Abiotic stress signalling in the fucus embryo

Cellular responses of 1-, 2and 4-d-old Fucus spiralis embryos subjected to a single dose of elevated pbotosynthetically active pboton ftux density (PPFD), witb or without ultraviolet (UV) radiation, were investigated by measuring the effects on tbe effective quantum yield of pbotosystem D (M I F m') and intracellular production of active oxygen species (AOS). Production of AOS was determined by tbe in vivo conversion of 5-(and-6)-cbloromethyl-2',7' -dicblorodibydroftuorescein diacetate (CMDCFH2-DA) to the ftuorescent compound dicbloroftuorescein (DCF) using confocal laser scan microscopy (CLSM) and image analysis. The role of xanthopbyU cycle pigments· in pbotoprotection was also assessed. A rapid decline in M I F m' was observed under aD elevated ligbt conditions. A correlation was found between non-pbotncbemical quenching and the de-epoxidation ratio zeaxanlhin/(zeaxanlhin + violoxantbin). Active oxygen formation increased with PPFD and was bigber in older embryos and wben UVB was present. Two pbotoinbibition responses were recognized: (i) a rapid decline of the PSD yield due to the violoxantbinzeaxanlhin cycle (pbotoprotection), and (ii) a slower second-pbase decline, correlated with active oxygen production. Electron transport rate (ETR) increased with embryo age, and was correlated with AOS production. As a result of enhanced AOS production, there was a slow recovery of the PSD yield, in particular witb increased effective UV dose. In general,"embryos were able to recover from the imposed ligbt conditions, but UVB bad a more damaging effect. OveraD, our data suggest that under natural conditions, embryos of F. spiralis are susceptible to elevated ligbt levels, and tbat UVB radiation is an important stress factor. Key-words: Active oxygen; chloromethyl dichlorodihydrofluorescein diacetate (CM-DCFH2-DA); confocal laser scan; oxidative stress; xanthophyll cycle. Abbreviations: L1F I Fm'• effective quantum yield of PSI!; AOS, active oxygen species; CLSM, confocal laserscan microscope; CM-DCFH2-DA, chloromethyl dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; PAM, pulse-amplitude modulation; PPFD, photosynthetically Correspondence: Murray T. Brown. £-mail: mtbrown@plymoulh.ac.uk @ 2001 Blackwell Science Ltd active photon flux density; PSI!, photosystem II; qP, photochemical quenching; qN, non-photochemical quenching; SA, sun+ UVA; SAB, sun+ UVA + UVB; shade, 15 pmol m-2 s-1 PPFD; sun, 300 pmol m-2 s-1 PPFD; UV, ultraviolet; V, violoxanthin concentration; Z, zeaxanthin concentratioiL INTRODUCTION Fucoid brown algae (Phaeophyceae) are important members of marine intertidal communities in the north Atlantic Ocean. Variability in their recruitment and regeneration of the Fucus canopy are major influences on the rate of succession and the abundance of other species (McCook & Chapman 1997). Despite the importance of stress tolerance in intertidal seaweeds, the underlying mechanisms that confer such tolerance are still poorly understood (Davison & Pearson 1996}: To date, most studies have focused on adult stages, but the mechanisms. by which Fucus embryos withstand the prevailing physical forces in the intertidal zone (light, tidal movement, waves) are key determinants of their ability to survive and establish a population. The impact on embryonic stages in the first days after settlement may be particularly severe. It has become apparent that the response of early life history stages to the environmental conditions cannot necessarily be predicted from knowledge of the adult canopy (Davison, Johnson & Brawley 1993). Although fucoid embryos are easily manipulated in laboratory culture and their early development has been well characterized (Kropf & Quatrano 1987; Kropf 1997), relatively little is known about their stress physiology. Fucus embryos develop under shade conditions, typically in rock crevices and under the protection of the adult canopy, so exposure to an elevated ligbt climate especially during emersion is likely to result in photoinhibition. Variable fluorescent techniques are suitable tools for studying photosynthetic inhibition as activity of the PSII reaction centre (i.e. the rate of production of electrons by the water-splitting system of PSII) is measured. The PAM method is based on the principle that under in vivo conditions, fluorescence changes originate almost exclusively from chlorophyll a and the associated antenna pigments in PSII (see Genty, Briantais & Baker 1989; Schofield, Evens & Millie 1998). The potential inhibition of photosynthesis due to light stress can be minimized through several physi801 802 S. Coelho et al. ological mechanisms (Schofield et al. 1998), including the involvement of the xanthophyll cycle pigment pool in dissipating excess energy from the reaction centres (Kroon 1994}. It is also known that excess light, cold, heat and drought can trigger increased production of harmful AOS such as hydrogen peroxide (H20 2), superoxide radicals (*02-) and hydroxyl radicals (*OH) (Foyer, Lelandais & Kunert 1994). Intracellular production of deleterious active oxygen species occurs in all organisms but is more problematic in phototrophs because they produce these metabolites during photosynthetic metabolism (Halliwell & Gutteridge 1989). Oxidative stress can be countered by a set of antioxidative enzyme-mediated reactions. How the oxiradical attack and the antioxidant defence evolve during the first (decisive) days after fertilization of Fucus eggs is not known. The aim of this study was to assess the impact of experimental light conditions on the physiology of developing Fucus spiralis L. embryos during the first 4 d after fertilization. This was achieved by investigating: (i) protection mechanisms against sunlight, using the PAM technique to measure photosynthetic performance and high-performance liquid chromatography (HPLC) to quantify the xanthophyll cycle and other relevant pigments; (ii) the intracellular production of active oxygen species in response to elevated PPFD using the fluorescent label CM-DCFH,DA in combination with CLSM and quantitative image analysis, and (iii) the ability of embryos to recover from oxidative stress under more favourable, dim-light, conditions. MATERIALS AND METHODS Plant material and growth conditions Receptacles of mature F. spiralis were collected from the compact intertidal seaweed belt, growing on concrete substrata along the shoreline of the Oosterschelde basin (29%o salinity) near Yerseke (south'westNetherlands), from September 1998 to April1999. Receptacles were stored at 4 •c in the dark until they were used (within 1 week). To achieve synchronous release of gametes, receptacles were incubated in filtered seawater (0·45 .urn) under strong white light at 15 •c. The gamete solution was then filtered through a 120 J.HI1 nylon mesh to discard debris and oogonia. Tune of fertilization was considered to be 30 min after gamete release. Fertilized eggs were then pipetted onto the surface of high-precision coverslips for CLSM (Assistent; Glaswarenfabrik Karl Hecht, Sondheim/Rhoen, Germany; 170 ± 10 Jlll1 thick, 25 mm diameter) and placed inside small Petri dishes containing 8 mL filtered sea water, where they attached and grew. For the analysis of chlorophyll fluorescence and xanthophyll cycle pigments, the embryos were grown onto cellulose nitrate filters in small Petri dishes. Attached zygotes were incubated at 15 •c under 15 .umol m-2 s-1 PPFD (4n-sensor QSL-100; Biospherical Instruments, Inc., San Diego, CA, USA) on a 12: 12 h light :dark cycle. Sea water was replaced daily. Replicate cultures (n = 3-5} were maintained for each treatment. Light conditions and experimental protocol In the south-west Netherlands, the reproductive season for F. spiralis is autumn/winter. The control/low PPFD (15 ,umol m-2 s-1} was selected on the basis of in situ mea' surements taken during a cloudy day at 1000 and 1600 h in February, when the adult canopy almost completely covers the embryos. These were considered to be the standard growth conditions for the first days after fertilization. The elevated PPFD (300 ,umol m-'s-1) was chosen based on in situ measurements at noon on a sunny day in February, when the adult canopy was partially removed and the embryos were exposed to higher light·levels. In nature, fertilization occurs during daytime low tide (Pearson & Brawley 1996), so the first potential exposure to high light is -24 h after fertilization, at the next daytime low tide. Experiments were conducted under four different light regimes: shade/control (Philips TLD18W33 fluorescent lamps; Philips, Eindhoven, The Netherlands), sun, SA and SAB. A ZABU UVD clear acrylate sheet (Wientjes BV, Roden, The Netherlands) was used to filter out UVC radiation (below 285 nm) under all light conditions, a Mylar filter was used to cut off UVB radiation (below 320 nm) and a plexiglass filter was used to remove UV A radiation (below 380 nm). A PPFD spectrum (250--700 nm) was scanned with a spectroradiometer equipped with a 2n. cosine-corrected sensor (MA CAM Photometries SR-9910PC; Macam Photometries Ltd, Livingston, UK). The biologically effective dose of Jones & Kok (1966) was 2·1 W m-2 (SAB}, 0·6 (SA), 0·1 (sun) and 0 (shade} and was calculated using a weighting factor, WF• = 1000*e-<HnJ*" (R2 = 1 ), derived from the plot in Forster & Ltining (1996) that sets WF300 = 1. For the calculation of this effective dose, A= 280--400 nm are considered, using the formula BED11K = • • 2B0-400.E (WF.•Q.), in which Q. is the PPFD (W m-2) at wavelength A (nm). · Fluorescence measurements In vivo light-modulated chlorophyll fluorescence was monitored in 1-, 2and 4-d-old photosynthetically competent embryos (Brawley, Quatrano & Wetherbee 1977) with a pulse-amplitude modulation apparatus (PAM 2000 Walz; Heinz Walz GmbH, Effeltrich, Germany), based on the principles described by Schreiber, Evens & Bilger (1986). The saturating pulse method provides information on processes r