a l y s i s

Fluorescence emission was measured using two excitation wavelengths, 510 nm to monitor PE (peak at 575 nm) and 600 nm to excite phycocyanin (PC) and allophycocyanin (APC) fluorescence emission (peak at 645 nm). The PE fluorescence emission maximum was reached after 7 h exposure for N. m u s c o r u m and after 20 h exposure for N. sp. A wavelength shift of the PE emission peak was observed after 2 h treatment for N. m u s c o r u m and after 4 h in N. sp. ( Fig. 2). The increase in the fluorescence measured for APC and PC in the case ofN. m u s c o r u m followed similar kinetics; the fluorescence maximum was reached after 4 h irradiation, and the peak shift to shorter wavelengths was observed after 1 h. For N. sp. the APC/PC fluorescence maximum was reached after 8 h exposure and the shift to shorter wavelengths after 1 h (Fig. 2(a,b) insets). 3.3. B i o c h e m i c a l a n a l y s i s

Phycobilisomes were extracted from control and UVexposed cells and purified by step sucrose density gradient to discriminate between intact phycobilisome structures and free phycobiliprotein fractions. There was a decrease in the phycobilisome concentration from both species submitted to

Fig. 1. Survival curve of

Nostocmuscorumand Nostocsp. submitted to

U V R . Cells were exposed as described in Section 2.1. D a t a are m e a n values f r o m three experiments, error bars indicate S.E.

UV-B, but phycobiliprotein loss was more pronounced in the case ofN. m u s c o r u m (Table 1). In parallel to phycobilisome degradation and/or disassembly, free phycobiliproteins within the cells increased notably (Fig. 3), which was confirmed by absorption and fluorescence emission spectra (data not shown). Free phycobiliproteins sediment to the interface between 0.5 and 0.75 M sucrose with soluble proteins. SDSPAGE analysis of this fraction showed that in the case of N. m u s c o r u m the intensity of the putative PE band increased slightly with irradiation time and decreased after 1 h exposure (Fig. 4 ( a ) ) . Densitometric data illustrate such variations: within control cells the putative PE band from N. m u s c o r u m represented 16% of the total protein. After 15 min of UV exposure the PE band represented 12%, after 30 min 18%, after 1 h 2% and after 2 h of exposure 9% of the total protein. In the case of N. sp. the increase in intensity of the putative PE band as a function of irradiation time was constant throughout the whole experiment (Fig. 4(b) ). The PE band represented 3% of total protein in control cells, 6% after 15 rain irradiation, 28% after 30 inn, 15% after 1 h and 26% after 2 h irradiation. UV-B-treated cells possess intact phycobilisomes as shown by step sucrose gradient centrifugation. The stability of phycobilisome protein composition suggested by sucrose density was confirmed for both species by electrophoretic analysis (Fig. 5(a,b)). The phycobiliprotein composition from both N o s t o c species was similar; however, the highmolecular-weight linker polypeptide (LcM) which attaches the phycobilisomes to the thylakoid membranes was different. N. m u s c o r u m presents only one LcM polypeptide with a MW of 64 kDa, while N. sp. possesses two LcM linker polypeptides of 90 and 94 kDa (not shown). The phycobiliprotein composition remained stable in N. m u s c o r u m and in N. sp. After 2 h of exposure a low-molecular-weight polypeptide appeared above the PE band in the latter species (Fig. 5 ( b ) ) .

i~, Arroz et a l , / Journal of Photochemistry and Photobiology B: Biology 44 (1998) 175-183

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Fig, 2. Fluorescence emission spectra c~fphycobiliproteins from Nostoc muscorum (a) arid Nostoc sp. (b) under UVR. Fluorescence emission of immobilized ceils in agarose was measured using an excitation wavelength ~f St0 nm to monitor phycoerythrin fluore~ence, Inset, fluorescence emission sl~eetta of phycocyanin and allophycocyaJain obtained by using ma excitation wavelength of 600 rim.

Table 1 Protein concentration in m g ml ~ per gram ceils (fresh weight) of Nostoc sp. and Nostoc muscorum during irradiatiort, Data are mean values from three independent experiments +2_S.E. Time [h]

0,00 0.25 13.50 1.00 2.~0

Nostoc sp. {rag mY* g ~+±_S.E.I

Nostoc muscorum [mg m l ~ g-~ ±S.E,] Total protein

Fraction I

PBS

Toral protein

Fraction I

PBS

12,0 5:1.05 6,1 5:0,22 6.Z ±B.45 6.5 ± O.80 5,5 ± 0.31

0.75 ± O, 10 1,00 ± 0.02 0.82±0.04 0.91 ± 0.07 1.24 ± 13.12

1,48 ± 0.15 0.55 ± 0.03 0.33_+,13.06 t3.27 i 13.05 0.14 ± 0.04

11.7 ± 0.9 7.5 ± l. 1 6,4±0.8 6.2 ± 1,43 6.7 ± 0.135

0.77 __,+0.07 0.45 ± 0.09 0.20±13.04 0.66 ± 0.18 0.96 -± 0.07

1.26 ± 0.22 0.80 ± 0.09 0,70±.0,11 0.95 ± 0.09 0.45 +_0.15

R. Ar&oz et al. / Journal of Photochemistry and Photobiology B: Biology 44 (1998) 175-183

i~iiiii, i i~iiii!~:

Control

iiiii;i! !

UV-B

Fig. 3. Sucrose density gradient of phycobilisome from control cells and UV-irradiated cells from Nostoc sp. (irradiation dose 30 kJ m 2).

179

To address the question of the observed variation (increase and/or decrease) in PE content during UV treatment, a monoclonal antibody anti-PE inmunospecific for PE forms R and r from Porphyra tenera and B and b forms from Porphyridium cruentum was used. The specificity of the antibody was tested against different concentrations of isolated PE from Nostoc muscorum and Nostoc sp. (Fig. 6). The monoclonal antibody anti-PE showed a similar degree of specificity for PE subunits from both species under investigation. The antibody was also tested for cross-reactions with other phycobiliproteins and soluble proteins, which were negative. The immunodetection of PE was used to monitor the synthesis of PE during the UV treatment following the phycobilisome extraction procedure. The homogenate before the sucrose gradient, the first fraction of the sucrose gradient as well as the phycobilisome fraction were probed. There was a decrease of PE in the homogenate after 15 min, followed by a detectable increase after 30 rain and 1 h exposure, dropping again afterwards in the case o f N o s t o c muscorum. ForNostoc sp. the PE concentration in the homogenate remained relatively stable with a detectable increase after 30 rain and 1 h of exposure. In the first fraction of the sucrose gradient the accumulation of PE following UV treatment was evident for both species but to a different extent; a peak was reached after 30 min of exposure for Nostoc muscorum, while the PE accumulation was more intense for Nostoc sp. As in the latter case, the PE concentration within phycobilisomes from Nostoc sp. was higher than that observed for Nostoc muscorum; however, it remained relatively constant through most of the experiment with a drop only after 2 h of exposure to UV for both species (Fig. 7).

(a)

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[min] Fig. 4. Electrophoretic patterns from the first fraction of sucrose density gradients. The protein samples were prepared as described in Section 2.5 Equal amounts of protein ( 10 Ixg) were loaded into each lane. After electrophoresis the gels were silver stained. (a) Nostoc muscorum and (b) Nostoc sp. The irradiation times are indicated. Arrows indicate the position of the putative phycoerythrin bands.

180

R. Ardoz et al. / Journal of Photochemistry and Photobiology B." Biology 44 (1998) 175-183

(a)

0

(b)

15

30

60

120

0

15

30

60

120

[min] Fig. 5. Electrophoreticpatterns from isolatedphycobilisomesextracted from UV-irradiatedcells.Equal amountsof protein (5 p,g) were loadedinto each lane. After electrophoresisthe gels were silver stained. (a) Nostoc muscorum and (b) Nostoc sp. Arrows indicatethe positionof the phycoerythrinbands.

N.

Homogenate

muscorum

N. m.

1

0.5

0.2

0.1

i ii:

N. sp. isolated from Yanaqocha

ili l

i'¸¸:,(;i'!

N. sp. 1

0,5

0.2 [pg protein]

0

0.1

Fig. 6. Standardizationofmonoclonalanti-PEagainstphycoerythrinsamples from Nostoc muscorum and Nostoc sp. The amountsof PE blottedin micrograms are indicated.

30

15

90

120

First fraction N, m.

:,

iil ,!i

i; i ~¸¸! ¸¸¸ > '~i:

4. Discussion One strategy of cells may be to avoid UV-B-induced damage instead of repairing it; however, protective mechanisms as well as UV-B-induced responses may not be sufficient to prevent damage to molecular targets such as proteins and nucleic acids. In the cyanobacteria A g m e n e l l u m q u a d r u p I i catum, A n a c y s t i s nidulans, S y n e c h o c o c c u s sp. and A n a b a e n a v a r i a b i l i s an intense photorepair activity as well as nucleotide excision repair and SOS activity were detected by analysis of survival curves after exposure to UV-B [30]. Ultraviolet radiation is known to affect many biochemical and physiological processes, including disruption of membrane structures, protein modification, proteolysis, enzyme inactivation, inhibition of transcription, mRNA stability and induction of DNA photoproducts and mutagenesis [31,32]. As a consequence, UVR may induce several pathways toward cell death when both protective and repair mechanisms are overloaded. Programmed cell death and apoptosis, which are actively regulated and require protein synthesis, are also induced by UVR [33]. The survival curve from both species studied here submitted to UVR followed a typical shoulder curve,

N. sp. 0

15

30

90

120

Phycobilisome fraction N. m.

I

I

0

15

I

I

I

N. sp. 30 90 [min]

120

Fig. 7. Dot-blot analysis of PE content of the protein fraction (in micrograms) after phycobilisomeextraction from Nostoc muscorum and Nostoc sp. submittedto UVR. The irradiationtimes are indicated.

R. Ar~Joz et al. / Journal of Photochemistry and Photobiology B: Biology 44 (1998) 175-183

indicating that a multi-hit phenomenon induced cell mortality. The results indicated that the DNA repair mechanisms, important to cope with enhanced UV-induced damage, are more efficient in the case of N o s t o c sp. than those in N. muscorum.

UV-B induces a decrease in the number of phycobilisomes in parallel with phycobilisome fragmentation, leading to phycobilisome substructures with different sedimentation coefficients. However, the increase in PE fluorescence emission not only reflects photodarnage but also a photoadaptation process. Phycobilisomes are light-harvesting complexes. The energy captured by PE565 is transmitted to PC6,), which in turn passes it to PC613 and afterwards to APC64o, which transfers the energy to PS II through the LcM polypeptide. Examination of fluorescence emission of dissociated phycobilisomes from different cyanobacteria has revealed several peaks, suggesting phycobilisome uncoupling at one or several sites of the energy-transfer chain [34]. UV-B excites the outermost phycobiliprotein PE; the excess energy, which cannot be transferred to PC, is released as fluorescence, accounting for the dramatic increase in PE fluorescence during UV irradiation. Following increasing exposure to UV-B, further damage to phycobilisome rods was evidenced by the shift of the fluorescence emission peak to shorter wavelengths, suggesting a structural change within the phycobilisomes. The parallel increase in the fluorescence at 650 nm is an indication of the energy overload. The increase in PE fluorescence (7 h for N. m u s c o r u m and 20 h for N. sp.) is also an indication of structural changes in the phycobilisome, e.g., PE accumulation on the rods induced by UV-B. Phycobiliprotein fluorescence under UVR was also studied in other cyanobacteria species [35,36]; in contrast to the N o s t o c strains analysed here, PE photobleaching takes place after shorter exposure times to artificial UV-B as well as to unfiltered solar radiation. A similar pattern of fluorescence increase/decrease and wavelength shift was observed when fluorescence emission spectra from isolated phycobilisomes were recorded (data not shown), suggesting that the stable protein composition within the phycobilisome structure from treated cells shown by gel electrophoresis does not necessarily mean that energy transfer is still functional. Even more, 2D-PAGE analysis of phycobilisomes extracted from artificial UVR-exposed cells [24] showed that the isoelectric point of the [3 PC subunit from N o s t o c sp. was shifted to basic pH values. The alteration of the electrical charge of the protein moiety explains the shift in the absorption and fluorescence emission of PC because the absorption of the bilin chromophore groups is strongly influenced by the conformation and the environment within the native polypeptide chain [37]. As a consequence, the energy cannot be transferred, protecting PS II from further photodamage. In order to address the question of whether the variations observed in PE are due to UV-B or not, a monoclonal antibody anti-phycoerythrin was used to follow the expression of PE under artificial UV-B. Consistent with fluorescence measurements in whole cells and isolated phycobilisomes

181

indicating accumulation of phycoerythrin following UV-B exposure, the dot-blot revealed the induction of PE synthesis reflected by the fact that its concentration within the phycobilisomes increased or remained stable and at the same time the accumulation of free PE in the first sucrose fraction increased for both species but to a different extent; PE concentration in N. sp. cells exposed to UV-B was significantly higher than in N o s t o c m u s c o r u m . In a context where transcription activity is repressed and proteins are degraded and/ or modified and the clonogenic capacity of the cells is seriously impaired, the induction of PE is lower than the response in N o s t o c sp. UV-B at low doses induces measurable damage to molecular targets such as DNA, mRNA and proteins, despite the existence of efficient UV absorbers in plants as well as in cyanobacteria [ 32,30 ]. Alternative UV-B screening systems may yield a better tolerance of organisms to UV-B. Phycoerythrin, as well as other phycobiliproteins, has a complex absorption spectrum with peaks at 280, 360 and 565 nm due to its protein-chromophore structure [37,38]. Phycobiliproteins can be excited by a broad band of wavelengths including UV-B, which causes excited states within the chromophores, and the energy is dissipated in the form of fluorescence. In addition, the phycobilisome concentration is very high in cyanobacterial cells. Phycobilisomes constitute up to 60% of the total soluble protein [39]; they are arranged over the thylakoid membranes which form several layers in the periphery around the DNA-containing centroplasm. These characteristics in association with its induction by UV-B make them a suitable absorber for UV-B radiation. Local populations as well as ecosytems may be adapted to natural fluctuations in UV-B within a certain range defined by the ozone column, solar elevation and scattering by air molecules. However, when ozone depletion brings the mean value of UV-B above its natural variation range, as is the case for certain regions of the planet, the potential biological effects might be important. Organisms living at highlands are additionally submitted to higher radiation due to the altitude effect. In fact, a 2 h exposure to unfiltered solar light at an altitude of ~ 1800 m above sea level under a cloudless sky induces as many pyrimidine dimers as 30 J m 2 UV-C radiation does in a cell culture [40]. N o s t o c sp. isolated from Yanaqocha has been shown to be more tolerant to UV-B than N o s t o c m u s c o r u m . In this paper we also present evidence that artificial UV-B induces PE synthesis and its accumulation in the phycobilisome, which increases cell tolerance against the detrimental effects of UV-B.

5. Abbreviations AE APC EDTA PAR

altitude effect allophycocyanin ethylenediaminetetraacetic acid photosynthetic active radiation

182

PC PE PMSF

SDS-PAGE UV-A UV-B UVR

R. Arfoz et aL / Journal of Photochemist~ and Photobiology B: Biology 44 (1998) 175-183

phycocyanin phycoerythrin phenylmethylsulfonyl fluoride sodium dodecyl sulfate polyacrylamide gel electrophoresis (315-400 nm) (280-315 nm) ultraviolet radiation

Acknowledgements

Rtmulo Ar~ioz was supported by a doctoral fellowship from the Deutscher Akademischer Austauschdienst (DAAD).

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