Photoinduction and photo-protective role of mycosporine-like amino acids
in the marine dinoflagellate Scrippsiella sweeneyae
海産渦鞭毛藻 Scrippsiella sweeneyae におけるマイコスポリン様アミノ酸の
光誘発性と光防御としての役割
00D5504
平良 ひとみ
指導教員
田口 哲
SYNOPSIS
マイコスポリン様アミノ酸（MAAs）の紫外線吸収物質としての役割を明らかにするために 、海洋の表層で
大量増殖する海産渦鞭毛藻 Scrippsiella sweeneyae を用いて紫外線照射実験を行った。MAAs の生合成は明期
の初めに最小、明期の中間において最大となり暗期に向けて減少していく日周性 を示し、渦鞭毛藻の日周鉛
直移動と同調した日周変化を示した。細胞内 MAA 含有量と細胞体積は、短波長紫外線（紫外線 B 域）や紫
外線照射強度に対して増加するという応答を示した。また紫外線照射下では、PSII 電子伝達の量子収率は減
少し、その減少は細胞内 MAA 量が高いほど低く抑えられ、細胞内 MAAs による防御性が高いことが示唆さ
れた。以上のことより S. sweeneyae は、紫外線照射によって細胞内により多くの MAAs を蓄積し、細胞内の
DNA までに透過する有害な紫外線の透過率を減少させる防御機構を保持していると示唆された。
Keywords: cell volume, dinoflagellate, DNA damage, mycosporine-like amino acids, ultraviolet radiation
Introduction
Reduced stratospheric ozone levels have caused to
increase levels of ultraviolet radiation (UVR), mainly
UVB (280-315 nm) incident on the surface of the Earth.
Solar UVB penetrates to biologically significant depths
within the euphotic zone in marine ecosystems.
Absorption of UVB photons causes organic molecules
such as nucleic acids, proteins, and pigments to undergo
conformational changes that subsequently interfere with
or destroy their ability to participate in vital metabolic
functions.
One possible mechanism to protect from those
disturbance is the synthesis of UV-absorbing compounds.
Most famous one has been known as mycosporine-like
amino acids (MAAs), which selectively absorb UVR
(310-360 nm) . Consequently, MAAs have long been
proposed to protect organisms from deleterious effect of
UVR. However, the evidence for screening role of
MAAs is limited. To provide the evidence, following two
capabilities; 1) inducibility and 2) resistibility of MAAs
due to UVR should be determined.
The regulation of MAA induction within
photosynthetic organisms has not been well understood.
The MAA induction has been postulated to be caused by
external light factors such as a total irradiance or
particular wavelengths within the solar spectrum e.g.,
photosynthetically available radiation (PAR: 400-700
nm), UVA (315-400 nm), and UVB. The cellular MAA
content in phytoplankton is positively related to the
amount of irradiance as an external light factor without
any relations among the intracellular physiological
properties. On the other hand, other physiological
properties such as pigments, cellular carbon, and cell
volume were entrained to each daily cycle. If MAA
production might be regulated by other physiological
properties possessing the intrinsic daily cycle, the
variation of cellular MAA content might reveal
conservative daily cycle by the internal factor without
relation to the amount of irradiance. As well as the link
between the effectiveness of protection by MAAs and
damages induced by UVR have been poorly
characterized.
The surface bloom forming dinoflagellates are known
as the appropriate experimental organisms to study the
protective mechanism under the effect of UVR in marine
ecosystem, since this taxonomic group is particularly
well adapted to marine environments experiencing high
PAR and UVR fluence rates and is also shown to posses
more inherent physiological trait of the capacity to
biosynthesize MAAs than other groups of phytoplankton.
Therefore, dinoflagellates are adequate to investigate the
link between the induction of MAAs and the protective
function of the induced MAAs by UVR.
This study was aimed at evaluating the screening role
of MAAs in dinoflagellate Scrippsiella sweeneyae,
which forms extensive red tides at sea surface in marine
coastal and estuarine waters. The objectives in this study
are (1) to elucidate the regulation factor of MAA
induction i.e., whether MAA induction could be
regulated by total irradiation of PAR and additional UVR
exposure as an external light factor or internal factor
revealing conservative variation pattern, (2) to
investigate the induction of MAAs in response to
changing intensities and spectral quality of UVR, and (3)
to examine the relationship between cellular MAA
content and UVR induced damage e.g., depletion of the
quantum yield of fluorescence for photosystem II (PSII).
1
Materials and Methods
Scrippsiella sweeneyae Balech (NIES-684) was obtained
from the National Institute of Environmental Science.
The algae were maintained in GPM medium containing
10-8 M sodium selenite prepared with filtered sea-water
at 25 o C and 30.8 W m-2 of cool white fluorescence light
on 12h light: 12 dark cycle.
First of all, MAAs in S. sweeneyae were identified by
HPLC analysis using authentic standards shinorine,
palythine, porphyra-334, and palythene, which were
obtained from the red alga Tichocarpus crinitus,
Chondrus yendoi, Porphyra yezoensis, and Palmaria
palmate, respectively. Mycosporine-glycine
was
obtained from cultures of the dinoflagellate Alexandrium
tamarense. Then the following experiments were carried
out.
were collected every day at the midday point of the light
cycle. They were analyzed for cell numbers, cell volume,
cellular Chl a, and MAA contents.
3. Identification of protective role of MAAs
To examine the relationship between cellular MAA and
UVR induced damage, UVR dose-response experiment
was performed.
Semi -continuous cultures with average cell density of
1600 ± 272 cells ml-1 were maintained at exponential
phase at high PAR (30.8 W m-2 ) in a 2 L bottle. To obtain
the different cellular MAA content, the incubation bottles
were covered by neutral density filters (NDF) and the
cultures were incubated for 6 h from beginning of the
light period. The light intensities were adjusted to 30.8
(HL), 15.2 (NDF1), and 7.7 W m-2 (NDF2). Then the
culture was placed in six quartz bottles in front of the
two UVR lamps under low background PAR (ca. 0.64 W
m-2 ) to prevent cell death during UVR exposure. Cells
were exposed to a constant UVR dose rate of 3.94 W m-2
(UVA: 1.43 W m-2 , UVB: 2.51 W m-2 ) for 12 min.
Sampling intervals corresponded to cumulative UVR
doses of 0, 0.47, 0.95, 1.42, 1.89, 2.36, and 2.84 kJ m-2 .
To ensure that the cultures were exposed to equal
amounts of light during incubation, the bottles were
rotated in a plankton wheel. Triplicate subsamples were
collected for the determination of cell numbers, cell
volume, cellular MAA contents, and fluorescence to
evaluate the reduction of the quantum yield of
fluorescence for PSII (Fv /Fm). To obtain the accurate data,
each experiment was conducted independently in
triplicate runs.
Sunscreen factor (S) depended on the wavelength (λ)
was estimated by the following equation,
1. Regulation factor of MAA induction
In Experiment 1, the response of cellular MAA content
to high PAR (HL: 30.8 W m-2 ) exposure was studied.
Cells acclimated to low PAR (LL: 7.7 W m-2 ) were
suddenly exposed to HL every 2 h from time zero to 10th
h of light period until the end of the 12 h light period.
Cultures exposed to HL were defined as HL 1 to HL 6
culture, respectively. LL culture was used as the control
for a comparison with the HL cultures. Duplicate
subsamples for cell number and cellular MAA contents
were harvested every 2 h for each culture.
In Experiment 2, the effect of sudden UVR exposure
on cellular MAA content in cells acclimated to HL was
investigated. Exponentially growing cells acclimated to
HL were cultured in two 16 L UV transmittable plastic
containers (>285 nm). Cellular MAA content was
monitored for 60 h of the acclimation process started at
midday of the beginning of exponential growth phase.
During the light period both UVA (0.80 W m-2 ) and UVB
(0.89 W m-2 ) were irradiated with PAR for 12 h with
Toshiba fluorescence tubes Model FL202E covered with
acetate film (<290 nm cut-off). Growing cultures under
HL exposure only was used as a control. Duplicate
subsamples
were
collected
for
microscopic
determinations of cell numbers and cell volume, HPLC
analyses of cellular Chl a and MAA contents, and flow
cytometry analysis of cell cycle every 2 h for 60 h.
S( λ) = 1−
Td (λ ) − 1
LnTd (λ )
(1)
where Td (λ) is the value of the transmittance at the distal
end of a cell with diameter d (µm). Td (λ) was calculated
from the following equation,
 V
d
Td (λ ) = exp− E ⋅ a rec [λ]⋅ 
l
 Vcell
(2)
where VE is the extraction volume (1 ml), Vcell is cell
volume (µm3 ), arec(λ) is the reconstructed absorption per
cell (µm2 cell-1 ) of total cellular MAAs and l is the quartz
cuvette length (1 cm). arec(λ) was calculated from
individual cellular MAA content and molar extinction
coefficients for each MAA.
Multiple linear regression analysis was conducted to
determine contribution of cellular MAA content and cell
volume to S value.
2. Photoinduction of MAAs by different spectral
compositions and intensities of UVA and UVB
The photoinduction of MAAs was examined under PAR
with UVR in exponentially growing cultures without a
loss in cellular growth rate and physiological activity.
Exponentially growing PAR-only (20.7 W m-2 )
cultures were subjected to four different combinations of
PAR and UVR (>410 nm, >313 nm, >290 nm, and >280
nm treatment) and five different intensities of UVR. The
UVR exposure was run over a period of logarithmic
growth phase that lasted 2 days. Triplicate subsamples
Results and Discussions
HPLC analysis of MAAs indicated the presence of a
mixture of MAAs in S. sweeneyae. Of these, five MAAs,
2
shinorine (λmax = 334 nm), mycosporine-glycine (λmax =
310 nm), palythine (λmax = 320 nm), porphyra-334 (λmax
= 334 nm), and palythene (λmax = 360 nm) were
identified.
cellular MAA content was not disrupted by UVR
exposure, indicating that this variation was not affected
by the spectral quality of light.
Cell cycle was also well phased to the light:dark cycle
and a distinct daily variation was observed for the
fraction of G1 , S, and G2 (Fig. 2). The each phase of cell
cycle in the UVR treatment was not disrupted by UVR
exposure, since similar variation in the cell cycle was
observed in the control. Daily variation of cellular MAA
content did not synchronized with that of cell volu me,
cellular Chl a content, and cell cycle (Fig. 1 and Fig. 2).
These results indicated that cellular MAA content
revealed independent daily variation from other
physiological properties in S. sweeneyae (Taira et al.
2004a).
1. Regulation factor of MAA induction
Experiment 1
The increase of cellular MAA content by HL was
observed in the first three cultures (HL1-3 cultures),
which were transferred to HL within 4 h. In contrast,
when the next three cultures past 6 h under LL were
transferred to HL (HL 4-6 cultures), no enhancement of
cellular MAA content due to HL was observed. In other
words, the MAA induction was not always occurred
anytime in the light period. This indicated that the timing
that cell could percept the HL signal was restricted.
Although the HL exposure could induce MAAs, MAA
induction might not be completely regulated by external
light factor.
Frequency (%)
100
Experiment 2
The cell volume and cellular Chl a content continuously
increased during the light period, and decreased during
dark period (Fig. 1a and b). The cell division also was
phased to the light:dark cycle and occurred during the
dark period. Those cycles were well phased each other.
However, the daily cycle of cellular Chl a content might
be disrupted by UVR exposure, because no pattern of
variability were discernible in the UVR treatment.
Cellular MAA content in both treatments began to
increase from dawn to maxima at midday, consequently
decreased toward to dusk (Fig. 1c). This repeatable
variation pattern indicated that cellular MAA content was
entrained to the daily cycle. This daily variation of
0
Cell volume x 103
(µm3)
Cellular Chl a concentrations
(pg Chl a cell-1 )
Concentration of total MAAs
(pmol MAA cell-1)
1.0
60
72
12
24
36
48
60
72
12
24
36
48
60
72
Frequency (%)
30.0
20.0
10.0
0.0
0
c
0.8
0.5
0.2
0.0
0
60
72
36
48
60
72
50
25
2. Photoinduction of MAAs by different spectral
compositions and intensities of UVA and UVB
Average cell densities at time zero were 392 ± 55 cells
ml-1 in all experiments. After 2 day UVR exposure, cell
densities increased to 1390 ± 251 and 1563 ± 147 cells
ml-1 in the PAR control and UVR treatments, respectively.
Growth rates did not differ appreciably between the UVR
treatments and control. Average cellular Chl a content at
time zero were 27.5 ± 6.6 pg Chl a cell-1 in all
experiments. Even after 2 day UVR exposure, cellular
Chl a content remained almost unchanged. Average cell
volume at time zero were 9.85×103 ± 153 µm3 in all
experiments. Cell volume increased significantly with
increasing UVR intensities and including shorter
wavelengths of UVR. This cell volume increase ranged
from 10 to 130 % over the controls . The highest increase
of 23.0 × 103 µm3 was obtained in the >280 nm treatment
at the highest intensity of UVR. Regulations in cell size
are considered to be a photoprotective strategy against
UVR and supraoptimum PAR where they become
detrimental in cell physiology and growth. An increase in
cell volume results in an increase in the pathlength of
light traveling through the cells. Such an increase in the
pathlength can afford the protection against UVR by
b
40.0
24
48
Figure 2. Percentages of S. sweeneyae i n G1 (circle), S (square) and G2
(triangle) phase of the cell cycle in the control (a) and UVR treatment (b),
respectively. Shade area indicates dark period.
0.0
48
12
36
Time (h)
5.0
36
24
b
75
0
10.0
24
12
0
a
12
25
100
15.0
0
50
0
20.0
50.0
a
75
Figure 1. Pattern of daily change in cell
volume
Time
(h) (a), daily change in cellular
Chl a content (b), and cellular MAA content (c) for 60 h. Circle and triangle
show the control and UVR treatment, respectively. Shade area indicates dark
period.
3
protecting sensitive sites, such as cellular DNA from the
harmful effects of UVR.
Average total cellular MAA content at time zero were
0.514 ± 0.114 pmol MAA cell -1 in all experiments. MAA
induction increased significantly with increasing UVR
intensities (Fig. 3b, c, and d). The induction of MAAs
also increased appreciably with UVR incorporating
shorter wavelengths. In the >280 nm treatment, the
highest MAA induction of 1.74 pmol total MAA cell-1
was revealed at highest UVR intensity. Those most
prominent increases were observed for the major MAAs
such as shinorine and mycosporine-glycine (Fig. 3d).
In S. sweeneyae both increases in cellular MAA
contents and cell volume due to UVR, without
compromising cell growth, might suggest that DNA was
protected from the harmful effects of UVR via a
two-pronged strategy of an increase in cell size and the
accumulation of MAAs within the cells (Taira et al.
2004b).
0.6
0.5
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
0.5
20.0
0.7
25.0 30.0 35.0
PAR (W m-2 )
c
0.6
0.6
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.5
1.0
1.5
UVB (>290 nm) (W m-2 )
0.0
0.0
b
95
75
90
85
80
50
25
75
0
70
0.00
1.00
2.00
3.00
0
0.60
0.0
UVR dose (kJ m - 2)
d
shinorine
mycosporine
-glycine
palythine
0.65
0.70
0.75
0.80
0.85
S (323)
Figure 4. (a): Effect of UVR on the optimum quantum yield for PSII (Fv/Fm ) in
Scrippsiella sweeneyae in HL ( ), NDF1 ( ), and NDF2 treatment ( ). Fv/Fm
was normalized at initial value. Vertical bars indicate one standard deviation (SD).
The solid line is a least square regression fit; y = -2.96x + 1 00, r2 = 0.984 for HL
treatment; y = -4.83x + 100, r2 = 0.986 for NDF1 treatment; y = -7.53x + 100, r2 =
0.996, n = 7 for NDF2 treatment. (b): Relationship between S (323) and damage
index obtained in HL ( ), NDF1 ( ), NDF2 treatment ( ). Vertical and
horizontal bars indicate one standard deviation (SD). The solid line is a least
square regression fit; y = -314x + 307, r2 = 0.997, n = 3.
1.0
1.5
2.0
UVA (W m-2)
0.7
0.5
0.0
b
100
a
Damage index (%)
0.7
a
0.6
Cellular MAA content (pmol cell-1)
100
Ratio of experimental to initial vF/Fm (%)
0.7
NDF1, and NDF2 treatment. S(323) values were 0.84 ±
0.00, 0.79 ± 0.01, and 0.66 ± 0.01 for HL, NDF1, and
NDF2 treatment, respectively.
NDF2 treatment which possessed the lowest S(323)
revealed the highest decrease in the normalized Fv /F m to
one at time zero during UVR exposure (Fig. 4a).
Decrease in the relative Fv /Fm was normalized to the
maximum decrease in NDF2 treatment, and defined as
the damage index. The damage index decreased
significantly with S(323) (Fig. 4b). Multiple linear
regression analysis revealed that only cellular MAA
content contributed significantly to S(323). These results
indicated that cellular MAA content might contribute
most significantly to the screening capability from
harmful UVR exposure.
porphyra
-334
palythene
Conclusions
For the bloom forming dinoflagellates near the surface
such as Scrippsiella sweeneyae, they might possess
evolved mechanisms for UV protection. One of the
evolved mechanisms was evidenced by the role of
MAAs as sunscreen compounds. Dinoflagellate could
protect from harmful UVR by inducing cellular MAAs
when they migrate to well lit sea surface at daytime. By
regulating cellular MAA content and cell volume, they
can effectively adapt to the different UVR intensity and
spectral composition due to sea water mixing, weather,
and changing of stratospheric ozone concentration. The
cellular MAA content could reduce the transmittance of
light impinging on individual cell, thus mitigating
possible UVR-mediated damage. These observations
indicated that MAAs could play the sunscreen role in S.
sweeneyae.
0.5
1.0
1.5 2.0
UVB (>280 nm) (W m-2)
Figure 3. Induction of MAAs in S. sweeneyae by different light intensities, (a)
>410 nm, (b) > 313 nm, (c) >290 nm, and (d) >280 nm treatments at Day 2 of
UVR exposure. Shinorine, mycosporine-glycine, palythine, porphyra-334, and
palythene are indicated with circles, diamonds, triangles, squares, and inversed
triangles, respectively.
3. Identification of protective role of MAAs
The different cellular MAA contents and cell volumes
were obtained among the three PAR treatments. Average
cellular MAA contents were 0.425 ± 0.005, 0.306 ±
0.010, and 0.176 ± 0.007 pmol cell-1 for HL, NDF1, and
NDF2 treatments, respectively. Cellular MAA content
was not enhanced during the UVR exposure in each
treatment. Cell volume increased to the maximum of
12 % during UVR exposure in all treatments. To
standardize the role of MAAs as sunscreen factor (S), S
was estimated. The S is defined as the fraction of the
incident radiation that is prevented from impinging upon
cell matter. The S varies between 0 (no sun-screen effect)
and 1 (all photons are prevented from hitting cell matter).
Estimated S at 323 nm (S[323]) did not change
significantly during UVR exposure of each treatment.
However, average S(323) were different among HL,
References
Taira, H., Aoki, S., Yamanoha, B. and Taguchi, S. (2004a) Journal of
Photochemistry and Photobiology B: Biology. 75:145-155.
Taira, H., Goes, I. J., Gomes, H. R., Yabe, K. and Taguchi, S. (2004b)
Plankton Biology and Ecology. 51:82-94.
4