UV-INDUCED DNA DAMAGE IN INTERTIDAL MARINE MACROALGAE FROM
THE COAST OF VALDIVIA, SOUTHERN CHILE, IN SUMMER
R. Rautenberger1, P. Huovinen1, I. Gómez1
1
Facultad de Ciencias Marinas y Limnológicas, Universidad Austral de Chile, Valdivia, Chile.
rrautenberger@uach.cl
Introduction
Ultraviolet radiation (UVR: 280-400 nm) is an abiotic environmental factor that may cause harmful damages to
many biologically relevant molecules and structures in macroalgal cells. UV-B radiation (280-315 nm) is absorbed
by DNA, inducing different types of mutagenic lesion by distorting the DNA helix: cyclobutane pyrimidine dimers
(CPDs) and 6-4 photoproducts (6-4 PPs) are formed with a ratio of 3:1. These damages restrict both replication and
transcription of DNA by hindering the progression of polymerases, which results in severe metabolic limitations
when they are not fully repaired.
Algae that are able to repair UV-induced DNA damages–even under UV-B exposure–can be regarded as UVtolerant. Macroalgae from the intertidal, which are regularly exposed to high irradiances of UV-B radiation in
summer, are generally tolerant to UV stress because they possess an effective UV shielding that attenuates the
penetration of UVR into the cells and/or have mechanisms that repair UV-induced damages efficiently (Gómez and
Huovinen 2010, Rautenberger et al. 2015).
The aim of our study was to study the UV tolerance of the most common intertidal macroalgae at the coastline of
Valdivia in summer on the molecular basis using DNA damage. Our results can help to broaden our understanding of
macroalgal strategies to tolerant UV stress.
Material and methods
Seven macroalgal species (Table 1) were collected from the rocky intertidal coast at Corinañco, near Valdivia, Chile,
in January 2015 and transported in a dark and cooled box to the laboratory at Universidad Austral de Chile in
Valdivia. After all macroalgae were carefully cleaned, they were acclimated to the laboratory conditions (40 µmol
photons m-2 s-1, 15 °C) for 24 h prior to the experiment.
For the radiation treatment, macroalgae were exposed to photosynthetically active radiation alone (PAR: 400-700
nm: 40 µmol m-2 s-1) as control and PAR + UVR (UV-A, 280-320 nm: 1.51 W m-2 + UV-B, 320-400 nm: 0.26 W m2
) for 24 h at the local summer seawater temperature of 15 °C. At the end of the experiment (24 h), samples of each
species were frozen in liquid nitrogen and stored at -80 °C until analysis.
CPDs and 6-4 PPs induced by the UV treatment were determined using an ELISA test (Mori et al. 1991, Gómez
and Huovinen 2010). Algal samples were ground in a mortar with liquid N 2 to a fine powder. DNA was extracted
using 2% (w/v) CTAB buffer containing 1.4 mM NaCl, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 0.2% (v/v)
β-mercaptoethanol at 37 °C for 30 min and, subsequently, with chloroform:isoamylalcohol (24:1). After DNA was
precipitated by 100% isopropanol and washed (76% ethanol containing 10 mM ammonium acetate), it was dissolved
in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Purified DNA was quantified photometrically (Varioskan
Spectrum, Thermo Scientific, USA) at 260 nm. Heat-denatured DNA samples (4 mg DNA mL-1; 10 min at 99 °C)
were applied to microplate wells and dried (90 °C). The plates were washed with phosphate-buffered saline-Tween
buffer (PBS-T) and blocked with 3% (w/v) BSA. After BSA was removed by washing (PBS-T), a primary antibody
(monoclonal anti-CPDs or anti-6-4 PPs antibodies, Kamiya Biomedical Company, Seattle, USA) was added and
incubated (30 min at 37 °C). A secondary antibody (monoclonal rabbit antimouse antibody conjugated with horse
radish peroxidase) was added after washing (PBS-T) and incubated (30 min at 37 °C). For detection of CPDs and 6-4
PPs, o-phenylenediamine–H2O2 was added and incubated (20-25 min at 37 °C). Colour reaction was stopped with
2M H2SO4. Absorbance, which was normalised to 4 mg DNA mL-1 in all samples, was measured at 492 nm
(Varioskan Flash, Thermo Scientific, USA).
Means and standard deviations (SDs) were calculated from three replicates per treatment (n = 3). Normal
distribution of raw data and residuals were tested by the Shapiro-Wilk W test. Levene’s test was performed to test on
homoscedasticity. If variances were equal, Student’s t-test was conducted to identify statistically significant
differences in absorbances at 492nm between controls (PAR alone) and UV treatments (PAR + UVR). Otherwise, a
1
Welch t-test was performed. A 5 % significance level (P = 0.05) was applied in all statistical tests performed using
the software package JMP 11.0 (SAS Institute Inc., Cary, North Carolina, USA).
Table 1: Macroalgal species used in the experiment, their taxonomic position and morpho-functional groups.
Species
Taxonomic position (order, class, phylum)
Mazzaella laminarioides (Bory de
Saint-Vincent) Fredericq
Sarcothalia crispata (Bory de SaintVincent) Leister
Pyropia columbina (Montagne)
W.A.Nelson
Lessonia nigrescens Bory de SaintVincent
Macrocystis pyrifera (Linnaeus)
C.Agardh
Durvillaea antarctica (Chamisso)
Hariot
Ulva sp.
Gigartinales, Florideophyceae, Rhodophyta
Morpho-functional groups of
thalli
Thick leathery
Gigartinales, Florideophyceae, Rhodophyta
Thick leathery
Bangiales, Bangiophyceae, Rhodophyta
Sheet-like
Laminariales, Phaeophyceae, Ochrophyta
Thick leathery
Laminariales, Phaeophyceae, Ochrophyta
Thick leathery
Fucales, Phaeophyceae, Ochrophyta
Thick leathery
Ulvales, Ulvophycaea, Chlorophyta
Sheet-like
Results
An increase in absorbance at 492 nm (Abs492nm), which indicates DNA damage either by formation of CPDs or 6-4
PPs, was detected in four out of the seven intertidal macroalgae exposed to UVR for 24 h. In the brown macroalga
Lessonia nigrescens, a significant increase in Abs492nm was only measured for CPD formation in PAR + UVRexposed specimens compared to PAR-controls (P = 0.011, Student’s t-test). In Macrocystis pyrifera, contents of both
CPDs and 6-4 PPs increased by 75.8% (P = 0.0001, Student’s t-test) and 10.6% (P = 0.020, Student’s t-test),
respectively, due to UV stress. In contrast, no DNA lesions were detected in Durviallaea antarctica (Fig. 1A and B).
Out of the three red macroalgae, Pyropia columbina was the only species that showed a significant increase in
Abs492nm by 64.2% for CPDs (P = 0.0001, Student’s t-test; Fig. 2A and B). However, an unexpected decrease in
Abs492nm in UVR-exposed samples compared to PAR-controls was detected in Sarcothalia crispata (Fig. 2B).
In the specimens of the green macroalga Ulva sp., Abs492nm remained unchanged for both CPDs and 6-4 PPs (Fig.
3).
0.7
0.7
Abs492nm (a.u.)
0.6
*
A
PAR
PAR + UVR
0.6
0.5
0.5
0.4
0.4
0.3
0.3
0.2
B
*
0.2
*
0.1
0.1
0.0
0.0
Lessonia nigrescens
Macrocystis pyrifera
Lessonia nigrescens
Durvillaea antarctica
Macrocystis pyrifera
Durvillaea antarctica
Species
Species
Figure 1: Formation of (A) cyclobutane pyrimidine dimers (CPDs) and (B) 6-4 photoproducts (6-4 PPs) in the
intertidal brown macroalgae Lessonia nigrescens, Macrocystis pyrifera, and Durvillaea antarctica after 24 h of
exposure to PAR (light columns) and PAR + UVR (dark columns) at 15 °C. Asterisks denote statistically significant
2
differences (P > 0.05) between PAR-control and treatments with UV radiation (i.e. PAR + UVR). Absorbance at 492
nm (Abs492nm) of all samples was normalised to 4 mg DNA mL-1.
0.7
0.7
PAR
PAR + UVR
A
0.6
Abs492nm (a.u.)
0.5
B
0.6
*
0.5
*
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
Mazzaella laminarioides Sarcothalia crispata
Mazzaella laminarioides Sarcothalia crispata
Pyropia columbina
Pyropia columbina
Species
Species
Figure 2: Formation of (A) cyclobutane pyrimidine dimers (CPDs) and (B) 6-4 photoproducts (6-4 PPs) in the
intertidal red macroalgae Mazzaella laminarioides, Sacrothalia crispata, and Pyropia columbina after 24 h of
exposure to PAR (light columns) and PAR + UVR (dark columns) at 15 °C. Asterisks denote statistically significant
differences (P > 0.05) between PAR-control and treatments with UV radiation (i.e. PAR + UVR). Absorbance at 492
nm (Abs492nm) of all samples was normalised to 4 mg DNA mL-1.
0.7
0.6
PAR
PAR + UVR
Abs492nm (a.u.)
0.5
0.4
0.3
0.2
0.1
0.0
6-4 photoproducts
CPDs
Damage
Figure 3: Formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) in the intertidal
green macroalga Ulva sp. after 24 h of exposure to PAR (light columns) and PAR + UVR (dark columns) at 15 °C.
Absorbance at 492 nm (Abs492nm) was normalised to 4 mg DNA mL-1.
Discussion
3
Tolerance of marine macroalgae to high irradiances of solar UV radiation is crucial for their survival and therefore
controls their vertical zonation in the field (Bischof et al. 1998). Macroalgae from higher tidal positions such as the
intertidal, which are frequently exposed to UV stress, are more tolerant to UV stress than species or even
conspecifics from lower positions on the shore, e.g. subtidal (Rautenberger et al. 2013). This can be attributed to an
increased UV shielding by UV-absorbing compounds and an efficient repair of UV-induced damages (Gómez and
Huovinen 2010, Rautenberger et al. 2015).
Intertidal macroalgae form the coast of Corinañco near Valdivia show a species-specific damage of DNA due to
UV stress. The brown macroalgae Macrocystis pyrifera exhibits the highest degree of DNA damage with formation
of both CPDs and 6-4 PPs as the only out of seven species. Therefore it can be characterised as highly UV sensitive
on molecular level, which might be attributed to ineffective UV shielding by phlorotannins and/or photorepair of
damaged DNA during UV exposure. The 20% increase in CPDs in Lessonia nigrescens is in accordance with
previous studies in which the same degree of formation of CPDs was detected (Gómez and Huovinen 2010). There it
has been shown that an increased UV screening due to UV-induced formation of phlorotannins prevented DNA (and
photosynthesis) from further damages and so, it probably did in our study. The red macroalgae Pyropia columbina is
characterised by high contents of MAAs, regardless if they are sun or shade-types. Although these MAAs are rapidly
induced or, were already high in P. columbina collected in summer, they could not protect DNA effectively, just
provide a basic UV protection. The other macroalgae, however, did not show any formation of DNA lesions due to
UV stress, indicating either an effective UV shielding and/or an efficient repair. The latter might be the case in Ulva
sp. because this species does not produce any UV shielding, indicating an efficient repair of damaged DNA
(Pescheck et al. 2010, 2014).
To understand UV tolerance of marine macroalgae it is important to include detection of DNA damage and put it
in relation to their UV shielding and their capability to repair UV-damaged damaged. This molecular study helps to
understand the physiological basis why macroalgae are able to survive UV stress in summer.
References
Bischof K, Hanelt D, Wiencke C (1998) UV-radiation can affect depth-zonation of Antarctic macroalgae. Mar Biol
131:597–605
Gómez I, Huovinen P (2010) Induction of phlorotannins during UV exposure mitigates inhibition of photosynthesis
and DNA damage in the kelp Lessonia nigrescens. Photochem Photobiol 86:1056–1063
Mori T, Nakane M, Hattori T, Matsunaga T, Ihara M, Nikaido O (1991) Simultaneous establishment of monoclonal
antibodies specific for either cyclobutane pyrimidine dimer or (6-4)photoproduct from the same mouse
immunized with ultraviolet-irradiated DNA. Photochem Photobiol 54:225–232
Rautenberger R, Wiencke C, Bischof K (2013) Acclimation to UV radiation and antioxidative defence in the
endemic Antarctic brown macroalga Desmarestia anceps along a depth gradient. Polar Biol 36:1779–1789
Rautenberger R, Huovinen P, Gómez I (2015) Effects of increased seawater temperature on UV tolerance of
Antarctic marine macroalgae. Mar Biol (in press), DOI: 10.1007/s00227-015-2651-7
Pescheck F, Bischof K, Bilger W (2010) Screening of UV-A and UV-B radiation in marine green macroalgae
(Chlorophyta). J Phycol 46:444–455
Pescheck F, Lohbeck KT, Roleda MY, Bilger W (2014) UVB-induced DNA and photosystem II damage in two
intertidal green macroalgae: Distinct survival strategies in UV-screening and non-screening Chlorophyta. J
Photochem Photobiol B: Biol 132:85–93
4