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Temperature stress-induced bleaching of the coralline alga Corallina officinalis: a role for the enzyme bromoperoxidase

Temperature stress-induced bleaching of the coralline alga Corallina officinalis: a role for the... Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn016 ......................................................................................................................................................................................................................................... Research article Temperature stress-induced bleaching of the coralline alga Corallina officinalis: a role for the enzyme bromoperoxidase Holly Latham* School of Biological Sciences, University of Plymouth, Plymouth, Devon, UK. * Corresponding author: 20 Wright Street, Codnor, Ripley, Derbyshire DE5 9RQ, UK. Email: underwater_babe@hotmail.co.uk Supervisor: Dr Les Jervis, School of Biological Sciences, University of Plymouth, Plymouth, Devon, UK. ........................................................................................................................................................................................................................................ Coralline algae are important components of coral reefs and are involved in reef building via calcification, cementation, the synthesis of anti-fouling compounds and the synthesis of allochemicals to aid recruitment, settling and metamorphosis of reefs species. Using Corallina officinalis we have shown that these algae undergo temperature-related bleaching at similar temperatures to those known to cause bleaching in corals. The bleaching appears to be associated with considerable increases in the vanadium-containing enzyme bromoperoxidase (VBPO). This enzyme is involved in hydrogen peroxide (H O ) elimination and generates the powerful bromi- 2 2 nating/oxidizing agent hypobromous acid (HOBr, probably present as Br ). This is used to synthesize volatile halogenated organic com- pounds (VHOCs) from a pool of organic acceptor molecules. Earlier in vitro work has shown this enzyme to be effective in bleaching the phycobilin photosynthetic accessory pigments and to be partly located in chloroplasts. The data presented here supports the suggestion that increases in temperature lead to an increase in the cellular production of H O and other reactive oxygen species that result in an 2 2 increase in VBPO, a subsequent increase in HOBr/Br followed by pigment bleaching when the capacity to produce VHOCs has been exceeded. Addition of the exogenous antioxidant mannitol decreases both pigment bleaching and VBPO induction. A scheme is pre- sented to illustrate proposals for the involvement of VBPO in the bleaching of coralline algae such as C. officinalis. The importance of these species in reef building and rebuilding is discussed. Key words: Corallina officinalis, coral reef, bromoperoxidase, phycobilins, bleaching, temperature stress. ........................................................................................................................................................................................................................................ the considerable volume of work carried out, there has been Introduction no definitive identification of the ROS directly responsible Coral reefs are under considerable threat globally from for pigment bleaching, although singlet oxygen generated anthropogenic and natural stress. A notable symptom of during the photosynthetic light reactions is a prime stress is bleaching of the pigments present in the coral suspect. The immediate effects of pigment bleaching polyps and their symbiotic photosynthetic zooxanthellae. appear to be reversible given a return to lower ambient sea The symptoms and causes of coral bleaching have been temperatures, as pigment loss appears to be due to either studied extensively and it is generally accepted that tempera- expulsion of zooxanthellae or a loss of their photosynthetic ture increases of 1 – 38C above the mean long-term annual pigments. Sustained temperature elevation, however, maximum for the geographical area induce oxidative stress leads to polyp death, precluding any recolonization by and the generation of reactive oxygen species (ROS) such zooxanthellae. as singlet oxygen, hydrogen peroxide (H O ) and the super- 2 2 Coralline algae, with their calcium carbonate outer coat, 3– 5 oxide radical anion. Interaction of ROS with photosyn- constitute major structural components of coral reefs and thetic pigments leads to bleaching and Lesser has shown are important in calcification, reef cementation and anti- 7– 9 that the addition of exogenous antioxidants such as ascorbic fouling processes. However, the effects of environmental acid and mannitol can lessen or prevent bleaching. In spite of stress on these reef species have been little studied. ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 104 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... 10 22, 25 Corallina officinalis, of the order Corallinales, is related products. H O produced in the cell is utilized to 2 2 to many important reef species. It has a crustose, discoid oxidize bromide anions (Br ) to a highly reactive intermedi- 10, 26 holdfast with erect, calcareous segmented and branched ate, probably either hypobromous acid (HOBr) or bro- þ 25 fronds, giving the alga a ‘feather-like’ appearance. Frond monium cations (Br ). The reactive species can then react colour is variable, with purple, red, pink and yellow in a number of ways, including to halogenate nucleophilic 24, 27 recorded, sometimes with white knuckles and extremities. organic compounds, or to react with excess H O to 2 2 11 28 Colours are often paler in more brightly lit sites and yield singlet oxygen in the absence of suitable substrates. colour alterations are considered to represent light-induced The typical reaction that occurs is illustrated below: stress and subsequent pigment degradation. Corallina growth rate is slow, becoming stunted when cold, and H O þ Br þ H ! HOBr þ H O 2 2 2 ceasing altogether at elevated temperatures. HOBr þ AH ! ABr þ H O C. officinalis is recorded widely throughout the northern ðA is representative of an organic, nucleophilic acceptorÞ Atlantic from northern Norway southwards to Morocco in the east and Greenland as far down as Argentina in the west, with scattered reports as far afield as Japan, China VBPO activity is considered to be rate limited by H O con- 2 2 and Australasia. The alga is found across a wide range of centration, with increases shown to be inducible by high light 23, 29 29 22 habitats, ranging from exposed, open areas coastline to stress, elevated pH and oxidative stress, suggesting calm, sheltered embayments, and has been recorded on the that environmental stressors capable of elevating H O 2 2 shore from the mid-littoral down to a maximum depth of levels are capable of causing elevated VBPO activity. almost 30 m, across a wide range of natural and artificial The main physiological roles suggested for VBPO are hard substrates. The morphology of Corallina varies, halohydrocarbon production, primarily as compounds for 7, 23, 27 dependent upon its position on the shore, occurring as a defence and competition, elimination of excess 12, 13 23 30 cushion, compact turf or as scattered clumps. H O and assistance in halide uptake. In a recent 2 2 Corallina is often the dominant organism in the habitat review, Dring has pointed to the two main locations of and provides structure in the community, supporting algal cells that generate increased ROS in response to diverse invertebrate communities and acting as substrata disease and stress. These are the chloroplast (via photo- for various epiphytes, sometimes resulting in overgrowth synthesis) and the plasma membrane (via the FADH- with subsequent mortality, particularly during summer dependent NADPH oxidase). Interestingly, VBPOs have months. been shown to be located within the chloroplast and on 31, 32 C. officinalis is one of a number of algal species that pos- the outer cell wall. The pigments bleached during 14 – 18 sesses phycobilisomes (PBS), highly organized aggrega- stress ( phycobilins, carotenoids, chlorophylls) are also tions of photosynthetic accessory pigments that are found located in the chloroplast. Recent work in our laboratory attached to the chloroplast thylakoid membranes in close suggests that HOBr is involved in pigment degradation and proximity to photosystem II (PSII). Within Corallina these bleaching, with both hypo-osmotic shock and ultraviolet serve to increase light capture efficiency and assist in uni- (UV) capable of altering accessory pigment concentrations, 15, 16 33, 34 directional energy transfer. Structurally they consist of and elevating VBPO activity. In addition, in vitro a series of peripheral rods surrounding a central core and studies have shown VBPO to be capable of producing contain three types of pigment proteins, ‘phycobiliproteins’; rapid bleaching responses in C. officinalis accessory pig- red fluorescent, phycoerythrins (PE, l ¼ 540 – 570 nm), ments. This would suggest that elevated temperatures, max blue fluorescent, phycocyanins (PC, l ¼ 610 – 620 nm), known to be capable of causing oxidative stress and associ- max 3, 4, 36, 37 bright blue fluorescent allophycocyanins (APC, l ¼ ated excess H O production, may be capable of max 2 2 14-16 650 – 660 nm), as well as associated linker polypeptides inducing increased VBPO activity, increased production 18, 19 for structure and assembly. Captured light energy is and accumulation of HOBr and subsequent degradation transported from high energy PE, through intermediate and bleaching of pigments. energy PC, to low energy APC before being transferred to Whilst in the UK we are located in a geographically remote chlorophyll a at the reaction centre of PSII. The number, location from major reef systems, coralline algae represent size and internal pigment ratio of the PBS are variable, and important local species. The aim of this study was to carry are known to be influenced by a number of environmental out preliminary investigations into the effects of elevated 19, 20 factors, allowing acclimation to various conditions. seawater temperatures and the presence of an exogenous anti- One response to environmental stress in many algae, oxidant mannitol on C. officinalis, exploring the responses including C. officinalis, is the production of volatile of accessory pigment concentrations and VBPO activity. The 21 – 24 halocarbons via vanadium bromoperoxidase (VBPO). temperature range we chose was 5 – 358C, the upper value This enzyme catalyses the addition of bromine or iodine to being representative of temperatures causing bleaching in organic substrates, producing a range of organic halogenated coral reefs in warmer oceanic regions. ......................................................................................................................................................................................................................................... 105 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... transferred to a mortar at room temperature, where 10 ml Materials and methods of 0.05 M ( pH 6.7) potassium phosphate buffer was added Sample collection and the sample was ground for a further minute. The liquid extract was pipetted into microcentrifuge tubes. The C. officinalis was collected in autumn 2006, from rock pools extract was then centrifuged in a microcentrifuge for 4 min in the mid to lower littoral zone, of Wembury Beach, Devon, at 13 000 rpm. The supernatant was removed, its exact UK (OS Grid Reference SX 517484). The Corallina was volume recorded, and transferred into labelled storage transported directly from Wembury Beach to the University tubes for use in assays. of Plymouth where it was transferred to aerated seawater tanks and maintained at 158C for three days prior to its Accessory pigment assay use to allow any transportation stress to subside. The pigment assay used to analyse PE, PC and APC concen- trations was based on that used by Rosenberg and Ramus. Standard experimental set-up A Phillips PU8720 spectrophotometer was set up to scan and Water baths were set up, at 58C, 158C, 208C, 258C, 308C record absorbance levels between 400 and 700 nm. and 358C. These were equilibrated for several days prior to Potassium phosphate buffer, 0.05 M ( pH 6.7), was used to the start of the experiment, until the chosen temperatures perform a baseline scan. Two, 1 ml pigment samples were had become settled and constant. Two fluorescent tubes taken from each extraction, scanned and the absorbance (one 18 W and one 20 W) were suspended on light frames values for 565, 615 and 650 nm were recorded. Pigment con- 6 in. above the tops of the tanks containing the Corallina, centrations were calculated from the absorbencies and set on a 12 h light, 12 h dark light regime. The units recorded. The Corallina extraction process is time- were then covered with black plastic to eliminate incident consuming therefore only basic one-way ANOVA statistical light. For each temperature, 35 g of Corallina, representative analyses were performed on the data. of the overall condition of the material collected and, where possible, free from epiphytic growth, were selected and Bromoperoxidase (VBPO) assay placed in 1 l, clear glass beakers, filled with natural seawater. The VBPO assay used was based on that used by de Boer Full water changes were performed every other day, ensuring et al. (1987), which follows the bromination of phenol that the temperatures were equal prior to exchange to red at 580 nm. Each assay contained 2 ml of 100 mM prevent thermal shock. phenol red, 0.2 ml of 1.0 M potassium bromide and 0.2 ml The extractions, pigment assays and VBPO assays were of 10 mM H O . Enzyme (0.1 ml) was added to start the 2 2 performed on Day 0, to establish control pigment concen- reaction. trations and VBPO activities prior to exposure to thermal stress. The 208C, 258C, 308C and 358C extractions, Bradford protein assay pigment assays and VBPO assays were performed on Days Protein was assayed using the Bradford dye binding assay 1 – 4 and Day 7, as it was not possible to access the labora- using bovine serum albumin as the standard reference tories over the weekend (Days 5 and 6) to extract samples. protein. The 58C and 158C samples, representative of ‘normal’ temp- erature ranges experienced by the Corallina collected, were Effect of exogenous antioxidants sampled only on Day 0 and Day 7. Following the extractions, To investigate whether the addition of an antioxidant would pigment assays and VBPO assays, the extracts were mixed prevent or delay pigment bleaching, two water baths were set with glycerol, in a 50:50 extract: glycerol ratio and stored up, as for the previous experiment, at the two highest temp- at 2208C for later analysis of protein. eratures, 308C and 358C. Each water bath contained two 1-l Photographs were taken on a Nikon Coolpix 5700 digital clear glass beakers, one containing just natural seawater and camera utilizing the flash, with all camera settings remaining the other made up to a 10 mM mannitol seawater solution. unchanged throughout the duration of the experiment. Six grams of Corallina, representative of the condition of the collected material, were transferred to each beaker. The Extraction seawater and mannitol solutions were exchanged daily, to The method used for phycobilin extraction was based on that prevent bacterial build up in the mannitol solutions, with of Rosenberg and Ramus. Two, 1 g, samples, each repre- the solutions brought up to equal temperatures prior to sentative of the overall condition of the Corallina batch, were exchange to prevent thermal shock. The experiment was removed from the containers, blotted dry, photographed and run for a total of four days and the extractions, pigment the exact weight of the samples recorded. The samples were assays and VBPO assays were all performed prior to the individually placed in a cooled mortar and freeze-thawed experiment start, on unexposed samples, to give control three times with liquid nitrogen while being ground to a values. They were then performed daily for the remaining fine powder. Each powdered sample was carefully three days, utilizing the same methods as detailed previously. ......................................................................................................................................................................................................................................... 106 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Statistical analyses production of ROS enough to enable the antioxidant defences of the Corallina to successfully regulate the Results for both pigment analyses and VBPO activity were 33 – 35 oxidant/antioxidant balance within the cell. Further checked to ensure that the data confirmed to the assumptions investigation using intracellular measures of ROS and a required for one-way ANOVA analysis. Probability plots, variety of irradiances, would enable confirmation of the drawn daily for the data sets, showed them all to be normally cause of this trend in the 208C and 258C PE and PC concen- distributed (P  0.05). As inequalities within the variances trations. It would allow verification of whether ROS levels may affect the outcome of the ANOVA, the significance were remaining stable, and establish that the pigment degra- level used was reduced to P ¼ 0.01, to reflect this possible dation was due to elevated temperature, not simply as a source of error. This reduction in significance level should result of the light levels under which the Corallina was reduce the likelihood of a type I error. Due to inequalities kept, which may also produce gradual elevations of H O 2 2 in the number of samples across the days, it was not possible concentrations and subsequent pigment degradation. to utilize a two-way ANOVA. It was considered to be most The higher temperatures, 308C and 358C, show far more appropriate to perform five separate one-way ANOVAs, drastic losses of PE across the 7 day period, although the across the five experimental days (Days 1 – 4 and 7). 308C mean values do show signs of leveling out over Days The null hypotheses under test are that there are no signifi- 3 to 7. Again, this may be due to the ability of the cant differences present between the means of the phycobilin Corallina defence mechanisms to cope with regulating ROS concentrations (H ) or the mean VBPO-specific activities 0 1 levels, once sufficient light energy input pathways have (H ) of the 208C, 258C, 308C, 358C, and control data sets. 0 2 been disabled by accessory pigment degradation. The 358C PE mean values show no signs of levelling off suggesting Results and discussion that once the temperature reaches this level, excessive pro- duction and accumulation of ROS has overwhelmed the anti- Visual bleaching oxidant defences of the Corallina. These unregulated levels Figure 1 shows the visual evidence of bleaching at 358Cover of ROS are likely to lead to complete pigment loss, damage 40, 41 the 7-day period of the experiment with the Corallina fronds to numerous cellular components, and cell death. changing from a dusky pink-purple at Day 0 to pale orange The APC mean concentrations do not produce the same at Day 1 through to complete bleaching by Day 3. All other clear trend as PE and PC pigment concentrations and, samples (208C, 258C, 308C) remained a healthy pink-purple while the 358C concentrations are consistently lowest out in colour throughout Day 2. By Day 3, the 308C samples of the four temperatures, the 208C, 258C and 308C APC con- were beginning to bleach to a pale orange, mainly around centrations actually increase in all but one mean (308C; Day the frond tips. The 208C and 258C, however, still remained 7). However, ANOVA analysis showed a clearly significant the same healthy pink-purple as the Day 0 samples. By fall in APC at 358C over all other temperatures. Day 4, the 308C samples remain similar in colour to the Bromoperoxidase-specific activity Day 3 samples, with partially bleached fronds. There was no visible difference between the 208C and 258C samples The plot of the mean VBPO-specific activities at the different of Day 4, and those on Day 0. By Day 7, the 358C exposure temperature exposures reflects the trend shown in Corallina samples were completely bleached white, with no the ANOVA results (Figure 3a), showing the correlation pale orange colouration visible, while the partial bleaching between elevated temperatures and increasing specific of the 308C samples had reached a larger proportion of the activity. There is a slight increase in specific activity mean fronds. values in the 308C samples, particularly towards the end of the experiment. The 99% confidence intervals suggested Phycobilin concentrations this difference not to be substantial. P-values of ,0.01 Both PE and PC concentrations showed clear trends of throughout the experiment were obtained, giving evidence decreasing concentration with increasing temperature for a significant difference between the data sets, allowing (Figure 2) confirming the visual observations. P-values of rejection of (H ) . 0 2 ,0.01 throughout the experiment were obtained, giving evi- There was a considerable decrease in the amount of dence for a significant difference between the data sets, soluble protein extracted from the 358C samples, giving allowing rejection of (H ) At lower temperatures, 208C rise to high VBPO-specific activities based on International 0 1. and 258C, only small initial decreases in PE and PC pigments Units (IU) per mg of protein. When specific activities were are visible and, following the initial drop, they remain rela- calculated based on IU per gram of Corallina extracted, the tively steady throughout the remainder of the experimental increases at 308C became more significant (Figure 3b). All period. It is probable that, at these temperatures, a small specific activities were declining by Day 7. decrease in photosynthetic energy input, caused by partial VBPO-specific activity has been shown to be primarily degradation of the accessory pigments, may reduce the rate-limited by the H O concentration, which has been 2 2 ......................................................................................................................................................................................................................................... 107 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 1. Photographs of bleaching Corallina treated at 358C. Samples of Corallina officinalis were treated at 358C as described in the methods. Photographs were taken at (a) prior to the start of treatment; (b) treatment at 358C for 24 h; (c) treatment at 358C for 48 h; (d) treatment at 358C for 7 days (complete bleaching). The orange colour in (b) and (c) is due to remaining carotenoids. 21, 22, 25, 27, 31, 32 demonstrated to be elevated by numerous environmental organic products. It is possible that under stressors including, high visible irradiance, UV radiation, sal- stress conditions, increased VBPO activity could lead to inity variation, nutrient and mineral deficiencies, exposure to excessive depletion of the pool of organic acceptor com- xenobiotics, heavy metal pollutants and extremes of temp- pounds drawn upon in the second stage of the reaction. 3, 6, 33, 35 – 37 eratures, This would suggest that the rise in Should this happen, and were the VBPO activity to remain VBPO-specific activity recorded in the higher temperature elevated with continued supply of excess H O and ready 2 2 samples is likely to be due to an increase in H O within availability of stored Br , a build up of the highly oxidizing 2 2 the Corallina. Increases in VBPO activity, in response the intermediate, HOBr, would occur. environmental stressors of altered salinity and increased Two routes are suggested, that may be possible mechan- UV radiation, have been previously reported to occur in isms by which temperature-induced bleaching of the Corallina in work in this laboratory by Dent (2006) and Corallina may occur (Figure 4). The HOBr may react directly Vesty (2006). with the pigments to cause bleaching, as suggested by The exact mechanisms by which elevated VBPO activity Thomas (2006), or the accumulated HOBr may react may cause bleaching within Corallina, and possibly other with excess H O , leading to the production of singlet 2 2 23, 26, 28 coralline algae, remains unconfirmed. However, a recent oxygen, which is also known to produce pigment 35 5 study in this laboratory by Thomas (2006) showed that bleaching. H O ,Br and VBPO can induce rapid bleaching in vitro The dramatic difference between the reaction of the 2 2 in pigment extracts from Corallina, whereas with H O VBPO-specific activity at 308C and at 358C, with the 2 2 and VBPO in the absence of Br , bleaching does not occur. higher temperature producing a much greater increase in It is known that the reaction of excess H O , produced by activity suggests the presence of a ‘tipping point’—a tempera- 2 2 the cell under stress, plus bromine (Br ), taken up from sea- ture at which the defence mechanisms of the Corallina fail to water, is catalysed by VBPO, and leads to the production of a regulate the production of ROS. Whether or not this tipping highly reactive intermediate, probably bromonium ions (Br ) point varies as a result of acclimation to local conditions is but usually represented as HOBr. Under normal conditions, unknown, although the distribution of Corallina across HOBr reacts with various organic compounds, either intra- equatorial, as well as temperate waters suggests that this or extracellularly, to produce a number of halogenated may be possible. Further investigation of possible ......................................................................................................................................................................................................................................... 108 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. Loss of phycobilins with increasing ambient temperature. Phycoerythrin and phycocyanin show a similar trend of decreasing pigment concen- trations, with the 208C and 258C mean values showing small initial decreases that stabilize after Day 2, while the 308C, and particularly the 358C, mean values drop considerably. In contrast, allophycocyanin levels increase initially before starting to decline. acclimation to thermal stress in Corallina and the possible samples, the control Day 0 samples, and those with added effects that global warming may have on its distribution mannitol, although the without mannitol mean values are, would no doubt yield interesting results. in all cases, very slightly higher than those from the mannitol treated samples. Low temperature (588888C and 1588888C) samples The mean PE concentrations showed dramatic falls at 358C with over 90% loss for the untreated samples against A comparison, on Day 7, of the samples kept at 58C and approximately 70% loss in the treated samples after 48 h 158C, with samples from the 208C and 258C Corallina showing the protective effect of mannitol. Losses were showed little variation in response between the lower temp- much less at 308C for both treated and untreated samples. eratures. The mean PE concentrations altered little over the 7 The mannitol added to the seawater acts as an antioxi- days, with relatively slight decreases noticeable across all dant, effectively removing ROS, preventing their accumu- four lower temperatures (results not shown). There was lation, and associated oxidative stress. The removal of also little difference in mean VBPO activity over the lower ROS both lowers the concentration of H O and removes temperature range (58Cto258C). 2 2 species that can be converted to H O , the main rate-limiting 2 2 Effect of exogenous antioxidant (mannitol) factor for VBPO. Lowering the available concentration is likely to maintain the VBPO activity at low levels, restricting The effect of adding mannitol show clear trends in the 358C the formation of HOBr, and preventing, or constraining the data. Over Days 1 – 3, the mean VBPO activity recorded for extent of, pigment bleaching. Limiting VBPO activity would the 358C without mannitol samples is consistently higher also help prevent excessive depletion of organic acceptor than those treated with mannitol (Table 1). The 308Cdata resources, ensuring sufficient substrate is available to react however, shows little difference between the standard ......................................................................................................................................................................................................................................... 109 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 3. Temperature-induced increase in bromoperoxidase-specific activity. (a) Mean specific activities, based on IU per mg protein, at 358C are far higher than all other samples, with an almost exponential rise in activity across the 7 days of the exposure. The 308C mean specific activities also increase notably by Day 3 but the 99% confidence intervals suggest this difference is not statistically significant. However, at 358C, there is a considerable reduction in the amount of soluble protein extracted, giving rise to very high specific activities based on protein. (b) Mean specific activities, based on IU per gram fresh weight of Corallina,at 358C are higher than all other samples. The 308C mean specific activities also increase notably by day 3 over the values at lower temperatures, showing a several fold increase in BPO catalytic activity. By day 7, all specific activities are declining. with the HOBr, therefore allowing successful conversion to at 308C suggests that, at this temperature, the antioxidant 22, 23 halohydrocarbons. defence mechanisms of the Corallina are efficiently scaven- The addition of mannitol to the Corallina samples pro- ging and removing ROS produced within the cell, maintain- duced results that appeared strongly dependent upon the ing low H O concentrations and preventing induction of 2 2 sample temperature. In the 308C Corallina samples the pre- elevated VBPO activity. The large differences in VBPO sence of mannitol in the seawater produced no real visible activities at 358C would suggest that, in the absence of man- difference in VBPO activity across the 4 days, while in the nitol, the antioxidant defence systems of the Corallina are 358C samples the added mannitol showed a strong protective failing, ROS are accumulating, and excess H O combined 2 2 effect, with untreated sample mean values reaching much with an increased rate of VBPO results in pigment bleach- higher levels than the treated sample rates. The similarity ing. At 358C, a considerable rise in activity is still visible in in VBPO activities in the absence and presence of mannitol the mannitol treated samples, this would suggest that the ......................................................................................................................................................................................................................................... 110 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... concentration of mannitol used, 10 mM, while sufficient to scavenge considerable levels of ROS, was not enough to com- pletely quench the levels produced. The PE, PC and APC pigment concentrations show similar trends in the 358C mean pigment concentrations, with the mannitol treated samples maintaining consistently higher concentrations in comparison with the untreated samples. This loss of pigments is likely to be due to the inability of cells to control ROS concentrations, raising the VBPO activity and causing degradation and pigment loss. Conclusions Wider ecological effects of bleaching in coralline algae Crustose coralline algae (CCA), the Corallinacea, contribute the most widespread and abundant benthic marine organ- 9, 42 isms to be found in the photic zone. Many species of CCAs are known to fulfill important roles in coral reef eco- systems, both in cementing the reef structure and laying 7– 9 down substantial amounts of calcareous material, and also in inducing settlement of numerous benthic reef organ- 8 43 isms, including corals. Though CCAs contribute signifi- cantly to the biotic cover and structure of a reef ecosystem, their ecological influences and the effects of environmental and anthropogenic impacts are relatively understudied, with most of the investigative focus resting on the corals themselves. The role of CCAs in recruitment of new corals also plays an vital role in recovery and resilience of the reef, to both natural and anthropogenic disturbances. Therefore, if temperature can produce such significant bleaching effects within Corallina, further study into Figure 4. Proposed mechanisms of phycobilin bleaching in Corallina offi- thermal bleaching of other coralline algae may prove immen- cinalis.(a) Shows the elimination of hydrogen peroxide via the formation of sely important in the future conservation of coral reefs, as þ þ HOBr (or Br ) by bromoperoxidase (VBPO). The HOBr/Br reacts with an adverse effects on the CCAs within a reef will subsequently organic acceptor to produce a volatile halogenated organic compound affect the entire reef ecosystem. Although the species (VHOC). Bleaching of phycobilin pigments is a minor side reaction. (b) Shows the impact of temperature-induced VBPO synthesis. The increased studied here is not involved in reef building, other coralline formation of HOBr/Br overwhelms the capacity of cells to form VHOC algae such as Lithophyllum yessoense are. This species has and the bleaching of pigments becomes a major reaction, either by been shown to contain VBPO and to produce volatile hydro- HOBr/Br directly (1) or by singlet oxygen formed by the reaction of þ carbons such as bromoform that are known to be involved HOBr/Br with hydrogen peroxide (2). Bleaching prevents energy transfer in inducing settlement of larval stages of benthic organisms, to PSII. Solid lines represent major reactions; dotted lines represent minor reactions; wavy lines represent incident light. and also in inducing metamorphosis of sea anenomies. Table 1. Effect of mannitol on induction of bromoperoxidase in response to temperature Temperature 308C þ Mannitol 308C 2 Mannitol 358C þ Mannitol 358C 2 Mannitol Day Fold increase in specific activity (IU/g fresh weight Corallina)overDay 0 01 1 1 1 1 1 1.28 1.11 21.8 2 1 1.38 3.14 24.8 3 1 1.06 4.38 27.2 ......................................................................................................................................................................................................................................... 111 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 15. D’Agnolo E, Rizzo R, Paoletti S et al. (1994) R-Phycoerythrin from the red alga Recognition of the importance of CCA as major reef building Gracilaria longa. Phytochem 35: 693–696. organisms warrants much more emphasis on the impact of 16. Kursar TA, Alberte RS (1983) Photosynthetic unit organisation in a red alga. environmental change on these organisms. They are clearly Plant Physiol 72: 409–414. as important in the re-building of damaged reefs as they 17. MacColl R (1998) Cyanobacterial phycobilisomes. J Struct Biol 124: 311–334. are in original reef construction. Their role needs to be 18. MacColl R, Guard-Friar D (1987) Phycobiliproteins. Boca Raton: CRC Press. more widely recognized. They deserve at least as much atten- 19. Liu L, Chen X, Zhang Y et al. (2005) Characterisation, structure and function tion as the more charismatic corals and their symbiotic of linker polypeptides in phycobilisomes of cyanobacteria and red algae: an zooxanthellae. overview. Biochimica et Biophysica Acta 1708: 133–142. 20. Lo´ pez-Figueroa F (1992) Diurnal variation in pigment content in Porphyra laciniata and Chondrus crispus and its relation to the diurnal changes of Acknowledgements underwater light quality and quantity. Mar Ecol 13: 285–305. 21. de Boer E, Plat H, Tromp MGM et al. (2005) Vanadium containing bromoper- Many thanks to Les Jervis for continued support and gui- oxidase: an example of an oxidoreductase with high operational stability in dance throughout the entire project. Also, thanks to Nick aqueous and organic media. Biotech Bioeng 30: 607–610. Crocker and Andy Atfield for advice on experimental 22. Littlechild J, Garcia-Rodriguez E (2003) Structural studies on the dodeca- design and techniques and assistance with experimental meric vanadium bromoperoxidase from Corallina species. Coord Chem Rev set-ups. 237: 65–76. 23. Ohsawa N, Ogata Y, Okada N et al. 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In A Post, NR Baker, JB Boyer, eds. methane on metamorphosis of larvae of the sea urchins Photoinhibition of Photosynthesis: From Molecular Mechanisms to the Field Strongylocentrotus nudus and Strongylocentrotus intermedius. Aquaculture Oxford, UK: BIOS, pp. 129–142. 251: 549–557. ........................................................................................................................................................................................................................................ Submitted on 30 September 2007; accepted on 28 January 2008; advance access publication 22 April 2008 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Temperature stress-induced bleaching of the coralline alga Corallina officinalis: a role for the enzyme bromoperoxidase

Bioscience Horizons , Volume 1 (2) – Jun 22, 2008

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Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn016 ......................................................................................................................................................................................................................................... Research article Temperature stress-induced bleaching of the coralline alga Corallina officinalis: a role for the enzyme bromoperoxidase Holly Latham* School of Biological Sciences, University of Plymouth, Plymouth, Devon, UK. * Corresponding author: 20 Wright Street, Codnor, Ripley, Derbyshire DE5 9RQ, UK. Email: underwater_babe@hotmail.co.uk Supervisor: Dr Les Jervis, School of Biological Sciences, University of Plymouth, Plymouth, Devon, UK. ........................................................................................................................................................................................................................................ Coralline algae are important components of coral reefs and are involved in reef building via calcification, cementation, the synthesis of anti-fouling compounds and the synthesis of allochemicals to aid recruitment, settling and metamorphosis of reefs species. Using Corallina officinalis we have shown that these algae undergo temperature-related bleaching at similar temperatures to those known to cause bleaching in corals. The bleaching appears to be associated with considerable increases in the vanadium-containing enzyme bromoperoxidase (VBPO). This enzyme is involved in hydrogen peroxide (H O ) elimination and generates the powerful bromi- 2 2 nating/oxidizing agent hypobromous acid (HOBr, probably present as Br ). This is used to synthesize volatile halogenated organic com- pounds (VHOCs) from a pool of organic acceptor molecules. Earlier in vitro work has shown this enzyme to be effective in bleaching the phycobilin photosynthetic accessory pigments and to be partly located in chloroplasts. The data presented here supports the suggestion that increases in temperature lead to an increase in the cellular production of H O and other reactive oxygen species that result in an 2 2 increase in VBPO, a subsequent increase in HOBr/Br followed by pigment bleaching when the capacity to produce VHOCs has been exceeded. Addition of the exogenous antioxidant mannitol decreases both pigment bleaching and VBPO induction. A scheme is pre- sented to illustrate proposals for the involvement of VBPO in the bleaching of coralline algae such as C. officinalis. The importance of these species in reef building and rebuilding is discussed. Key words: Corallina officinalis, coral reef, bromoperoxidase, phycobilins, bleaching, temperature stress. ........................................................................................................................................................................................................................................ the considerable volume of work carried out, there has been Introduction no definitive identification of the ROS directly responsible Coral reefs are under considerable threat globally from for pigment bleaching, although singlet oxygen generated anthropogenic and natural stress. A notable symptom of during the photosynthetic light reactions is a prime stress is bleaching of the pigments present in the coral suspect. The immediate effects of pigment bleaching polyps and their symbiotic photosynthetic zooxanthellae. appear to be reversible given a return to lower ambient sea The symptoms and causes of coral bleaching have been temperatures, as pigment loss appears to be due to either studied extensively and it is generally accepted that tempera- expulsion of zooxanthellae or a loss of their photosynthetic ture increases of 1 – 38C above the mean long-term annual pigments. Sustained temperature elevation, however, maximum for the geographical area induce oxidative stress leads to polyp death, precluding any recolonization by and the generation of reactive oxygen species (ROS) such zooxanthellae. as singlet oxygen, hydrogen peroxide (H O ) and the super- 2 2 Coralline algae, with their calcium carbonate outer coat, 3– 5 oxide radical anion. Interaction of ROS with photosyn- constitute major structural components of coral reefs and thetic pigments leads to bleaching and Lesser has shown are important in calcification, reef cementation and anti- 7– 9 that the addition of exogenous antioxidants such as ascorbic fouling processes. However, the effects of environmental acid and mannitol can lessen or prevent bleaching. In spite of stress on these reef species have been little studied. ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 104 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... 10 22, 25 Corallina officinalis, of the order Corallinales, is related products. H O produced in the cell is utilized to 2 2 to many important reef species. It has a crustose, discoid oxidize bromide anions (Br ) to a highly reactive intermedi- 10, 26 holdfast with erect, calcareous segmented and branched ate, probably either hypobromous acid (HOBr) or bro- þ 25 fronds, giving the alga a ‘feather-like’ appearance. Frond monium cations (Br ). The reactive species can then react colour is variable, with purple, red, pink and yellow in a number of ways, including to halogenate nucleophilic 24, 27 recorded, sometimes with white knuckles and extremities. organic compounds, or to react with excess H O to 2 2 11 28 Colours are often paler in more brightly lit sites and yield singlet oxygen in the absence of suitable substrates. colour alterations are considered to represent light-induced The typical reaction that occurs is illustrated below: stress and subsequent pigment degradation. Corallina growth rate is slow, becoming stunted when cold, and H O þ Br þ H ! HOBr þ H O 2 2 2 ceasing altogether at elevated temperatures. HOBr þ AH ! ABr þ H O C. officinalis is recorded widely throughout the northern ðA is representative of an organic, nucleophilic acceptorÞ Atlantic from northern Norway southwards to Morocco in the east and Greenland as far down as Argentina in the west, with scattered reports as far afield as Japan, China VBPO activity is considered to be rate limited by H O con- 2 2 and Australasia. The alga is found across a wide range of centration, with increases shown to be inducible by high light 23, 29 29 22 habitats, ranging from exposed, open areas coastline to stress, elevated pH and oxidative stress, suggesting calm, sheltered embayments, and has been recorded on the that environmental stressors capable of elevating H O 2 2 shore from the mid-littoral down to a maximum depth of levels are capable of causing elevated VBPO activity. almost 30 m, across a wide range of natural and artificial The main physiological roles suggested for VBPO are hard substrates. The morphology of Corallina varies, halohydrocarbon production, primarily as compounds for 7, 23, 27 dependent upon its position on the shore, occurring as a defence and competition, elimination of excess 12, 13 23 30 cushion, compact turf or as scattered clumps. H O and assistance in halide uptake. In a recent 2 2 Corallina is often the dominant organism in the habitat review, Dring has pointed to the two main locations of and provides structure in the community, supporting algal cells that generate increased ROS in response to diverse invertebrate communities and acting as substrata disease and stress. These are the chloroplast (via photo- for various epiphytes, sometimes resulting in overgrowth synthesis) and the plasma membrane (via the FADH- with subsequent mortality, particularly during summer dependent NADPH oxidase). Interestingly, VBPOs have months. been shown to be located within the chloroplast and on 31, 32 C. officinalis is one of a number of algal species that pos- the outer cell wall. The pigments bleached during 14 – 18 sesses phycobilisomes (PBS), highly organized aggrega- stress ( phycobilins, carotenoids, chlorophylls) are also tions of photosynthetic accessory pigments that are found located in the chloroplast. Recent work in our laboratory attached to the chloroplast thylakoid membranes in close suggests that HOBr is involved in pigment degradation and proximity to photosystem II (PSII). Within Corallina these bleaching, with both hypo-osmotic shock and ultraviolet serve to increase light capture efficiency and assist in uni- (UV) capable of altering accessory pigment concentrations, 15, 16 33, 34 directional energy transfer. Structurally they consist of and elevating VBPO activity. In addition, in vitro a series of peripheral rods surrounding a central core and studies have shown VBPO to be capable of producing contain three types of pigment proteins, ‘phycobiliproteins’; rapid bleaching responses in C. officinalis accessory pig- red fluorescent, phycoerythrins (PE, l ¼ 540 – 570 nm), ments. This would suggest that elevated temperatures, max blue fluorescent, phycocyanins (PC, l ¼ 610 – 620 nm), known to be capable of causing oxidative stress and associ- max 3, 4, 36, 37 bright blue fluorescent allophycocyanins (APC, l ¼ ated excess H O production, may be capable of max 2 2 14-16 650 – 660 nm), as well as associated linker polypeptides inducing increased VBPO activity, increased production 18, 19 for structure and assembly. Captured light energy is and accumulation of HOBr and subsequent degradation transported from high energy PE, through intermediate and bleaching of pigments. energy PC, to low energy APC before being transferred to Whilst in the UK we are located in a geographically remote chlorophyll a at the reaction centre of PSII. The number, location from major reef systems, coralline algae represent size and internal pigment ratio of the PBS are variable, and important local species. The aim of this study was to carry are known to be influenced by a number of environmental out preliminary investigations into the effects of elevated 19, 20 factors, allowing acclimation to various conditions. seawater temperatures and the presence of an exogenous anti- One response to environmental stress in many algae, oxidant mannitol on C. officinalis, exploring the responses including C. officinalis, is the production of volatile of accessory pigment concentrations and VBPO activity. The 21 – 24 halocarbons via vanadium bromoperoxidase (VBPO). temperature range we chose was 5 – 358C, the upper value This enzyme catalyses the addition of bromine or iodine to being representative of temperatures causing bleaching in organic substrates, producing a range of organic halogenated coral reefs in warmer oceanic regions. ......................................................................................................................................................................................................................................... 105 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... transferred to a mortar at room temperature, where 10 ml Materials and methods of 0.05 M ( pH 6.7) potassium phosphate buffer was added Sample collection and the sample was ground for a further minute. The liquid extract was pipetted into microcentrifuge tubes. The C. officinalis was collected in autumn 2006, from rock pools extract was then centrifuged in a microcentrifuge for 4 min in the mid to lower littoral zone, of Wembury Beach, Devon, at 13 000 rpm. The supernatant was removed, its exact UK (OS Grid Reference SX 517484). The Corallina was volume recorded, and transferred into labelled storage transported directly from Wembury Beach to the University tubes for use in assays. of Plymouth where it was transferred to aerated seawater tanks and maintained at 158C for three days prior to its Accessory pigment assay use to allow any transportation stress to subside. The pigment assay used to analyse PE, PC and APC concen- trations was based on that used by Rosenberg and Ramus. Standard experimental set-up A Phillips PU8720 spectrophotometer was set up to scan and Water baths were set up, at 58C, 158C, 208C, 258C, 308C record absorbance levels between 400 and 700 nm. and 358C. These were equilibrated for several days prior to Potassium phosphate buffer, 0.05 M ( pH 6.7), was used to the start of the experiment, until the chosen temperatures perform a baseline scan. Two, 1 ml pigment samples were had become settled and constant. Two fluorescent tubes taken from each extraction, scanned and the absorbance (one 18 W and one 20 W) were suspended on light frames values for 565, 615 and 650 nm were recorded. Pigment con- 6 in. above the tops of the tanks containing the Corallina, centrations were calculated from the absorbencies and set on a 12 h light, 12 h dark light regime. The units recorded. The Corallina extraction process is time- were then covered with black plastic to eliminate incident consuming therefore only basic one-way ANOVA statistical light. For each temperature, 35 g of Corallina, representative analyses were performed on the data. of the overall condition of the material collected and, where possible, free from epiphytic growth, were selected and Bromoperoxidase (VBPO) assay placed in 1 l, clear glass beakers, filled with natural seawater. The VBPO assay used was based on that used by de Boer Full water changes were performed every other day, ensuring et al. (1987), which follows the bromination of phenol that the temperatures were equal prior to exchange to red at 580 nm. Each assay contained 2 ml of 100 mM prevent thermal shock. phenol red, 0.2 ml of 1.0 M potassium bromide and 0.2 ml The extractions, pigment assays and VBPO assays were of 10 mM H O . Enzyme (0.1 ml) was added to start the 2 2 performed on Day 0, to establish control pigment concen- reaction. trations and VBPO activities prior to exposure to thermal stress. The 208C, 258C, 308C and 358C extractions, Bradford protein assay pigment assays and VBPO assays were performed on Days Protein was assayed using the Bradford dye binding assay 1 – 4 and Day 7, as it was not possible to access the labora- using bovine serum albumin as the standard reference tories over the weekend (Days 5 and 6) to extract samples. protein. The 58C and 158C samples, representative of ‘normal’ temp- erature ranges experienced by the Corallina collected, were Effect of exogenous antioxidants sampled only on Day 0 and Day 7. Following the extractions, To investigate whether the addition of an antioxidant would pigment assays and VBPO assays, the extracts were mixed prevent or delay pigment bleaching, two water baths were set with glycerol, in a 50:50 extract: glycerol ratio and stored up, as for the previous experiment, at the two highest temp- at 2208C for later analysis of protein. eratures, 308C and 358C. Each water bath contained two 1-l Photographs were taken on a Nikon Coolpix 5700 digital clear glass beakers, one containing just natural seawater and camera utilizing the flash, with all camera settings remaining the other made up to a 10 mM mannitol seawater solution. unchanged throughout the duration of the experiment. Six grams of Corallina, representative of the condition of the collected material, were transferred to each beaker. The Extraction seawater and mannitol solutions were exchanged daily, to The method used for phycobilin extraction was based on that prevent bacterial build up in the mannitol solutions, with of Rosenberg and Ramus. Two, 1 g, samples, each repre- the solutions brought up to equal temperatures prior to sentative of the overall condition of the Corallina batch, were exchange to prevent thermal shock. The experiment was removed from the containers, blotted dry, photographed and run for a total of four days and the extractions, pigment the exact weight of the samples recorded. The samples were assays and VBPO assays were all performed prior to the individually placed in a cooled mortar and freeze-thawed experiment start, on unexposed samples, to give control three times with liquid nitrogen while being ground to a values. They were then performed daily for the remaining fine powder. Each powdered sample was carefully three days, utilizing the same methods as detailed previously. ......................................................................................................................................................................................................................................... 106 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Statistical analyses production of ROS enough to enable the antioxidant defences of the Corallina to successfully regulate the Results for both pigment analyses and VBPO activity were 33 – 35 oxidant/antioxidant balance within the cell. Further checked to ensure that the data confirmed to the assumptions investigation using intracellular measures of ROS and a required for one-way ANOVA analysis. Probability plots, variety of irradiances, would enable confirmation of the drawn daily for the data sets, showed them all to be normally cause of this trend in the 208C and 258C PE and PC concen- distributed (P  0.05). As inequalities within the variances trations. It would allow verification of whether ROS levels may affect the outcome of the ANOVA, the significance were remaining stable, and establish that the pigment degra- level used was reduced to P ¼ 0.01, to reflect this possible dation was due to elevated temperature, not simply as a source of error. This reduction in significance level should result of the light levels under which the Corallina was reduce the likelihood of a type I error. Due to inequalities kept, which may also produce gradual elevations of H O 2 2 in the number of samples across the days, it was not possible concentrations and subsequent pigment degradation. to utilize a two-way ANOVA. It was considered to be most The higher temperatures, 308C and 358C, show far more appropriate to perform five separate one-way ANOVAs, drastic losses of PE across the 7 day period, although the across the five experimental days (Days 1 – 4 and 7). 308C mean values do show signs of leveling out over Days The null hypotheses under test are that there are no signifi- 3 to 7. Again, this may be due to the ability of the cant differences present between the means of the phycobilin Corallina defence mechanisms to cope with regulating ROS concentrations (H ) or the mean VBPO-specific activities 0 1 levels, once sufficient light energy input pathways have (H ) of the 208C, 258C, 308C, 358C, and control data sets. 0 2 been disabled by accessory pigment degradation. The 358C PE mean values show no signs of levelling off suggesting Results and discussion that once the temperature reaches this level, excessive pro- duction and accumulation of ROS has overwhelmed the anti- Visual bleaching oxidant defences of the Corallina. These unregulated levels Figure 1 shows the visual evidence of bleaching at 358Cover of ROS are likely to lead to complete pigment loss, damage 40, 41 the 7-day period of the experiment with the Corallina fronds to numerous cellular components, and cell death. changing from a dusky pink-purple at Day 0 to pale orange The APC mean concentrations do not produce the same at Day 1 through to complete bleaching by Day 3. All other clear trend as PE and PC pigment concentrations and, samples (208C, 258C, 308C) remained a healthy pink-purple while the 358C concentrations are consistently lowest out in colour throughout Day 2. By Day 3, the 308C samples of the four temperatures, the 208C, 258C and 308C APC con- were beginning to bleach to a pale orange, mainly around centrations actually increase in all but one mean (308C; Day the frond tips. The 208C and 258C, however, still remained 7). However, ANOVA analysis showed a clearly significant the same healthy pink-purple as the Day 0 samples. By fall in APC at 358C over all other temperatures. Day 4, the 308C samples remain similar in colour to the Bromoperoxidase-specific activity Day 3 samples, with partially bleached fronds. There was no visible difference between the 208C and 258C samples The plot of the mean VBPO-specific activities at the different of Day 4, and those on Day 0. By Day 7, the 358C exposure temperature exposures reflects the trend shown in Corallina samples were completely bleached white, with no the ANOVA results (Figure 3a), showing the correlation pale orange colouration visible, while the partial bleaching between elevated temperatures and increasing specific of the 308C samples had reached a larger proportion of the activity. There is a slight increase in specific activity mean fronds. values in the 308C samples, particularly towards the end of the experiment. The 99% confidence intervals suggested Phycobilin concentrations this difference not to be substantial. P-values of ,0.01 Both PE and PC concentrations showed clear trends of throughout the experiment were obtained, giving evidence decreasing concentration with increasing temperature for a significant difference between the data sets, allowing (Figure 2) confirming the visual observations. P-values of rejection of (H ) . 0 2 ,0.01 throughout the experiment were obtained, giving evi- There was a considerable decrease in the amount of dence for a significant difference between the data sets, soluble protein extracted from the 358C samples, giving allowing rejection of (H ) At lower temperatures, 208C rise to high VBPO-specific activities based on International 0 1. and 258C, only small initial decreases in PE and PC pigments Units (IU) per mg of protein. When specific activities were are visible and, following the initial drop, they remain rela- calculated based on IU per gram of Corallina extracted, the tively steady throughout the remainder of the experimental increases at 308C became more significant (Figure 3b). All period. It is probable that, at these temperatures, a small specific activities were declining by Day 7. decrease in photosynthetic energy input, caused by partial VBPO-specific activity has been shown to be primarily degradation of the accessory pigments, may reduce the rate-limited by the H O concentration, which has been 2 2 ......................................................................................................................................................................................................................................... 107 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 1. Photographs of bleaching Corallina treated at 358C. Samples of Corallina officinalis were treated at 358C as described in the methods. Photographs were taken at (a) prior to the start of treatment; (b) treatment at 358C for 24 h; (c) treatment at 358C for 48 h; (d) treatment at 358C for 7 days (complete bleaching). The orange colour in (b) and (c) is due to remaining carotenoids. 21, 22, 25, 27, 31, 32 demonstrated to be elevated by numerous environmental organic products. It is possible that under stressors including, high visible irradiance, UV radiation, sal- stress conditions, increased VBPO activity could lead to inity variation, nutrient and mineral deficiencies, exposure to excessive depletion of the pool of organic acceptor com- xenobiotics, heavy metal pollutants and extremes of temp- pounds drawn upon in the second stage of the reaction. 3, 6, 33, 35 – 37 eratures, This would suggest that the rise in Should this happen, and were the VBPO activity to remain VBPO-specific activity recorded in the higher temperature elevated with continued supply of excess H O and ready 2 2 samples is likely to be due to an increase in H O within availability of stored Br , a build up of the highly oxidizing 2 2 the Corallina. Increases in VBPO activity, in response the intermediate, HOBr, would occur. environmental stressors of altered salinity and increased Two routes are suggested, that may be possible mechan- UV radiation, have been previously reported to occur in isms by which temperature-induced bleaching of the Corallina in work in this laboratory by Dent (2006) and Corallina may occur (Figure 4). The HOBr may react directly Vesty (2006). with the pigments to cause bleaching, as suggested by The exact mechanisms by which elevated VBPO activity Thomas (2006), or the accumulated HOBr may react may cause bleaching within Corallina, and possibly other with excess H O , leading to the production of singlet 2 2 23, 26, 28 coralline algae, remains unconfirmed. However, a recent oxygen, which is also known to produce pigment 35 5 study in this laboratory by Thomas (2006) showed that bleaching. H O ,Br and VBPO can induce rapid bleaching in vitro The dramatic difference between the reaction of the 2 2 in pigment extracts from Corallina, whereas with H O VBPO-specific activity at 308C and at 358C, with the 2 2 and VBPO in the absence of Br , bleaching does not occur. higher temperature producing a much greater increase in It is known that the reaction of excess H O , produced by activity suggests the presence of a ‘tipping point’—a tempera- 2 2 the cell under stress, plus bromine (Br ), taken up from sea- ture at which the defence mechanisms of the Corallina fail to water, is catalysed by VBPO, and leads to the production of a regulate the production of ROS. Whether or not this tipping highly reactive intermediate, probably bromonium ions (Br ) point varies as a result of acclimation to local conditions is but usually represented as HOBr. Under normal conditions, unknown, although the distribution of Corallina across HOBr reacts with various organic compounds, either intra- equatorial, as well as temperate waters suggests that this or extracellularly, to produce a number of halogenated may be possible. Further investigation of possible ......................................................................................................................................................................................................................................... 108 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. Loss of phycobilins with increasing ambient temperature. Phycoerythrin and phycocyanin show a similar trend of decreasing pigment concen- trations, with the 208C and 258C mean values showing small initial decreases that stabilize after Day 2, while the 308C, and particularly the 358C, mean values drop considerably. In contrast, allophycocyanin levels increase initially before starting to decline. acclimation to thermal stress in Corallina and the possible samples, the control Day 0 samples, and those with added effects that global warming may have on its distribution mannitol, although the without mannitol mean values are, would no doubt yield interesting results. in all cases, very slightly higher than those from the mannitol treated samples. Low temperature (588888C and 1588888C) samples The mean PE concentrations showed dramatic falls at 358C with over 90% loss for the untreated samples against A comparison, on Day 7, of the samples kept at 58C and approximately 70% loss in the treated samples after 48 h 158C, with samples from the 208C and 258C Corallina showing the protective effect of mannitol. Losses were showed little variation in response between the lower temp- much less at 308C for both treated and untreated samples. eratures. The mean PE concentrations altered little over the 7 The mannitol added to the seawater acts as an antioxi- days, with relatively slight decreases noticeable across all dant, effectively removing ROS, preventing their accumu- four lower temperatures (results not shown). There was lation, and associated oxidative stress. The removal of also little difference in mean VBPO activity over the lower ROS both lowers the concentration of H O and removes temperature range (58Cto258C). 2 2 species that can be converted to H O , the main rate-limiting 2 2 Effect of exogenous antioxidant (mannitol) factor for VBPO. Lowering the available concentration is likely to maintain the VBPO activity at low levels, restricting The effect of adding mannitol show clear trends in the 358C the formation of HOBr, and preventing, or constraining the data. Over Days 1 – 3, the mean VBPO activity recorded for extent of, pigment bleaching. Limiting VBPO activity would the 358C without mannitol samples is consistently higher also help prevent excessive depletion of organic acceptor than those treated with mannitol (Table 1). The 308Cdata resources, ensuring sufficient substrate is available to react however, shows little difference between the standard ......................................................................................................................................................................................................................................... 109 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 3. Temperature-induced increase in bromoperoxidase-specific activity. (a) Mean specific activities, based on IU per mg protein, at 358C are far higher than all other samples, with an almost exponential rise in activity across the 7 days of the exposure. The 308C mean specific activities also increase notably by Day 3 but the 99% confidence intervals suggest this difference is not statistically significant. However, at 358C, there is a considerable reduction in the amount of soluble protein extracted, giving rise to very high specific activities based on protein. (b) Mean specific activities, based on IU per gram fresh weight of Corallina,at 358C are higher than all other samples. The 308C mean specific activities also increase notably by day 3 over the values at lower temperatures, showing a several fold increase in BPO catalytic activity. By day 7, all specific activities are declining. with the HOBr, therefore allowing successful conversion to at 308C suggests that, at this temperature, the antioxidant 22, 23 halohydrocarbons. defence mechanisms of the Corallina are efficiently scaven- The addition of mannitol to the Corallina samples pro- ging and removing ROS produced within the cell, maintain- duced results that appeared strongly dependent upon the ing low H O concentrations and preventing induction of 2 2 sample temperature. In the 308C Corallina samples the pre- elevated VBPO activity. The large differences in VBPO sence of mannitol in the seawater produced no real visible activities at 358C would suggest that, in the absence of man- difference in VBPO activity across the 4 days, while in the nitol, the antioxidant defence systems of the Corallina are 358C samples the added mannitol showed a strong protective failing, ROS are accumulating, and excess H O combined 2 2 effect, with untreated sample mean values reaching much with an increased rate of VBPO results in pigment bleach- higher levels than the treated sample rates. The similarity ing. At 358C, a considerable rise in activity is still visible in in VBPO activities in the absence and presence of mannitol the mannitol treated samples, this would suggest that the ......................................................................................................................................................................................................................................... 110 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... concentration of mannitol used, 10 mM, while sufficient to scavenge considerable levels of ROS, was not enough to com- pletely quench the levels produced. The PE, PC and APC pigment concentrations show similar trends in the 358C mean pigment concentrations, with the mannitol treated samples maintaining consistently higher concentrations in comparison with the untreated samples. This loss of pigments is likely to be due to the inability of cells to control ROS concentrations, raising the VBPO activity and causing degradation and pigment loss. Conclusions Wider ecological effects of bleaching in coralline algae Crustose coralline algae (CCA), the Corallinacea, contribute the most widespread and abundant benthic marine organ- 9, 42 isms to be found in the photic zone. Many species of CCAs are known to fulfill important roles in coral reef eco- systems, both in cementing the reef structure and laying 7– 9 down substantial amounts of calcareous material, and also in inducing settlement of numerous benthic reef organ- 8 43 isms, including corals. Though CCAs contribute signifi- cantly to the biotic cover and structure of a reef ecosystem, their ecological influences and the effects of environmental and anthropogenic impacts are relatively understudied, with most of the investigative focus resting on the corals themselves. The role of CCAs in recruitment of new corals also plays an vital role in recovery and resilience of the reef, to both natural and anthropogenic disturbances. Therefore, if temperature can produce such significant bleaching effects within Corallina, further study into Figure 4. Proposed mechanisms of phycobilin bleaching in Corallina offi- thermal bleaching of other coralline algae may prove immen- cinalis.(a) Shows the elimination of hydrogen peroxide via the formation of sely important in the future conservation of coral reefs, as þ þ HOBr (or Br ) by bromoperoxidase (VBPO). The HOBr/Br reacts with an adverse effects on the CCAs within a reef will subsequently organic acceptor to produce a volatile halogenated organic compound affect the entire reef ecosystem. Although the species (VHOC). Bleaching of phycobilin pigments is a minor side reaction. (b) Shows the impact of temperature-induced VBPO synthesis. The increased studied here is not involved in reef building, other coralline formation of HOBr/Br overwhelms the capacity of cells to form VHOC algae such as Lithophyllum yessoense are. This species has and the bleaching of pigments becomes a major reaction, either by been shown to contain VBPO and to produce volatile hydro- HOBr/Br directly (1) or by singlet oxygen formed by the reaction of þ carbons such as bromoform that are known to be involved HOBr/Br with hydrogen peroxide (2). Bleaching prevents energy transfer in inducing settlement of larval stages of benthic organisms, to PSII. Solid lines represent major reactions; dotted lines represent minor reactions; wavy lines represent incident light. and also in inducing metamorphosis of sea anenomies. Table 1. Effect of mannitol on induction of bromoperoxidase in response to temperature Temperature 308C þ Mannitol 308C 2 Mannitol 358C þ Mannitol 358C 2 Mannitol Day Fold increase in specific activity (IU/g fresh weight Corallina)overDay 0 01 1 1 1 1 1 1.28 1.11 21.8 2 1 1.38 3.14 24.8 3 1 1.06 4.38 27.2 ......................................................................................................................................................................................................................................... 111 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 15. D’Agnolo E, Rizzo R, Paoletti S et al. 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In A Post, NR Baker, JB Boyer, eds. methane on metamorphosis of larvae of the sea urchins Photoinhibition of Photosynthesis: From Molecular Mechanisms to the Field Strongylocentrotus nudus and Strongylocentrotus intermedius. Aquaculture Oxford, UK: BIOS, pp. 129–142. 251: 549–557. ........................................................................................................................................................................................................................................ Submitted on 30 September 2007; accepted on 28 January 2008; advance access publication 22 April 2008 .........................................................................................................................................................................................................................................

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Bioscience HorizonsOxford University Press

Published: Jun 22, 2008

Keywords: Key words Corallina officinalis coral reef bromoperoxidase phycobilins bleaching temperature stress

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