Photoacclimation of antarctic marine diatoms to solar ultraviolet radiation

Journal of Experimental Marine Biology and Ecology,

ELSEVIER

204 (1996) 85-101

Photoacclimation E. Walter Helbling”, “PolarResearch ‘Australian

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

of antarctic marine diatoms to solar ultraviolet radiation

Bruce E. Chalkerb, Walter C. Dunlapb, Osmund HolmHansena,* , Virginia E. Villafafie

Scripps Institution of Oceanography, Universiry of California at San Diego, La Jolla, CA, 9209.3-0202, USA Institute r?f Marine Sciences, PMB No. 3, Townsville M.C., Queensland 4810, Australia Program,

Received IO August 1995; revised 30 January

1996; accepted

29 February

1996

Abstract The present study was carried out at Palmer Station (64.7” S, 64.1” W), Antarctica, during the austral spring-summer of the years 1993 and 1994. Two centric diatom species (Thulussiosiru sp. and Corethron criophilum Castracane) and two pennate species (Pseudonitzschia sp. and Frugiluriopsis cylindrus (Grunow) Krieger) were isolated from natural phytoplankton assemblages and exposed to solar radiation to study long term (more than 1 week) photoacclimation to ultraviolet radiation (UVR). At the beginning of the experiments, three of the cultures had relatively low concentrations of UV-absorbing compounds (i.e., mycosporine-like amino acids) and photosynthetic rates were significantly inhibited by UVR. At the end of the experiments (8- 12 days), however, the two centric diatom species had high contents of mycosporine-like amino acids (MAAs) and did not show any significant differences in photosynthetic rates when exposed to either UVR + PAR or just to PAR. The synthesis of MAAs was slightly less when samples were exposed only to PAR than when exposed to UVR in addition to PAR. The rates of synthesis of MAAs, relative to phytoplankton carbon, for the two centric diatoms were 0.001 and 0.008 p.g of MAAs . (pg C) ’ . day ’ for shinorine and porphyra-334, respectively. The concentrations MAAs in Pseudonitzschiu sp., and Ftqiluriopsis cylindrus at the end of the experiments were much lower (less than one tenth) than that in the centric diatoms and the cultures were still inhibited by UVR. In the pennate diatoms MAAs increased in concentration as a response only to UVR and not to PAR. The loss rates of MAAs in Thalussiosiru sp. after transferring the culture from high (1200 pE.m-‘.ss ) to low irradiance (250 pE. mm’ s I) were 0.0002 and 0.0023 pg MAAs.(pg C)~ ’ .dayy for shinorine and porphyra-334, respectively. These results provide further evidence that MAA compounds are synthesized in response to high light conditions and that they do decrease the photoinhibitory effects of UVR. Keywords:

*Corresponding

UVR; Diatoms; Photosynthesis;

MAAs; Photoacclimation

author. Fax: ( + I-61 9) 534-73 13; e-mail: [email protected]

0022-0981/96/$15.00 0 1996 Elsevier PII SOO22-098 I (96)0259 l-9

Science B.V. All rights reserved

1. Introduction Short term studies (1 day or less) done with Antarctic phytoplankton (Helbling et al., 1992; Smith et al., 1992; Holm-Hansen et al., 1993; Prezelin et al., 1994) have shown that rates of photosynthesis are significantly decreased by exposure to solar ultraviolet radiation (UVR, < 400 nm). Various species of Antarctic phytoplankton have shown different sensitivities to UVR, with diatoms generally being more resistant to UVR than flagellates and dinoflagellates (Davidson and Marchant, 1994; Helbling et al., 1994; Karentz, 1994). One of the mechanisms whereby phytoplankton can decrease the damage incurred by UVR is by the synthesis of mycosporine-like amino acids (MAAs) which absorb UVR (Carreto et al., 1990; Karentz et al., 1991 a; Karentz, 1994: Vernet et al., 1994). There are many reports in the literature supporting the view that these compounds can act as a protective mechanism against UVR (Dunlap and Chalker, 1986; Dunlap et al., 1989, 1995). Long term (l-2 wk) cultures of natural assemblages of Antarctic phytoplankton have shown that relatively high concentrations of MAAs can be synthesized by phytoplankton after 1 wk of exposure to UVR (Villafane et al., 1995). These authors also showed that there was a change in species composition, from flagellates to diatoms, in samples that were exposed to UVR; treatments not exposed to UVR were still dominated by flagellates. That study led us to further investigate the photoacclimation of marine diatoms and to determine if both pennate and centric diatoms are equally resistant to UVR, and also if species from both groups can synthesize MAAs in response to UVR exposure. In this paper we present data on such photoacclimation of unialgal cultures of Antarctic diatoms (two centric and two pennate) after exposure to natural solar radiation.

2. Materials and methods The study was carried out at Palmer Station (64.7” S, 64.1” W), Antarctica, during the austral spring-summer (October-December) of 1993 and 1994. Two centric diatom species (Thalussiosiru sp. and Corethron criophilum Castracane) and two pennate diatom species (Pseudonitzschiu sp. and Fragilm-iopsis c$indrus (Grunow) Krieger). were isolated from natural phytoplankton assemblages and grown in f/2 medium (Guillard and Ryther, 1962). The cultures were kept in illuminated incubators at a temperature between 0 to 2 “C, with the illumination being provided by daylight fluorescent tubes (250 pJ_E.m ’ s ’ ) with a light:dark period of 15:9 h. Before being used in any experiment, the cultures were placed in 4 1 polycarbonate bottles (not aerated but shaken occasionally) and exposed to natural solar radiation for 12 h in an outside water bath with flowing seawater to maintain temperature between 0 to 2 “C. After this period, the cultures were transferred to 2 1 round quartz vessels and exposed to natural solar radiation under two experimental treatments: ( 1) vessel with no optical pre-filter so that the cultures received both UVR ( < 400 nm) and Photosynthetic Available Radiation (PAR, 400-700 nm), and (2) vessel covered with a Plexiglas UF-3 tilter, which allowed the samples to receive only PAR.

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At the beginning and at the end of each experiment (which lasted between 8 to 12 days), a subsample (about 350 ml) was drawn from each vessel to determine the impact of UV-B (280-320 nm) and UV-A (320-400 nm) radiation on photosynthetic rates, as measured by radiocarbon incorporation during a short term incubation period (6-8 h centered around local noon). This was done by transferring the samples to 50 ml quartz tubes, and after the addition of 5 pCi (0.185 Mbq) of NaHr4C0, to each tube, the cultures were exposed to solar radiation under three different treatments: (a) samples that received all UVR in addition to PAR (quartz tubes), (b) samples that received UV-A and PAR (quartz tubes wrapped in Mylar film which has 50% transmission at 323 nm) and (c) samples that received only PAR (quartz tubes covered with a Plexiglas UF-3 filter, with 50% transmission at 400 nm). During all incubations triplicate samples were used for each treatment. After the incubation period the samples were filtered through Whatman GF/F glass fiber filters (25 mm), the filters placed in scintillation vials, exposed to HCl fumes for 3-4 h, and then dried overnight in a vented hood. The amount of 14C incorporated by the samples was determined by standard liquid scintillation counting techniques. In order to test the differences in the rate of photosynthesis statistically between treatments, a non-parametric Kruskal-Wallis test was applied to the data, and when there were significant differences (P < 0.05), an a posteriori test (Nemenyi procedure) was performed (Zar, 1984). Growth of the cultures in the 2 1 quartz vessels was monitored by measurement of chlorophyll-a (chl-a), which was done every day by filtering a variable amount of sample (lo-50 ml) through a GF/F glass fiber filter, extracting chl-a in absolute methanol (Holm-Hansen and Riemann, 1978) for at least 1 h, and using fluorometric techniques to determine chl-a concentrations (Holm-Hansen et al., 1965). Samples (15-25 ml) of the cultures were also taken every other day and fixed with boratebuffered formalin (final concentration of formaldehyde was 0.4%) for floristic analysis, which was carried out using an inverted microscope (Utermohl, 1958). The phytoplankton carbon content was obtained by applying the equations of Strathmann (1967) to the cell volumes (Kovala and Larrance, 1966). Samples of variable volume ( 100-200 ml) were drawn from each culture every l-3 days, filtered through a GF/F filter (25 mm) and the filter with the particulate material extracted in 10 ml of absolute methanol at 4 “C overnight. After centrifugation, the clear supernatants were used for analyses and quantification of UV-absorbing compounds, which was done by both absorption spectra and high-performance liquid chromatography (HPLC) techniques. The absorption spectra of the extracts (250 to 750 nm) were determined using a 10 cm pathlength quartz cuvette and a Perkin Elmer Lambda 6 UV-VIS Spectrophotometer with automatic baseline correction. For HPLC analyses the methanol extracts were concentrated IO-fold by drying 2.0 ml of sample extract (2 X 1.0 ml, successively) using a Savant (Model DNA100 Speedvac) rotary vacuum concentrator in 1.5 ml sample tubes and redissolving the dry residue in 200 pl of 80% methanol with the aid of an Eppendorf (model 5432) vortex mixer. Suspended insoluble material was removed using an Eppendorf model 5415C microcentrifuge (5 min at 14 000 rpm). MAAs were separated and quantitated by reversephase, isocratic HPLC as previously described (Dunlap and Chalker, 1986; Dunlap et al., 1989; Karentz et al., 1991a; Stochaj et al., 1994) using a Brownlee RP-8 column

88

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(Spheri-5, 4.6 mm I.D. X 25 cm) protected with an RP-8 guard cartridge (Spheri-5, 4.6 mm I.D. X 3 cm). Strongly acidic MAAs (shinorine, porphyra-334 and mycosporine2Gly) were quantified using an aqueous mobile phase consisting of 55% methanol and 0.1% acetic acid (0.8 ml/min) and the more weakly acidic MAAs (mycosporine-Gly: Val, palythine, asterina-330, palythinol and palythene) were quantified using a mobile phase consisting of 25% methanol and 0.1% acetic acid (0.8 ml/min). MAAs were identified by comparison with authenticated standards (prepared by W.C. Dunlap) and quantified by dual wavelength absorbance at 3 13 nm and 340 nm (Waters model 440 detector) and dual channel, peak-area integration (Spectra-Physics model 4400 integrator). Mycosporine-Gly was best analyzed by ion-exchange chromatography (Dunlap and Yamamoto, unpubl.) on a Brownlee bonded-phase amino column (4.6 mm I.D. X 25 cm) protected with a Brownlee amino cartridge (4.6 mm I.D. X 3 cm) using a mobile phase consisting of 40 mM ammonium acetate and 17.5 mM acetic acid in aqueous methanol (80%). However, only variable quantities of Mycosporine-Gly in trace concentrations were found throughout this study (data not presented). During the study period, natural solar radiation was monitored continuously with a spectroradiometer (PUV-5 IO, Biospherical Instruments) which records it-radiance at four wavelengths in the ultraviolet region of the electromagnetic spectrum (305, 320, 340 and 380 nm), and also for PAR (400 to 700 nm). The data were stored in a 386 computer at a frequency of once per min. Ozone concentration data were obtained from the National Science Foundation Polar-UV Network site at Palmer Station (Booth et al.. 1994).

3. Results The rates of photosynthesis of the four diatom species when exposed to three radiation treatments (UVR + PAR, UV-A + PAR, and PAR) are shown in Fig. 1 for incubations done at the beginning (Fig. 1A, C, E, and G) and at the end of the experiments (Fig. 1B, D, F, and H). At the beginning of the experiments there was an increase in photosynthetic rates (expressed as assimilation numbers) in all treatments that received UV-A + PAR or just PAR as compared with the control (UVR + PAR). There was much variation in the inhibition observed for each species, with C. cri@zil~lm (Fig. IC) being the species that showed the least amount of inhibition. Irradiance conditions during all experimental periods are shown in Fig. 2 and Table IA. At the end of the experiments those samples that had been grown with exposure to UVR showed the same or higher assimilation numbers than those that had grown receiving only PAR. Comparing the rates of photosynthesis among the three treatments for the samples that had been grown with UVR (hatched bars) it is seen that the centric diatoms (Fig. IB and D) did not show significant differences between the three radiation regimes (P > 0.05) and the assimilation numbers were equal or higher than those in the cultures exposed to just PAR at the beginning of the experiment. However. Pse~~donit~schiu sp. and F. cylindrus (Fig. IF and H) did show signiticant differences (P < 0.05) between the three radiation treatments. The assimilation numbers of Pseudonitzschia sp. were comparable, within each treatment, at the beginning and at the

E.W. Helhling

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85-101

1.6 1.2 0.8 0.4 0

$1.6 5

” 1.2

Pseudonitzschia

sp.

go.8 0.4 0 1.6 1.2 0.6 0.4 0 UVR+PAR Fig.

I. Mean

UVA+PAR

PAR

UVR+PAR

UVA+PAR

photosynthetic rates, shown as assimilation numbers (mg C. [mg chl - a]-’

species under three different

radiation treatments: (i) UVR + PAR; (ii) WA

PAR h ‘), for four diatom

+ PAR and (iii) PAR. A. C, E,

and G; incubations done at the beginning of each experiment. B, D, F, and H; incubations done at the end of each experiment, with hatched bars indicating samples that had received UVR during the growth period while dark bars indicate samples that had not received any UVR.

The lines on top of the bars indicate one standard

deviation. Dates of incubations and mean irradiance received by the samples are in Table 1.

end of the experiment. However, the assimilation numbers of F. cylindrus at the end of the experiment were slightly lower than those at the beginning. Of the four cultures that had been grown with exposure only to PAR (dark bars), only Thalassiosira sp. did not show any significant difference (P > 0.05) between the radiation treatments (Fig. 1B). The other three diatom species did show a significant (P < 0.05) increase when UVR was screened. The percent enhancements of photosynthesis when UVR is screened-off (by the use of selective filters) in these cultures are shown in Table 1. The absorption characteristics of the methanol-soluble compounds at the beginning

90

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December

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8.5% 101

4 3 2 1 0 60 46 30 15 0 1600 t 0 1200 ; a

800

LOO $

0

November

December

Fig. 2. Local noon solar irradiance data for the months of November and December of 1993 and 1994. A and B, UV-B

(290-320

nm); C and D, UV-A

Foundation Monitoring

Network

(320-400

nm); E and F (PAR).

Data from the National

Science

at Palmer Station.

and at the end of each experiment are shown in Fig. 3. At the beginning of the experiments the cultures had very small amounts of UV-absorbing compounds, with the exception of C. criophilum (Fig. 3B), which showed high values as represented by its absorption values in the UVR region of the spectrum. Centric diatoms (Fig. 3A and 3B), showed an increase in the concentrations of UV-absorbing compounds with time in both treatments (UVR + PAR and PAR) but the increase was greater in those samples exposed to UVR. The pennate diatoms (P.seudonit=sc,hiLl sp. and F. c~1indru.s) showed very low concentrations of UV-absorbing compounds (Fig. 3C and 3D), both at the start and end of the growth period. These UV-absorbing compounds, identified as MAAs, were separated and quantified for each diatom species. A representative chromatogram from each diatom culture and the changes in concentrations of the major MAA compounds per unit phytoplankton carbon biomass with time are shown in Figs. 4-7. For Thalussiosiru sp. two different MAAs were identified: shinorine and porphyra-334 (Fig. 4A). Both compounds showed an increase in concentration with time, with slightly lower values in the vessel that received only PAR as compared to the one that received also UVR. The concentration of

Corethron

sp.

15.8

23.2

15.8

44.1

102

200

27

217

31

53

5

7

138

150

12

13

No UVR

28

56

22

12

No UV-B

120

156

44

19

No UVR

Last day (culture grown with PAR)

700

360

I .4 0.9

700

1320

FE.m-2.s-’

PAR

0.9

2.2

UV-B

W.m-?

Last day

30.2

22

30.2

54.6

UV-A W. mm2

1.4

s

7

3

G

!z ?

3 :

e

5

3 ?

B

t? 1.2

;

I .4

z 2 2 R

Z 1

h E

2.6

UV-B W.rn-’

A. Mean daily PAR and mean total column ozone during the experiments, and mean irradiances (PAR, UV-A and UV-B) during the 6-8 h periods to determine rates of carbon uptake. B. The percent enhancement (percent increase in photosynthetic rate when UVR is screened-off by filters as compared to control culture exposed to PAR and UVR) of photosynthesis in the 6-8 h incubations on the tirst and last days of the experiments.

Fragilariopsis

31

26

116

criophilum

Pseudonitzschia

cylindrus

100

Thalassiosira

UV-A

W.m-’

No UV-B

s-’

No UV-B

340

490

340

1050

PAR pR. m-*.

Last day (culture grown with UVR + PAR)

No UVR

297

261

297

296

Ozone D.U.

First day

ofphotosynthesis

21.6

sp.

B. Percent enhancement

Frugilariopsis

cylindrus

22.3

Pseudonitzschiu

sp.

30.1

27.6

sp.

PAR E.mm’.dayy

First day

by UVR on four diatom species

and ozone concentrations

of photosynthesis

criophilum

Thalassiosiru

Corethron

conditions

of inhibition

A. Irrudiance

Table I Magnitude

92

0.12 0.09

c

_-j_------. P&donitzsc;hia

I

250

350

450

Wavelength Fig. 3.

Spectral

550

sp.

I

650

750

(nm)

absorption curves of four Anrarcbc diatom cultures before and after exposure to wlar radiatwn

Dashed lines Indicate spectral characteristw

at the heginning of the experiments. Solid lines indicate wnples

at the end of the experiments after exposure to solar radiatmn. Thin wlid hne UVR + PAR treatment; heavy solid line PAR treatment.

shinorine factor

(Fig. 4B) was lower

of 7. The most abundant

than that of porphyra-3.34 MAAs

in C. criophilum

(Fig.

4C) by approximately

(Fig. 5) were also shinorine

a and

porphyra-334, but other MAA compounds were also found in this species in low concentration (Fig. 5A). The concentrations of the two major MAAs (Fig. SB, C) were higher in the samples that received UVR than in the samples that received only PAR. The rates of synthesis of each of these compounds, relative to phytoplankton carbon, were quite similar in Thulassiosiro sp. and C. criophilum, with values of approximately respectively. 0.001 and 0.008 kg MAA (~g C) ’ day ’ for shinorine and porphyra-334, In the pennate diatom Psruclonitzschia sp. (Fig. 6) only porphyrd-334 was detected. although in much lower concentrations than in the centric species. The concentration of porphyra-334

increased

slightly

on day 3 for both radiation

treatments,

after which

the

E.W. Helbling

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I

0.06

OL

0

I

I

I

2

4

6

8

Time (days) Fig. 4. Mycosporine-like amino acids in Thalmsiosircl sp. Experiment started on 29.Nov.1993 and ended on 6-Dee-1993. A, HPLC chromatogram showing the peaks and retention time (in min) of shinorine (1 I.21) and porphyra-334 (12.20). B, C, Concentrations of MAAs (in pg MAA/kg phytoplankton C) as a function of time for samples exposed to UVR + PAR (0) or just to PAR (0).

values decreased to about 0.001 pg MAA (yg C)- ‘. In F. cylindrus (Fig. 7A) three main MAA compounds (shinorine, porphyra-334 and mycosporine glycine-valine) were found, but their concentrations were relatively low throughout the experiment. However, the concentrations of shinorine and porphyra-334 (Fig. 7B and C) in the samples

94

Shinorine 7.05

6.49i Porphyra-334

,s,uPalyihene

g 0.009 ‘C

e 0.006 I_ 6 0.003

4

2

0

8

6

12

10

Time (days) Fig. 5. Mycosporine-like on 2.Da-1994.

amino acids in Corrt/vwl

cr-wphilwn. Experiment started on 22.Now

I994 and ended

A, HPLC chromatogram showing the peak\ and retention time (in min) of shinorine (6.49).

porphyra-334

(7.05).

Concentration

of the major MAAs

mycosporine-2

exposed to UVR + PAR (3)

glycine

(8.21).

(in p_g MAAlp+

palythine

(I I .03),

phytoplankton

and palythene

( 18.43).

R,

C.

C) ah a function of ttme for wmpleh

or just to PAR (0).

receiving UVR + PAR increased with time, but the values of mycosporine glycine-valine (Fig. 7D) increased until day 4, after which its concentration decreased. The concentrations of these MAAs in samples that had received only PAR were generally low and constant

throughout

the experiment.

4. Discussion Our data

indicate

that there

is a differential

response

and sensitivity

to UVR

by the

E.W. Hrlhling

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9s

8.%101

A

0.006

0.001

0

2

6 4 Time (days)

8

10

Fig. 6. Mycosporine-like amino acids in P.wudonitz.schia sp. Experiment started on IX-Now1993 and ended on 26.Nov- 1993. A, HPLC chromatogram showing the peak and retention time (in min) of porphyra-334 (7. I I ). B, Concentration of MAA (in )~g MAA/pg phytoplankton C) as a function of time for samples exposed to UVR + PAR (0) or just to PAR (0).

four Antarctic diatoms used in our experiments. Previous studies done with Antarctic phytoplankton have shown that diatoms were more resistant to UVR exposure as compared to other phytoplanktonic groups (Karentz et al., 1991b; Davidson and Marchant, 1994; Helbling et al., 1994; Karentz, 1994). This resistance to UVR has been hypothesized to be due, at least in part, to the synthesis of UV-absorbing compounds (Karentz et al., 1991a; Karentz, 1994; Villafaiie et al., 1995). However, the synthesis of UV-absorbing compounds is not exclusive to diatoms as species from other groups of phytoplankton, such as flagellates (Marchant et al., 1991) and dinoflagellates (Carreto et al., 1990; Vernet et al., 1994), have also been shown to produce MAAs. The synthesis of UV-absorbing compounds seems to be closely related to increased resistance to UVR of the marine diatoms used in our experiments. It is seen (Fig. 1 Fig. 3 and Table 1) that when cellular MAA concentrations were low, the inhibition of photosynthesis was significantly greater than when MAA concentrations were high. Similar results have been found for natural phytoplankton assemblages in Antarctic waters (Dunlap et al., 1995; Villafaiie et al., 1995).

96

A

E

s

Porphyra-334

._ I-

0.0006

0.0006

Mycosporine

,

Gly-Val

I

/

,

2

0

4

6

8

10

12

Time (days) Fig. 7. Mycosporine-like ended

on

(4.22). MAA/pg

2.Dec.1994.

porphyra-374

amino acid\ an ~r-~r~~~/urio~~.t~~ c~v/iudrrc.\. Expcrtment

arted

on 22-NO\- I YY4 and

A, HPLC chromatogram hhowing the peak\ and retention time (in minutes) of \hlnorinr (4.X8).

phytoplankton

and mycosporine glycincvaline

(8.34).

B. C. Concentration

C) as a function of time of the major MAAs

compound\

of MAA\

(in pg

for samples exposed to

UVR + PAR (Cl) or ju\t to PAR (0).

The UV-absorbing compounds can effectively absorb UVR at wavelengths that range from about 3 10 nm to 360 nm (Karentz et al., 1991 b; and Fig. 3 in this paper). This region within the UVR spectrum has been shown to be the most damaging when

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convolving the incident solar irradiance with a plant action spectrum (Caldwell, 197 1; Green et al., 1974; Coohill, 1989). Due to their absorption characteristics and a decrease in the MAA concentrations with depth in corals, these compounds have often been assumed to serve a protective role against UVR (Dunlap et al., 1986). Our results indicate that the synthesis of MAA compounds in the centric diatom species was related not only to UVR exposure but also to an increase in PAR (Fig. 3 Fig. 4 Fig. 5), while in F. cylindnts MAAs were synthesized primarily as response to UVR (Fig. 7). The chromophores responsible for initiation of synthesis of MAA compounds have not been determined. Due to the more rapid attenuation in the water column of UVR as compared to PAR (Smith and Baker, 1979, 198 1; Kirk, 1994) the depth of the upper mixed layer (UML) would seemingly play an important role in the rate of synthesis of MAA compounds by phytoplankton. If the depth of the UML is much greater than the depth to which significant amounts of UVR can penetrate, species that can synthesize MAAs in response to PAR as well as to UVR would conceivably be better able to cope with UVR than species that produce MAAs only in response to UVR. Previous studies (Helbling et al., 1994; Vernet et al., 1994) have shown that phytoplankton within the UML have higher concentrations of UV-absorbing compounds than phytoplankton found below the UML. Also, the concentrations of these compounds in phytoplankton within the UML were higher in water columns with shallow UMLs as compared with water columns with deep UMLs. Since the UML in Antarctica generally varies from about 15 to more than 80 m (Mitchell and Holm-Hansen, 1991; Helbling et al., 1995), with a mean value of 55 m for the region around Elephant Island (Helbling et al., 1995), phytoplankton will thus be exposed to large changes in irradiances. With a UML depth of 55 m, nearly all UV-B and the shorter wavelengths of UV-A would be essentially removed well above the base of the UML, while PAR would penetrate to greater depths. This condition would favor those species that can synthesize MAAs in response to PAR. In the Antarctic, diatoms are usually the dominant group in terms of carbon biomass during blooms (HolmHansen et al., 1989), although in some areas blooms can be dominated by Phaenc~~tis sp. (Garrison et al., 1987; Fryxell and Kendrick, 1988). Diatoms are usually found in relatively shallow upper mixed layers, in which phytoplankton are exposed to high solar radiation, while flagellates tend to be dominant in deep UMLs (Kopczynska, 1992; Helbling et al., 1994). Sudden changes and deepening of the depth of the UML are often found in Antarctic waters due to changes in wind stress (Mitchell and Holm-Hansen, 199 1) and these changes can be fairly large considering the relatively low stability of the upper water column. If phytoplankton remain for a sufficiently long time in a shallow UML, the cellular concentrations of MAAs should be fairly high. If the cells are then suddenly mixed to greater depths (due to a deepening of the UML), and thus are exposed to lower irradiances than previously, one of the questions that arises is how the MAA concentrations will change in the process of dark acclimation of the cells. We simulated this change of irradiance associated with the deepening of an UML by reducing PAR from 1200 to 250 yE.m-*.s-’ and following the concentrations of MAAs within the cells of Thalussiosira sp. with time (Fig. 8). The concentrations of shinorine and porphyra-334 decreased with time and after 10 days their concentrations per unit carbon

0.06 z! tE tf

0.05

0.04

0.03

0

2

4

6

a

10

Time (days)

incubators. A, >hinorine. B. porphyra-334.

were

about

61% of their value at time Aero. This indicates a loss rate of about 0.0002 for shinorine and porphyra-334. respectively. kg MAA (kg C))‘.day-’ rates are about 4-S times less than the rates of synthesis for these two compounds in Thultrssiosircr sp. Considering the opposite situation, in which the depth of the UML is getting shallower (for example due to thermal heating), phytoplankton would then receive

and 0.0023 These loss

higher irradiance than in the previous condition (e.g., deep mixed layer) and might thus be strongly inhibited by solar UVR. The low loss rate for MAAs (at least in Thalctssiosir~~ sp.) as noted above suggests that phytoplankton could retain sufficient concentrations of MAAs to alleviate UVR stress associated with the new higher solar relationship irradiance. Other studies (Dunlap et al., 1995) have found an inverse between total MAA concentrations and degree of inhibition of photosynthesis in natural populations of phytoplankton. For example, with a MAA concentration of about 0.6 fq MAA (kg chl-a))‘. these authors found that the relative increase of photosynthesis was less than 35% when all UVR was excluded from the samples. In our case of T~7czlassio,ri~-r~ sp., which had a Cl&-u ratio of approximately 42, and a rate of synthesis

E.W. Helbling

et al. I J. Exp. Mar.

Biol.

Ecol.

204 (1996)

85-101

99

of about 0.01 pg MAAe(pg C)-’ *day-’ it would take approximately one day to reach a cellular concentration of 0.6 kg MAA. (kg chl-a))’ and thus give significant UV protection to the cells. On the other hand, considering a loss rate of about 0.002 kg MAA.(p,g C))’ .day-‘, phytoplankton with an initial concentration of MAAs similar to that in Thalassiosira sp. (Fig. 8), it would take at least 20 days before the MAA compounds reached a cellular concentration of 0.6 pg MAA (pg chl-a)-’ when in a deep UML with a irradiance of 250 FE. cm-* . s ’ Our data thus support the concept that MAAs can serve a protective function in phytoplankton for minimizing cellular damage by solar UVR. Although many reports have suggested that diatoms as a group are more resistant to UVR than other phytoplanktonic groups, our results show that there is much variability within diatom species in regard to MAA concentrations and sensitivity to UVR.

Acknowledgments This work was supported by National Science Foundation Grant OPP #92-20150. We thank Antarctic Support Associates personnel and L. Sala, R. Marguet, G. Rae and H. Diaz for their generous help during our field work at Palmer Station. We also thank two anonymous reviewers for their helpful comments. This paper is AIMS Contribution No. 759.

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