J. Phycol. 47, 603–614 (2011)  2011 Phycological Society of America DOI: 10.1111/j.1529-8817.2011.00998.x SPOROGENESIS UNDER ULTRAVIOLET RADIATION IN LAMINARIA DIGITATA (PHAEOPHYCEAE) REVEALS PROTECTION OF PHOTOSENSITIVE MEIOSPORES WITHIN SORAL TISSUE: PHYSIOLOGICAL AND ANATOMICAL EVIDENCE 1 Ansgar Gruber2, Michael Y. Roleda3,4 Biologische Anstalt Helgoland, Alfred Wegener Institute for Polar and Marine Research, 27498 Helgoland, Germany Inka Bartsch Section Functional Ecology, Department Seaweed Biology, Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany Dieter Hanelt Biozentrum Klein Flottbek, University of Hamburg, Ohnhorst-Str. 18, 22609 Hamburg, Germany and Christian Wiencke Section Functional Ecology, Department Seaweed Biology, Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany To study the effect of different radiation conditions on sporogenesis of Laminaria digitata (Huds.) J. V. Lamour., excised disks were induced to form sporangia under PAR (P), PAR + ultraviolet-A (UVA) (PA), and PAR + UVA + ultraviolet-B (UVB) (PAB) conditions in the laboratory. Vitality of meiospores, released from sori induced under different radiation conditions in the laboratory and from sori of wild sporophytes acclimated to in situ solar radiation in the presence and absence of ultraviolet radiation (UVR), was measured in terms of their germination capacity. Sorus induction in disks of laboratory-grown sporophytes was not hampered under light supplemented with UVR, and sorus area was not significantly different among P, PA, and PAB. Vitality and germination rate of meiospores released from sori induced under different radiation treatments was comparable. Likewise, screening of UVR of the natural solar radiation did not promote higher germination rates of meiospores released from wild sporophytes. Germination rates were, however, higher in meiospores released from laboratory-induced sori compared to sori of wild sporophytes. Higher DNA damage (formation of cyclobutane pyrimidine dimers, CPDs) was observed in laboratory-grown nonsorus compared to sorus tissue, while CPDs were nondetectable in both sorus and nonsorus tissue of wild sporophytes. To explain the apparent protection of developing meiospores and the unexpected UV resistance of soral tissue, concurrent anatomical investigations of sporogenic tissue were performed. We observed the previously unreported existence of two types of sterile paraphysis cells. One type of paraphysis cells, the most frequent type, contained several red-fluorescing plastids. The other type, less frequently occurring, was completely filled with substances emitting blue fluorescence under violet excitation, presumably brown algal phenolic compounds (phlorotannins). Cells of this type were irregularly scattered within the sorus and did not contain red-fluorescing plastids. Meiospore-containing sporangia were positioned embedded between both types of paraphysis cells. In vegetative tissue, blue autofluorescence was observed only in injured parts of the blade. Results of our study suggest that the sorus structure with phlorotannins localized in the specialized paraphysis cells may be able to screen harmful UVR and protect UV-sensitive meiospores inside the sporangia. Key index words: cyclobutane pyrimidine dimers; DNA damage; germination; hyaline cells; kelp; Laminaria digitata; Laminariales; meiospore; paraphysis cell; phlorotannin; physode; plastids; sorus; ultraviolet radiation Abbreviations: CPD, cyclobutane pyrimidine dimer; UVA, ultraviolet A (320–400 nm wavelength); UVB, ultraviolet B (280–320 nm wavelength); UVR, ultraviolet radiation 1 Received 15 May 2010. Accepted 14 January 2011. Present address: Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany. 3 Author for correspondence: e-mail michael.roleda@botany. otago.ac.nz. 4 Present address: Department of Botany, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. 2 The reproductive biology of seaweeds is generally regulated by seasonal environmental factors to ensure recruitment success (De Wreede and Klinger 1988). For example, short-day sorus induction and 603 604 ANSGAR GRUBER ET AL. winter peak in reproduction can minimize meiospore exposure to adverse environmental conditions, such as high temperature and high solar irradiance, and thereby possibly enhance meiospore viability. Kelp sporogenesis (e.g., in Laminaria hyperborea) usually occurs during the season of slow or no growth in autumn and winter (Kain 1979). In some species where year-round reproduction occurs, a bimodal peak in seasonal maximum sorus formation and abundance of released meiospores is reported (reviewed by Bartsch et al. 2008). L. digitata is one of the species of the genus Laminaria sporulating during summer when reproductive tissue can be observed from late spring to early winter (May–December; Lüning 1982, Roleda et al. 2005b) in the population around Helgoland (North Sea). Recently, a detailed phenological study revealed that the abundance of mature sori of L. digitata at Helgoland peaked in August to September, while maximal sorus area is found from September to October (Gehling 2006), a time when ambient solar radiation just passed its peak but still is relatively high (http://coast.gkss.de/ data/helgo_rad.html). Sorus formation in investigated Laminariales is inducible under a broad range of daylengths (8– 16 h Æ d)1) and temperatures (1C–17C) (Lüning 1988, Buchholz and Lüning 1999, Bartsch et al. 2004), but higher temperatures are inhibitory (I. Bartsch, unpublished data). Moreover, sporogenesis also depends on the critical internal nitrogen and phosphorus content of the reproductive tissue, reported in several Pacific Laminariales species, indicating the relatively high nutritional cost of reproduction (Nimura et al. 2002, Kumura et al. 2006). Light quality is also reported to influence sporogenesis. In Saccharina japonica (=Laminaria japonica), red light inhibits sporangium formation, and blue light promotes sporogenesis compared to white light (Mizuta et al. 2007). The morphogenesis of reproductive organs in Laminariales has been described in detail for many species (e.g., Nishibayashi and Inoh 1958, 1960, Ohmori 1967) and follows a common pattern: All sori originate from meristodermal cells, which divide periclinally into a lower and an upper cell. The upper cell elongates into the paraphysis without division, and the lower cell gives rise to a unilocular sporangial mother cell (Ohmori 1967). Although the occurrence of paraphysis cells has been described in a number of Laminariales and Tilopteridales (Cribb 1953, Toth 1974, Henry 1987), detailed anatomical descriptions of paraphysis cells are confined to a few studies (Griggs 1907, Ohmori 1967 and references therein, Walker and Bisalputra 1977, Martinez and Correa 1993, Motomura 1993). Sporangia arranged between paraphysis cells have been illustrated in Nereocystis (Walker and Bisalputra 1977), while elongated paraphysis cells towering over the sporangia were described in Cymathere (Griggs 1907). Other paraphyses described in many Japanese Laminariales (Nishibayashi and Inoh 1958, 1960, Ohmori 1967) form a dense protective cover with club-shaped heads and a thick hyaline outer wall probably composed of sulphated polysaccharides (Motomura 1993). While most paraphysis cells are reported to contain nuclei and several chromatophores, paraphyses of Saccharina cichorioides (=Laminaria cichorioides) and Saccharina yendoana (=Laminaria yendoana) contain ‘‘a nucleus and many granules’’; with the granules thought to be physodes by their staining characteristics (Ohmori 1967). Motomura (1993) also describes the presence of a large number of physodes and active Golgi bodies in the cytoplasm of the paraphyses of Saccharina angustata (=Laminaria angustata). The functional significance of paraphysis cells based on these previous findings, however, remains to be explored. A possible role of paraphyses has recently been described in a Dictyotales species, Stoechospernum polypodioides (=Stoechospernum marginatum). Its paraphyses are rich in sulfated polysaccharides (fucoidan) and contain polyphenols. They undergo histochemical changes during tetrasporogenesis pointing to both structural and protective roles (Bhamrah and Kaur 2005). A pressure is thought to be exerted on sporangia between tightly packed paraphyses to aid spore release. Coordinated release of phenols and sulfated polysaccharides may serve as protection and prevent desiccation, respectively (Bhamrah and Kaur 2005). The impact of UVR has been investigated in all life-history stages of kelps, from meiospores, gametophytes, and juvenile and adult sporophytes (Dring et al. 1996, Roleda et al. 2007a, Müller et al. 2008, Miller et al. 2009). However, the effect of UVR on sporogenesis itself and the vitality and viability of spores released from sorus developed under different radiation (presence and absence of UVR) conditions has not been subjected to detailed investigation. Moreover, few studies have directly compared differential responses in wild versus cultured algae to UVR. One study showed that wild Gigartinales gametophytes are less susceptible to UVR-induced DNA damage compared to culture-grown gametophytes (Roleda et al. 2004b). This indicates that wild algae acclimated to high solar radiation in the field are more robust compared to low-lightacclimated algae grown in culture. Among cultured young kelp sporophytes, increasing thallus thickness and opacity in relation to available cell-bound UVabsorbing compounds, can minimize UVR effects on growth and DNA damage (Roleda et al. 2005a, 2006b,d). Generally, the nonuniformly shaped and unevenly spaced cells in macroalgal tissue can cause multiple scattering and intracellular self-shading (Grzymski et al. 1997). This cellular architecture can attenuate up to 95% of the incident UVB radiation and yet transmit between 70% and 80% of the visible radiation (Robberecht and Caldwell 1983). Radiation is thus selectively filtered, removing the P H O T O P R O T E C T I V E R O L E O F P A R A PH Y S E S short UV wavelengths before reaching UV-sensitive organelles such as nuclei and plastids. However, the remaining 5% incident UVB radiation may still have a significant impact on development, growth, and eventual reproduction of the organism. For example, photosynthesis of young kelp sporophytes was able to acclimate to UVR but not growth, affecting cellular differentiation and gross morphology as shown in the curling and thickening of meristematic lamina (Roleda et al. 2004a). Phlorotannins are localized in physodes, small vesicles in the cytoplasm, and are also a constituent of cell walls (Ragan and Glombitza 1986) that can selectively filter short UV wavelengths (van Alstyne et al. 1999). On the other hand, there is an indication that an extracellular sheath, as for example the gametangium wall of Ascoseira mirabilis, may also function as a UV filter (Roleda et al. 2007b) somewhat comparable to scytonemin-containing extracellular sheaths in cyanobacteria (Dillon and Castenholz 1999). In sporophytes of several Laminariales, most phlorotannin-containing physodes or polyphenol-rich cells occur in the outer cortex and meristodermal cells; some physodes are also present in the epidermis and the medulla (Tugwell and Branch 1989, Shibata et al. 2004, Halm et al. 2011). Phlorotannin allocation seemed to follow patterns predicted by the optimal defense theory, which states that chemical defenses are allocated preferentially toward thallus parts that are highly valuable to the organism (Iken et al. 2007). There are indications that in some kelp species reproductive tissues are better furnished with polyphenols than vegetative parts of the blade (Tugwell and Branch 1989), but the protective function against high PAR and UVR remains to be explored. In this study, we investigated the effect of UVR on sporogenesis, and the corresponding vitality of meiospores released. The anatomy of different tissues (sorus and nonsorus) was investigated to address their differential susceptibility to UVR. The following questions were addressed: 1. Is sorus formation influenced by different radiation conditions? 2. Do meiospores developed in sori under different radiation conditions in the laboratory and meiospores from wild sporophytes show different viability? 3. Are sorus and nonsorus tissues equally susceptible to UVB-induced DNA damage? 4. Does sorus tissue possess specialized anatomical structures and chemical compounds that can potentially screen high PAR and UVR? MATERIALS AND METHODS Effect of artificial UVR on sporogenesis of laboratory-grown material (experiment 1). L. digitata sporophytes were grown from stock male and female gametophytes (AWI culture number 3157 and 3158, respectively; isolated from Helgoland, North Sea), 605 according to the method of tom Dieck (1992). After 12 weeks, young sporophytes were transferred into flasks and aerated with membrane-filtered air. Cultures were maintained at 10C and 50 lmol photons Æ m)2 Æ s)1 of PAR (Osram L58W ⁄ 954 Lumilux de Luxe, Munich, Germany) under a 16:8 light:dark (L:D) photoperiod. The sterile Provasoli-enriched seawater (PES; Provasoli 1968) was changed weekly. To accommodate growing sporophytes, cultures were transferred from 1 to 10 L flasks as they increased in size. After 22 months, sporophytes had a length of 19–44 cm. From each nonsporogenic sporophyte, four 2 cm diameter disks were punched out 7–12 cm above the blade ⁄ stipe transition. More than 450 disks obtained from 117 individuals were pooled and maintained in several aerated 5 L flasks for 13 d to heal the marginal wounds. These disks were then used to artificially induce sporogenesis (Buchholz and Lüning 1999). Transparent polyethylene mesh (Diamant Gaze; Haver & Boecker, Oelde, Germany) was tailored into a pouch with 10 separate enclosures (2 · 5, see Fig. S1 in the supplementary material) to accommodate individual disks. Two pouches with 10 disks and one with six disks were laid at the bottom of each basin (40 cm · 30 cm · 13 cm); each pouch was fixed on an acrylic glass plate with vanadium nails to keep it in a fixed position. They were submerged under 6 L of PES, flooding 6 cm in height. A total of six basins were prepared. Inside a 10C temperature-controlled room, two shelves were separately provided with four white fluorescent tubes (L65 Watt ⁄ 25S; Osram) and three UV lamps (Q-Panel UVA-340, 40 W, Cleveland, OH, USA) arranged alternately (Fig. S1). Each shelf accommodated three basins, each covered with different filter foils. These were Ultraphan transparent (Digefra GmbH, Munich, Germany), Folanorm (Folex GmbH, Dreieich, Germany), and Ultraphan URUV (Digefra GmbH) corresponding to different radiation treatments consisting of PAR (400–700 nm wavelength, P), PAR + UVA radiation (320– 400 nm, PA), and PA + UVB radiation (280–320 nm, PAB), respectively. To create a setup with lower irradiance of PAR, UVA, and UVB, basins under another shelf were covered by two layers of neutral black mesh (Diamant Gaze; Haver & Boecker). PAR and UVR were measured below the cut-off filters and mesh covers using a quantum sensor (LI-192SA) attached to a LICOR datalogger (LI-1400; LI-COR Biosciences, Lincoln, NE, USA) and a cosine sensor connected to a UV-VIS Spectrometer (Marcel Kruse, Bremerhaven, Germany), respectively. Irradiances of PAR, UVA, and UVB, and corresponding daily doses are tabulated in Table S1 (in the supplementary material). Moreover, the biologically effective doses between 280 and 320 nm applied were calculated using action spectra for DNA damage in Escherichia coli (280–320 nm; Setlow 1974). To provide water movement, each basin was furnished with a submersible water pump (Eheim Type 1005, Deizisau, Germany). To avoid temperature increase under the filter foils, ventilators were used to circulate air through the side of the basin under the filter foils. This effectively maintained water temperatures between 10C and 12C. The blade disks secured inside the polyethylene mesh pouch were acclimated for 4 d under PAR at 16:8 L:D photoperiod. At the start of the experiment, daily PAR was supplemented with 4 h of UVR in the middle of the light phase. PES was changed weekly. The disks inside the polyethylene bags were regularly reversed (from daily to 2–3 times a week) to evenly irradiate both sides of each disk. This simulates natural conditions, where kelp blades are continuously shifted by water movement, exposing both sides to direct solar radiation. Formation of sori on the excised blade disks was regularly checked and recorded for 12.5 weeks (87 d). At the end of the experiment, disks were photographed with a digital camera (Coolpix 4500; Nikon, Tokyo, Japan), and sorus areas were 606 ANSGAR GRUBER ET AL. calculated using an area-calibrated imaging software (Program developed at the University of Konstanz, Germany; cf. Gruber 2004). After termination of the sorus-induction experiment, disks were processed for germination experiments and DNA damage analysis. Effect of natural UVR on sporogenic wild material (experiment 2). Thirty fertile L. digitata sporophytes were collected during low tide in July 2004 at Helgoland. They were kept hanging in shaded natural seawater conditions for 1 week until the start of the experiment. Ripe sori were present on the distal parts of each sporophyte. To determine the effect of UVR on the DNA of soral and nonsoral tissue, and on the germination capacity of released meiospores, whole sporophytes were transplanted into a mesocosm and exposed under different radiation treatments using cut-off filter foils. The mesocosm (1.5 m · 1.0 m · 0.35 m basin) was established on the rooftop of the Biologische Anstalt Helgoland. The basin was equally divided into three compartments representing the P, PA, and PAB treatments. The mesocosm was provided with continuous flow-through seawater supply circulated into a cooling unit (Aqua Medic, Bissendorf, Germany) with temperature set at 15C. During the period of experiment (Fig. S2a in the supplementary material), daily min.–max. daytime (06:00–18:00) air temperature was lowest at 12.5C–16C (25 July 2004) and highest at 18.5C–24C (31 July 2004), while min.–max. nighttime (18:01–05:59) air temperature was lowest at 12.5C–14C and highest at 18C–22C, on days following and preceding the above dates, respectively (Fig. S2a, values approximately derived from http://coast. gkss.de/data/helgo_rad.html). Spot measurements of water temperature ranged between 16C ± 2C during daytime and 14C ± 2C during nighttime. Additional water circulation inside the basin was generated using three submersible water pumps (Eheim Typ 1060). The holdfasts of each of the five sporophytes were tied to concrete blocks (30 cm · 30 cm · 4 cm) using plastic cable ties. Two concrete blocks (n = 10 sporophytes) were submerged into each radiation treatment. Fertile blade parts, floating below the air–water interface, were exposed to the full solar radiation. At times, they can be partially shaded by blades of neighboring plants as may occur under natural conditions. The experiment was terminated after 8 d. Thereafter, sorus and nonsorus tissues were processed for DNA damage analysis (n = 5) and sori for meiospore release and germination experiment (n = 5). Air and underwater PAR was measured continuously during the entire experimental period using a cosine (LI 192, UWQ 6534) and spherical (LI 193, SPQA 3428) sensor, attached to a LI-COR datalogger (LI-1400; LI-COR Biosciences). Surface UVR was measured by a multichannel UV-Spectroradiometer (iSiTEC GmbH, Bremerhaven, Germany; Hanken and Tüg 2002) installed at the rooftop of the institute. The unweighted irradiances during experiment 2 are compiled in Table S2 (in the supplementary material). Tissue DNA damage. At the end of experiments 1 and 2, small tissue fragments (5 mm diameter) were obtained from the sporogenic (n = 8) and proximate nonsporogenic (n = 8) part of the same disk (experiment 1) and sporophyte (experiment 2). Samples were flash frozen in liquid nitrogen and stored in )80C until analysis. They were homogenized in liquid nitrogen, and DNA was extracted using 2% CTAB extraction buffer and quantified fluorometrically using the PicoGreen assay (Molecular Probes, Eugene, OR, USA) and a Cary Eclipse Fluorescence Spectrophotometer (Variance Scientific Instrument, Palo Alto, CA, USA) (Roleda et al. 2005b). The accumulation of CPDs was determined following a two-step antibody assay using antithymine dimer H3 (Affitech, Oslo, Norway) and rabbit antimouse immunoglobulins (conjugated with horseradish peroxidase; DakoCytomation, Glostrup, Denmark). Chemiluminescent detection was subsequently performed using ECL Western blotting detection reagent (Amersham, Buckinghamshire, UK) (Roleda et al. 2005b). Developed films (using X-ray film developer) were scanned using Biorad imaging densitometer (Model GS-700; Bio-Rad Laboratories, Hercules, CA, USA), and grayscale values were quantified using Multi-Analyst (Macintosh Software for Bio-Rad’s Image Analysis Systems). A calibration series of UVirradiated calf thymus DNA (Serva Electrophoresis GmbH, Heidelberg, Germany) supplemented with unexposed DNA was included giving 1 lg Æ mL)1 DNA for each calibration point. The UV-irradiated DNA (45 min exposure to 2 TL 20W ⁄ 12 lamps; Philips, Eindhoven, the Netherlands) was previously calibrated against UV-irradiated HeLa DNA with known amounts of CPDs (kindly provided by A. Vink). CPDs were quantified by comparing the grayscale values within the linear range of the film. Germination. At the end of each experiment, disks with induced sorus (experiment 1) and disks obtained from wild sporophytes (experiment 2) were blotted dry and kept dark overnight in a moist chamber at 5C ± 1C. Spores were released by immersing each disk in separate petri dishes (85 mm · 15 mm) at 12C and 50 lmol photons Æ m)2 Æ s)1 in PES. To facilitate meiospore release, the petri dishes were agitated (80 rotations Æ min)1) using a shaker (Unimax 1010; Heidolph Instruments, Schwabach, Germany). After 30 min, the disks were removed from the dishes. The spore density was counted using a Neubauer Chamber (Brand GmbH, Wertheim, Germany). To ensure that the density of spores per unit area was similar between each experimental unit, two to four drops of each spore suspension were transferred into new culture dishes (53 mm · 12 mm) and filled with 11 mL PES. Spores were allowed to germinate for 3 d under a 16:8 L:D photoperiod and a photon fluence rate of 10 lmol photons Æ m)2 Æ s)1 and 10C. Germination rate was scored by counting 300 cells per replicate using a light microscope (Axioplan; Carl Zeiss, Oberkochen, Germany) equipped with a ·20 seawater immersion objective (Achroplan; Carl Zeiss) under phase contrast illumination. Only living spores that had formed a germ tube were classified as germinated; dead and living nongerminated spores were not differentiated. Cellular structure. Transverse (cross) and surface sections of sori and nonsori tissues from wild sporophytes were handmade using a sharp razor blade and observed under an Olympus BX51 epifluorescence microscope equipped with a Nikon DXM1200 digital camera (Olympus Europe, Hamburg, Germany). Köhler’s illumination or Nomarski’s differential interference contrast illumination were used to view transmitted light images (·40 UplanFL objective; Olympus). Red autofluorescence (as emitted by chls) was visualized using the mirror unit U-MWSG2 (Olympus), while blue autofluorescence (as emitted by phlorotannins, Schoenwaelder and Clayton 1998, Schoenwaelder 2002a) was visualized using a set of filters consisting of exciter D436 ⁄ 10, dichroic 455DCLP and emitter D470 ⁄ 30 (Chroma Technology Corp, Bellows Falls, VT, USA), resulting in excitation at 431–441 nm and fluorescence detection at 455–485 nm. Corresponding pictures of different illuminations of the same specimen were taken using the software LUCIA (Nikon GmbH, Düsseldorf, Germany). Merged fluorescence images were assembled with the software AxioVision (Carl Zeiss). In parallel, sorus tissue was also fixed with chromic-acetic solution and stained with methylene blue. Statistical analysis. Data were tested for homogeneity (Levene Statistics) of variance. Corresponding transformations (square roots) were performed to heteroskedastic data. Sorus size, DNA damage of sorus and nonsorus blade parts, and meiospore germination after preexposure to varying fluence rates of different combinations of spectral irradiance (P, PA, and PAB) were tested using analyses of variance (ANOVA, 607 P H O T O P R O T E C T I V E R O L E O F P A R A PH Y S E S RESULTS Sorus formation under different radiation conditions. Sorus development started with an initial darkening and thickening of the tissue. The maturing sorus was covered by a skin-like tissue (Fig. S3a in the supplementary material). The ripe sorus tissue appeared convexly embossed, with brown matt (dull and roughened) surface compared to the surrounding nonsorus tissue (Fig. S3b). Under the high-radiation treatment, the margin between sorus and nonsorus tissue was more distinct in UV-irradiated disks than those exposed to PAR only, where sorus areas appeared indistinct and diffused (Fig. S3c). Under the low-radiation treatment, sorus contour and area between different radiation treatments was relatively similar (Fig. S3d). First sori were observed after 48 d in all treatment combinations except under low-P treatment where first sori were observed at day 57 (Fig. 1a). A higher number of sporogenic disks was formed under high- 30 radiation conditions compared to low-radiation conditions between days 48 and 69 (arrows, Fig. 1b). There was a general trend that sorus formation was slightly delayed under P compared to PA and PAB conditions. At day 90, nearly all disks had formed a distinct sorus in all treatment combinations. Sorus area was not significantly different between treatments (Fig. 2; ANOVA, P = 0.064). Tissue DNA damage and meiospore germination. UVRinduced DNA damage under laboratory conditions was significantly higher in nonsorus compared to sorus parts of the disks (Fig. 3; ANOVA, P < 0.001). In wild sporophytes, which had been exposed to different in situ solar radiation conditions for 8 d, tissue DNA damage was nondetectable in both the sorus and nonsorus parts of the tissue. P a PAB ns 2.0 1.5 1.0 0.5 0.0 P 25 PA 2.5 Sorus area (cm2) P < 0.05). All analyses were followed by Duncan’s multiple range test (P = 0.05). Statistical analyses were performed using SPSS (SPSS, Chicago, IL, USA). PA PAB Low High Irradiance FIG. 2. Area of sorus formed on excised blade disks exposed to different low- and high-light intensities consisting of PAR (P), PAR + UVA (PA), and PAR + UVA + UVB (PAB) after 87 d induction experiment. Vertical bars are standard deviations (SD, n = 16–17). Bars grouped under the same horizontal lines are not significantly different, ns. UVA, ultraviolet A; UVB, ultraviolet B. 15 10 5 0 0 10 20 30 40 50 60 70 80 90 30 b DNA damage (CPD · Mb-1) Number of sporogenic disks 20 25 20 15 10 5 0 0 10 20 30 40 50 60 70 80 90 Time (d) FIG. 1. Time-course observation on the initiation and development of sorus in excised blade disks under low (a) and high (b) irradiance of PAR (P), PAR + UVA (PA), and PAR + UVA + UVB (PAB). Arrows pointing to day 48 show the first appearance of sorus, while arrows pointing to day 69 show higher number of disks with sorus under high compared to low P, PA, and PAB treatments. At the end of the experiment, 100% of P- and PAexposed disks formed sorus compared to 96% of PAB-exposed disks. UVA, ultraviolet A; UVB, ultraviolet B. Low PAB High PAB 50 40 30 20 ** 10 0 Sorus Nonsorus Tissue FIG. 3. UVR-induced DNA damage in soral and nonsoral sections of each disk induced to sporulate for 87 d; exposed to 18:6 light:dark photoperiod, with 4 h UVR supplement in the middle of the light phase. Disks were exposed under low and high PAR + UVR corresponding to low PAB and high PAB, respectively. Vertical bars are standard deviations (SD, n = 8). Bars grouped under the same horizontal lines are not significantly different; group with ** is significantly different from the other. CPD, cyclobutane pyrimidine dimer; UVR, ultraviolet radiation. 608 ANSGAR GRUBER ET AL. TABLE 1. Germination rates (n = 5, ±SD) of meiospores released from induced sori of disks exposed under different radiation treatments. P PA PAB Laboratory experiment (cultured sporophytes disks) Rooftop experiment (wild sporophytes) 88.7 ± 3.4 91.5 ± 1.4 91.5 ± 1.1 27.0 ± 9.4 41.8 ± 7.2 38.7 ± 8.5 Analysis of variance (ANOVA, P = 0.05) showed insignificant differences within treatments in both laboratory and rooftop experiments. P = PAR; PA = PAR + UVA; PAB = PAR + UVA + UVB. UVA, ultraviolet A; UVB, ultraviolet B. Meiospores released from sori induced under different radiation treatments in the laboratory were equally viable. Germination rates under low white light were between 89% and 92% (Table 1). Meiospores released from sori of field-collected sporophytes, which had been acclimated under different in situ radiation conditions for 8 d also showed no differences in viability and germination rates in P, PA, or PAB conditions. Germination rates of meiospores released from wild sporophytes in the rooftop experiment were, however, significantly lower (ANOVA, P < 0.05; 27%–42%) compared to meiospores of laboratory-induced sori (89%–92%; Table 1). Cellular structure of sorus and vegetative tissue. Sorus tissue consists of coniform sporangia that contain the developing meiospores and sterile clubbed paraphysis cells (Fig. 4, a–d). Within the sporangia, plastids (olive-green, Fig. 4a, black arrow with M) and physodes (turquoise-green, Fig. 4a, white arrow with M) of the developing meiospores were visible. We found two distinct types of paraphysis cells. One type contains a structured cytoplasm and several plastids (Fig. 4a, transmitted light image, red arrow), which show the typical red autofluorescence of chl (Fig. 4, a and d, red arrows). Another type of paraphysis cell had a hyaline appearance and was devoid of plastids (Fig. 4, a and d, blue arrows). These cells exhibited a bright blue fluorescence upon violet excitation (Fig. 4d, blue arrow), presumably containing phenolic compounds. Among the plastid-containing paraphysis cells, the presence of blue-fluorescing compounds was weak and restricted to the tiny physodes (Fig. 4d, white arrow with P). The hyaline paraphysis cells are part of the general palisade-like structure of the sporangium (Fig. 4, e–h). The sporangia feature a cap-like structure that appears transparent in fresh material (Fig. 4, a and d, yellow arrows), and the paraphysis cells also produce a thickened transparent outer layer (Fig. 4, a and d, green curly braces). Both structures color intensely upon chromic-acetic fixation and staining FIG. 4. Structure of the Laminaria digitata sorus I. Cross-section of fresh sorus tissue, (a) bright-field illumination, (b) red autofluorescence (long pass 590 nm), (c) blue autofluorescence (band pass 455–485 nm), and (d) false color merged image (autofluorescence channels shown in their respective colors, transmitted light image shown in grayscale) of the same specimen; showing plastid-containing (red arrows) and plastid-free hyaline paraphysis cells filled with blue-fluorescing compounds (blue arrows), olive-green plastids (black arrows) and turquoise-green physodes (white arrows) in developing meiospores (arrows marked with M) and paraphysis cells (arrows marked with P), transparent cap-like structures of sporangia (yellow arrows), transparent regions at apex of the paraphysis cells (green curly braces). Overview on fresh sorus tissue in cross-section, (e) bright-field illumination, (f) red autofluorescence (long pass 590 nm), (g) blue autofluorescence (band pass 455–485 nm), and (h) false color merged image (colors as in a–d) of the same specimen shown from left to right, hyaline paraphysis cells protrude plastid-containing paraphysis cells. Scale bars, 40 lm. P H O T O P R O T E C T I V E R O L E O F P A R A PH Y S E S 609 FIG. 5. Structure of the Laminaria digitata sorus II. (a) Cross-section of sorus tissue after chromic-acetic fixation and staining with methylene blue, cap-like structures of sporangia (yellow arrow) and deposit at apex of the paraphysis cells (green curly brace) are intensely stained. (b) Surface view of fresh sorus tissue, paraphysis cells with plastids forming a ring (red arrow) and plastid-free hyaline paraphysis cells (blue arrow). Overview of fresh sorus tissue in surface view, (c) transmitted light image, (d) red autofluorescence (long pass 590 nm), (e) blue autofluorescence (band pass 455–485 nm), and (f) false color merged image (autofluorescence channels in their respective colors, transmitted light image in grayscale) of the same specimen from left to right, plastid-free hyaline paraphysis cells are unevenly distribution within the sorus tissue. Scale bars, 40 lm. with methylene blue (Fig. 5a, yellow arrow and green curly brace). The plastid-containing as well as the hyaline paraphysis cells were also observed in surface view on fresh sorus tissue (Fig. 5b, red and blue arrows, respectively). The distribution of the hyaline paraphysis cells among the sorus tissue is uneven; they occur either in small groups or loosely interspersed within the sorus tissue (Fig. 5, c–f). While blue-fluorescing compounds were detected intracellularly in the sorus tissue (Figs. 4 and 5), vegetative blade tissue did not contain such bluefluorescing compounds inside intact cells (Fig. 6, a– d). Plastids of the vegetative cells show the typical red autofluorescence, while blue-fluorescing compounds were observed extracellularly, forming a ‘‘plug’’ in an injured section of the tissue (Fig. 6, a–d, region between white curly braces). In surface view, the vegetative tissue also showed a line of blue-fluorescing material. Most probably the tissue was injured and became scarred (Fig. 6, e–h, region between white curly braces). All blue-fluorescing structures are considered to contain phenolic compounds. DISCUSSION Sorus induction under different experimental radiation conditions revealed that sporogenesis of L. digitata is not affected by UVR. Germination capacity of meiospores released from sorus developed under different radiation conditions was also not significantly different, suggesting some degree of photoprotection inside the sporangium. The lower DNA damage in the sorus compared to vegetative tissue further indicates effective UVB screening within the sporogenic tissue. This protection of meiospores within the sorus tissue may be explained by the presence of paraphysis cells within the sporogenic tissue that we detected in two distinct types that differ in their cell content and hence radiation absorbance. Taken together, our results justify the late spring to autumn sporogenesis of L. digitata inhabiting the upper sublittoral, where blades can be exposed to naturally high solar radiation especially during low tides in summer. Okamura (1927) described in detail the occurrence and development of sori of several Japanese Laminariales. He showed some phenological evidence that high irradiance may hamper sporogenesis, describing a larger extent of sorus formation on the ‘‘underside’’ compared to the ‘‘upper surface’’ of the blade of S. japonica and S. cichorioides (Okamura 1927). Although an ‘‘under surface’’ and ‘‘upper surface’’ cannot be easily distinguished in the field, the differential sorus development previously described may indicate irradiation stress. 610 ANSGAR GRUBER ET AL. FIG. 6. Vegetative tissue of fertile Laminaria digitata fronds. Cross-section of fresh vegetative tissue, (a) transmitted light image, (b) red autofluorescence (long pass 590 nm), (c) blue autofluorescence (band pass 455–485 nm), and (d) false color merged image (autofluorescence channels in their respective colors, transmitted light image in grayscale) of the same specimen are shown; blue-fluorescing compounds are not present intracellularly in healthy vegetative tissue but may be deposited extracellularly in regions recovering from injury (marked between white curly braces). Overview of fresh vegetative tissue in surface view, (e) transmitted light image, (f) red autofluorescence (long pass 590 nm), (g) blue autofluorescence (band pass 455–485 nm), and (h) false color merged image (colors as in a–d) of the same specimen shown from left to right; occurrence of blue-fluorescing compounds within the vegetative tissue is restricted to regions of injury (scratch of injury marked between white curly braces). Scale bars, 40 lm. The sorus is thought to be completely opaque, shielding the developing spores from high PAR and UVR. Hence, germination under low light (PAR) is equally successful regardless of the light condition during sorus induction. In the field, to avoid sudden and drastic change in light environment (high PAR ⁄ UVR shock), timing of meiospore release during dawn or dusk (Amsler and Neushul 1989) can be important to enhance spore viability, as reported in the nighttime spore release in S. japonica [=L. japonica] (Fukuhara et al. 2002). Considering that meiospores can swim within the euphotic layer for up to 120 h (Reed et al. 1992), release during the day can expose the naked, unwalled spores to high PAR and UVR. However, losing their flagella and swimming competency, spores can sink passively to the low-light environment of the seafloor under macroalgal canopies with enhanced germination success (Amsler et al. 1992, Fredriksen et al. 1995). When released meiospores are further exposed to PA and PAB treatments, they are observed to be highly sensitive to UVR, forming DNA lesions inhibiting photosynthesis (Roleda et al. 2005b, 2006c) and germination (Roleda et al. 2006e, 2010, Wiencke et al. 2007). The presence of cellular phlorotannin-containing physodes can protect meiospores and enhance germination capacity, a response reported to be species specific (Wiencke et al. 2004, Roleda et al. 2006a). While formation of thin cell walls is initiated within 8–10 h after release (Steinhoff et al. 2008), this fine structure and the few intracellular phlorotannin-containing physodes in the developing gametophytes may not be sufficient to offer as much UV-screening protection as to spores enclosed inside the sporangium wall. Alternatively, swimming meiospores can be protected by low concentrations of phlorotannin exudates in coastal environments. Phlorotannins are persistently released from healthy and stressed brown algae (e.g., kelps and rockweeds) directly via exudation through slime channels (Halm et al. 2011), and indirectly via tissue erosion and mechanical damage (Sieburth and Jensen 1969). Moreover, phlorotannin exudation rates reportedly increased with high PAR and emersion (Ragan and Jensen 1978, Carlson and Carlson 1984). Together with phlorotannin exuded during spore release, low P H O T O P R O T E C T I V E R O L E O F P A R A PH Y S E S phlorotannin concentration in contact liquid (>0.84 lg Æ mL)1 released into the seawater) can already reduce the impact of UVB exposure to UV-sensitive kelp meiospores (Swanson and Druehl 2002). For example, seawater containing phlorotannin exudates of Macrocystis increased the survivorship of germinating Saccharina groenlandica (=Laminaria groenlandica) meiospores exposed to PAB (Swanson and Druehl 2002). As exudation of phlorotannins is a mechanism of protecting the vulnerable unicellular stages of the kelp life cycle, it seems possible that the specialized plastid-free paraphyses identified in our study provide a reservoir for the exudation of phlorotannins upon meiospore release. The higher germination rate of meiospores from laboratory-induced sorus compared to the sorus of wild sporophytes can be attributed to the sorus quality and seasonal variation. The sorus of sporophytes collected in July 2004 (this study) was observed to be obscure and ‘‘unripe,’’ probably resulting from the high solar radiation and high temperature during the season (see Fig. S2a). Previously, sporophytes collected in a cooler month (May 2002; see Fig. S2b) have distinct ripe sorus and did show up to 90% germination of the meiospores released (Roleda et al. 2005b). The more opaque appearance of sorus compared to vegetative tissue suggests the presence of a relatively higher amount of UV-screening pigments. This observation corresponds to the photoprotective capacity revealed by the lower amount of DNA damage in the sorus compared to the nonsorus tissue of cultured sporophytes. The wild sporophytes acclimated to natural solar radiation showed no measurable CPDs in both the sporogenic and nonsporogenic tissue parts, indicating enhanced overall UVR protection. This finding could also be related to the fact that exposure to high PAR in nature can enhance UVB tolerance by increasing activity of photorepair enzymes (Warner and Caldwell 1983). Similarly, DNA damage was also not detectable in thalli of wild Gigartinales (Roleda et al. 2004b). Conversely, cultured young kelp sporophytes grown at relatively low PAR were more susceptible to UVB-induced DNA damage. Accumulation of CPDs in nonsporogenic tissue is related to thallus thickness and optical property relative to the available cell-bound pigments and UV-absorbing compounds (Roleda et al. 2005a, 2006b,d, 2007a). Our anatomical investigation further supports our view that the architecture and chemical composition of the sorus protects the photosensitive meiospores. Anatomical investigation of sporogenic tissue of L. digitata revealed for the first time the presence of two distinct types of paraphysis cells. The hyaline paraphysis cells were devoid of plastids and filled with blue-fluorescing substances, as revealed by epifluorescence microscopy under violet excitation, thereby indicating the presence of 611 phlorotannins (Hura et al. 2008). Early investigators already described paraphysis cells that deviate from the normal plastid-rich type in S. cichorioides. This species possesses paraphyses with many ‘‘granules’’ as stained with cresyl blue, neutral red, and vanillin hydrochloric acid (Ohmori and Inoh 1963, Ohmori 1967). Vanillin hydrochlorid acid is a histochemical marker for tannins (Gardner 1975), and thus the ‘‘granules’’ can be regarded as polyphenol-rich physodes. The hyaline polyphenol-rich paraphysis cells in L. digitata were randomly interspersed among paraphysis cells containing several plastids—a type of paraphysis that has routinely been described for Laminariales (Ohmori 1967 and references therein). Furthermore, cross-sections through sori of L. digitata revealed that sporangial and paraphysis caps were highly stained after chromic-acetic fixation and methylene-blue treatment. As methylene blue stains anionic hydrocolloids (Soedjak 1994) and algal polysaccharides (Jiao and Liu 1999), this staining corroborates findings of Motomura (1993) for S. angustata. He suggested that the caps are composed of sulfated polysaccharides (=fucoidan) by their intense pink staining with toluidene blue-O. Combined with the histochemical evidence for the contents of both types of paraphysis cells, it is thereby probable that sori possess a UVR-screening capacity in the newly described polyphenol-rich hyaline paraphyses, high PAR screening in the plastid-containing paraphyses, and a protection against desiccation by the polysaccharide-rich cap structures of sporangia and paraphysis cells. These findings suggest a similar function of paraphyses in Laminaria and in the Dictyotalean species S. marginatum (Bhamrah and Kaur 2005). Whether polyphenolrich paraphyses in sori are inducible by high irradiances and ⁄ or PAB and whether they are a common phenomenon in other kelps remains to be investigated. The localization of polyphenols in paraphyses also conforms with previous studies reporting higher phlorotannin contents in sporogenic compared to vegetative tissues in a number of Laminariales (Tugwell and Branch 1989, van Alstyne et al. 1999). In other Laminariales, phlorotanninrich cortical layers and meristoderm cells that can selectively filter short UV wavelength are present as well (Tugwell and Branch 1989, Lüder and Clayton 2004, Shibata et al. 2004). Apart from the phlorotannins in soral paraphysis cells, we also observed for the first time the accumulation of phlorotannins in the wound-healing process of naturally injured tissue of L. digitata. This response was previously reported in Ecklonia radiata and L. hyperborea subjected to simulated herbivory in the laboratory (Lüder and Clayton 2004, Halm et al. 2011). The present study supports the general view that phlorotannins have multiple functions in brown 612 ANSGAR GRUBER ET AL. algae. They offer protection against UVR and are involved in wound-healing processes (Amsler 2001, Schoenwaelder 2002b, Lüder and Clayton 2004, Amsler and Fairhead 2006, Halm et al. 2011). In the intertidal kelp Alaria marginata, higher concentrations of phenolic compounds were found in the reproductive fronds (sporophylls) than in vegetative blades. Consequently, the sporophylls were consumed by herbivorous snails at a lower rate than were the vegetative blades (Steinberg 1984). Such chemical defense can also effectively protect nutrient- and lipid-rich kelp meiospores (Brzezinski et al. 1993, Nimura et al. 2002, Kumura et al. 2006, Steinhoff et al. 2008) inside the sporangium against herbivory. Formation of specialized phlorotannin-rich paraphyses during sorus development might also be a strategy to accumulate phenolic compounds in the soral tissue as a chemical defense against herbivory. Synthesis and localization of phlorotannin in the paraphysis cells for UV protection can be energetically expensive (Purrington 2000), as observed in the patchy distribution of the hyaline paraphyses among the plastid-containing paraphyses. The higher irradiance of PAR encountered in the field may present a more adverse effect for meiospores inside the sporangium compared to UVR. Therefore, our observation suggests that the production of more plastid-containing paraphyses for the screening of high PAR might be of more importance and may also be energetically less expensive than the production of phlorotannin-containing paraphyses. This response probably is essential for the successful sporogenesis of L. digitata and the maintenance of its depth range in the infralittoral fringe, where adult sporophytes are often exposed to the full solar irradiation. 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Sensitivity and acclimation to UV radiation of meiospores from five species of Laminariales from the Arctic. Mar. Biol. 145:31–9. Wiencke, C., Lüder, U. H. & Roleda, M. Y. 2007. Impact of ultraviolet radiation on physiology and development of zoospores of the brown alga Alaria esculenta from Spitsbergen. Physiol. Plant. 130:601–12. Supplementary Material The following supplementary material is available for this article: Figure S1. Experimental setup for induction of sporogenesis in Laminaria digitata under artificial radiation consisting of PAR only (P), PAR + UVA (PA), and PAR + UVA + UVB (PAB). Figure S2. (a) Time-series air temperature data from GKSS Station Helgoland during the experimental period (24 July to 1 August 2004, this study), compared to (b) air temperature during collection period of Roleda et al. (2005b), when higher spore vitality was observed. Figure S3. Sporogenesis in Laminaria digitata. Table S1. Laboratory radiation treatments with corresponding daily dose and radiation ratios. Table S2. Ambient solar radiation measured during the in situ rooftop experiment. This material is available as part of the online article. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.