View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Plymouth Electronic Archive and Research Library Annals of Botany doi 10.1093/aob/mcz042 A pinch of salt: Response of coastal grassland plants to simulated seawater inundation treatments Hanley ME*, Sanders SKD, Stanton H-M, Billington RA, Boden R School of Biological and Marine Sciences, University of Plymouth, Drake’s Circus, Plymouth PL4 8AA, United Kingdom. *Author for correspondence: Dr Mick Hanley Tel: +44 (0) 1752 584631 E-mail: mehanley@plymouth.ac.uk Orchid ID 0000-0002-3966-8919 Running Head: Plant responses to simulated seawater flooding treatments 1 Annals of Botany doi 10.1093/aob/mcz042 1 Summary 2  Background and Aims The combination of rising sea-levels and increased storm 3 frequency and intensity is predicted to increase the severity of oceanic storm surge 4 events and impact of flooding on coastal ecosystems globally. Understanding how 5 plant communities respond to this threat necessitates experiments involving plant 6 immersion in saline water, but logistical issues and natural variation in seawater 7 composition, mean that pure NaCl solutions or marine aquarium salts (MS) are widely 8 used. Nonetheless, their comparative impact on plant ecophysiology, and thus 9 relevance to understanding ‘real-world’ flooding scenarios, is unknown. 10  Methods In the first of two experiments, we examined how six ecophysiological 11 responses in white clover (Trifolium repens) varied when plants were subjected to 12 five different inundation treatments; i.e. deionised water, natural seawater, a MS 13 solution, and two NaCl solutions. In a second experiment, we examined how 14 immersion in deionised water, MS solution, and natural seawater affected six 15 European perennial herb species, three native to Spanish sand dunes, and three from 16 British coastal grasslands. 17  Results The two NaCl solutions induced exceptional Trifolium mortality, but 18 responses varied little between MS and seawater treatments. In experiment 2, although 19 leaf tissue necrosis and proline concentrations increased, and growth decreased 20 compared to untreated controls, only one response in one species varied between MS 21 and seawater treatments. Chemical speciation modelling revealed major variation in 22 free Na+ and Cl- between NaCl solutions and seawater, but minor differences between 23 MS and seawater. 2 Annals of Botany doi 10.1093/aob/mcz042 24  Conclusions We show that NaCl solutions are unsuitable surrogates to investigate 25 plant response to elevated environmental salinity. Although responses to natural 26 seawater and MS were consistent within species, there was notable between species 27 variation. Consequently, the first steps to elucidating how these species-specific 28 responses influence coastal plant community recovery following storm surge, can 29 likely be achieved using commercial marine aquarium salts as substitutes for natural 30 seawater. 31 32 33 Key Words – Coastal plants, Flooding; Instant Ocean, Ionic Stress; Osmotic Stress; 34 NaCl, Salinity, Sand dunes, Sea-level rise; Storm surge 35 3 Annals of Botany doi 10.1093/aob/mcz042 36 INTRODUCTION 37 The past, present, and likely future impacts of anthropogenic climate change (ACC) on 38 plant species and communities are widely reported and reasonably well understood 39 (Parmesan & Hanley, 2015). Most studies to date however, focus on the long-term, 40 chronic impacts of ACC (e.g. elevated CO2, variation in precipitation regimes, and 41 temperature increase), whereas much of the environmental threat is likely to stem from 42 stressors and disturbances linked to an increased frequency and intensity of acute, extreme 43 events (Rahmstorf & Coumou, 2011; Vasseur et al., 2014). Of these, coastal flooding 44 represents one of the most significant challenges; a combination of increased sea-surface 45 temperatures coupled with sea-level rise is predicted to increase the frequency and 46 severity of oceanic storm surges globally (Vousdoukas et al., 2016; Vitousek et al., 2017). 47 As a result, many low-lying coastal areas face an increased risk of seawater inundation 48 (Nicholls & Cazenave, 2010) with supra-littoral habitats such as sand dunes, upper salt 49 marshes, and grasslands likely subject to periodic seawater immersion for the first time 50 (Hoggart et al., 2014). Such habitats are both economically and ecologically important 51 since they provide a natural sea defence and important refuge for many species excluded 52 from intensive agriculture (Fisher et al., 2011; Duarte et al., 2013; Hanley et al., 2014). 53 Consequently, understanding the response of coastal vegetation to any increase in the 54 frequency and duration of seawater inundation is critical to ensure effective coastal 55 management (Hoggart et al., 2014; Hanley et al., 2014; Christie et al., 2018). 56 The impact of freshwater flooding on plants is well understood, but in addition to soil 57 anoxia and reduced access to atmospheric O2 and CO2 (Colmer & Voesenek, 2009; Perata 58 et al., 2011), seawater flooding imposes additional stresses. Most obviously, this is 4 Annals of Botany doi 10.1093/aob/mcz042 59 elevated salinity since seawater typically contains around 35 gL-1 (35 ‰) salt, of which 60 chloride and sodium contribute 19 gL-1 and 11 gL-1 respectively. Together, Na+ and Cl- 61 cause both osmotic (limiting the plant's ability to absorb water) and ionic (increased 62 toxicity) stresses, although for most species, Na+ seems to exert more obvious (certainly 63 better studied) toxic stress than Cl- and (Maathuis & Amtmann, 1999; Munns & Tester, 64 2008). As noted by Kronzucker et al., (2013), this stress is widely associated with a 65 detrimental shift in cytosolic K+/Na+ ratios and the disruption of cellular and whole-plant 66 potassium homeostasis by Na+. As a general response, plants synthesise and accumulate 67 stress metabolites (e.g. proline) and ions (i.e. K+) to exclude or compartmentalize Na+ and 68 Cl- and re-establish homeostatic function (Flowers & Colmer, 2008; Munns & Tester, 69 2008). Even if successfully achieved however, this likely imposes a cost on plant growth 70 and reproductive potential (Munns & Tester, 2008; White et al., 2014; Hanley et al., 71 2020) with concomitant implications for subsequent population and community-level 72 interactions. Understanding these ecophysiological and ecological responses to seawater 73 inundation is consequently, critical to understanding post-flooding community recovery, 74 assembly and function (Tolliver et al., 1997; Tate & Battaglia, 2013; Hoggart et al., 2014; 75 Lantz et al., 2015; Hanley et al., 2017). 76 Nonetheless, remarkably few studies have examined the response of coastal plant 77 communities and their constituent species to acute seawater flooding, likely due in part 78 to the difficulty in conducting realistic experiments. It is for example impossible to predict 79 exactly where and when storm surges will occur and extremely unlikely that any two 80 flooding events would be the same. As a result, our ability to examine the ‘before and 81 after’ impacts of real-world flood events in the field is extremely limited (Middleton, 82 2009; Lantz et al., 2015). Similarly, manipulative field studies where supra-littoral coastal 5 Annals of Botany doi 10.1093/aob/mcz042 83 vegetation is experimentally flooded with seawater are rare (Tate & Batiglia, 2013); 84 logistical and even ethical considerations are limiting. Even when achieved, most 85 deliberately flooded sites experience long-term inundation over natural tidal cycles 86 (Neubauer et al., 2013; Hopfensperger et al., 2014; Masselink et al., 2017), rather than 87 acute, short-duration inundation of the kind experienced in the aftermath of storms. The 88 lack of suitable field sites and scenarios necessitates a focus on controlled ‘flooding’ in 89 laboratory and greenhouse experiments using locally collected seawater (Camprubi et al., 90 2012; Hanley et al., 2013, 2017, White et al., 2014). This raises a further issue however 91 in that even if the ratio of the major elements remains ‘nearly constant’ (Levington, 2001), 92 there is marked seasonal and regional salinity variation in seawater (Dessier & Donguy, 93 1994; Donguy, 1994; Donguy & Meyers, 1996). 94 Given the most significant impact of short-duration seawater immersion on plant 95 metabolism and physiology seems to be associated with the effects of Na+ and Cl- 96 (Flowers & Colmer, 2008; Munns & Tester, 2008), the simplest experimental approach 97 would be to use a sodium chloride solution made up to typical seawater strength (i.e. 98 35‰) using deionised water. In addition to Cl- (± 55% of total chemical content) and Na+ 99 (± 31%) however, seawater also contains the major ions SO 2-4 (7.8%), Mg 2+ (3.7%), Ca2+ 100 (1.2%) and K+ (1.1%), and minor and trace elements (together less than 0.2%) including 101 bromine, carbon, strontium, boron, silicon, fluorine, nitrogen, phosphorous and iron 102 (Levington, 2001). The relative concentration of many of these other elements is much 103 more variable than Na+ and Cl- (Levington, 2001; Wheeler et al., 2016) and their impact 104 on plant metabolism and function less clear; some, e.g. K+, may have direct toxicological 105 or osmotic effects while also having the potential to mitigate or amplify the impact of 106 other elements (Flowers & Colmer, 2008). 6 Annals of Botany doi 10.1093/aob/mcz042 107 One possible solution is to use commercially available marine aquarium salt compounds, 108 which closely approximate typical inorganic chemical composition of seawater and offer 109 a relatively consistent ‘seawater’ surrogate (Flynn et al., 1995; Tolliver et al., 1997; 110 Mopper et al., 2016). Nonetheless, some chemical seawater constituents (e.g. nitrogen 111 and sulphur) are mobilised rapidly by biological processes and so their concentration is 112 spatially and temporally variable (Levington, 2001). Indeed, much of the solute content 113 of seawater is derived from organic matter (living and dead), highlighting the important 114 biological contribution to seawater chemistry (Levington, 2001). This biological 115 variability may impose additional impacts on terrestrial plant response to seawater 116 inundation beyond the chemical effects alone. 117 The aim of this study was to elucidate how the response of common coastal plant species 118 to simulated flooding varied according to the ‘seawater’ options available. Specifically, 119 we test the hypothesis that the most commonly applied simulated seawater treatments all 120 elicit similar plant physiological responses. In the first experiment we subjected white 121 clover (Trifolium repens) to immersion in 1: (deionised) water, 2: natural seawater, 3: 122 commercially available marine aquarium salt, 4: sodium chloride solution balanced to 123 average oceanic salinity (hereafter SalNaCl), and 5: sodium chloride solution balanced to 124 average ionic concentration of Instant Ocean (hereafter IonNaCl). We then examined 125 subsequent mortality, plant growth, flowering, and association with N-fixing bacteria to 126 determine whether each treatment resulted in similar, or varying plant responses. In 127 experiment 2, we subjected six different coastal plant species to immersion in 1: deionised 128 water, 2: natural seawater, and 3: aquarium salt solution, quantifying immediate post- 129 inundation proline accumulation, and subsequent longer-term leaf necrosis and growth as 130 measures of plant response. 7 Annals of Botany doi 10.1093/aob/mcz042 131 MATERIALS AND METHODS 132 Plant collection and cultivation 133 Native to Europe, North Africa and Asia, white clover (Trifolium repens L. Fabaceae) is 134 by virtue of its value as a nitrogen-fixing pasture crop, now globally distributed. In its 135 native range however, it is a common component of coastal plant communities such as 136 sand dunes, upper salt marshes, and grasslands (Grime et al., 2007). In June 2011 we 137 collected 12 large (± 100 mm diameter), branched plant fragments with multiple rooting 138 points from the upper section (700m from a seawall) of a grassland pasture at South 139 Efford Marsh near Aveton Gifford, Devon, England (50°18'14"N, 03°50'59"W). All 140 samples were taken from distinct patches separated by at least 5 m to reduce the likelihood 141 of collecting material from the same individual (Ab-Shukor et al., 1988). The plant 142 fragments were transplanted into 110 × 110 × 120 mm plastic pots containing John Innes 143 No. 2 potting compost and cultivated in a sheltered outdoor area. See White et al. (2014) 144 for full details. 145 In late summer 2016, we collected seeds of Centaurea nigra (Asteraceae), Lotus 146 corniculatus (Fabaceae), and Plantago lanceolata (Plantaginaceae) from coastal 147 grasslands located across southern England (Table 1). In late spring 2017 seeds of their 148 congeners Centaurea polycantha, Lotus creticus, and Plantago coronopus were collected 149 from sand dunes located near Zahara de los Atunes, Andalucía, Spain. Seeds of all species 150 were collected from mature inflorescences from a minimum of 30 maternal plants, and 151 after drying and cleaning, stored in airtight containers at room temperature until 152 germination. 8 Annals of Botany doi 10.1093/aob/mcz042 153 Experiment 1 154 In early December 2014 stolon fragments of white clover (approximately 10mm long and 155 with discernible roots) were cut from each of eight plants and used to cultivate 24 clones 156 from each parent. Initially planted into 50-mm diameter pots containing John Innes No. 157 2 compost and retained in an unheated greenhouse with natural illumination (mean daily 158 Max 21.8 ± 0.7 °C, Min 4.3 ± 0.3 °C), in March 2015, daughter rametes were transplanted 159 into 75 × 75 × 80 mm plastic pots containing John Innes No. 2 compost. Plants were 160 arranged randomly on trays with capillary matting (mean daily Max 32.4 ± 1.1 °C, Min 161 7.4 ± 0.3 °C), and watered twice weekly to pot capacity with tap water until the start of 162 the experiment. 163 Experimental Treatments 164 Class A volumetric glassware and glass-distilled deionised water (ddH2O) were used for 165 preparation of all treatments to ensure reproducibility. Approximately 30 L of seawater 166 was collected from Wembury, Devon, England (50°19'03"N, 04°05'03"W) in mid-March 167 and stored in large, sealed plastic containers outdoors in the dark for 74-d until use to 168 reduce the pool of labile dissolved organic carbon compounds present. Conductivity at 169 the time of use was 42.4 mS cm-1, and salinity 34.9 ‰. Aged seawater (hereafter SW) was 170 one of our five main treatment groups, along with a no-salt immersion treatment of ddH2O 171 (DW) and one using a commercially available marine aquarium salt (MS) ‘Instant Ocean®’ 172 (Aquarium Systems, Blacksburg, Virginia, USA). MS solutions using Instant Ocean have 173 been used in studies on plant response to both saltwater flooding (Tolliver et al., 1997; 174 Mopper et al., 2016) and increased soil salinity (Naumann et al., 2007, 2008), but its 9 Annals of Botany doi 10.1093/aob/mcz042 175 effects on plant growth and physiological responses have never been compared against 176 natural seawater. 177 We dissolved 33.3 gL-1 of Instant Ocean into deionised water to achieve a salinity of 178 35.1 ‰. The balance of major cations (Na+ K+, Ca2+ Mg2+, Sr2+) and anions (Cl−, SO 2-4 ) 179 in this MS approximates closely seawater salts, falling within 10 % of typical seawater 180 concentrations by mole for most of the major anions and cations, but has 5-fold higher 181 nitrate and 50-fold higher ammonium (Atkinson & Bingman, 1997). Many trace anions 182 (e.g. Cu2+, Co2+) are also present at low (μM) level, although these variations relate only 183 to total concentrations and do not take into account speciation, ion pair formation, or 184 actual bioaccessibility (Atkinson & Bingman, 1997). Different salts however, exert 185 variable ionic charges, such that saline solutions made up from different constituent salts 186 can have the same salinity but different ionic strength. Consequently, we prepared two 187 different sodium chloride solutions; one the same salinity as typical seawater (SalNaCl), 188 (Atkinson & Bingman 1998), the other the same ionic strength (IonNaCl), based on 189 Debye-Hückel theory (Debye & Hückel, 1923). We prepared 25.0 L of SalNaCl solution 190 using Trace Metals Grade (>99.99 %) sodium chloride (Sigma) in ddH2O using Class A 191 volumetric glassware (5-L) to a final salinity of 35 ‰. A similar volume of IonNaCl was 192 prepared with the same constituents, but assuming an average seawater ionic strength of 193 0.7 M (i.e. 38.7 g NaCl/L ddH2O). All ‘salt’ solutions, plus deionised water were stored 194 in sealed, dark plastic containers in the experimental greenhouse for two days prior to use 195 for temperature equilibration. 196 In early-June 2015, six established ramets were selected from each of the eight parent 197 ‘stock’ plants. Each ramet, uniform in size and appearance, was assigned at random to 198 one of the five treatment groups, or a no-immersion control treatment. In so doing, we 10 Annals of Botany doi 10.1093/aob/mcz042 199 ensured that each treatment group received genetically identical material. Although 200 seawater flooding following storms can persist for up to 96-hrs, a 24 h duration is typical 201 for low-lying UK coastline habitats following tidal-surge events (Environment Agency, 202 2014). By immersing to pot-level (in large plastic tubs) we simulated short-term soil 203 waterlogging; while we recognise that seawater inundation following storm-surge would 204 likely result in shoot submergence, we were able to separate the effect of ionic imbalance 205 in the root-zone rather than the impact of oxygen deficiency caused by full immersion 206 that our treatments would impose. 207 Immediately after immersion, pots were arranged randomly on a wire mesh-topped bench 208 inside the greenhouse; the wire mesh allowing free drainage and prevention of cross- 209 contamination between treatment groups. 48-hr after immersion, and thereafter every two 210 days for a further 90 d, the pots were watered to capacity (with rain water). Mean daily 211 greenhouse temperatures during this phase of the experiment were: 36.9 °C (± 0.8) max 212 and 13.2 °C (± 0.2) min. 213 Post-immersion plant response and recovery 214 Following immersion, one randomly selected shoot on each plant was marked at a 215 terminal node with loosely tied cotton thread (‘Stolon Growth’). This was used to quantify 216 subsequent stolon elongation 35-d post-immersion, when we also estimated the 217 proportion of above-ground necrotic tissue (‘Necrosis’). Mortality was checked daily 218 from the start to the end of the experiment 90-d post-immersion, when after counting the 219 number of fully matured inflorescences present, surviving plants were harvested (late 220 August 2015). Plants were cleaned of any adhering compost before roots and shoots were 221 separated and oven-dried at 50 °C for 24-hr. Total dry weight biomass (roots and shoots 11 Annals of Botany doi 10.1093/aob/mcz042 222 combined) attained during the period after immersion was taken as a measure of plant 223 growth. We also selected the longest root branch on each plant to quantify the number of 224 rhizobia nodules per unit root length. 225 The effects of ‘Immersion Treatment’ on ‘Necrosis’ and ‘Stolon Growth’ at 35-d-post 226 immersion and ‘Growth’, ‘Flowering Effort’ and ‘Nodules’ at harvest, were examined 227 using One-Way ANOVA; all data were Logit (ln(x+1)) transformed prior to analysis to 228 ensure heterogeneity of variance, and Tukey pairwise comparisons used to locate 229 differences between treatment means. 230 Experiment 2 231 In mid-June 2017, seeds of all six species were set to germinate in 225mm × 165mm × 232 50mm (covered) propagator trays containing John Innes seed compost. One week after 233 germination, 150 individual seedlings per species were transplanted into 50mm diameter 234 pots containing John Innes seed compost. All initial plant cultivation was conducted in a 235 controlled growth room set at 15°C and a 12-hour day/night illumination regime. When 236 the plants were 6 weeks old (early August), 150 individuals from each of the UK species 237 were transplanted into 70mm × 70mm × 80mm square pots containing John Innes seed 238 compost and moved to an elevated, outdoor ‘hard standing’ area on the University of 239 Plymouth campus. A similar procedure was used for the Spanish species, except that they 240 were transplanted into horticultural sand (Westland Horticulture Ltd, Dungannon, UK) to 241 better simulate sediment in their native sand dune habitat. 242 Experimental Treatments 243 In early-October 2017, 119 individual plants (checked for health and similar size) of each 244 species were allocated at random to one of three treatment groups (DW, MS or SW), 12 Annals of Botany doi 10.1093/aob/mcz042 245 subdivided into 24- or 96-hrs immersion times, such that there were 17 replicate plants 246 per treatment/immersion time combination, or a no-immersion control treatment,. 247 Seawater was collected from Plymouth Sound, Devon, England (50°19'03"N, 248 04°05'03"W) in October 2017; conductivity at the time of use was 41.6mS cm-1, and 249 salinity 34.0‰. The MS solution using Instant Ocean was prepared to a salinity of 34‰. 250 Immediately after immersion, pots were arranged randomly on a wire mesh-topped bench 251 inside a greenhouse. 252 Post-immersion proline accumulation 253 Seventy-two hours after immersion, five plants per treatment/immersion time group were 254 selected at random for proline analysis. From these, fully expanded, healthy leaves were 255 harvested and “flash-frozen” in liquid nitrogen before storage at -80°C. Proline analysis 256 was adapted from Shabnam et al., (2016). Briefly, c. 50 mg of leaves were ground in 40% 257 v/v EtOH at a ratio of 20µl/mg of leaf material in a cold pestle and mortar. The extract 258 was stored at 4°C overnight to allow extraction of proline before storage at -20°C. Proline 259 standards or extract (50µl) were heated with 100µl reaction mix (1.25% w/v ninhydrin in 260 glacial acetic acid) at 100 °C in a covered polypropylene 96 well plate for 30 minutes 261 before centrifugation of the plate at 1300 rpm for 2 minutes. The supernatant fluid was 262 transferred to clean plates and absorbances determined at 520 nm using an Omega 263 Fluostar platereader (BMG Labtech). 264 Post-immersion plant recovery 265 All remaining plants were cultivated for a further 100 d, with pots watered weekly to 266 capacity with rainwater. Mean daily greenhouse temperatures during this phase of the 13 Annals of Botany doi 10.1093/aob/mcz042 267 experiment were: 6.1°C (± 0.03) minimum and 18.9°C (± 0.06) maximum. At 28-d post- 268 immersion, we estimated the proportion of above-ground necrotic tissue (‘Necrosis’) 269 present on each plant. Mortality was checked daily until the end of the experiment (early 270 January 2018) when all surviving plants were harvested and processed as describe above 271 (Experiment 1). 272 The effects of ‘Immersion Treatment’ on ‘Proline’, ‘Necrosis’, and ‘Growth’ were 273 examined using One-Way ANOVA on each species; all data were ln(x+1) transformed 274 prior to analysis and Tukey pairwise comparisons used to locate differences between 275 treatment means. Due to the relatively large number of tests generated (i.e. six per 276 response, three responses), we adopted P < 0.01 to avoid Type I error. 277 Solute speciation modelling 278 Since the true levels of free ions, and ion pairs, in the solutions used here vary from the 279 amounts of solute added (based on formation of ion pairs and precipitating minerals), it 280 was necessary to model the chemical interactions within the solutions. In so doing, we 281 were able to understand how the actual ion concentrations affected plants, rather than 282 estimating effects from, e.g. total sodium added. The speciation of ions, ion pairs and 283 precipitates etc. was modelled using the MS composition given by Atkinson & Bingman 284 (1997) and the SW composition given by Nordstrom et al., (1979). The PHREEQC 285 Interactive 3.3.12 package (Parkhurst & Appelo, 1999) was used with the Lawrence 286 Livermore National Laboratory database (llnl.dat), which is based on the EQ3/6 model 287 of Wolery (1979). The model was run at 20 °C on the basis of 10 kg solution under test 288 with a headspace of 100,000 L of air comprising (% v/v) water vapour (1.00, since 289 experiments were conducted c. 1 km from the coast), carbon dioxide (0.04), oxygen 14 Annals of Botany doi 10.1093/aob/mcz042 290 (20.95), methane (0.00018), argon (0.93), neon (0.002), helium (0.0005), balanced with 291 nitrogen. Liquid and gas were at atmospheric pressure and the liquid was equilibrated 292 with the headspace mixture. 293 294 RESULTS 295 Experiment 1 296 Plant mortality was exceptionally high in the IonNaCl and SalNaCl treatment groups 297 where all except one individual in SalNaCl died within three weeks of immersion. By 298 contrast, no more than one plant died in any of the other treatment groups. As a result, 299 all further analysis focussed solely on the remaining DW, MS and SW treatment groups. 300 At 35-days post immersion, Trifolium repens exhibited increased necrosis following MS 301 or SW treatment (Fig 1), but DW had no effect (F3,27 = 12.08, P<0.001) compared to the 302 ‘no immersion’ control. Stolon elongation however, did not vary between treatment 303 groups (F3,27 = 2.52, P= 0.079). By the end of the experiment, plants in both the MS and 304 SW treatments were considerably smaller than those in untreated controls (F3,26 = 5.78, 305 P=0.004). Both ‘Flowering Effort’ (F3,26 = 2.43, P=0.087) and root colonisation by 306 rhizobia (F3,26 = 2.14, P=0.12) were unaffected by immersion treatment (Fig 1). Post- 307 hoc examination of treatment means showed no variation in plant necrosis or final 308 biomass between MS and SW treatments (Fig 1). 309 Experiment 2 310 No more than two plants of twelve in any of the species/treatment group combinations 311 died over the course of the experiment and we attempt no further analysis on mortality. 15 Annals of Botany doi 10.1093/aob/mcz042 312 The effects of immersion treatments on initial proline accumulation varied between 313 species and treatments (Fig 2). For the two Centaurea species, although DW96 had no 314 effect on leaf proline concentrations compared to untreated controls, the MS and SW 315 immersion treatments resulted in significant accumulation (C. nigra – F5,24 = 22.6, 316 P<0.001; C. polycantha – F6,28 = 4.45, P=0.003). The effect was however, more 317 marked for C. nigra, where 96-hrs MS and SW immersion yielded a 3- and 5-fold 318 respectively increase in proline concentrations (note the DW24 sample for this species 319 was lost prior to analysis). For C. polycantha, post-hoc analysis suggested that SW24 320 was the only treatment to stimulate significantly increased proline synthesis, even 321 though concentrations more than doubled in all MS and SW treatments compared to the 322 control. Lotus creticus (F6,28 = 5.43, P<0.001) exhibited a similar response to C. nigra, 323 i.e. higher proline levels in the longer MS and SW immersion treatments. Lotus 324 corniculatus however (F6,28 = 5.78, P<0.001), had significant increased proline 325 concentrations only in MS96 and SW96. Neither of Plantago lanceolata (F5,28 = 1.65, 326 P=0.163) or P. coronopus (F6,28 = 1.67, P=0.165) showed any variation in post- 327 immersion proline levels. Consistent for all species, we found no variation in proline 328 accumulation response between ‘time-equivalent’ MS or SW treatments. 329 At 28-days post immersion, all six species exhibited increased necrosis following MS or 330 SW treatment (Fig 3); DW had no effect. For Centaurea nigra (F6,77 = 13.01, P<0.001), 331 immersion in MS96 and both SW treatments increased leaf necrosis compared to the 332 control, while for C. polycantha (F6,77 = 17.47, P<0.001), all MS and SW treatments 333 elicited this effect. Lotus corniculatus (F6,77 = 20.68, P<0.001), was the only species 334 exhibiting significant variation between time-equivalent (i.e. 24-hr) MS and SW 335 treatments, where SW24 did not vary from untreated controls. Although unaffected at 16 Annals of Botany doi 10.1093/aob/mcz042 336 shorter durations, L. creticus (F6,77 = 4.59, P=0.001) displayed more necrosis in both 337 IO96 and SW96 treatments. Both Plantago species suffered increased necrosis 338 following MS and SW immersion; all treatments, except MS24, caused increased 339 necrosis in P. lanceolata (F6,77 = 7.97, P<0.001), while for P. coronopus (F6,77 = 5.27, 340 P<0.001), elevated tissue necrosis was common throughout. 341 Five of the six species exhibited reduced growth (final plant dry biomass) following MS 342 or SW treatment (Fig 4); DW had no effect. For both Centaurea nigra (F6,76 = 20.03, 343 P<0.001) and C. polycantha (F6,74 = 16.74, P<0.001), all MS and SW treatments 344 resulted in reduced size. Plantago coronopus (F6,75 = 6.10, P<0.001), exhibited a similar 345 response, while P. lanceolata (F6,77 = 5.02, P<0.001) plants in all MS and SW 346 treatments, except MS96, were smaller than controls. For the two Lotus species (L. 347 corniculatus - F6,77 = 6.95, P<0.001; L. creticus - F6,73 = 2.77, P=0.018) however, we 348 observed few treatment-specific effects; L. creticus did not achieve our P<0.01 349 criterion, while for L. corniculatus, post-hoc tests suggested that only plants in the 350 MS24 treatment were smaller than controls. Nonetheless, consistent for all six species, 351 there was no variation in final dry biomass between ‘time-equivalent’ MS or SW 352 treatments. 353 Solute speciation modelling 354 Modelling of MS composition compared with SW showed that overall available Na+ and 355 Cl- concentrations were broadly similar; i.e. Instant Ocean 430 mM and 488 mM, 356 respectively, SW 434 mM and 523 mM, respectively. For an NaCl solution that was 357 salinity-matched to MS (i.e. SalNaCl), concentrations of free Na+ and Cl- were 358 substantially higher (both 572 mM), with most of the c. 25 mMol per litre that was not 17 Annals of Botany doi 10.1093/aob/mcz042 359 present as free ions (since 596 mM total Na+ and Cl- added) found as the NaCl ion pair 360 in solution. In both SW and MS, free K+ was present at 6.3 mM and 9.0 mM, 361 respectively; a slight increase in the key ion used by plants to re-establish homeostatic 362 function after exposure to NaCl (Munns & Tester 2008). 363 364 DISCUSSION 365 Our study presents three major conclusions. First, exceptionally high Trifolium mortality 366 in the IonNaCl and SalNaCl treatments (experiment 1) shows that ‘pure’ NaCl solutions 367 are unsuitable surrogates to study the effect of seawater immersion on plant physiology. 368 Second, except one instance (necrosis in 24-hr treatments for Lotus corniculatus), all MS 369 vs SW comparisons suggest that a commercially available marine aquarium salt elicits 370 similar plant ecophysiological responses to natural seawater. Finally, all species 371 responded negatively to simulated seawater flooding (MS or SW treatments). 372 Although the greatest impact of seawater flooding on plant performance may stem from 373 the ionic and osmotic stress imposed by Na+ and Cl-, our results suggest that other 374 seawater constituents moderate these effects. From the methodological perspective, this 375 is important because a number of studies have attempted to mimic the impact of salt- 376 spray and/or sea water immersion using NaCl solutions applied directly onto the plant or 377 soil surface (Ab-Shakor et al., 1988; Sykes & Wilson, 1989; van Puijenbroek et al., 2017; 378 Varone et al., 2017). In so doing, these experiments fail to account for the likelihood that 379 the ionic and osmotic stresses they ascribed to elevated Na+ and Cl- are in fact, influenced 380 or moderated by other salts. One area for further investigation (specifically in comparison 381 with NaCl solutions) is to determine whether K+ in seawater (1.1% of total salt 18 Annals of Botany doi 10.1093/aob/mcz042 382 concentration) helps mitigate deleterious changes in cytosolic K+/Na+ ratios and 383 disruption of potassium homeostasis (Maathuis & Amtmann, 1999; Kronzucker et al., 384 2013). Similarly, changes in the cytoplasmic balance of Na+/SO 2-, Na+/Mg2+4 , and 385 Na+/Ca2+ ratios also have deleterious effects on plants grown in high salinity, effects 386 likely magnified when ‘pure’ NaCl solutions are used rather than seawater that naturally 387 contains these SO 2-, Mg2+4 , and Ca 2+ ions (Maas & Grattan, 1999; Maathuis & Amtmann, 388 1999; Shabala et al., 2005). Our Trifolium response data (experiment 1) certainly call into 389 question the biological relevance of the many studies that seek to assess crop plant 390 response to increased soil salinity using NaCl solutions (e.g. Dai et al., 2018; Flam- 391 Shepherd et al., 2018; Wu et al., 2018; Zhang et al., 2018). Salinized irrigation waters for 392 example, contain a range of cations and anions beyond Na+ and Cl- (Maas & Grattan, 393 1999) and our speciation modelling shows that a NaCl solution matched to average 394 seawater salinity contains considerably more free Na and Cl ions than seawater (i.e. an 395 increase of 32% and 9% in SalNaCl respectively). 396 Although commercial aquarium salts have been used to determine how salinity affects 397 coastal plants (Tolliver et al., 1997; Mopper et al., 2004; Naumann et al., 2008), these 398 studies have assumed, rather than demonstrated, that observed effects were compatible 399 with those produced by natural seawater. Our results suggest that this assumption may be 400 valid. In comparisons of six different biochemical, growth and reproductive responses 401 involving seven different plant species, we found only one significant difference between 402 time-equivalent SW and MS immersion treatments; i.e. above-ground tissues necrosis in 403 Lotus corniculatus was twice the amount in 24-hr MS immersion compared to 24-hr SW 404 plants. This necrosis response seems to have carried over into final plant biomass where 405 24-hr MS was the only treatment to display significantly reduced growth in comparison 19 Annals of Botany doi 10.1093/aob/mcz042 406 to the untreated control. The fact that these necrosis and biomass differences was not 407 apparent in the 96-hr treatments also suggests however, that any response is at best short- 408 lived and may even be a statistical artefact. The general consistency of observed 409 biological responses, corroborates our modelling of the compositions of MS and SW in 410 that concentrations of free Na+ (less than 1% difference) and Cl- (7% higher in SW) ions 411 are remarkably similar. In-fact given its role in counteracting cytoplasmic Na+ 412 accumulation, the (42%) higher K+ availability in MS might suggest that plants subjected 413 to MS rather than SW would recover better from simulated flooding. No plant response 414 observed in our experiments corroborated this suggestion however. 415 Although in experiment 2, all six species were affected negatively by (simulated) 416 seawater immersion for at least two of the responses examined, there were some 417 interesting patterns of response. First, and as might be expected, congenerics tended to 418 react in broadly similar ways. For example, while neither Plantago species showed any 419 variation in leaf proline concentrations, proline responses to all immersion treatments in 420 the two Lotus species were remarkably similar. In Centaurea, necrosis and final plant 421 biomass also showed very similar treatment-specific responses. More interesting than any 422 indication of phylogenetic conservation, was perhaps the general commonality of 423 response of congenerics grown in different media (i.e. English species in commercial 424 potting compost; Spanish species in horticultural sand). When coupled with the dramatic 425 response of Trifolium repens to SalNaCl and IonNaCl solutions in experiment 1, this 426 observation suggests that achieving a field-relevant salinity treatment, is a more important 427 methodological consideration than what growing media is used to cultivate plants. 428 Second, in terms of the overall lack of plant mortality, all species showed a remarkable 429 tolerance to up to 4 days simulated seawater flooding. Finally, the consistency of all other 20 Annals of Botany doi 10.1093/aob/mcz042 430 plant responses to MS and SW treatments nonetheless highlights the negative impact 431 seawater flooding exerts on coastal vegetation, underscoring growing concerns about the 432 predicted increase in the frequency and severity of oceanic storm surges on low-lying 433 coastal areas (Nicholls & Cazenave, 2010). 434 An important consideration here is that all experiments were performed on plants grown 435 in monoculture in greenhouse conditions, free from competition and environmental 436 stressors. Indeed, even in controlled greenhouse experiments, the responses of plants to 437 simulated seawater flooding in monoculture changed when the same species were grown 438 together (Hanley et al., 2017). Consequently, even apparently minor species-specific 439 differences in plant response to seawater inundation are likely to be magnified in sand 440 dunes, salt marshes, and other coastal habitats following actual flood events such that 441 species composition is modified after the event (see Engels & Jensen, 2010; Guo & 442 Pennings, 2012; Schile et al., 2017). For example, a study on long-term tundra recovery 443 following a major storm surge in the Canadian Arctic (Lantz et al., 2015) reported 444 species-specific variation in plant recovery; specifically, graminoids exhibiting greater 445 resilience than shrubs. This is important because any reduction in species diversity or loss 446 of key plant functional groups stemming from increased flood severity or frequency may 447 reduce community resilience to further perturbation. Ford et al., (2016) for example, 448 recently described how reductions in salt marsh diversity led to increased erosion 449 potential, particularly where sandy, low organic content soils predisposed these habitats 450 to sediment loss. The global importance of plant communities to coastal defence, at a time 451 when they also face increased flood risk (Duarte et al., 2013; Morris et al., 2018), gives 452 urgency to our need to better understand how acute seawater inundation affects 453 component species and ecosystem processes. Our inability to predict where and when 21 Annals of Botany doi 10.1093/aob/mcz042 454 flooding will happen, and difficulties associated with conducting manipulative 455 experiments on natural communities, means plant biologists may be constrained to work 456 in more highly controlled systems to achieve this aim. 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Calculation of chemical equilibrium between aqueous solutions and 620 minerals - The EQ3/6 software package. Lawrence Livermore National 621 Laboratory Report UCRL-52658. Livermore, California, USA. 622 Wu H, Shabala L, Azzarello E, et al., 2018. Na+ extrusion from the cytosol and tissue- 623 specific Na+ sequestration in roots confer differential salt stress tolerance between 624 durum and bread wheat. Journal of Experimental Botany 69: 3987–4001 625 Zhang M, Cao Y, Wang Z, et al., 2018. A retrotransposon in an HKT1 family sodium 626 transporter causes variation of leaf Na+ exclusion and salt tolerance in maize. New 627 Phytologist 217: 1161-1176. 628 629 630 27 Annals of Botany doi 10.1093/aob/mcz042 631 Table 1. Details of seed collection sites for six coastal dune and grassland species from 632 SW Spain and southern England used to compare plant performance following 633 simulated seawater flooding treatments. 634 Region Species Site name Lat:Long Centaurea nigra L. Saltash, Cornwall 50°23`37``N 04°13`40``W Southern Lotus corniculatus L. Wembury, Devon 50°18`59``N 04°06`14``W England Plantago lanceolata L. Sandwich, Kent 51°16`48``N 01°21`42``E Centaurea polyacantha Willd. Atlanterra, Cadiz 36°05`39``N 05°48`44``W South West Lotus creticus L. Zahara, Cadiz 36°08`15``N 05°51`01``W Spain Plantago coronopus L. Zahara, Cadiz 36°07`35``N 05°50`23``W 635 636 28 Annals of Botany doi 10.1093/aob/mcz042 637 Figures 638 639 Figure 1. Responses of Trifolium repens to simulated seawater flooding (MS – a marine 640 aquarium salt solution (‘Instant Ocean®’); SW – natural seawater) compared with 641 immersion in deionised water (DW) or untreated controls. Panels show effects on; 642 above-ground tissue necrosis and stolon extension at 28-d post immersion, and final 643 plant dry weight biomass, inflorescence number, and root colonisation by Rhizobia at 644 90-d-post immersion. 29 Annals of Botany doi 10.1093/aob/mcz042 645 646 Figure 2. The effect of simulated seawater (marine aquarium salt solution ‘Instant Ocean®’ and natural ’Seawater’) and freshwater 647 (‘Deionised’) flooding on mean (±SE) leaf proline concentrations for six European coastal grassland species 3-d after root-zone immersion. 30 Annals of Botany doi 10.1093/aob/mcz042 648 649 Figure 3. The effect of simulated seawater (marine aquarium salt solution ‘Instant Ocean®’ and natural ’Seawater’) and freshwater 650 (‘Deionised’) flooding on mean (±SE) above-ground tissue necrosis for six European coastal grassland species 35-d after root-zone 651 immersion. 31 Annals of Botany doi 10.1093/aob/mcz042 652 653 Figure 4. The effect of simulated seawater (marine aquarium salt solution ‘Instant Ocean®’ and natural ’Seawater’) and freshwater 654 (‘Deionised’) flooding on mean (±SE) total plant dry weight biomass for six European coastal grassland species 100-d after root-zone 655 immersion. 32