Emission of volatile halogenated compounds, speciation and localization of bromine and iodine in the brown algal genome model Ectocarpus siliculosus

This study explores key features of bromine and iodine metabolism in the filamentous brown alga and genomics model Ectocarpus siliculosus. Both elements are accumulated in Ectocarpus, albeit at much lower concentration factors (2-3 orders of magnitude for iodine, and < 1 order of magnitude for bromine) than e.g. in the kelp Laminaria digitata. Iodide competitively reduces the accumulation of bromide. Both iodide and bromide are accumulated in the cell wall (apoplast) of Ectocarpus, with minor amounts of bromine also detectable in the cytosol. Ectocarpus emits a range of volatile halogenated compounds, the most prominent of which by far is methyl iodide. Interestingly, biosynthesis of this compound cannot be accounted for by vanadium haloperoxidase since the latter have not been found to catalyze direct halogenation of an unactivated methyl group or hydrocarbon so a methyl halide transferase-type production mechanism is proposed.

EXAFS Extended X-ray absorption fine structure GG Oligo-guluronates ICP-MS Inductively coupled plasma-mass spectrometry NIST National Institute of Standards and Technology PMA Phorbol myristate acetate vBPO Vanadium bromoperoxidase vIPO Vanadium iodoperoxidase VHPO Vanadium haloperoxidase XAS X-ray absorption spectroscopy This article is dedicated to the memory of Yasuyuki Muramatsu (January 9, 1950-July 2, 2016), a dear friend and outstanding scientist in the fields of Radioecology, Radiochemistry, Geochemistry and Analytical Chemistry in Japan, who untimely passed away during the writing of this article, and to Alison Butler (University of California, Santa Barbara) on the occasion of her Bader Award.

Introduction
Two centuries ago, the elements bromine and iodine were discovered in sea water and seaweed (Laminaria and Fucus) ashes, respectively [1,2]. Due to their unique evolutionary history and phylogenetic distance from other important eukaryotic lineages [3,4], brown algae present some remarkable chemical and physiological adaptations which are also reflected at the genome level [5], making them fascinating experimental models, not only for phycologists, but for a community of interdisciplinary researchers. This includes the function of iodide as extracellular antioxidant protecting the surface of Laminaria digitata (Hudson) Lamouroux against oxidative stress [6]. Indeed, L. digitata is the strongest iodine accumulator currently known among living organisms [7]. In fact, this constituted the first documented case of an inorganic antioxidant in a living system-and the chemically simplest antioxidant known. Key to the antioxidant function is the mobilization of iodide in the apoplast and its efflux into the surrounding seawater upon oxidative stress [6,8].
In this context, communities of the giant kelp Macrocystis have been found to impact iodine speciation in coastal sea water [9]. The role of bromine is less well investigated, but a recent study [10] highlights that Laminaria accumulates bromide, which complements iodide as an antioxidant especially for the detoxification of superoxide, but with an overall more diverse role.
In contrast to kelps such as Laminaria and Macrocystis, which have a large size (up to around 3 m for Laminaria, and 70 m for Macrocystis, respectively), very complex morphology and which can attain a high standing stock in rocky coastal seabed ecosystems, Ectocarpus is a genus of filamentous brown algae with a worldwide distribution along temperate coastlines, and is a nuisance as a "fouling" organism on many man-made surfaces in the sea. It has some significant advantages as an experimental model compared to Laminaria or Macrocystis and constitutes one of the best-studied seaweeds [11,12]: due to its small size of only a few mm or cm and its fast growth it can easily be cultivated in small volumes of seawater media both axenically and with associated bacteria [13]; it belongs to a sister group of the ecologically and economically very important kelps [14]; its entire, well-known life cycle can be completed within a few months in culture, many molecular tools are available, including mutant collections, microarrays and proteomics. It has also become the first seaweed of which the entire genome has been sequenced and thus offers unprecedented opportunities for study [5]. Features of its inorganic biochemistry include a remarkable, non-ferritin mode of iron storage [15]. Ectocarpus is also well studied with regards to its pathologies which include viruses [16,17], fungi [18], oomycetes [18] and plasmodiophoraleans [19].
While brown algae of the genus Laminaria are the strongest accumulators of iodine in life on Earth [20][21][22] and a major contributor to the biogeochemical flux of iodine [23] and, to a lesser extent, of brominated and iodinated halocarbons to the atmosphere [24], hardly anything is known about Ectocarpus in this respect. This is remarkable considering the major role of brown algae in global halogen cycling, but also how well other areas of physiology and biochemistry have been studied in Ectocarpus. However, a single gene of a vanadium haloperoxidase (VHPO) and a number of enzymes related to halogen metabolism have been identified in the Ectocarpus genome. This includes at least three different families (21 loci) of haloacid dehalogenase (HAD) and two haloalkane dehalogenase families [5,25]. It is tempting to hypothesize that the dehalogenases protect Ectocarpus against halocarbons which act as alkylating, toxic agents and are being produced by kelps and other potential substrate algae on which Ectocarpus occurs. The presence of a single VHPO gene, a bromoperoxidase, in the Ectocarpus genome contrasts with the presence of multigenic families of VHPOs, of both bromo-and iodoperoxidases in the kelps Laminaria [26,27] and Macrocystis [28].
The uptake of iodide from seawater in Laminaria involves VHPOs [21] and its strongest accumulation in this species seems to be linked to the presence of a particular VHPO subclass, the iodoperoxidases, specific for iodide oxidation [26,27]. In Laminaria, most of the iodine is accumulated in the apoplast of the cortical cell layer [29].
The first line of defense against pathogens in Laminaria is an oxidative burst [30], which is considered a central element of eukaryotic defense in general [31] and which, in the case of kelp species, serves to control bacterial biofilms [32]. Among its triggers are oligoguluronates (GG; [30,32]), bacterial lipopolysaccharides [33], prostaglandin A 2 [34], methyl jasmonate, and polyunsaturated free fatty acids [35]. In L. digitata, early transcriptional defense responses are similar to those in land plants but also involve tightly regulated halogen metabolism which might play roles in more sophisticated chemical defense reactions including distance signaling [36,37]. In brown algae, bromide and VHPOs have also been shown to catalyze oxidative cross-linking between cell wall polymers, suggesting a role in spore and gamete adhesion and cell-wall strengthening (for reviews see [38,39]). Much less is known about defense mechanisms in Ectocarpus. Recent studies observed strong differences in susceptibility to infection by Eurychasma dicksonii among different Ectocarpus strains [40], and Eurychasma infection [41] as well as Cu stress [42] were found to result in overexpression of the single VHPO encoded in the Ectocarpus genome.
In our previous studies [6,10,43], X-ray absorption spectroscopy (XAS) has proven to be a suitable, non-invasive tool to probe the chemical state and solution environment of accumulated iodine and bromine in kelps. Likewise, we have used energy-dispersive X-ray analysis (EDX) for localization studies of Fe in Ectocarpus [44].
Following our recent study which included Ectocarpus among a number of taxa from various algal lineages [43], this study explores the speciation and localization of bromine and iodine metabolism in Ectocarpus. First iodine and bromine levels in Ectocarpus are determined using ICP-MS. Then the localization of bromine and iodine is investigated using energy-dispersive analysis of X-rays. K-edge XAS is applied to probe the stored chemical state of these elements under different physiological conditions in vivo (both in healthy cultures and in the presence of the oomycete Eurychasma dicksonii). Finally, the production of volatile halocarbons is explored.

Biological material
A well-characterized strain of E. siliculosus which had served for sequencing the Ectocarpus genome [5] was obtained from the Culture Collection of Algae and Protozoa (CCAP 1310/4) and was used for all experiments described here. Ectocarpus was grown in half-strength Provasolienriched sea water as described previously [13,45,46], i.e. at 18.

ICP-MS analysis of total iodine and bromine
Iodine and bromine contents in freeze-dried Ectocarpus filament samples were analyzed by ICP-MS after pyrohydrolysis separation as described previously [48,49]. Concentrations obtained are given in ppm (i.e. μg halogen g −1 of freeze-dried material).

Energy-dispersive analysis of X-rays
Ectocarpus siliculosus was harvested from cultures prior to fixation, dehydration, and embedding. Filaments were fixed in a 0.1 M phosphate buffer solution containing 2% (w/v) paraformaldehyde, 1% (w/v) glutaraldehyde, and 1% (w/v) caffeine for 2 h. The fixed cells were then washed with 0.1 M phosphate buffer and dehydrated in successive ethanol baths of 30, 50, 75, 85, 95, and 100% (three times). The cells were then embedded in 1:1 (v/v) ethanol/LR White resin (LWR; EMS, Hatfield, PA, USA) for 3 h followed by 100% LWR overnight in gelatin capsules under vacuum. Sections of 3 μm were cut on a Leica EMUC6 microtome and deposited on glass slides. Slides were coated with platinum in a Quorum Technologies Q150T ES sputter coater. platinum-coated samples were analyzed under high vacuum in a Quanta 450 FEG environmental scanning electron microscope (ESEM) equipped with an Oxford Instruments INCA energy dispersive X-ray (EDX) microanalysis system.

X-ray fluorescence spectroscopy
Ectocarpus filaments were washed briefly with 100 µM ethylenediaminetetracetic acid (EDTA) followed by distilled water to mitigate calcium ion interference in the fluorescence spectrum. Washed filaments were placed on Ultralene™ film (Spex Sample Prep, USA), allowed to dry, and then placed in the GEOCARS X-ray beam of the Advanced Photon Source at Argonne National Laboratory (Lemont, IL).
The selected region of interest was rastered at a resolution of 2 µm with an X-ray beam energy of 5.0 keV, flux of 1.206 × 10 12 photons s −1 , and dwell time of 20 ms pixel −1 . The resulting iodine L β1 X-ray fluorescence signal was mapped using the Larch Mapviewer™ software version 0.9.32 [50]. By virtue of the intensity and broadness of the Ca K β1 signal, calcium ion interference precluded mapping of both the iodine L α1 and L β1 fluorescent signals from untreated Ectocarpus filaments. Only after EDTA washing was the I L β1 signal resolved from Ca K β1 , while the I L α1 remained confounded by calcium due to proximity of their respective fluorescent energies.

X-ray absorption spectroscopy
Bromine and iodine K-edge XAS measurements and extended X-ray absorption fine structure (EXAFS) data reduction were carried out at the EMBL Outstation Hamburg at DESY, Germany as described previously [51].

Halocarbons
Ectocarpus was sub-cultured into three 1 L flasks filled to 800 mL with sterilized coastal seawater enriched with Guillard's F/2. Three identical flasks of the media (excluding the Ectocarpus culture) were incubated as controls. Incubation conditions were 20 °C at 100 μmol m −2 s −1 and a 12:12 light/dark cycle. Flasks were stoppered with sterilized cotton in hessian plugs and covered with foil.
For Ectocarpus emission experiments, the solid contents of a culture flask was divided into two 200 mL flasks, filled completely to eliminate headspace with purged, filtered seawater obtained from the Atlantic Ocean at 3000 m depth and sealed with glass stoppers. A third flask was prepared without Ectocarpus addition as a control. The flasks were stored at 15 °C in the dark, sampled and immediately analyzed every 2 h over a 12 h period. 10 mL sample aliquots were taken each time using autoclaved perfluoroalkoxy (PFA) tubing into borosilicate gas syringes, filtered through a standard glass-fiber filter (GFF) and 10 mL of blank seawater added to compensate and eliminate the headspace. The blank seawater used was continuously purged throughout the course of the experiment to minimize VOC content. At the end of the 12 h period the flasks were filtered and the solid mass of Ectocarpus recorded. This was then allowed to air dry and the dry weight was recorded. For investigating the effects of oxidative stress, the experiment was performed as described above except for the addition of 2 mM H 2 O 2 to simulate oxidative stress.
10 mL sample aliquots were analysed using purge and trap coupled to thermal desorption-gas chromatographytime of flight mass spectrometry (P&T-TD-GC-TOFMS, Markes Unity2 CIA8 TDU, Agilent 6890, Markes Bench-Tof MS) with a custom built purge and trap unit [52]. The TOF-MS allowed high sensitivity monitoring of all ions from 35 to 500 amu simultaneously. Halocarbons were calibrated using NOAA standard SX-3570, accounting for the purge and trap efficiencies of each gas [52]. The control flask was analysed first at each time point to check for carry-over in the analysis and/or production in the blank seawater. No significant change in the analysed VOC content of the blank seawater was observed throughout the emission experiments.

Total halogen levels (ICP-MS)
Initially, halogen levels in cultured Ectocarpus were determined in order to establish whether there is any significant accumulation of iodine and bromine in this organism. Freeze-dried thalli of the brown alga Ectocarpus siliculosus cultivated in normal PES medium contained mean concentrations of 196 ± 11 ppm iodine and 738 ± 3 ppm bromine. In contrast, freeze-dried Ectocarpus siliculosus thalli cultivated in PES medium supplemented with 50 µM KI contained 804 ± 3 ppm iodine, but only 186 ± 3 ppm bromine.

Localization of halogens (EDX and XFS)
Next, the localization of halogens was explored in order to get first insight into potential function. Based on XFS and EDX, most iodine was concentrated in physode-like vesicles (Fig. 1), even though some was also detectable in the cytosol and/or apoplast (Fig. 2). The EDX signal from bromine ( Fig. 2) was considerably weaker than that of iodine, showing some localization to the apoplast, cytosol, and possibly intracellular structures (Fig. 2).

XAS
XAS is the method of choice for determining the chemical environment and redox state of halogens in seaweeds [51]. The X-ray Absorption Near-Edge spectra (XANES) at the Br K-edge in Fig. 3 show that non-infected Ectocarpus (trace C) is quite similar to an aqueous solution of NaBr (trace D); it appears that Br is present in Ectocarpus as hydrated bromide [43]. The spectrum of Ectocarpus siliculosus infected with Eurychasma dicksonii (trace B) is subtly different and resembles more that of Br incorporated in an aromatic system, such as in the model compound 4-bromophenylalanine (trace A). The spectra show that in uninfected algae, bromine is present in its reduced anionic form, bromide, whereas upon infection prevailing over several months, incorporation of a major fraction into organic compounds occurs, concomitant with general senescence of the cultures. However, this was not detectable in experiments with a shorter time span of infective pressure.
It was shown earlier [43,51] that the Extended X-ray Absorption Fine Structure (EXAFS) of hydrated bromide could be simulated with a shell of oxygen atoms at approx. 3.3 Å; although the hydrogen atoms cannot be observed in EXAFS, it can be safely assumed that these oxygens are noncovalently bound to bromide by hydrogen bonds. For noninfected Ectocarpus this is the only contribution (Fig. 4a, b, bottom trace in both panels, attempts to simulate the weaker peaks in the Fourier transform did not lead to significant improvements). For infected Ectocarpus (Fig. 4a, b, top traces in both panels) the carbon peak in the Fourier transform is the strongest, and the pattern of peaks is reminiscent of that of aromatic amino acids such as 4-bromophenylalanine and 3,5-dibromotyrosine. The Br-C distance found in the simulation with both a phenyl group and a shell of H-bonded heteroatoms is 1.90 Ǻ, which is in between the ranges considered typical for bonds with sp 2 (1.87-1.88 Ǻ) and sp 3 (1.92-1.96 Ǻ) C; the good agreement between experiment and simulation for the other atoms of the phenyl ring strongly indicates sp 2 C in this case (Table 1). For the incorporation of Br in organic compounds to be observed by XAS, it is necessary that the infection lasts for a prolonged duration of time (> 1 month in a batch culture); experiments in which the infection was left for only 3 weeks did not result in EXAFS other than that of hydrated bromide.
The iodine K-edge spectra (Fig. 5) showed hardly any characteristic features. The EXAFS was so weak that it could not be reliably extracted. The XANES of noninfected Ectocarpus (Fig. 5, trace C) remotely resembles that of an aqueous solution of NaI (trace E) and probably represents iodide in the form of I − . In its lack of features it resembles the XANES of lyophilized Laminaria [6] which contains iodide surrounded by a rather disordered shell of hydrogen-bonded heteroatoms, probably from biomolecules rather than water. Addition of 50 μM iodate (trace B) resulted in an increase of the edge step by a factor of approx. 2 prior to normalization (not shown). This addition did however not result in a significant change in the appearance of the edge, as might have been expected on the basis of the spectrum of pure iodate (trace A), suggesting that the added iodate is reduced to iodide upon contact with Ectocarpus. Addition of 50 μM iodide (trace D) resulted in a much larger edge step prior to normalization (not shown), viz. by a factor of 5. The resulting spectrum is featureless like that of the control, except for the more pronounced shoulder at 33,230 eV. In spite of the addition of a considerable amount of hydrated iodide, the features are not as sharp as that of the NaI solution (trace E). This implies that the iodide was taken up by Ectocarpus and that a relatively ordered hydration shell was replaced by biomolecules.

Discussion
Overall, the results of this study show that Ectocarpus has an active bromine and iodine metabolism. Both elements are actively accumulated from surrounding seawater, albeit (especially in the case of iodine) at much lower concentration factors than Laminaria [20,21]: This study observed concentration factors of 714 for iodine and 2.6 for bromine (i.e. 2-3 and < 1 order of magnitude, respectively, based on fresh weight) compared to normal seawater. This compares to up to 5 orders of magnitude for iodine [21] and around 1 order of magnitude for bromine in Laminaria [10], and 3-4 orders of magnitude for iodine in Macrocystis [28]. Interestingly, artificial iodine supplementation (50 µM KI) in the culture medium resulted in a much lower bromine tissue concentration (186 ± 3 ppm) than in Ectocarpus cultures grown in medium without iodide supplementation (738 ± 3 ppm). This suggests that iodide and bromide compete for the same uptake and storage system, in which the sole V-bromoperoxidase detected in Ectocarpus [5,25] is likely the key driver. Iodide is well established as being the preferred substrate of V haloperoxidases [53], viz. of both bromo-and iodoperoxidases [27]. Our recent work had already shown that Ectocarpus accumulates bromide [43], similar to morphologically more complex brown algae (Laminariales and Fucales). Here, XAS has shown that, like Laminaria [6], Ectocarpus also accumulates iodine in its reduced form, iodide, bound to H-bonding heteroatoms (O-H, N-H) in biomolecules, with the hydration shell removed, as in lyophilized Laminaria. Again, similar to Laminaria [29], the present study also shows that Ectocarpus accumulates bromide and iodide in the apoplast (iodine storage is exclusively apoplastic, while smaller amounts of bromine can also be detected in the cytosol- Fig. 1). Our observation that the addition of iodate to the Ectocarpus growth medium results in an increased (estimated from X-ray absorption intensity) uptake of the element as iodide implies a reduction, which is in line with recent observations concerning the iodine uptake by marine macroalgae [9]. Interestingly, the reductant could be iodide, to form elemental iodine, which may be considered besides HOI to be the neutral species that is formed as an intermediate in VHPO-mediated iodine uptake. According to Küpper & Kroneck [54] the equilibrium is towards disproportionation at high pH and temperature, so that one might argue that starting at lower T and pH with only IO 3 − and I − , some I 2 might be formed by comproportionation, and removed from the equilibrium by uptake in Ectocarpus and subsequent reduction, potentially explaining the enhanced iodine uptake upon addition of iodate.
Instead, it is tempting to hypothesize that an S-adenosylmethionine-dependent methyl transferase-type mechanism is operative here, similar to that described for methyl halide transferase, a class of enzymes which is responsible for chloromethane production in the higher plant Mesembryanthemum crystallinum [57] and other salt-tolerant higher plants [58]. Considering that halocarbon production in seaweeds has usually been associated with oxidative stress and, in    Table 1 particular, with hydrogen peroxide, it has to be highlighted that this reaction is independent of any reactive oxygen species: It should be noted that (like for V haloperoxidases), iodide is the preferred substrate for methyl halide transferases, with usually much lower K M and higher V max values reported for iodide over bromide and chloride [58]. While a very moderate increase of methyl iodide production was observed in this study under oxidative stress conditions, this may arguably not be due to the direct participation of H 2 O 2 in the biosynthetic reaction(s), but rather due to the mobilization of iodide from its apoplastic store due to H 2 O 2 , analogous to the situation in Laminaria [6], thus merely increasing substrate availability for a hypothetical methyl transferase-type enzyme. It should be noted that Ectocarpus genome annotation has at present not uncovered any S-adenosyl-methionine-dependent methyl transferase-but also that, at present, many open reading frames in the Ectocarpus genome S-adenosyl-L-methionine + halide → S-adenosyl-L-homocysteine + methyl halide.
encode for genes of still unknown function [25]. A BLAST search of the complete cDNA sequence of Beta maritima [AF084829; 58] against the Ectocarpus genome (11×) and latest (as of November 2017) cDNA database did not yield any significant matches (not shown). Other halogen-related enzymes that have been identified in the Ectocarpus genome include at least three different families (21 loci) of haloacid dehalogenase (HAD) and two haloalkane dehalogenases. The HADs belong to a large superfamily of hydrolases with diverse substrate specificity, including phosphatases and ATPases. The dehalogenase enzymes may contribute to protect Ectocarpus against halogenated compounds produced as defence metabolites by kelps [6] allowing it to successfully grow as an epiphyte or endophyte on kelp thalli [59,60]. gratefully acknowledged. We are also grateful to Claire M.M. Gachon (Scottish Association for Marine Science) for her assistance with the Ectocarpus/Eurychasma cultures. EPM and CJC are grateful to the Advanced Photon Source at Argonne National Laboratory for facilitating the GSECARS X-ray experiments and Tony Lanzirotti for his assistance on the GSECARS beamline and with subsequent data analysis. Furthermore, the authors are grateful for support from the European Community in the framework of the Access to Research Infrastructure Action of the Improving Human Potential Program to the EMBL Hamburg Outstation. Finally, we would like to thank Yuka Uchida (Department of Chemistry, Gakushuin University, Tokyo) for her help with the ICP-MS analyses mentioned here.