A Physiological Study on Photoacclimation of Marine ...

Aus dem Alfred-Wegener-Institut für Polar- und Meeresforschung Bremerhaven

A Physiological Study on Photoacclimation of Marine Planktonic Algae to Different Ultraviolet-B Irradiances

Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. nat am Fachbereich 2 (Biologie/Chemie) der Universität Bremen

vorgelegt von

Rüdiger Röttgers

Bremen, September 1999

Gutachter: Prof. Dr. PD Dr. habil.

Viktor Smetacek (1. Gutachter) Dieter Hanelt (2. Gutachter)

Contents

Contents

DANKSAGUNG · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · v ABBREVIATIONS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · vii SUMMARY/ZUSAMMENFASSUNG · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 1 Summary · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 1 GENERAL INTRODUCTION· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 3 Preface · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 3 Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 3 Photosynthesis as a function of irradiance · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 4 Photoinhibition of photosynthesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 5 Photoinhibition and UV radiation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 6 Photoacclimation · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 6 Photosynthesis-irradiance response, photoacclimation and sensitivity to UVB radiation· · · · 8 Thesis outline and introduction to the publications · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 9 PUBLICATIONS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 11 List of Publications· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 11 Erklärung · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 11 Publication 1: The use of simulated solar-irradiance spectra (290-750 nm) for . measurements of phytoplankton photosynthesis · · · · · · · · · · · · · · · · · · · · 13 Publication 2: Phytoplankton photoacclimation to ultraviolet radiation and the role of . mycosporine-like amino acids · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 27 Publication 3: Physiological evidence that planktonic algae do not acclimate to . alterations in ultraviolet-B irradiance · · · · · · · · · · · · · · · · · · · · · · · · · · · · 39 Publication 4: Short-term UVB effects on photosynthesis of the diatom Thalassiosira . weissflogii under variable irradiance · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 63 GENERAL DISCUSSION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 77 Impact of UVB on photosynthesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 77 Impact of UVB on growth · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 82 Species-specific differences · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 82 Correlation of UVB sensitivity in growth and photosynthesis· · · · · · · · · · · · · · · · · · · · · · 83 Photoacclimation to UVB?· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 84 Ecological considerations · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 86 Conclusion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 91 REFERENCES · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 93

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Contents

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Danksagung

Danksagung

Mein herzlicher Dank geht an Prof. Dr. Viktor Smetacek für die Annahme als Doktorand und für die Begutachtung dieser Arbeit. Dr. Dieter Hanelt danke ich für die Erstellung des zweiten Gutachtens und für seine hohe Diskussions- und Erklärungsbereitschaft während der gesamten Promotionszeit. Prof. Dr. Gunter-Otto Kirst danke ich für seine Teilnahme an der Prüfungskommision. Mein besonderer Dank geht an Marcus Baumann, der mich schon als Student unterstützt hat, mir das Thema zur Doktorarbeit nahelegte und in den ersten Monaten die Arbeit betreute. Genauso dankbar bin ich Bernd Kroon, der anschließend die Betreuung übernahm und der jederzeit für Fragen und Diskussionen zur Verfügung stand. Gerade in den letzten Monate war er ständig damit beschäftigt die Arbeit Korrektur zu lesen und weitere Anregungen zu geben. Vielen Dank dafür! In einem nichtabzuschätzende Maße hilfreich war auch die Unterstützung von Lilo Riegger und Dale Robinson, von denen ich viel gelernt habe und deren Anregungen viel zum Gelingen dieser Arbeit beigetragen haben. Vielerlei Hilfe bekam ich auch von allen anderen Mitglieder der BioOptik-Gruppe, ein Dankeschön an Erika Allhusen, Astrid Bracher, Nicole Dijkman, Noga Stambler und Marika Sündermann. Robin Brinkmeyer und Patrick Eden danke ich für das Korrigieren des englischen Textes. Allen anderen AWI-Angehörigen danke ich für ihre vielfältige Unterstützung während meiner Promotionszeit, allen voran: Doris Abele, Anke Dauelsberg, Elisabeth Helmke, Helga Schwarz, Helmut Tüg und Uschi Wellbrock. Eine sehr großen Einfluß auf das Gelingen der Arbeit hatten viele meiner Freunde, die mein Interesse an der Wissenschaft wachgehalten haben, ohne es manchmal überhaupt zu wollen. Dazu gehören: Frederico Brandini, Jörg Dutz und Bärbel Hönisch, Patrick Eden, Leo Goeyens, Claudia Grahl und Uwe Hoppe, Rubens Lopes, Thomas Sawall, Michael Tibcken und Martina Kaczmarek, Markus von Wangelin, und Katrin Wagenitz. Letztendlich konnte die Arbeit nur gelingen aufgrund der Akzeptanz und Unterstützung durch meine Lebensgefährtin Gabi Pollmeyer. Ihr und unserer gemeinsamen Tochter Lucie ist die Arbeit gewidmet.

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Danksagung

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Abbreviations

Abbreviations α α

*

acell (λ) ap (λ) β BWF DU E E0 Eg Ek Em EPAR, E0,PAR EUVA EUVB Emax, E0,max E0,min rETR rETRm rETR vs. E0 φII F0 Fm Fm´ Ft Fv kd MAAs PAR P PT PTpot * P P*g P*m P* vs.E0 PSI PSII PSU tmix UML UVA UVB UVC UVR zmix

maximum light utilization coefficient for rETR vs. E0 curves [µmol e (µmol photons)-1] maximum light utilization coefficient normalized to chlorophyll a for P* vs.E0 curves -1 -2 -1 -1 [µgC (µgChl a h) (µmol photons400-700 nm m s ) ] 2 -1 cellular absorption coefficient [m cell ] -1 2 -1 particular absorption [m ] or [m cell ] photoinhibition parameter for rETR vs. E0 curves Biological Weighting Function -1 Dobson Unit (=atmospheric ozone concentration) [0.01 mm atm ] -2 -1 -2 irradiance [µmol photons m s ] or [Wm ] -2 -1 scalar irradiance [µmol photons m s ] or [Wm-2] growth irradiance irradiance at the onset of light saturation [µmol photons400-700 nm m-2 s-1] -2 -1 irradiance at the onset of photoinhibition [µmol photons400-700 nm m s ] irradiance of PAR [µmol photons400-700 nm m-2 s-1] irradiance of UVA [Wm-2] irradiance of UVB [Wm-2] maximum E0,PAR during a light cycle [µmol photons400-700 nm m-2 s-1] minimum E0,PAR during a light cycle [µmol photons400-700 nm m-2 s-1] relative Electron Transport Rate maximum relative Electron Transport Rate relative Electron Transport Rate as a function of irradiance quantum yield of photosystem II steady state fluorescence yield maximum fluorescence yield maximum fluorescence yield under actinic irradiance fluorescence yield under actinic irradiance variable fluorescence vertical attenuation coefficient for downward irradiance [m-1] Mycosporine-like Amino Acids Photosynthetically Available Radiation (400-700 nm) photosynthetic rate daily photosynthesis integrated through the water column [mg C m-2 d-1] potential, i.e. uninhibited PT photosynthetic rate normalized to chlorophyll a [µgC (µgChl a h)-1] photosynthetic rate at Eg [µgC (µgChl a h)-1] maximum photosynthetic rate [µgC (µgChl a h)-1] photosynthetic rate as a function of irradiance PhotoSystem I PhotoSystem II PhotoSynthetic Unit mixing time [min] Upper Mixed Layer UltraViolet-A radiation (320-400 nm) UltraViolet-B radiation (280-320 nm) UltraViolet-C radiation (200-280 nm) UltraViolet Radiation (200-400 nm) mixing depth [m]

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Abbreviations

viii

Summary

Summary

Short- and long-term reactions to ultraviolet radiation (UVR) of marine planktonic algae were investigated with respect to the influence of an enhancement of ultraviolet-B radiation (UVB) caused by stratospheric ozone depletion. In order to assess these reactions under ecologically relevant irradiances, experiments were conducted under controlled laboratory conditions. Realistic irradiances were applied by simulating solar-like spectral distributions under various stratospheric ozone concentrations in a „solar-irradiance simulator“. The simulator was used as an illumination source to grow cultures and to determine photosynthesis of planktonic microalgae. As previously observed under natural conditions as well as with strong artificial UV irradiances, UVR had a pronounced influence on microalgal photosynthesis under the here applied moderate and realistic irradiances. Reduction in photosynthesis was observed with a simulation of enhanced UVB corresponding to 30% ozone depletion under constant irradiances. Under a light-cycle simulating a variable irradiance (such as induced by vertical mixing of the water column) photosynthesis was signif icantly reduced at 13% ozone depletion. Under both conditions, the total effect induced by enhanced UVB irradiance was mainly related to the applied maximum incident irradiance not to the applied cumulative dose. Effects under variable irradiances by vertical mixing were similar to effects under static irradiance conditions. There were also reductions in photosynthesis and growth by enhanced UVB irradiance for various temperate and polar algal species after long-term exposures. Species-specific

differences were observed, but to a lower degree than previously reported by other authors. The detected species-specific differences in UVB-sensitivity were not related to growth temperature or cell-size, but to taxonomic differences. Within a single species effects were cell-size dependent. UVB enhancement reduced photosynthesis and growth simultaneously to the same degree, which indicates a close-coupling of photosynthesis and growth. On the basis of this closecoupling the possibility and capacity of a UVB-related photoacclimation in algae that could counteract the effects of changes in UVB irradiance by ozone depletion was evaluated . Photoacclimation to irradiance of ultraviolet A (UVA) and photosynthetically available radiation (PAR) was observed by pronounced changes in photosynthetic and photoprotective pigments, including UV-absorbing compounds, and by changes in photosynthetic capacity and growth. UV-absorbing compounds (mainly mycosporine-like amino acids, MAAs) diminished the detrimental effects of UVB and hence, provided some but not full protection against UVB radiation. On the other hand, enhanced or reduced UVB irradiance did not change the pigment contents or pigment composition, but influenced photosynthesis and growth. However, effects on photosynthesis and growth were the same during short and long exposures, and long-term pre-exposure did not reduce the short-term UVB-sensitivity. Hence, short-term exposures can be used to predict the expected influences during long-term exposures, which would not be possible if photoacclimation was to reduce these short-term effects. There were

1

Summary

no indications of photoacclimation to UVB irradiance. Algae acclimated to high irradiances have a higher relative content of MAAs and are less sensitive to UVB radiation. However, this lower UVB sensitivity is established by an acclimation to a higher UVA and PAR, not to a higher UVB irradiance. Under natural conditions vertical mixing will induce photoacclimation to low irradiance by subjecting phytoplankton to a low mean UVA/PAR irradiance. Phytoplankton of high turbulent waters will be acclimated to a low irradiance and, hence, be more sensitive to UVB irradiance at the surface. Additionally, vertical mixing will not reduce the effects of UVB radiation by limiting the time of exposure to high surface irradiances. Ambient UVB radiation influences plankton productivity instantaneously and this influence will only be mitigated by photoacclimation to the concomitant UVA and PAR irradiance. In contrast, effects of ozone depletion which only increases UVB irradiance, will not be counteracted by an acclimation to the enhanced UVB irradiance.

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General Introduction

General Introduction

Preface The discovery of a seasonal strong ozone depletion over the Antarctic - referred to as the Antarctic "ozone hole" - in the early 70´s and the connection of the anthropogenic release of chlorofluorocarbons (CFC) with ozone layer destruction, encouraged scientific interest in the effects of ultraviolet radiation on terrestrial and marine environments. Ozone (O3) strongly absorbs ultraviolet-B radiation (UVB: 280-320 nm) and the stratospheric ozone concentration influences the UVB irradiance incident on the earth´s surface (Figure 1). Ozone depletion is highest at higher latitudes and negligible in the tropics (Madronich 1994, Madronich et al. 1995, 1998, WMO 1992). However, incident UVB irradiance in the tropics is about an order of magnitude stronger than at higher latitudes due to the higher solar elevation and naturally lower ozone concentrations. Ambient UV radiation was discovered to have a strong impact on marine photosynthesis and is seen as a stress factor for many marine organisms (Cullen & Neale 1994, Franklin & Forster 1997, Häder 1997). The ecological consequences for marine environments by enhanced UVB irradiance due to ozone depletion are discussed (e.g. Kramer 1990, Holm-Hansen et al. 1993, Weiler & Penhale 1994, Häder 1997). This thesis focuses on the impact of UVB enhancement on photosynthesis and growth of marine phytoplankton. It examines in detail the ability of planktonic algae to acclimate to a given UVB irradiance. UV effects on photosynthesis of marine planktonic algae are explained as a function of incident irradiance (E0). This relationship, the P vs. E0 curve, is an appropriate tool for examining photosynthesis with respect to changes in

irradiance. Photoinhibition is illustrated by its influence on the P vs. E0 curve. This is followed by explanations of photoacclimation and its consequences for growth and photoinhibition.

Introduction Life on Earth depends mainly on a single process which converts light energy of the solar irradiance into organic carbon compounds: photosynthesis. Hence, photosynthesis is one of the most extensively studied biological mechanism. Aquatic phototrophic organisms, less than 1% of the total plant biomass, are responsible for approximately 40% of annual global photosynthesis (Berger et al. 1989, Falkowski 1994). Marine microalgae, like all plants, are essentially dependent on wavelengths between ~350 and 700 nm for photosynthesis (Figure 1). On the other hand, the concomitant incident ultraviolet radiation (UVR: ~290-400 nm) affects algal physiology adversely and subsequently photosynthesis and growth. Since UV radiation penetrates to considerable depths into clear oceanic waters (Jerlov 1950, Smith & Baker 1979, 1981, Smith et al. 1992), UV radiation can impact aquatic environments. However, the ecological consequences of these impacts are difficult to determine due to the complexity of the underwater light field. Spectral quality of irradiance changes strongly with solar elevation, atmospheric conditions (including ozone), and depth in the water (see Kirk 1994). It is shown in a number of studies that ambient UV radiation significantly inhibits planktonic photosynthesis and growth (reviewed by Smith & Baker 1989, Holm-Hansen et al. 1993, Cullen & Neale 1994). How extreme this

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General Introduction

PAR

UVR A B

solar spectrum

Absorbance

Irradiance (log)

physiological mechanisms which regulate photosynthesis and growth in terms of irradiance, i.e. photoacclimation. This thesis focuses on the question of whether algae are able to counteract the influence of enhanced UVB irradiance by UVB-induced photoacclimation and whether through that the impact of ozone depletion is diminished.

absorption spectrum

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Wavelength [nm] Figure 1. A typical absorption spectrum of an methanolic extract made from the diatom Thalassiosira antarctica and a solar irradiance spectrum (note the logarithmic scale for irradiance). Indicated are the different wavebands of photosynthetically available radiation (PAR: 400-700 nm) and of ultraviolet radiation (UVR): UVA (320-400 nm) and UVB (280-320 nm). The absorbance maximum at 336 nm corresponds to an absorption by mycosporine-like amino acids (MAAs), those at 440 nm and 664 nm to absorption by photosynthetic pigments (mainly chlorophyll a). The dotted line indicates an arbitrary border between wavelengths mainly positive (>365 nm) and wavelengths mainly negative for photosynthesis (360 nm for photoacclimation. Regulative and acclimative responses to wavelength 1/2 E0,max for 12 min. This duration was enough to obtain significant differences when E0,max to E0,min curves under different irradiance spectra were compared. A steady state fluorescence signal (Ft) in response to a change in irradiance was determined after 90 to 100 sec, therefore minimum time intervals had to be set to 2 minutes. Figure 5 shows typical rETR vs. E0,PAR curves of T. weissflogii cultured at 15° C.

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Irradiance [µmol photons400-700 nm m-2 s-1] Figure 4. Pulse Amplitude Modulated fluorescence measurements (PAM) under light-cycles simulating vertical mixing but using irradiances of different spectral quality during the cycle. (A) Modeled irradiance of circular vertical mixing (line) and actually used irradiances (bars) in 2-min intervals for PAM-measurements. (B) The measured photosystem II quantum yield (φII) at the end of each interval determined under two different irradiance spectra (see legend). (C) Corresponding curves of relative electron transport rate (±SD) as a function of incident irradiance (rETR vs. E0). Measurements are conducted with irradiance spectra under a GG 420 cut-off filter (squares) and with a spectra simulating 30% ozone depletion (circles). For spectral distribution see Fig. 1b,c. Each measurement resulted into two rETR vs. E0,PAR curves: a first (dotted line) from low irradiance to maximal irradiance (Emin – Emax) and a second (solid line) from maximal to low irradiance (Emax – Emin).

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Photosynthesis measurements with simulated solar irradiance

A 80

60

rETR

The data showed that an enhancement of ultraviolet radiation always decreased rETRm and effects by simulated 30% ozone depletion were measurable. Algae grown under fluorescence tubes (Figure 5a) showed a higher decrease of rETRm due to UVA of longer wavelengths, whereas algae grown in the solar-irradiance simulator under normal ozone conditions (Figure 5b) showed higher absolute values of rETRm and greater effects by short UVA and UVB. UVA had also effects on the light limited slope of photosynthesis, α, that increased with decreasing UVA. An increase of β (the parameter for decrease of rETR at higher light intensities) was only visible when both, UVB and UVA, were excluded (Figure 5).

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GG 420 WG 360 WG 335

0

Discussion

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B 80

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rETR

The lamp-filter system (solar-irradiance simulator) provides realistic solar-like irradiances which simulates incident irradiance spectra corresponding to different atmospheric conditions, especially ozone concentrations and water depth (here: 2 m). Simulation of ozone depletion was similar to naturally occurring irradiance changes by ozone depletion (Figure 2). We found that the overall stability of irradiance was good enough to provide longterm exposures (days) under low irradiances (E0,PAR = 200 µmol photons400-700 nm m-2 s-1). However, small changes occurring during this time or during exposures to higher irradiance could be controlled in short intervals and adjusted accurately back to the original irradiance spectra. Care should be taken in controlling the spectral output of the lamp-filter system, wherefore a good spectroradiometer should always be available. During an experiment or during culturing, irradiance spectra were constant and for different experiments the same irradiance spectra could be used. We simulated an experimental setup with cut-off plates under in situ-irradiance conditions such as frequently used in other studies

normal ozone 16% ozone depletion 30% ozone depletion

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Irradiance [µmol photons400-700 nm m-2 s-1] Figure 5. Relative electron transport rate as a function of incident irradiance (rETR vs. E0) of Thalassiosira weissflogii determined under different irradiance spectra. For a culture grown under daylight fluorescence tubes (A) and grown under an irradiance simulating normal ozone conditions (B). Corresponding spectra are shown in Fig. 1 b,c. Shown are results of the corresponding measurements during the E0,max to E0,min part of the light-cycle.

Publication 1

(e.g. Helbling et al. 1992, Boucher & Prézelin 1996). Additionally, we simulated an enhancement of UVB irradiance. Physiological mechanisms and overall reactions to UV radiation can be elucidated by such cut-off experiments. Due to the exponential increase in damage with shorter UVB wavelengths it is difficult to predict the influence of enhanced UVB irradiance in experiments comparing normal with lower UVB irradiances. Therefore a realistic simulation of UVB enhancement was done here by a wavelength dependent increase in UVB irradiance, such as provoked by ozone depletion. Many studies showed that UVB has measurable effects on marine photosynthesis during standard exposures in situ or under artificial UVB treatments (see Cullen and Neale 1994). Nevertheless, effects were highly variable, possibly due to differences in algae physiology, phytoplankton composition, natural variability in irradiance and experimental treatments, in particular high and unrealistic UVB levels might produce a different physiological response. Our study confirmed measurable effects of total UV and enhanced UVB under ecological relevant irradiance levels with standard incubation times. The influences of ultraviolet radiation on 14C-incorporation by algae was similar to results using a so-called ”photoinhibitron” (Cullen et al. 1992; Neale et al. 1994, Neale et al. 1998a), for determination of BWF, but provides more accurate P* vs. E0 curves. Excluding shorter wavelengths (UVB, then UVA) increased the maximal rate of photosynthesis, probably by decreasing chronic photoinhibition or photodamage. An expected high influence on photosynthesis by UVA during the incubation is visible. PAR only has no detrimental effect on P* at higher irradiance levels. The realistic change in UVB, much lower than used in other studies, showed nevertheless significant differences in the observed maximum photosynthesis (P*m).

The data presented here showed that the light history of the algae had a significant impact on photosynthetic performance under different irradiance spectra. Algae grown under UVB-free and low UVA conditions (fluorescence tubes) react different than algae grown under a solar-like spectrum. Photoacclimation to the given irradiance, i.e. irradiance and spectral quality, has significant influences on the photosynthesis and the amount of UV/UVB effects. Acclimation to a solar-like irradiance spectrum (~290-700 nm) decreased the sensitivity to longer UVA (differences in P*m: 19% to 10%) and the alga was now less affected by the same amount of UVR. An increase of UV radiation, by shorter UVA and UVB wavelengths and by enhanced UVB irradiance did now further decrease photosynthesis and implies a higher sensitivity to these wavelengths (Figure 3). We used a standard incubation time of 4 h for the 14C – P* vs. E0 curves, a comparable long time of constant intensities, which will rarely occur under natural condition where short time scale variations of incident irradiance might be experienced in mixing water columns. Relative to damage and repair, changes in incubation time may change the amount of differences found during incubation. If the law of reciprocity holds, i.e. effects of UVB were related to cumulative exposure and not irradiance, shorter exposures under the same irradiances should decrease the overall effect until it would not be measurable. Moderate irradiances and changes in UVB would require longer exposure times to evaluate significant effects, whereas high UVB irradiance would need shorter times or would result in more pronounced effects. We were therefore interested in whether UVB would have measurable effects even at realistic levels and during very short exposures (min). This situation is comparable to natural irradiance conditions during vertical mixing. PAM-fluorometry was used for measure-

23

Photosynthesis measurements with simulated solar irradiance

ments during incubations under fast changing light intensities, simulating vertical mixing with mixing rates shorter than one hour (40 min). During the simple experiments shown here we did not modify the spectral distribution in the course of light cycle, what occurs in situ with changes in water depth. Starting from a depth with low irradiances an algae will be exposed to increasing irradiances until it reached the surface and decreasing irradiances on the way down. During an irradiance increase, φII will decrease and vice versa, related to the down-regulation of PSII performance. This reversible down-regulation is usually termed dynamic photoinhibition as defined by Osmond (1994). In so far PAR is used during the light-cycle, the two resulting curves (upward [E0,min to E0,max] and downward [E0,max to E0,min]) are mostly identical. UV radiation should change this pattern by damaging PSII centers (chronic photoinhibition) and decrease φII, and thus should inhibit the relative electron transport rate (rETR). The algae will experience the highest damage at the surface and, dependent on the balance between damage and repair (=recovery), take some of this damage back into the deeper water. Thus, any inhibition occurring at the surface could be brought down into deeper water, where ambient level of UV would be too low to have direct effects. Our data showed that this method can indeed accurately determine this short-time effect by UV, even at E0,max of only 750 µmol photons400-700 nm m-2 s-1 (E0,max can naturally be >1500 µmol photons400-700 nm m-2 s-1 at the local noon) and when exposure to E0,max was only 4 min. UV radiation with increasing shorter wavelengths increased the impact on rETR before and after reaching E0,max, from no difference, when PAR only was used, to a maximal impact, when a solar-like irradiance was used. Actually, PAR light only increased rETR for each irradiance after reaching E0,max due to photoacclimation occurring during the measurements of 40 min (Figure 4c).

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Overall reactions of the used algae under the different growth conditions (fluorescence tubes and solar simulator) are rather similar to the results of the 14C-incubations with longer incubation time and, thus, higher doses (see above). This indicates that the law of reciprocity failed under the here applied conditions and that the observed effects of UV radiation are mainly determined by the applied irradiances. This failure of reciprocity was observed also in other studies (Helbling et al. 1994, Cullen and Lesser 1991). The cumulative dose might not be a proper measure for compare of different UV exposures. With both methods the effects of a realistic enhancement of UVB radiation are directly measurable and can be related to different ozone concentrations, simulated by the setup. The methods are easily employed in lab and during ship cruises, and will be used to examine short-term and long-term effects of UV radiation on planktonic algae, including photoacclimation to UVB, UVA, and PAR under realistic, i.e. ecological relevant irradiance conditions. References Abele-Oeschger D, Tüg H & Röttgers R (1997). Dynamics of UV-driven hydrogen peroxide formation on an intertidal sandflat. Limnol Oceanogr 42:1406-1415 Boucher NP, Prézelin BB (1996) An in situ biological weighting function for UV inhibition of phytoplankton carbon fixation in the Southern Ocean. Mar Ecol Prog Ser 144:223-236 Bracher AU, Wiencke C (1999) Simulation of naturally enhanced UV-radiation on photosynthesis of Antarctic phytoplankton. Mar Ecol Prog Ser (in press) Cullen JJ, Lesser MP (1991) Inhibition of photosynthesis by ultraviolet radiation as a function of dose and dosage rate: results for a marine diatom. Ma. Biol 111:183-90 Cullen JJ, Neale PJ (1994) Ultraviolet radiation, ozone depletion, and marine photosynthesis. Photosynth Res 39:303-20 Cullen JJ, Neale PJ, Lesser MP (1992) Biological weighting functions for the inhibition of phytoplankton photosynthesis by ultraviolet radiation. Science 258:646-50 Eilers PH, Peeters JCH (1988) A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton. Ecol Modelling 42:199-215 Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH

Publication 1

(eds) Culture of marine invertebrate animal. Plenum Press, New York, p 29-60 Helbling EW, Villafañe V, Ferrario M, Holm-Hansen O (1992) Impact of natural ultraviolet radiation on rates of photosynthesis and on specific marine phytoplankton. Mar Ecol Prog Res 80:89-100 Helbling EW, Villafañe V, Holm-Hansen O (1994) Effects of ultraviolet radiation on Antarctic marine phytoplankton photosynthesis with particular attention to the influence of mixing In: Weiler CS, Penhale PA (eds) Ultraviolet radiation in Antarctica: measurements and biological effects. American Geophysical Union, Washington, DC, p 207-227 Herrmann H, Häder D-P, Köfferlein M, Seidlitz HK, Ghetti F (1996) Effects of UV radiation on photosynthesis of phytoplankton exposed to solar simulator light. J Photochem Photobiol B:Biol 34:21-28 Holm-Hansen O, Lorenzen CJ, Holmes RW, Strickland JDH (1965) Fluorometric determination of chlorophyll. J Cons int Explor Mer 30:3-15 Holm-Hansen O, Lubin D, Helbling EW (1993) UVR and its effects on organisms in aquatic environments. In: Young AR et al. (eds), Environmental UV photobiology. Plenum, New York, p 379-425 Holm-Hansen O, Villafañe VE, Helbling EW (1996) Photoinhibition in Antarctic phytoplankton by ultraviolet-B radiation in relation to column ozone values. Ant J 31:229-230 Hurtubise RD, Havel JE, Little EE (1998) The effects of ultraviolet-B radiation on freshwater invertebrates: experiments with a solar simulator. Limnol Oceanogr 43:1082-1088 Karentz D (1994) Ultraviolet tolerance mechanisms in Antarctic marine organisms. In: Weiler CS, Penhale PA (eds) Ultraviolet radiation in Antarctica: measurements and biological effects. American Geophysical Union, Washington, DC, p 93-110 Kirk JTO (1994) Light and photosynthesis in aquatic ecosystems. Cambridge University Press, Cambridge Lewis MR, Smith JC (1983) A small volume, short-incubation-time method for measurement of photosynthesis as a function of incident irradiance. Mar Ecol Prog Ser 13:99-102 Marchant HJ, Davidson AT, Kelly GJ (1991) UV-B protecting compounds in the marine alga Phaeocystis pouchetii from Antarctica. Mar Biol 109:391-395 Molina LT, Molina MJ (1986) Absolute absorption cross-sections of ozone (185-350 nm). J Geophys Res 91(D13):14501-14508 Neale PJ, Lesser MP, Cullen JJ (1994) Effects of ultraviolet radiation on the photosynthesis of phytoplankton in the vicinity of McMurdo Station. In: Weiler CS, Penhale PA (eds) Ultraviolet radiation in Antarctica: measurements and biological effects. American Geophysical Union, Washington, DC, p 125-142 Neale PJ, Cullen JJ, Davis RF (1998a) Inhibition of marine photosynthesis by ultraviolet radiation: variable sensitivity of phytoplankton in the Weddell-Scotia Sea during austral spring. Limnol Oceanogr 43:433-448 Neale PJ, Davis RF, Cullen JJ (1998b) Interactive effects of ozone depletion and vertical mixing on photosynthesis of Antarctic phytoplankton. Nature 392:585-589 Osmond CB (1994) What is photoinhibition? Some insights from comparisons of shade and sun plants. In Baker

NR, Bowyer NR (eds) Photoinhibition of Photosynthesis, from Molecular Mechanisms to the Field. BIOS Scientific, Oxford, p 1-24 Platt T, Gallegos CL, Harrison WG (1980) Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J Mar Res 38:687-701 Prézelin BB, Boucher NP, Smith RC (1994) Marine primary production under the influence of the Antarctic ozone hole: Icecolor ’90. In: Weiler CS, Penhale PA (eds) Ultraviolet radiation in Antarctica: measurements and biological effects. American Geophysical Union, Washington, DC, p 159-186 Riegger L, Robinson D (1997) Photoinduction of UV-absorbing compounds in Antarctic diatoms and Phaeocystis antarctica. Mar Ecol Prog Ser 160:13-25 Schindler D, Schmidt RV, Reid RA (1972) Acidification and bubbling as an alternative to filtration in determining phytoplankton production by the 14C method. J Fish Res Bd Can 29:1627-1631 Schreiber, U 1994 New emitter-detector-cuvette assembly for measuring modulated chlorophyll fluorescence of highly diluted suspensions in conjunction with the standard PAM fluorometer. Z Naturforschung 49c:646-656 Smith RC, Baker KS (1989) Stratospheric ozone, middle ultraviolet radiation and phytoplankton productivity. Oceanogr Mag 2:4-10 Smith RC, Prézelin BB, Baker KS, Bidigare RR, Boucher NP, Coley T, Karentz D, MacIntyre S, Matlick HA, Menzies D, Ondrusek W, Wan Z, Waters KF (1992) Ozone depletion: Ultraviolet radiation and phytoplankton biology in Antarctic waters. Science 255:952-959 Steemann-Nielsen E (1952) The use of radioactive carbon (14C) for measuring organic production in the sea. J Cons perm int Explor Mer 18:117-140 Strid A, Chow WS, Anderson JM (1994) UV-B damage and protection at the molecular level in plants. Photosynth Res 39:475-489 Tüg H (1978) Absolut- und Relativanschluß südlicher Standardsterne. Dissertation, Universität Bochum Vincent WF, Roy S (1993) Solar ultraviolet-B radiation and aquatic primary production: damage, protection and recovery. Environ Res 1:1-12

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Photosynthesis measurements with simulated solar irradiance

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Publication 2: Phytoplankton photoacclimation to ultraviolet radiation and the role of mycosporine-like amino acids Rüdiger Röttgers & Lieselotte Riegger Key words: MAAs, ozone depletion, UV-absorbing compounds, ultraviolet-A radiation,UVA, ultraviolet-B radiation, UVB

Abstract. Many marine algae exhibit mycosporine-like amino acids (MAAs) which absorb ultraviolet (UV) radiation between 310-360 nm. Marine phytoplankton are sensitive to inhibition of photosynthesis by solar UV radiation, and MAAs are considered to provide protection against this radiation and against an enhancement of UVB (280-320 nm) irradiance by ozone depletion. Photoacclimation to higher UV as well as to higher PAR irradiance induces accumulation of MAAs and reduces UV sensitivity in planktonic algae. The diatom Thalassiosira antarctica accumulates MAAs under high irradiance (200 µmol photons400-700 nm m-2 s-1), whereas Thalassiosira weissflogii exhibits only negligible amounts of MAAs. Sensitivity to enhanced UVB irradiances is reduced with higher growth irradiance for T. antarctica, but not for T. weissflogii, indicating some protection provided by MAAs. The absolute influence of enhanced UVB depends on the growth irradiance for T. antarctica as well as on the spectral quality of this growth irradiance for both species. These experiments showed again that MAAs act as UV sunscreens, but do not provide full protection against enhanced UVB irradiance. Photoacclimation to irradiance consisting of wavelengths >350 nm alters UVB sensitivity and this photoacclimation has to be considered in experiments investigating UVB effects.

Introduction The discovery of stratospheric ozone depletion due to anthropogenic inputs of chlorinated fluorocarbons encouraged scientific interest on the possible influences of the related enhancement of UVB (280-320 nm), as well as on effects by natural UV (~290–400 nm) radiation on marine phytoplankton. UV penetrates to considerable depth in oceanic waters (Jerlov 1950, Smith and Baker 1979, Smith et al. 1992) and it became clear that the ambient UV irradiance has detrimental effects on the biology of planktonic algae (reviews: HolmHansen et al. 1993, Cullen and Neale 1994).

UV radiation inhibits planktonic photosynthesis, and it is of ecological importance to know whether an ozone-related increase in UVB would lead to a decrease in planktonic primary production. Short-term incubations were conducted to measure the extent of photoinhibition by UV and to predict losses in photosynthetic production by an UVBenhancement (Smith et al. 1992, Helbling et al. 1994, Prézelin et al. 1994). However, algae have a number of protective strategies and are able to adapt and acclimate to a wide range of the incident irradiance. Adaptation and acclimation may reduce the impact of solar UV radiation (Karentz 1994, Strid et al. 1994, Vincent and Roy 1993). Like all plants algae are able to adjust photosynthesis in response to the incident irradiance conditions. This includes short-term (sec-min) adjustments and long-term (h-days) regulations (photoacclimation) to changes in the flux and spectral distribution of the incident irradiance (Falkowski and Raven 1997). These regulations are, for example, changes in the PSII performance (dynamic photoinhibition, Osmond 1994), changes in photosynthetic and protective pigments, and changes in photosynthesis-related enzyme (e.g. RuBisCO) (see Falkowski & Raven 1997). Many marine algae exhibit UV-absorbing compounds, mainly mycosporine-like amino acids (MAAs; Carreto et al. 1990a, Karentz 1991, Davidson et al. 1994) which absorb radi-

27

Ultraviolet radiation and the role of MAAs

ation between 310 and 360 nm. Accumulation of MAAs in algae is correlated with exposure to PAR and UV irradiance (Carreto et al. 1989, 1990b, Marchant et al. 1991) and this photoinduction is wavelength dependent, with highest efficiency at wavelengths between 350 and 470 nm (Carreto et al. 1990b, Riegger and Robinson 1997). Natural phytoplankton samples collected in the Southern Ocean showed highest concentrations of MAAs at the surface where light intensity were highest (Mitchell et al. 1989, Vernet et al. 1989, 1994, Helbling et al. 1994). These substances were therefore suggested as possible protective agents (sunscreens) against UV radiation (e.g. Shibata 1969, Dunlap and Chalker 1986, Karentz et al. 1991) and were considered to be a characteristic feature of UV-related photoacclimation (Karentz 1994, Villafañe et al. 1995, Helbling et al. 1996). UV sensitivity has been reported to be dependent on cell size (Karentz 1994). Smaller species have a higher UV sensitivity due to higher surface to volume ratios (Karentz et al 1991). With the same intracellular concentration of MAAs larger cell will have a higher cellular MAA content by having larger volume. Protection by MAAs is dependent on the optical pathlength by these substances, i.e. also on cell size (see discussion in Riegger and Robinson 1997). The pathlength will be shorter in smaller planktonic algae and the protection here less effective. Despite the variability in species-specific UV-sensitivity (e.g. Karentz 1994), diatoms are less sensitive to UV than other taxa (e.g. Smith et al. 1992). The colonial form of the prymnesiophyte Phaeocystis, for example, exhibits high amounts of MAAs but is more sensitive to UV radiation than all examined diatoms (Davidson et al. 1994, Davidson and Marchant 1994). Studies with different diatom species under natural irradiance conditions revealed that MAAs are accumulated and the amount of photoinhibition by UV is decreased in

28

response to higher irradiances (Helbling et al. 1996). However, it is not clear whether this lower sensitivity is caused by the higher MAA content itself or by other photoacclimative responses (e.g. changes in photosynthetic and photoprotective pigments). Protection against photoinhibition by MAAs should be visible in a change of the related biological weighting functions (BWFs) in that waveband where those substances absorb light. Studies on the dinoflagellate Prorocentrum micans found a wavelength independent change in BWFs of cultures exhibiting low and high amounts of MAAs, suggesting incomplete protection against UV (Lesser 1996a, b). In contrast, studies on the dinoflagellate Gymnodinium sanguineum found strong changes in BWFs between 320 and 360 nm in cultures exhibiting low and high amounts of MAAs (Neale et al. 1998). Here the wavelengths at which protection was provided correlates to wavelengths absorbed by MAAs. These cultures were grown under PAR only at low and high irradiances, whereas the former were grown under constant PAR with and without UV radiation. Ozone depletion resulted in a wavelengthdependent increase in UVB radiation with relatively higher increases at shorter wavelength (40 generations). Cultivation under a solar-like spectrum (~295-750 nm) was performed in a so-called “solar-irradiance-simulator” consisting of a 400-W metallogen lamp (Philips MSR 400 HR) and a system of three liquid filters to adjust the spectral output to a given solar spectrum (further descriptions see Publication 1). We used irradiances simulating a solar spectrum of 300 Dobson Units (DU) (normal ozone) in 2 m water depths at 60° solar elevation (Figure 1). Algae were maintained under this 300 DU spectrum in 100-ml quartz-glass beakers inside a specially designed incubator (see Publication 1). Cultures were carefully mixed in regular intervals using a slow-moving magnetic stirrer connected to an electronic timer. This prevented sinking of the cells and provided a homogeneous exposure.

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Ultraviolet radiation and the role of MAAs

Table 1. Irradiance energy [Wm-2] of EUVB, EUVA and EPAR for the three spectra used, corresponding to 100 µmol photons400-700 nm m-2 s-1. For spectral distribution of each irradiance see Fig. 1.

[Wm-2] EUVB EUVA EPAR

30% ozone depletion 0.14 1.57 20.1

Temperature was the same as in the normal PAR culturing (see above) with an accuracy of ±0.1° C. Experiments were conducted after at least 7 days of culturing under these conditions (>10 generation). Photosynthesis as a function of incident irradiance (P* vs. E0) was measured under two simulated solar-irradiance spectra (300 DU and 220 DU) as described in Publication 1. We used the same spectral distributions (300 DU normal ozone) previously used during culturing and a second spectrum simulating 30% ozone depletion to test the UVB sensitivity in photosynthesis of the algae (220 DU). For one set of curves a sample of the culture (ca. 90 ml) was diluted with fresh medium, resulting in 400 ml algae suspension of less than 10 µg Chl a l-1. 30 ml is used for chlorophyll a-determinations. Chlorophyll a (Chl a) concentrations were determined in quadruplicate using the method of Holm-Hansen et al. (1965). The remaining volume was spiked with NaH14CO3 (Amersham-Buchler) and than dispensed into thirty-four 10-ml narrow-necked quartz-glass bottles with glass stoppers and three completely darkened glass bottles, which were used as dark controls. Two sets of 17 bottles were exposed for 4 hours at 17 different incident irradiances (E0) and under the two different spectra, respectively. Handling of samples before exposure took about 30 min. Temperature during incubation was the same as during culturing and constant at ±0.1° C. The data of each P* vs. E0 curve were fit to the model of Eilers & Peeters (1989), resulting

30

normal ozone

culture chamber

0.07

0.00

1.55 20.1

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into four parameters: the maximal photosynthetic capacity, P*m [µgC (µgChl a h)-1], the light limited slope, α* [µgC (µgChl a h)-1 (µmol photons400-700 nm m-2 s-1)-1], the onset of light saturation, Ek [µmol photons400-700 nm m-2 s-1], and the onset of photoinhibition at higher irradiances, E m [µmol photons 400-700 nm m -2 s -1]. Differences between the two curves in these parameters were analyzed by multiple nonlinear regression using the NLIN procedure of the SAS program (SAS Institute). This provided 95%-confidence intervals for P*m and Em and Ek, and significance of differences in these parameter (p < 0.05). Spectral distributions (Figure 1) of all irradiances were checked regularly using a fastscanning spectroradiometer (UV 320 D, Instruments System, Munich, Germany). Table 1 shows the energy of each waveband, EUVB, EUVA, and EPAR, corresponding to 100 µmol photons400-700 nm m-2 s-1 for these spectra. Spectral absorption (300-750 nm) of an algae extract was determined in a scanning spectrophotometer (Cary 3e, Varian). A total of 25 ml algae suspension was centrifuged (2500 rpm, 4 min, rotor diameter 140 mm) in a glasscentrifugation vial. The resulting pellet was dispersed in 4 ml 80% methanol and extracted for 1 h. The sample was filtered through a 0.2µm membrane-filter (Spartan 13A) using a glass-syringe and scanned against pure 80% methanol. Cell-counts and cell-size measurements were made by inverted microscopy (Utermöhl 1958).

Publication 2 Thalassiosira antarctica -6 Cellular Absorbance [10-2 m2 cell-1]

50 µmol photons400-700 nm m-2 s-1 100 µmol photons400-700 nm m-2 s-1 200 µmol photons400-700 nm m-2 s-1

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Wavelength [nm] Figure 2. Relative absorption of a methanolic extracts from cultures grown under simulated solar spectra of normal ozone conditions but different irradiances (see legend). For Thalassiosira antarctica grown at 4° C (upper graph) and for Thalassiosira weissflogii grown at 15° C (lower graph). All spectra are normalized to total absorption.

Results Light absorption. Methanolic extracts of Thalassiosira antarctica grown at 200 µmol photons400-700 nm m-2 s-1 showed a high absorption maximum at 336 nm (Figure 2, upper graph). This maximum corresponded to a high cellular content of mycosporine-like amino acids (MAAs) (Riegger & Robinson 1997). Extracts of Thalassiosira weissflogii showed no pronounced maximum at 336 nm (Figure 2, lower graph) under each growth irradiance, showing negligible cellular contents of MAAs under all growth irradiances. T. antarctica decreased the cellular MAA content when grown under lower irradiances, visible in a

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Wavelength [nm] Figure 3. Cellular absorption of a methanolic extract of Thalassiosira weissflogii grown at different irradiances (see Fig. 2 for legend) under daylight fluorescence tubes (upper graph) and under a simulated irradiance of normal ozone conditions (lower graph).

reduction of relative absorbance at 336 nm (Figure 2, upper graph). Relative absorbance of T. weissflogii grown at 50, 100 and 200 µmol photons400-700 nm m-2 s-1 were rather similar (Figure 2, lower graph). For both species the absorption showed a relative increase of absorbance at 470 nm with increasing growth irradiance, which corresponds to a typical increase in photoprotective carotenoids. As a typical response to higher growth irradiances absorption per cell (shown for T. weissflogii only) decreased with increasing growth irradiance under daylight fluorescence tubes (Figure 3, upper graph), and to a lower degree under a simulated spectrum of normal ozone (Figure 3,

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Ultraviolet radiation and the role of MAAs

32

5

P* [µgC (µgChl a h)-1]

lower graph). In each case absolute cellular absorption was distinctly lower under a simulated solar irradiance than under fluorescence tubes (Figure 3). Considering the spectral quality for each irradiance (Figure 1), relative light absorption by the photosynthetic pigments was higher under a simulated solar irradiance. The algae responded to this higher absorption by reducing the cellular content of photosynthetic pigments. Photosynthesis-irradiance curves. Previous experiments had shown (data not published) that under the here used conditions short-term incubations under different UV-B irradiances had no effect on the light limited slope of the P* vs. E0 curve (α*), due to the very small absolute difference in UV-B at lower irradiances (2%. Since the two species were cultured under different temperatures, we first tested the influence of temperature on the UVB-sensitivity of T. weissflogii. Cultures grown at 10°, 15°, and 20° C were incubated during P* vs. E0 measurements under the same temperature and simultaneously exposed to two different irradiance spectra (simulated normal ozone vs. simulated 30% ozone depletion [enhanced UVB]). Alterations in temperature induced typical changes in the corresponding P* vs. E0 curves (Figure 4). With decreasing temperature, from 20° to 10° C, Em decreased from 364 to 153 µmol photons400-700 nm m-2 s-1 and P*m from 5.1 to 2.8 µgC (µgChl a h)-1. UVB enhancement at 10°, 15° and 20° C resulted in 8%, 6%, and 8% lower P*m, respectively. Corresponding changes in Em were 15% (10° C, p350 nm) has some regulatory functions (for example in photoreactivation) and is used for acclimation to changes in incident UV, as shown for the photoinduction of UV-absorbing mycosporine-like amino acids (MAAs) in planktonic algae (Carreto et al. 1989, 1990, Riegger & Robinson 1997). An increase in the cellular MAA-content increases the UV absorption by these substances and reduces damage to other molecules. It was shown that photoacclimation to higher PAR and UVA irradiance simultaneously reduces the sensitivity to UVB by increasing the cellular MAA content (Neale et al. 1998, Publication 2). However, Lesser (1996a, b) could not find full protection by MAAs by comparing algae grown with and without UV in a study on the dinoflagellate Prorocentrum micans. Photoacclimation as well as short-term adjustments optimize photosynthetic performance and growth under different irradiances. This only includes regulations for photoinhibition at excess irradiances, whereas a possible UVB-related photoacclimation would solely be directed to lower photoinhibition. As can be seen in P* vs. E0 curves, photoinhibition at excess irradiance distinctly decreases with acclimation to higher PAR irradiances (e.g. Steemann-Nielsen 1962, Takahashi et al. 1971). There is yet no indication that algae are able to acclimate to changes in the incident UVB irradiance. This paper addresses the question of whether algae are able to acclimate to an enhanced UVB irradiance on time scales of days to weeks. By that the impact of UVB would be lower than it would be without such acclimation, hence, the UVB sensitivity would decrease with prolonged exposures, similar as that described for acclimation to higher PAR. However, it is questionable if a visible change is attained by photoacclimation to UVB or

Publication 3

caused by UVB damages, e.g. pigment bleaching by high amounts of UVB. To date, UVrelated experimental studies adopted unrealistic irradiances during exposures, including exposures to UV only and to too high UVB:PAR ratios. Reviewing UV-related studies on photosynthesis, Cullen & Neale (1994) stated that it is not certain that the amount of inhibition by UVB found in these studies would be obtained under more realistic conditions. Therefore experiments executed here were conducted under most realistic and "moderate" irradiance conditions and were focussed on physiological alterations by ecologically relevant UVB changes. A so-called "solar-irradiance simulator" was used to expose algae to solar-like spectra (290750 nm). These spectra could be adjusted to simulate different irradiances, corresponding to different stratospheric ozone concentrations (for details see Publication 1). Experimental results from temperate and polar algae exposed to different UVB irradiances (EUVB), without changes in UVA or PAR are presented. Long-term (days) as well as short-term (h) exposures were used to examine reactions to realistic changes in incident UVB. The data show measurable effects of simulated 30% ozone depletion on photosynthesis and growth, but there is no indication for acclimation to changes in EUVB. Materials & Methods Phytoplankton species. Various temperate and Antarctic species from the collection at the Alfred Wegener Institute were used. Temperate diatoms (Bacillariophyceae): Phaeodactylum tricornutum (Bohlin), Thalassiosira weissflogii (Grunow) Fryxell & Hasle; temperate cryptophyte (Cryptophyceae): Rhodomonas balthica Karsten; polar diatoms: Thalassiosira antarctica Comber, Chaetoceros sp., Corethron pennatum (Grunow) Ostenfeld

- prior C. criophilum -; polar prymnesiophyte (Prymnesiophyceae): Phaeocystis antarctica Karsten. Temperate algae were originally isolated from the North Sea, polar species from the Weddell Sea and around Elephant Islands, Antarctica. Two different isolates of T. antarctica were used, one with a cell size of 22 µm valve diameter, another with 40 µm. P. antarctica was cultured in the colonial form of 32 to 128 cells per colony (700 µmol photons400-700 nm m-2 s-1 was reduced under solar-like irradiance to 66% of the photosynthetic rate under PAR only (Publication 1, Figure 3b). The reduction by UVB was about 15% and the rest (19%) was by UVA. Under these conditions UVB caused less than 50% of the total photoinhibition which is equal to what is observed in situ (Holm-Hansen et al. 1993). Simulation of 30% ozone depletion induced a further 12-15%

General Discussion

photoinhibition (Publication 1, Figure 3b). The other species examined also showed an increase in photoinhibition by enhanced UVB irradiances. Reduction of P*m ranged from 2 to 19% (Publication 3, Figure 4). The overall effects of UVA and UVB irradiance observed in the studies presented were similar to the effects obtained by Cullen, Neale and coworkers using a so-called „photoinhibitron“ to determine BWFs (Cullen et al. 1992, Neale et al. 1994, Neale et al. 1998b,c). However, the applied UVB irradiances of current experiments were lower than in the „photoinhibitron“, total spectral quality of the irradiances was more similar to in situ irradiances, and the obtained P vs. E curves had a higher accuracy. Photoinhibition under variable irradiance. The time scale of exposure is a critical point in relating photoinhibition determined in situ or under laboratory conditions to natural conditions. In situ, bottles are suspended in water for a couple of hours, usually around local noon, and laboratory studies regularly use exposures of 30 min to 4 h. Photoinhibition is thought to be time-dependent and to occur in situ only during longer exposures. Some studies demonstrated that phytoplankton withstand short exposures (10-15 min) to surface irradiances by showing no decrease in photosynthesis at high irradiances (e.g. Marra 1978b). Due to vertical mixing the natural irradiance regime for a single algal cell is highly variable. This limits exposure to high irradiance at the sea surface and hence, may also limit photoinhibition or even protect phytoplankton against photoinhibition. To examine photoinhibition of T. weissflogii under short-term exposures, photosynthesis was measured under variable irradiances to simulate vertical mixing. The same set of irradiance spectra as used for experiments under constant irradiances was also used here. Similar effects of UV radiation were found for the electron transport rate (rETR) as a func-

tion of irradiance. Maximum rETR (rETRm) decreased with successive shorter UV wavelengths (Publication 1 and 4), similar to the effects on P*m during 4 h incubations under constant light (Publication 1). For PAR it is generally accepted that rETR is directly related to photosynthesis (e.g. Genty et al. 1989). The experimental setup was used to evaluate the influence of vertical mixing on the impact of enhanced UVB irradiance. Not surprisingly, the maximum attained irradiance (Emax) had a strong influence on overall effects on rETRm. The reduction of rETRm by enhanced UVB increased linearly with increasing E max (Publication 4, Figure 5). Mixing time (tmix), mixing depth (zmix) and overall attenuation (kd) showed influences only when Emax was already high (Publication 4, Figure 5). Increases in zmix (= k d ) increased the reduction of rETR m slightly. tmix showed the highest impact at 80 min, with a lower impact at 40 and 120 min. The overall UVB dose to which an algae is exposed during one mixing cycle is determined by all of these parameters. This cumulative dose is lower under deeper mixing, shorter mixing times, or higher attenuation. The minimum dose in the experiments was attained by zmix of 50 m, kd of 0.08 m-1 and tmix of 40 min. Under these conditions, irradiances in excess of the growth irradiance of 50 µmol photons400-700 nm m-2 s-1 were only attained for 20 min when Emax was 750 µmol photons400-700 nm m-2 s-1 (see Publication 4, Figure 2+3a). The influence on rETRm by enhanced UVB (simulating 30% ozone depletion) was around 17% (Publication 4, Figure 5, upper graph). Experiments with lower E max (500 µmol photons400-700 nm m-2 s-1) showed an influence of only 6% by the same UVB enhancement, independent of dose (Publication 4, Figure 5, upper + middle graph). Due to the fast increase and decrease in irradiance under a light-cycle with Emax of 750 µmol photons400-700 nm m-2 s-1 irradiances above 500 µmol photons400-700 nm m-2 s-1 was attained for only 8 min (Publication 4,

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General Discussion

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(1998c) concluded that fast and shallow vertical mixing enhances the effect of UVB compared to a static condition. The overall effect was stronger under higher irradiances. The laboratory data presented here support the findings of the influence of higher irradiance but reveal that the influences of mixing times and mixing depth are considerably complex. The overall effect of enhanced UVB is dependent on induced damage and concurrent repair. Both are a function of irradiance and time and are influenced by all parameters (zmix, tmix, Emax). Dose- or irradiance-dependence of UVB induced photoinhibition. Photosynthesis and photoinhibition by excess light are usually reported as a function of irradiance (Neale 1987), whereas effects by UV radiation are commonly related to the cumulative dose. As noted above, photoinhibition by UVB is a time- and dose-dependent process, requiring long exposures and high total doses. It is seen here that 14C-measurements of photoinhibition

% Difference in P*m

Figure 3a). In summary, due to the setup of the light cycle, a 11% (17% - 6%) reduction in rETRm was attained by exposure for only 8 min to a moderate irradiance of 600-750 µmol photons400-700 nm m-2 s-1. Some experiments suggest (data not shown) that even shorter exposures (400 µmol) the curve became more non-linear and reached a maximum. However, this is mainly provoked by the mathematical formulation of the used P vs. E model (Eilers & Peeters 1988). Between 100 and 400 µmol photons 400-700 nm m -2 s -1, photoinhibition by enhanced UVB is a function of irradiance and therefore irradiance-dependent. Photosynthesis measured with a fluorometric method (PAM) allows faster determinations under variable irradiances from 0 to 750 µmol photons400-700 nm m-2 s-1. The total differences in UVB dose by enhanced UVB (30% ozone depletion) were considerably low with values of 250 to 1100 J m-2 UVB, corresponding to only 19 to 84 J m-2 DNA300 using the DNA action spectra of Setlow (1974) and 44 to 193 J m-2 EXP300 using the BWF of Behrenfeld et al. (1993)(both are normalized to 300 nm [e300 nm = 1]). During P vs. E measurements using 4 h incubation the total difference in UVB dose would already be >1000 J m-2 for an irradiance of 100 µmol photons400-700 nm m-2 s-1. Variation of irradiance can be used to evaluate dose-

dependence. No pure dose-dependence was found, however, some indications for an irradiance-dependence were observed (Publication 4, Figure 7). Both measurements (14C and PAM) clearly showed that photoinhibition induced by enhanced UVB is mainly a function of irradiance and not of dose. Cullen and Lesser (1991) also showed that photoinhibition by UVB is not dose-dependent for nutrient-replete cultures of Thalassiosira pseudonana for exposure longer than 30 min. Effects of UV occur in the first 30 min of the UV exposure (Cullen & Lesser 1991, Herrmann et al. 1996, 1997). As photosynthesis is the sum of various mechanisms, which can partly be dose and partly be irradiance-dependent due to the kind of repair and protection mechanisms involved, no pure dependence on dose or irradiance is expected. In some studies with natural communities, pronounced dose-dependence was found (Behrenfeld et al. 1993, Neale et al. 1998b), while other studies did not (Helbling et al. 1994). In summary, both methods used in this thesis showed that the influence of enhanced UVB (as well as of total UVB) under nutrient-replete conditions is pronouncedly irradiance-dependent, especially at low UVB irradiances. Future studies on ecological effects of UVB or total UV should therefore focus on realistic total irradiances and spectral qualities and not on realistic UVB doses without examining the dose- or irradiance-dependence. Under natural conditions repair can be reduced by nutrient-limitation, temperature etc., then a dose-dependence will predominate (e.g. Neale et al. 1998b). Under laboratory nutrient-replete conditions it is evident (Cullen & Lesser 1991, this work) that repair capacities of algae are high enough to prevent any relation to the total amount of UVB when exposed to realistic irradiances. Nevertheless, most UV studies thus far have focussed on differences in dose. Reciprocity of dose and the related effect was assumed. Reciprocity will

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General Discussion

only occur when extreme high UVB doses are given and repair activity is comparably low, or even prevented, i.e. under unrealistic irradiance conditions.

Impact of UVB on growth UVB as well as UVA radiation affects growth of many planktonic algae (review: see Publication 3). Some studies observed reduced growth after acute exposure to high levels of UVB and showed that UVB effects were increased by an irradiance lacking UVA and blue light which prevents repair of UVB induced DNA-damage by photoreactivation (Karentz 1991, 1994). More important, growth is also affected by natural UVA and UVB radiation, whereas excluding UV increases growth rates (Jokiel & York 1984, Smith et al. 1992). Enhancement of UVB with lamps decreased growth rates (e.g. Behrenfeld et al. 1992). Studies with moderate UVB irradiances were conducted by Behrenfeld and coworkers (Behrenfeld et al. 1992, 1993 and 1994), who reported the spectral quality and dose used in their experiments. Spectral quality and irradiance of UVB were realistic, however, UVB:UVA and UV:PAR ratios were not. For instance, UVB irradiance of less than 40% of the near noon solar irradiance in summer was given for a short period (3 h). UVA (7 h) and PAR (12 h) was given during a simulated daily light-dark cycle at less than 20% of this irradiance only (see Behrenfeld et al. 1994). The biologically effective UVB dose weighted by the action spectra (290-320 nm) of Behrenfeld et al. (1993) was 6285 J m-2 d-1 under a treatment of enhanced UVB and 2133 J m-2 d-1 under a treatment of normal UVB. Experiments done in this thesis using continuous illuminations (24 h) and an irradiance corresponding to 100 µmol photons400-700 nm m-2 s-1 had a biologically effective UVB dose weighted with the same action spectra of only

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2300 J m-2 d-1 under enhanced UVB, 1200 J m-2 d-1 under normal UVB (normal ozone) and 700 J m -2 d -1 under less UVB (WG 320). The increase in UVB irradiance by enhanced UVB was consistently lower than that used by Behrenfeld et al. (1994). Using the DNA action spectra of Setlow (1974), the corresponding biologically effective dose for the three treatments were 630, 170 and 40 J m-2 d-1 at 100 µmol photons400-700 nm m-2 s-1, respectively. Decreases in growth rates by increasing UVB was found for all species examined. The decrease was greater under higher total irradiance. The mean effect of enhanced UVB (30% ozone depletion) was about 10% (max: 36%) (Publication 3, Figure 6) and was thus comparable to other studies (for review see Publication 3). When growth rates under solarlike irradiances were compared to growth under daylight fluorescence tubes (data not shown), UVA radiation significantly inhibited growth of Phaeocystis antarctica and Corethron pennatum. Comparing the UVB doses with those of others (see above), the effects were much higher than expected. Growth is significantly affected by UVB and by UVB enhancements for all examined species, however, some species were able to withstand UVB changes at lower irradiance (40-µm Thalassiosira antarctica), whereas others were highly affected and are expected to be sensitive in growth at UVB irradiance corresponding to a PAR irradiance of lower than 100 µmol photons400-700 nm m-2 s-1.

Species-specific differences Effects of enhanced UVB on growth and photosynthesis were found to be highly species-specific. The species-specific differences were mainly seen in the extent of the UVB-sensitivity. All species were affected in growth and photosynthesis.

General Discussion

What is responsible for these species-specific differences? Karentz et al. (1991 and 1994) found a size-dependence in UVB sensitivity: greater species were less affected by UVB than smaller species. This is consistent with the idea that greater species could be protected more effectively by high amounts of UV-absorbing compounds (MAAs) (GarciaPichel 1994). On the other hand, El-Sayed (1988) noted that larger diatoms seemed to be killed by ambient UV radiation. In this thesis, no general dependence on size of different temperate and polar species was found. The greatest species (C. pennatum) showed high UVB sensitivity in growth under long-term experiments, but was in contrast reported by Karentz et al. (1991) to be insensitive to UVB in short-term experiements. However, two observations point to a size- or MAA-dependence: a) the difference in UVB-sensitivity by the 40-µm and 22-µm T. antarctica, the latter being much more sensitive (Publication 3, Figure 6) and b) the difference between the polar 40-µm T. antarctica, which has high amounts of UV-absorbing compounds and the small T. weissflogii, which has nearly no UVabsorbing compounds (Publication 2). Photosynthesis of T. weissflogii was affected by UVB under all growth irradiances, whereas for the 40-µm T. antarctica the effect decreased with higher growth irradiance. This indicates that UV-absorbing compounds provide a significant protection against enhanced UVB for short-term exposures and that size is regulating the maximum amount of these compounds, hence protection. Effects on growth increased with increasing growth irradiance for both species. Temperature may also influence UVB sensitivity by limiting repair capacities and increasing photoinhibition (e.g. Roos & Vincent 1998). No general differences were found between temperate species grown at 15° C and polar species grown at 4° C (Publication 3). Photosynthesis measurements of T. weissflogii

grown at different temperatures showed that decreasing temperature increased photoinhibition and decreased P*m, but the percentage of a further decrease in P*m by enhanced UVB did not vary with temperature (Publication 2, Figure 4). Temperature did have an effect on PAR/UVA induced photoinhibition, but did not affect the influence of UVB. The species-sensitivity can also be influenced by taxonomic differences. Diatoms were already known to be less affected by UV (e.g. Smith et al. 1992), and also in the current study the two non-diatom species (Rhodomonas balthica [Cryptophyceae] and P. antarctica [Prymnesiophyceae]) were found to be more sensitive to enhanced UVB during short-term P vs. E measurements than diatoms (Publication 3, Figure 4). The influence in growth was comparable to the most sensitive diatom species (Publication 3, Figure 6). No general factor was observed to be responsible for species-specific UVB-sensitivity. However, for taxonomically related species, size and the amount of UV-absorbing compounds may play an important role in providing protection against enhanced UVB. For diatoms, which decrease their cell size with cell-doubling, protection against UV would change as size alters. Contrary to MAAs and size, temperature had no pronounced effect on UVB-sensitivity, but affects influences of excess PAR and UVA.

Correlation of UVB sensitivity in growth and photosynthesis UVB effects on growth and photosynthesis of nutrient-replete culture were determined using the same total irradiances and the same spectral distributions for both measurements. A close coupling between photosynthesis and growth was expected and a reduction in photosynthesis should also be observable by a reduction in growth.

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General Discussion

Species which were highly affected by enhanced UVB in growth (µ) showed a high photoinhibition of photosynthesis by enhanced UV. This showed a good correlation between UVB sensitivity in photosynthesis and growth (Publication 3). A linear correlation was obtained, i.e. a proportional difference in P*g leads to a similar proportional difference in µ. Even for P*m which is attained at an irradiance far higher than growth irradiance, a good 1:1 correlation was found. For most species examined, the differences in photosynthesis induced by enhanced UVB during short-term exposures were similar to the respective differences determined after a long-term exposure to enhanced UVB. For more sensitive species, photosynthesis was affected stronger by a long-term exposure than previously observed during a short-term exposure. Thus, for a single culture, short-term measurements of photosynthesis conducted under various UVB irradiances can be used to predict minimum differences in growth, which would be observed when samples of this cultures would be grown for a longer time under these various UVB irradiances.

Photoacclimation to UVB? Photoacclimation is a general feature of all photosynthetic organisms and is easily observed in planktonic algae (Falkowski & Raven 1997). It can be studied by evaluation of P vs. E curves (e.g. Prézelin 1981, Sukenik et al. 1990). Many other biochemical and physiological changes are accompanied with alterations in irradiance (see Falkowski & Raven 1997), and photoacclimation is regulated by changes in total irradiance and spectral quality (Falkowski & LaRoche 1991, Nielsen & Sakshaug 1993). In this thesis, photoacclimation to PAR and UVA was visible during all experiments (e.g. Publication 3). This includes changes in light

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absorption (hence pigments), in α, P*m and Em, and in the maximum PSII quantum yield. Pronounced changes were observed with a change of spectral quality during growth (daylight fluorescence tubes vs. simulated solar irradiance). The fluorescence tubes used had a 2-fold higher ratio of red to blue light (see Publication 2, Figure 1). Due to this strong change in spectral quality of PAR, the cellular pigment content decreased to less than 50% of the former values with a transfer to a solar-like irradiance. Photoacclimation to PAR generally responds to the amount of absorbed light (e.g. Nielsen & Sakshaug 1993). Since total integrated PAR irradiance (400-700 nm) was held constant under both illuminations (fluorescence tubes vs. solar-like irradiance), the much higher blue and UVA radiation under simulated solar irradiances increased the amount of light absorbed by the algae (350700 nm) to ~25%, which in turn was downregulated by a decrease in cellular pigment content. When acclimated, the in vivo cellular absorption was equally reduced to 75% of the former value by reducing the total pigment content to less than 50%. Changes by increasing irradiance from 50 to 200 µmol photons400-2 -1 s were also significant but less 700 nm m pronounced than the changes by spectral quality. For those species exhibiting UV-absorbing compounds (MAAs), an increase of relative absorption of MAAs with increasing irradiance could be observed. Since cell-specific light absorption decreased under solar-like irradiances and the ratio of absorbance at 336 to absorbance at 664 nm (a330 nm:a664 nm) did not significantly change (personal observations), cellular absorption by UV-absorbing compounds also decreased under solar-like irradiances. This shows that irradiance induced MAAs, but that the relative absorption of MAAs is not enhanced by a higher amount of UV. However, changes in MAAs by UVA were not examined in detail and other studies could

General Discussion

show that the MAA content and the related absorption was higher in species grown under PAR + UV radiation than in species grown under PAR only (Helbling et al. 1996, Lesser 1996a, b). These studies did not further examine differences induced by UVB and UVA and as indicated by the study of Riegger and Robinson (1997) the increase in MAAs is induced by longer UVA radiation not by UVB. To summarize the changes by growth irradiance, algae grown under daylight fluorescence tubes had higher total levels of photosynthetic pigment, higher cellular in vivo absorption and higher total levels of UV-absorbing compounds than algae grown under a simulated solar irradiance. Cultures grown under an irradiance of fluorescence tubes were much less affected by changes in UVB during short-term exposures (Publication 1, Figure 3 and Publication 2, Figure 7). Results of preliminary experiments were confused by the fact that some species were not affected by enhanced UVB when grown under fluorescence light, but showed significant effects when acclimated to a solarlike irradiance (personal observations). However, algae grown under solar-like irradiance, but with wavelengths >360 nm only, did likewise increase their UVB sensitivity (personal observations). These observations can only be explained by changes in pigment content accompanied with the changes in growth irradiance: algae grown under fluorescence tubes and than transferred to solar-like irradiance have much higher levels of photosynthetic pigments, hence more PSUs, than needed under that irradiance. A much higher number of PSUs is needed to be damaged by UVB to reduce the photosynthetic rate, thus a lower UVB sensitivity seems to be obtained. These differences were even higher for those species exhibiting a high amount of UVabsorbing compounds (Publication 2), as the cellular content of these substances (=protection) were also higher under irradiance of

fluorescence tubes (see above). This was a special problem during the preliminary experiments clearly showing the need to use controlled solar-like irradiances in laboratory studies to examine effects by UV radiation. PAR-related photoacclimation can be seen in a culture grown under low irradiance which is exposed to a high irradiance. Photoacclimation was visible some days after this shift to a high irradiance in alterations in pigment composition, cellular pigment content, P vs. E characteristics, PSII quantum yield etc. An increase in growth irradiance (PAR) will induce an increase in P*m and a decrease in the susceptibility of photoinhibition during shortterm exposures (see Neale 1987), i.e. a lower sensitivity to enhanced PAR irradiance will be observed. A similar reaction is expected when algae are able to acclimate to enhanced UVB irradiance. After a prolonged exposure to enhanced UVB, the sensitivity to short-term UVB enhancements should decrease and the obtained P*m should be higher than that obtained for algae not exposed to enhanced UVB for longer times (=normal UVB). Behrenfeld et al. (1992), who examined UVB-effects on growth of Phaeodactylum tricornutum under natural irradiances did not observe a decrease in the influence on growth by enhanced UVB over a period of more than 30 days. The influence (reduction) on growth even increased during prolonged exposure. However, the ambient irradiance was changing due to a decrease in maximum solar elevation during the experimental period. So far, no study on photoacclimation to UVB has been conducted under controlled laboratory conditions. In this thesis, different species were studied under laboratory conditions, especially under well controlled UVB irradiances. There was no indication for photoacclimation to enhanced UVB irradiance because no alterations in pigments, UV-absorbing compounds, or PSII quantum yield were observed (Publication 3, Table 2). Significant changes in P vs. E curves

85

General Discussion

* were found, mainly as a decrease in Pm by enhanced UVB. However, this UVB-sensitivity was not altered under prolonged exposure to enhanced UVB (Publication 3, Figure 4). The proportional change by an alteration in UVB was the same for a culture under normal UVB when incubated under enhanced UVB, or for a culture grown under enhanced UVB and incubated under normal UVB. The obtained P*m values did not increase with longer exposure to enhanced UVB irradiance. For less sensitive species, seen in the correlation of UVB influence on P*m and growth (see above), P*m determined under normal UVB (or under enhanced UVB) was the same for a culture grown under normal UVB and for another grown under enhanced UVB (Publication 3, Figure 4 a-f). For very sensitive species (22-µm T. antarctica, P. antarctica) this proportional change was also similar, but the obtained absolute values of P*m were lower when grown under enhanced UVB (Publication 3, Figure 4 g + h). The results are unequivocal for T. weissflogii since the same situation was found for effects on P*m as well as for effects on rETRm with different methods and different experimental setups (Publication 3 and 4). Therefore, it is concluded that algae are not able to acclimate to enhanced UVB irradiances, which will occur with ozone depletion at time scales up to 14 days. As observed here (Publication 2 and 3), many other studies showed a decrease in UVBsensitivity of algae with increasing total growth irradiances either directly in culture grown at different irradiances (Neale et al. 1998a) or indirectly in samples from different depths (e.g. Helbling et al. 1992, Neale et al. 1998b). However, this change in UVB sensitivity is due to photoacclimation to PAR and UVA and can partly be a result of the relative increase in UV-absorbing compounds (Publication 2, Neale et al. 1998a, Helbling et al. 1996). A change in UVB-sensitivity by increasing PAR/UVA will not decrease the

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impact of enhanced UVB by stratospheric ozone depletion. Such an enhancement of UVB irradiance is only accompanied by a insignificant increase in PAR and UVA irradiance.

Ecological considerations Significant effects of UVB on photosynthesis and growth of different algal species were obtained in this thesis by applying realistic solar-like irradiances and relevant ozonerelated changes in UVB. The applied doses of UVB were distinctly lower than in other studies but the amount of changes were about equal. There are two possible explanations. First, enhanced UVB was realistically simulated by a wavelength-dependent increase in UVB irradiance, and second, full spectral irradiance with realistic UVB:UVA:PAR ratios were given. However, results showed that the cumulative dose is not a proper parameter to compare different experimental setups. Temperature is thought to influence the UVB-related photoinhibition and/or repair, and hence, polar species are possibly more affected by UVB and more sensitive to enhanced UVB (Ross & Vincent 1998). In this thesis, no pronounced differences were found between temperate species, cultured at 15° C, and polar species, cultured at 4° C. But, the most sensitive were among the polar species. The results of this study cannot support a temperature dependence of UVB sensitivity since less and high sensitive species were found in both groups. The experiment conducted with T. weissflogii grown at different temperature (Publication 2, Figure 4) showed that photoinhibition by excess irradiance increased with decreasing temperature and P*m was reduced at lower temperatures. Similar observation were reported in other studies (e.g. Ross & Vincent 1998). The proportional reduction in P*m by enhanced UVB was, nevertheless, independent

General Discussion

to temperature. Whereas PAR and possible UVA related photoinhibition is seen to be dependent on temperature for a single species, UVB photoinhibition is not. In addition, the individual P vs. E curves of different species did not show a higher susceptibility to excess irradiance in polar species. Even P. antarctica which was highly affected by enhanced UVB did not show pronounced photoinhibition by excess irradiance. In contrast, the temperate cryptophyte R. balthica showed the highest photoinhibition at irradiances >500 µmol photons400-700 nm m-2 s-1. Polar species seem to be adapted to the low temperature in their environment. It is evident that temperature is involved in PAR/UVA related photoinhibition for a single species. The results indicate again that UVB has different targets and mechanisms for damage and/or recovery (repair) than PAR and UVA radiation. UVB effects are caused by temperature-independent photochemical reactions of the highly destructive UVB radiation. Repair is an enzymatic process and hence, temperature-dependent. However, for photosynthesis, algae are most probably adapted to low temperatures in their repair capacities. In summary, temperature appears not to be responsible for UVB sensitivity of polar species as previously suggested (Ross & Vincent 1998). Species-specific differences in UVB-sensitivity will probably lead to a change in the phytoplankton community structure by enhanced UVB under ozone depletion. This change could still alter productivity. The species-specific differences observed in this thesis are not as pronounced as found elsewhere (e.g. Karentz 1991, 1994). Only one diatom species (T. antarctica) of larger cell-size was less affected in growth but was highly sensitive at smaller cell-size (Publication 3). Changes in community structure to a higher number of less-sensitive species will, therefore, decrease the impact of UVB but will not completely counteract the loss in production. Community

structure is controlled by many other factors like nutrient-availability, grazing and PARirradiance. A change will occur only if UVB is a major controlling factor of this structure. So far, phytoplankton are mainly thought to be controlled by limitation of nutrients and PAR or by grazing. For instance, in a long-term study on a freshwater benthic diatoms communities an increase in diatom biomass by UV exposure was observed (Bothwell et al. 1994). Grazing by larval chiromids (Diptera) was considerably reduced, since these insects were more affected by UV radiation than the algae. UVB radiation is one of the most variable parameters in pelagic environments because in a given water mass it alters with solar elevation (time of day, time of the year), water depths and stratospheric ozone concentration, whereas total PAR is not influenced by ozone and to a lesser degree by solar elevation and depth. Solar elevation influences UVB pronouncedly because the pathlength for light through the atmosphere, hence, absorption by ozone, is increasing with decreasing elevations. Maximal UVB irradiances are reached at the sea surface during local noon together with maximal total irradiances. Even this maximal UVB irradiance is changing on time scales of some days by normal variations in atmospheric ozone concentration. In the Antarctic, when the „ozone hole“ is present, changes of more than 50% in ozone will occur during 3-4 days by a circular movement of the „ozone hole“ around the southern pole. UVB and UVB:PAR ratios are therefore changing on time scales of minutes, hours and days. Changes in UVB by ozone depletion induce changes a) on longer time scales (years) and b) increase differences in the maximum obtained UVB:PAR ratio. The already existing short-term (min) and longterm (days) changes in natural UVB irradiance do obviously have significant effects on planktonic photosynthesis. UVB enters the atmosphere since million of years and live has developed under oxygen free, hence, ozone

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General Discussion

free, conditions, when UVB irradiance was far higher. It could be assumed that algae have evolved photoacclimative regulations to mitigate the influences of UVB. Astonishingly, as this thesis indicates, this is not the case. Algae cultures, grown under a solar-like irradiances, are not able to acclimate to alterations in UVB. Photoacclimation studied with single algal cultures fairly well describes photoacclimation of natural phytoplankton (Falkowski & Woodhead 1992), thus, UVB photoacclimation is probably not established in natural phytoplankton. Photoacclimation to high irradiance of UVA and PAR leads to a concomitant decrease in short-term UVB-sensitivity, e.g. by an increase in protection by MAAs. Some results indicate that the long-term influence in growth by higher UVB are even enhanced under higher total irradiances, despite photoacclimation. The good correlation between influence in photosynthesis and influence in growth by UVB, and the fact that some species were even more affected in growth than in photosynthesis, leads to the conclusion that shortterm incubations measuring photosynthesis could sometimes underestimated the potential effects on growth. However, growth of natural phytoplankton is often limited by nutrients and not directly related to photosynthesis. As noted above, natural UV or UVB irradiance inhibited photosynthesis when measured under constant irradiances in situ. It was discussed whether photoinhibition of photosynthesis occurs under natural conditions of highly variable irradiances induced by vertical mixing. The conclusion that photoinhibition in nature would have a lower effect on primary productivity was drawn by two reasons. First, photoinhibition was thought to influence photosynthesis only at strong irradiances and only after relatively long exposures, and second, if exposure is considerably long, photoinhibition will be counteracted by regulations to the higher irradiance by photoacclimation. This is valid for photoinhibition by PAR but seems not

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to be true for that by UVB radiation. Photoinhibition by PAR is found always with excess irradiance, i.e. with irradiances far higher than the mean irradiance the phytoplankton are acclimated to. Under in situ conditions photoinhibition by PAR is rarely observed and possibly insignificant in surface sample. UV radiation, especially UVB, does however affect photosynthesis at lower than saturating irradiances. It even reduces photosynthesis of algae significantly at constant growth irradiance (Eg). Influences on P*m are more pronounced than on P*g, but P*m is normally reached at a higher irradiance than Eg. Lastly, photoacclimation which is mainly a reaction to PAR, cannot completely counteract photoinhibition by shorter UV, especially by UVB. By photoacclimation photosynthesis, as well as the susceptibility to photoinhibition, can be altered by the range and frequency of variations in irradiance recently experienced by the algae (see Falkowski 1983, Cullen & Lewis 1988, Lande & Lewis 1989). Besides the daily light-cycle, a determinant of incident irradiance is depth, and vertical motion through the water column is a major source of fluctuations in irradiance. Changes in depth of an organism is attained actively by the motility of the organism, passively by sinking or by motions of the water column itself. These movements are induced by differences in temperature or salinity or by tidal waves. In oceanic regions, wind shear on the surface has the greatest impact on vertical mixing. Considerable fast vertical mixing is achieved by turbulent eddy mixing, internal waves and Langmuir circulation (Denman & Gargett 1983). Time scales considered are a few minutes to several hour, the mixing depth can be a few meters shallow or reaching several tens of meters. Since the driving force is mainly by wind stress, deeper mixing includes successively longer mixing times, however, the motion of single algae will be more or less turbulent or even chaotic. For Langmuir cells a circular motion is pro-

General Discussion

posed ranging vertically (depths) from 2 to >10 m and horizontally 5 to 300 m with mean cycling times less than 30 min (Denman & Gargett 1983). Denman and Gargett (1983) showed that vertical displacements of 10 m through the UML vary from 0.5 h to hundreds of hours. As vertical mixing is thought to reduce the exposure time to high irradiance at or near the surface, it was concluded that phytoplankton are protected against UV-related photoinhibition by strong vertical mixing. Strong and deep mixing decreases the mean irradiance experienced by the phytoplankton. By that their UVsensitivity is increased since they are acclimating to low irradiances. Simulated vertical mixing did however increase total integrated production compared to static conditions, when photoinhibition was visible under static conditions (Marra 1978a). No change was observed with vertical mixing in the opposite situation (no visible photoinhibition under static conditions; Yoder & Bishop 1985, see Neale 1987). Helbling et al. (1994) found positive and negative effects by simulated vertical mixing, depending on the maximal subjected irradiance. Compared to static incubations, higher irradiances were accompanied by a decrease in total integrated primary production with vertical mixing, whereas under low irradiance mixing increased total primary production. Similar results were revealed by modeling the influence of vertical mixing on ozone-related photoinhibition on Antarctica phytoplankton using different BWFs attained for natural phytoplankton from the Weddell-Scotia Confluence (Neale et al. 1998b). Deeper mixing (lower mean irradiance) increased water-column integrated daily photosynthesis (PT) compared to uninhibited photosynthesis (PTpot), whereas shallow mixing (higher irradiances) decreased PT. 50% ozone depletion did decrease PT. This decrease was always enhanced by vertical mixing. The change was again greatest at short and

shallow mixing and lowest at deep and long mixing. A maximum change of -8.5% was observed, average values were around -2.7 to -2.8% and quite low in comparison to other factors influencing PT with vertical mixing, for instance zmix (-27 to +21%) or the physiological variability (±28%). Short mixing times (10 to 20 min) always produced the highest effects but the model was based on data of 0.5 to 3.5 h static incubations. Additionally, during determination of BWFs no recovery (low repair) was observed (Neale et al. 1998b). Results of the presented thesis (Publication 4) are not used to model integrated photosynthesis but revealed that even under conditions of high repair the potential effect of ozone depletion would be more dependent on irradiance and less dependent on zmix (25 to 75 m) and tmix (40 to 120 min). Scales of zmix and tmix were equal to that of the study by Neale et al. (1998c), but unfortunately, incubation times