Inès Sahraoui

Melliti Ben

Garali Sondes

Chakroun Zoubaida

Catherine Gonzalez

Olivier Pringault

Sakka Hlaili

Monoculture and co-culture tests of the toxicity of four typical 1 herbicides on growth, photosynthesis 2 and oxidative stress responses of the marine diatoms 3 Pseudo-nitzschia mannii and Chaetoceros decipiens 4 5

Keywords: diatom, herbicide, growth, photosynthesis, antioxidant enzyme activity. 55

The toxicity of four herbicides in mixture (alachlor, diuron, des-isopropyl-atrazine and 31 simazine) on the growth and the photosynthesis parameters of two marine diatoms Pseudo-32 niszchia mannii and Chaetoceros decipiens have been investigated for 9 days in monoculture 33 and co-culture tests. The catalase (CAT) and guaiacol peroxidase (GPX) were also monitored 34 to assess the oxidative stress response. In single-species assays, while both species displayed 35 no affected instantaneous growth rate by herbicides, their physiological responses were 36 different. Chl a content of P. mannii significantly decreased upon herbicide exposure, due 37 probably to pigment destruction or inhibition of their synthesis. This decrease was associated 38 with a reduction in the chlorophyll fluorescence parameters (ABS0/RC, TR0/RC, ET0/RC and 39 DI0/RC). In contrast, C. decipiens maintained an effective photosynthetic performance under 40 herbicide exposure, as Chl a per cell content and the specific energy fluxes per reaction center 41 remained unchanged relative to control values. GPX activity was significantly higher in 42 contaminated P. mannii and C. decipiens monocultures than in controls at early herbicide 43 exposure (1 day), whereas a significant induction of CAT activity occurred later (from day 3 44 for C. decipiens and at day 9 for P. mannii) in response to herbicides. In control co-culture, P.

45 mannii was eliminated by C. decipiens. As observed in the monoculture, the herbicides did not 46 affect the photosynthetic performance of C. decipiens in co-culture, but significantly reduced 47 its instantaneous growth rate. The oxidative stress response in co-culture has similar trends to 48 that of C. decipiens in monoculture, but the interspecies competition likely resulted in higher 49 CAT activity under herbicide exposure. Results of this study suggest that herbicide toxicity for 50 marine diatoms might be amplified by interspecies interactions in natural communities, which 51 might lead to different physiological and growth responses.

Introduction 56

The expansion and intensification of agricultural activities over the last few decades has 57 led to a substantial increase in herbicide use [START_REF] Mhadhbi | Evaluating polar 681 pesticide pollution with a combined approach: a survey of agricultural practices and POCIS 682 passive samplers in a Tunisian lagoon watershed[END_REF]de Suza et al. 2020). These 58 toxicants are not readily adsorbed by soil [START_REF] Moncada | Environmental fate of diuron Environmental Monitoring Branch Department of 689 Pesticide Regulation[END_REF] and therefore they can reach rivers, 59 lakes and then marine waters, through spraying, leaching and runoff [START_REF] Carter | How pesticides get into water -And proposed reduction measures[END_REF]Ramezani, 60 2008). Some herbicides, belonging to triazines and phenylureas are forbidden or restricted in 61 several Mediterranean countries (e.g. atrazine, 2004/141/CE ; simazine 2004/247/CE and 62 diuron, 2007/417/CE) but they are still found in aquatic ecosystems [START_REF] Nödler | Polarorganic micropollutants in the coastal environment of 705 different marine systems[END_REF] 63 [START_REF] Bancon-Montigny | Seasonal changes of chemical contamination in coastal waters during 507 sediment resuspension[END_REF]. Herbicides are persistent pollutants in seawater, since they are 64 weakly adsorbed by marine sediment [START_REF] Thomas | Antifouling paint booster biocides in UK coastal waters: 783 Inputs occurrence and environmental fate[END_REF][START_REF] Tissier | Les substances 792 prioritaires de la Directive cadre sur l'eau (DCE) Fiches de synthèse Rapport IFREMER[END_REF]. Moreover, their 65 degradation is limited and can potentially produce more toxic and/or persistent secondary 66 metabolites (Ortiz-Hernández et al. 2013). Therefore, herbicides constitute a potential threat for 67 marine ecosystems and their phytotoxicity has already been demonstrated on several 68 phototrophic organisms, including phytoplankton (e.g. glyphosate, [START_REF] Smedbol | Phytoplankton growth and PSII 766 efficiency sensitivity to a glyphosate-based herbicide (Factor 540 ® )[END_REF]Lam 69 et al. 2020;triazine, Yang et al. 2019), microphytobenthic biofilms (e.g. diuron and atrazine, 70 [START_REF] Legrand | Inhibition of microphytobenthic 630 photosynthesis by the herbicides atrazine and diuron[END_REF]glyphosate, Lima et al. 2014) and macrophytes (e.g. norflurazon and 71 fluridone, [START_REF] Chalifour | Combined effect of temperature and bleaching 537 herbicides on photosynthesis pigment and fatty acid composition of Chlamydomonas reinhardtii[END_REF]paraquat, Moustakas et al. 2016;glyphosate, Mudge and Houlahan, 72 2019). Most herbicides, including triazines and phenylureas, are photosynthetic inhibitors.

73

Their active molecules bind to the D1 protein that blocks electron transport from photosystem 74 II [START_REF] Schuler | Aquatic risk assessment of herbicides in freshwater ecosystems of South 758 Florida[END_REF], thus inhibiting Hill's reaction [START_REF] Leboulanger | Effect of atrazine and nicosulfuron on 622 freshwater microalgae[END_REF]Dorigo et 75 al. 2004;[START_REF] Macedo | Effects of the herbicide bentazon on growth 646 and photosystem II maximum quantum yield of the marine diatom Skeletonema costatum[END_REF]. This further provokes oxidative stress with reactive oxidative 76 species (ROS) formation and accumulation leading to the damage of pigments, proteins, and 77 lipids of the photosynthetic apparatus [START_REF] Gomes | Temperature and Light Modulation of Herbicide Toxicity on Algal and 576 Cyanobacterial Physiology[END_REF]. Other herbicides belonging to chloroacetamide family, such as alachlor and metalachlor, inhibit the biosynthesis of amino-79 acids and long chain fatty acids (Boger et al. 2003;[START_REF] Yang | The action of 804 herbicides on fatty acid biosynthesis and elongation in barley and cucumber[END_REF][START_REF] Busi | Resistance to herbicides inhibiting the biosynthesis of Very Long Chain Fatty Acids[END_REF].

80

Several studies have assessed the impact of herbicides on benthic and planktonic 81 microalgae, particularly on diatoms. These silicified microalgae are ubiquitous photosynthetic 82 organisms in marine and fresh waters that account for 20% of global primary production and 83 they are the major contributors to sedimentary organic matter [START_REF] Nelson | Production and dissolution 698 of biogenicsilica in the ocean: revised global estimates comparison withregional data and 699 relationship to biogenic sedimentation[END_REF]Thornton, 84 2014). Diatoms are at the base of marine food webs, particularly the herbivorous pathway 85 (Sakka Halili et al. 2014;[START_REF] Meddeb | A new type of plankton food web functioning in coastal waters revealed by coupling 667 Monte Carlo Markov Chain Linear Inverse method and Ecological Network Analysis[END_REF]) and thus the effects of toxicants on these primary 86 producers can be cascaded to higher trophic levels consequently impacting the function of the 87 whole ecosystem (Relyea, 2009;[START_REF] Pesce | Effects of organic herbicides on phototrophic microbial 721 communities in freshwater ecosystems[END_REF][START_REF] Hashimoto | Effects of a herbicide on paddy predatory insects depend on their microhabitat use and an 585 insecticide application[END_REF]. Diatoms quickly 88 respond to environmental pollutants and hence they are often used as efficient bioindicators to 89 assess the ecological status of aquatic ecosystem [START_REF] Stevenson | Assessing environmental conditions in rivers and streams with diatoms 772 The Diatoms: Applications for the Environmental and Earth Sciences Second Edition 773[END_REF][START_REF] Berthon | Using diatom life-forms and ecological guilds to assess 528 organic pollution and trophic level in rivers: a case study of rivers in south-eastern France[END_REF]; 90 [START_REF] Pandey | The use of diatoms in ecotoxicology and bioassessment: Insights advances and 711 challenges[END_REF][START_REF] Pandey | Evaluating features of periphytic 713 diatom communities as biomonitoring tools in fresh brackish and marine waters[END_REF]. Exposure to herbicides at concentrations ranging from µg 91 L -1 to mg L -1 , was often followed by either significant decrease in Chl a, growth rate, 92 photosynthetic efficiency or lipid content for several marine diatoms (Schmitt-Jansen and 93 Altenburger, 2005;[START_REF] Gatidou | Evaluation of single and joint toxic effects of two antifouling 573 biocides their main metabolites and copper using phytoplankton bioassays[END_REF][START_REF] Bao | Acute toxicities of five commonly used 510 antifouling booster biocides to selected subtropical and cosmopolitan marine species[END_REF][START_REF] Ebenezer | Physiological and biochemical responses of the marine dinoflagellate 567 Prorocentrum minimum exposed to the oxidizing biocide chlorine[END_REF] 94 [START_REF] Coquillé | The influence of natural dissolved organic matter on herbicide 546 toxicity to marine microalgae is species dependent[END_REF]. Deformation of diatom frustules and DNA alteration can be also often 95 observed (Schmitt-Jansen and Altenburger, 2005;[START_REF] Debenest | Herbicide effects on freshwater benthic 552 diatoms: Induction of nucleus alterations and silica cell wall abnormalities[END_REF][START_REF] Roubeix | Impact of the herbicide metolachlor on 735 river periphytic diatoms: Experimental comparison of descriptors at different biological 736 organization levels[END_REF].

96

However, it is possible to observe no adverse effects on diatoms by herbicides. For example, 97 [START_REF] Debenest | Effects of pesticides on freshwater diatoms[END_REF] have not detected any impact of atrazine (25 µg l -1 ) on a freshwater 98 diatom community biomass and [START_REF] Hourmant | Effect of Bentazon on 590 Growth and Physiological Responses of Marine Diatom: Chaetoceros gracilis[END_REF] even found increase in pigment content, 99 photosynthetic rate, and ATP synthesis for Chaetoceros gracilis exposed to bentazon with 100 concentration of 0 to 100 µg L -1 . In fact, there is a considerable variability in diatom species 101 sensitivity to herbicides [START_REF] Larras | Linking Diatom Sensitivity to Herbicides 619 to Phylogeny: A Step Forward for Biomonitoring?[END_REF], even towards the same herbicide (Larras et al. 102

2012).

Most of the data on the sensitivity of diatoms to herbicides are provided from single-104 species bioassays. These allow a wide assessment of herbicide impacts at the molecular, cellular 105 and population levels [START_REF] Wood | How benthic diatoms within natural communities 798 respond to eight common herbicides with different modes of action[END_REF][START_REF] Mohr | Defense mechanisms in phytoplankton: traits and trade-offs Technical University of 687 Denmark 99[END_REF][START_REF] Chamsi | 540 Effects of herbicide mixtures on freshwater microalgae with the potential effect of a safener[END_REF][START_REF] Demailly | Impact of diuron and S-metolachlor on the freshwater 560 diatom Gomphonema gracile: Complementarity between fatty acid profiles and different kinds of 561 ecotoxicological impact-endpoints[END_REF].

106

However, there are insufficient conclusive data in view of the effects of herbicides at the 107 community level. In natural communities, interactions between coexisting species may alter 108 their ability to respond to perturbations. Indeed, it has been shown that diatoms respond 109 differently to environmental stress depending on whether they are tested in laboratory 110 monocultures, mixtures or in natural communities [START_REF] Bérard | Tolerance of Oscillatoria limnetica Lemmermann to atrazine 521 in natural phytoplankton populations and in pure culture: influence of season and temperature[END_REF][START_REF] Schabhüttl | Temperature and species 752 richness effects in phytoplankton communities[END_REF] 111 [START_REF] Koedooder | 604 Diatom-Bacteria Interactions Modulate the Composition and Productivity of Benthic Diatom 605[END_REF].

112

The present study aimed to assess the toxicity of four herbicides (diuron, des-isopropyl-113 atrazine (DIA), alachlor and simazine) in mixture on two ecologically important marine 114 diatoms: the pennate species Pseudo-nizschia mannii and the centric species Chaetoceros 115 decipiens. The first species, known as a neurotoxic producer, was recently reported in 116 Mediterranean waters [START_REF] Hlaili | Toxic and potentially toxic diatom 749 blooms in Tunisian (SW Mediterranean) waters: Review of ten years of investigations[END_REF]) and the second is a potentially noxious diatom 117 that can form intense blooms [START_REF] Smayda | Autecology of bloom forming microalgae: Extrapolation of laboratory results to 763 field populations and the Redfield -Braarud debate revisited p 215-270[END_REF][START_REF] López-Cortés | Mass fish die-off during a diatom bloom in the Bahía de La Paz Gulf of California[END_REF]. These diatoms were Nonaxenic cultures were grown in f/2 medium [START_REF] Guillard | Studies on Marine Planktonic Diatoms I Cyclotella nana Hustedt and 578 Detonula confervacea (Cleve) Gran[END_REF] Then, in a second step, transmission electron microscopy was used to identify the two diatoms 144 at the species level. For this, 20 mL subsamples of cultures were filtered then washed with 145 250 mL of distilled water through a 3 μm pore size polytetrafluoroethylene (PTFE) membrane 146 using a Millipore vacuum filtration apparatus. The diatom frustules were then re-suspended in 147 5 mL distilled water and cleaned by adding 10 mL each of concentrated sulfuric and nitric acid 148 in a boiling water bath for 60 min. Frustules were then washed once more with 250 mL of 149 distilled water in the filtration apparatus and re-suspended in a final volume of 5 mL. For 150 electron microscopy examination, one drop of cleaned sample was diluted 20:1 (for cultures) 151 and single drops were dried onto 400-mesh copper grids bearing pure carbon support films, and 152 examined using a Tecnai G2 TEM operating at 60 kV, at the Electron Microscopy Unit (Faculty of Sciences of Bizerte, Tunisia). The species Pseudo-nitzschia mannii and Chaetoceros 154 decipiens Cleve were identified following identification keys of [START_REF] Amato | Morphology phylogeny and sexual cycle of Pseudo-nitzschia 500 mannii sp nov (Bacillariophyceae): a pseudo-cryptic species within the P 501 pseudodelicatissima complex[END_REF] 155 and of [START_REF] Lee | Critical criteria for identification of the genus Chaetoceros 625 (Bacillariophyta) based on setae ultrastructure II Subgenus Hyalochaete[END_REF] containing 1000 mL of F/2 medium and inoculated with diatom strain cultures in the 166 exponential growth phase. Cultures were set at the initial cell densities of 5 x 10 3 mL -1 for P. 167 mannii and 5 x 10 2 cells mL -1 for C. decipiens, respecting the in situ concentrations during 168 bloom periods [START_REF] Sahraoui | Blooms of the diatom 738 genus Pseudo-nitzschia H Peragallo in Bizerte lagoon (Tunisia SW Mediterranean)[END_REF]2012). For each species in monoculture or co-culture, two

174

All treatments (in mono-and co-cultures) were incubated for 9 days in the temperature-175 controlled chamber under the same light and temperature conditions described above.

176

Subsamples were taken from all treatments at six time points (t= 0, 1, 2, 3, 6 and 9 days) for 177 growth, physiological, photochemical and biochemical analyses.

Chlorophyll a 180

For chlorophyll a (Chl a), 10 ml samples were filtered through Whatman GF/F filters.

181

Pigment concentrations were determined using the standard spectrophotometric method 182 [START_REF] Parsons | A Manual of Chemical and Biological Methods for Seawater 716 Analysis[END_REF], following extraction with 10 ml 90% acetone overnight at 4°C in the dark. Samples (100 ml) were collected by centrifugation at 6000 g for 30 min at 4°C and then 203 broken down in the phosphate buffer (pH = 7.0) by sonication. Samples were observed under a 204 microscope to make sure the breaking ratio was more than 95%. Finally, the sonicated solution 205 was centrifuged again at 6000 g for 30 min at 4°C and the supernatants were collected to 206 measure the antioxidant enzyme activities.

207

Catalase activity was determined according to the method of Beers and Sizer (1952) by 208 following the H2O2 disappearance kinetic for 5 minutes at 240 nm. The reaction medium 209 contained 50 mM phosphate buffer at pH 7.5 and 10 mM H2O2. The extinction coefficient was 210 0.036 mM -1 cm -1 and the reaction was initiated by the addition of the protein extract.

211

The guaiacol peroxidase (GPX) activity was determined according to the method of 212 [START_REF] Jiang | Selective oxidation of enzyme extracts for improved quantification of 593 peroxidase activity[END_REF] based on the increase in the absorbance at 470 nm due to the 213 polymerization of guaiacol to tetragaiacol in the presence of hydrogen peroxide for 5 minutes.

214

The reaction medium contained 0.1 M sodium phosphate at pH 6.5, 0.135 mM H2O2 and 3.6 215 mM guaiacol. The molar extinction coefficient of tetragaiacol was 26.6 mM -1 cm -1 . The 216 reaction was initiated by the addition of the protein extract.

230

The statistical analyses were performed with the software XLSTAT 18.

231

RESULTS

232

Response of diatom growth 233

Instantaneous growth rate 234

At day 1, the instantaneous growth rate of P. mannii monoculture was about ~1.10 d -1 for 235 the control and the herbicide treatments (Fig. 1a). Values then increased to about 1.25 d -1 at day 236 2 and then decreased to ~ 0.39 d -1 at day 9 for both treatments. The herbicide mixture had no 237 significant effect on the growth of P. mannii maintained in monoculture (two-way ANOVA, P 238 > 0.05, Fig. 1a). However, the co-culture had a negative effect on the growth of P. mannii, 239 which displayed constantly negative values over the experiment for the control treatment (-0.35 240 to -0.07 d -1 ) (two-way ANOVA, P < 0.05, Fig. 1a). Negative growth rates were also registered 241 for P. mannii contaminated co-culture, with similar values to those of the control co-culture.

242

The herbicide mixture did not affect the growth of C. decipiens in monoculture (two-way 243 ANOVA, P > 0.05, Fig. 1b) since comparable instantaneous growth rate values were registered 244 between the control and the herbicide treatment (from ~0.5 at day 1 to ~0.2 at day 9). In control 245 co-culture, C. decipiens maintained similar growth rates to those of the control monoculture 246 (two-way ANOVA, P > 0.05, Fig. 1b). Conversely, the herbicide mixture has significantly 247 impacted C. decipiens when the species was growing in co-culture (two-way ANOVA, P < 248 0.05, Fig. 1b) and its instantaneous growth rate values were reduced by 35.41 %, 41.39 % and 249 61.11 % compared to the control co-cultures, at days 3, 6 and 9, respectively.

250

Chlorophyll a content

Under monoculture conditions, the Chl a per cell content of P. mannii (initially of 252 9.34 ± 0.4 pg cell -1 ) increased significantly in the control to reach a maximum on day 3 253 (18.77 ± 5.75 pg cell -1 ) and then decreased sharply to attain 2.09 ± 0.01 pg cell -1 at the end of 254 the experiment (Fig. 2a). The herbicide mixture induced a significant reduction in Chl a cell For the diatom co-culture under control conditions, the initial Chl a per cell content (9.86 261 ± 0.8 pg cell -1 ) slightly diminished at the beginning (until day 3) to increase thereafter 262 significantly by 2 to 3-fold compared to the initial level (Fig. 2c). The herbicide mixture induced 263 a significant increase in Chl a cell content compared to the control (Student's t-test, P < 0.05) 264 with the most important rise at day 3 (increase by 8 folds compared to the control) (Fig. 2c).

265

Physiological response of diatoms 266 267

Photosynthetic parameters 268

Whether for the diatom monocultures or co-culture, the maximum quantum yield of PSII 269 (Fv/Fm) did not show any significant difference between the control and herbicide treatment 270 (Student's t-test, P > 0.05, Fig. 3a). This parameter declined from the beginning (~ 0.7 -0.8 271 a.u.) to the end of the experiment, reaching values of -20 to -30 % relative to the initial level 272 (Fig. 3a).

273

For P. mannii in monoculture, the herbicide mixture induced a significant sharp decrease 274 of ABS0/RC (Student's t-test, P < 0.05, Fig. 3b). This reduction was noticeable from day 1, 275 such that values of this parameter (in the range of 1.38-1.98 a.u. for control) approached the 276 zero at day 2 (-98.01 % relative to control) Values recovered to control levels at day 3 to decrease again to approximately -50 % relative to the control at day 9. This ratio was unchanged 278 by the herbicide mixture in the monoculture of C. decipiens (Student's t-test, P > 0.05, Fig. 3b) 279 with values ranging in general between 2 and 3 a.u. For the diatom co-culture, there was also 280 no significant effect of the herbicide mixture on ABS0/RC (Student's t-test, P > 0.05, Fig 3b)

281
and values were often in the range of 2 to 3 a.u.

282

Likewise, the ratios of trapping of excitation energy per reaction centre (TR0/RC) and 283 electron transport per active reaction centre (ET0/RC) were significantly reduced by herbicide 284 for P. mannii monoculture (Student's t-test, P < 0.05, Fig. 3c,d) as both parameters decreased 285 at day 2 by 80% and 89 % relative to control, respectively. Conversely, TR0/RC and ET0/RC 286 were unchanged by the herbicide mixture in C. decipiens monoculture (Student's t-test, P > 287 0.05) with values mostly of about 1.5-1.8 a.u. and 1.12-2.1 a.u., respectively (Fig. 3c,3d). For 288 the diatom co-culture, there was no significant effect of the herbicide mixture on both ratios 289 (Student's t-test, p > 0.05, Fig. 3c,d).

290

Herbicide treatment significantly impacted the dissipated energy flux per active reaction 291 center (DI0/RC) for P. mannii monoculture (Student's t-test, P < 0.05, Fig. 3e), causing 33-292 98% reduction relative to control. Conversely, no significant effect was observed for this 293 physiological parameter for the C. decipiens monoculture (range of values: 0.78-1.31 a.u.) nor 294 for the diatom co-culture (range of values: 0.74-1.18 a.u.) (Student's t-test, P > 0.05, Fig. 3e).

295

Antioxidant enzyme activity 296

For the P. mannii monoculture, the catalase activity was relatively low in the control 297 (range of values: 0.42 -1.05 U mg -1 Protein, Fig 4a). The herbicide mixture did not affect the 298 enzyme activity (Student's t-test, P > 0.05) except at the end of the experiment where a great 299 increase was observed (roughly 20 times the control value, Fig. 4a). In the C decipiens control 300 monoculture the catalase activity was significantly greater than that of P. mannii control 301 monoculture (range of values: 7.14-10.52 U mg -1 Protein) (Student's t-test, P < 0.05). Exposure Fig. 4b) and values increased by 3-5 folds relative to control after 3-days treatment. The same 304 response to herbicides was observed for the diatom co-culture (Student's t-test, P < 0.05, 305 Fig. 4c) as catalase activity increased by 4-7 folds in comparison to control after 3-days 306 treatment.

307

Overall, the GPX activity, measured in monocultures and co-culture, was not affected by 308 the herbicide mixture addition (Student's t-test, P > 0.05, Fig. 4d,e,f) except on day 1, when 309 the enzyme showed 2-3 folds higher activity in herbicide treatment. The GPX activity values 310 generally varied within the range of 1.83-22.41 U mg -1 Protein, for all type of cultures (Fig. 4d, 311 e, f). [START_REF] Demailly | Impact of diuron and S-metolachlor on the freshwater 560 diatom Gomphonema gracile: Complementarity between fatty acid profiles and different kinds of 561 ecotoxicological impact-endpoints[END_REF]. All these authors have used herbicides at higher 323 concentrations than used in this study (∑ herbicides ~ 5 µg L -1 ), which could explain the lack 324 of herbicide impact on the growth of P. mannii and C. decipiens monocultures. However, other 325 studies have reported positive impact of herbicides on diatom growth (50 µg L -1 atrazine, 326 [START_REF] Kalopesa | Atrazine effects on growth of the diatom 599 Rhizosolenia setigera (Ehrenberg) brightwell[END_REF]; 30 µg L -1 isoproturon, [START_REF] Debenest | Sensitivity of 555 freshwater periphytic diatoms to agricultural herbicides[END_REF]; and 7 μg L -1 diuron, [START_REF] Ricart | Effects of low concentrations of the phenylurea herbicide diuron on biofilm 733 algae and bacteria[END_REF]). In our experiment, P. mannii and C decipiens, growing as monocultures, seemed 328 to tolerate the herbicide mixture at the tested concentration. Both species originated from 329 Bizerte Lagoon, where noticeable contamination by herbicides has been reported for waters and 330 sediment [START_REF] Pringault | Consequences of contaminant mixture on the dynamics and functional diversity of 726 bacterioplankton in a southwestern Mediterranean coastal ecosystem[END_REF][START_REF] Bancon-Montigny | Seasonal changes of chemical contamination in coastal waters during 507 sediment resuspension[END_REF][START_REF] Mhadhbi | Evaluating polar 681 pesticide pollution with a combined approach: a survey of agricultural practices and POCIS 682 passive samplers in a Tunisian lagoon watershed[END_REF]). The 331 ecosystem has long been under the influence of water runoff from adjacent land areas used for 332 agricultural crops cultivation with intensive use of herbicides [START_REF] Salem | First Survey of Agricultural Pesticides Used for 515 Crops in Ichkeul Lake-Bizerte Lagoon Watershed (Tunisia)[END_REF].

333

Consequently, it is likely that P. mannii and C. decipiens isolated from this contaminated 334 ecosystem might have developed tolerance to herbicides through adaptation mechanisms that 335 make them physiologically able to resist to these pollutants.

336

In monoculture, the Chl a per cell content of P. mannii was significantly affected by Herb 337 treatment (P < 0.05, Fig. 2a), although growth was not affected (Fig. 1a). This suggests that the 2b), suggesting that the two species have different 349 levels of tolerance to herbicides. In fact, studies carried out on Chaetoceros species reported 350 different results. An increase in Chl a content was observed for C. gracilis exposed to 50 to 351 60 µg l -1 of Bentazon, while a drastic decrease was found for Chaetoceros sp. as a short-term response to 2,4-D dimethylamine (50 to 300 mg l -1 ) and endosulfan (20 to 100 mg l -1 ) exposure 353 (Mine and Matsunaka 1975;[START_REF] Hii | Physiological responses of Chaetoceros sp. and 587 Nannochloropsis sp. to short-term 2 4-D dimethylamine and endosulfan exposure[END_REF]. suggesting a relative integrity of photosystem II under the experimental conditions. However, 358 herbicide-induced changes were clearly visible in all the other OJIP parameters (ABS0/RC, 359 TR0/RC, ET0/RC and DI0/RC) for P. mannii monoculture (Fig. 3). Several authors have already 360 pointed out that Fv/Fm is probably not the best parameter to evaluate toxic effects of herbicides 361 due to its relatively low sensitivity (e.g. [START_REF] Juneau | Use of chlorophyll fluorescence as a tool for determination of 596 herbicide toxic effect[END_REF][START_REF] Kargar | Chlorophyll Fluorescence -a 601 Tool for Quick Identification of Accase and ALS Inhibitor Herbicides Performance[END_REF]. Fv/Fm measures 362 the optimal photosynthetic efficiency and does not reflect the true nature of PSII-activity under 363 normal light conditions [START_REF] Juneau | Use of chlorophyll fluorescence as a tool for determination of 596 herbicide toxic effect[END_REF]). The decrease in the photosynthetic parameters in 364 P. mannii monoculture may indicate an increase in activated fraction of RCs or in antenna size 365 by P. mannii to counterbalance the chlorophyll pigment deterioration by herbicides (cf. Fig. 2a) 366 to maintain an efficient light energy transfer. In fact, these ratios are influenced by the ratio of 367 active/inactive RCs of light harvesting complex (LHC) and as the number of active centers 368 increased, they decreased [START_REF] Mathur | Analysis of high temperature stress on the dynamics of 663 antenna size and reducing side heterogeneity of Photosystem II in wheat leaves (Triticum 664 aestivum)[END_REF]Kumar et al. 2019). None of the specific energy suggesting that the species could maintain an effective photosynthetic performance under 371 herbicide exposure, which could explain the lack of effect on its Chl a content. Therefore, C. 372 decipiens seemed to be physiologically more tolerant to herbicides than P. mannii. This could 373 be due to the antenna organization, which differs significantly between pennate and centric 374 diatoms [START_REF] Gundermann | Fucoxanthin-chlorophyll protein complexes of 581 the centric diatom Cyclotella Meneghiniana differ in Lhcx1 and Lhcx6_1 content[END_REF].

375

The enzymes involved in oxidative damage repair have significantly increased under 376 herbicide contamination. However, this stimulation was different between the diatom species. Indeed, the CAT activity has increased for P. mannii only by the end of the experience, whereas 378 C. decipiens showed a considerably higher CAT response to the herbicide mixture (P < 0.05, 379 Fig. 4). CAT enzyme has been possibly activated to regulate toxic levels of reactive oxygen 380 species (ROS) which production was probably induced by herbicides [START_REF] Bai | Effects of atrazine on photosynthesis 503 and defense response and the underlying mechanisms in Phaeodactylum tricornutum[END_REF] [START_REF] Maronić | S-metolachlor 659 promotes oxidative stress in green microalga Parachlorella kessleri -A potential environmental 660 and health risk for higher organisms[END_REF]. At short term (day 1), the 386 herbicide mixture promptly stimulated the GPX activity for P. mannii and C. decipiens (Fig. Under control conditions, diatom species in co-culture have evolved in a different way 397 than when maintained in monoculture, with a slower growth for C decipiens and a decline for 398 P. mannii. (two-way ANOVA, P < 0.05, Fig. 1). This suggests that C. decipiens achieved 399 growth advantage by dominating P. mannii due to either a better competition for resources (e.g., 400 nutrients, light) and/or a chemically mediated interference (i.e. allelopathy). In our study, the 401 mono-and co-cultures bioassays were performed under similar physico-chemical conditions, using the same non-limiting nutrient concentrations, that the exclusion of P. mannii in co-403 culture might probably be explained by allelopathy. Allelopathy was found to play a significant 404 role in dominance, succession, and formation of natural diatom communities (Leflaive and Ten 405 Hage, 2009;[START_REF] Zhang | The study on the influencing factors of Chaetoceros curvisetus 810 allelopathic effect on Skeletonema costatum and the primary research on the properties of 811 Chaetoceros curvisetus allelochemical[END_REF]). In addition, the small size of P. mannii may increase its 406 sensitivity to allelopathic interactions due to a high surface/volume ratio (Lyczkowski and 407 [START_REF] Lyczkowski | Allelopathic effects of Alexandrium fundyense (Dinophyceae) 643 on Thalassiosira cf gravida (Bacillariophyceae): a matter of size[END_REF].

408

The single-species exposure to the herbicide mixture, at the tested concentration, showed 409 that P. mannii and C. decipiens were tolerant, with no significant impact on their instantaneous 410 growth rates (two-way ANOVA, P ˃ 0.05, Table 2). However, growth of C. decipiens in co-411 culture was significantly impacted by herbicides (two-way ANOVA, P < 0.05, Table 2), 412 suggesting that its resistance to herbicide exposure could be modulated when the species 413 competes in co-culture. This trend may be explained by the classical trade-off theory between 414 tolerance ability and competitive-response ability in phytoplankton [START_REF] Mohr | Defense mechanisms in phytoplankton: traits and trade-offs Technical University of 687 Denmark 99[END_REF]Lürling, 415

2021).

416

The differences in sensitivity to herbicides among P. mannii and C. decipiens under co-417 culture conditions might be also explained by differences at the genetic level or in the trophic 418 mode. In fact, some authors have observed that herbicides may cause a decrease in growth of 419 some diatoms, specifically from the centric genera, whereas it has the opposite effect on pennate 420 diatoms [START_REF] Tang | Differential toxicity of atrazine to selected freshwater 781 algae[END_REF][START_REF] Bérard | 524 Comparison of the ecotoxicological impact of the triazines Irgarol 1051 and atrazine on 525 microalgal cultures and natural microalgal communities in Lake Geneva[END_REF]. In this sense, [START_REF] Larras | Linking Diatom Sensitivity to Herbicides 619 to Phylogeny: A Step Forward for Biomonitoring?[END_REF] have found a 421 strong phylogenetic signal for diatom sensitivity to herbicides with a major separation within 422 the phylogenetic tree. These authors observed that the most sensitive species were mainly 423 centrics and araphid diatoms whereas the most resistant species were mainly pennates. Other 424 authors asserted that sensitivity differences among diatom species to herbicides is more likely 425 related to their trophic mode [START_REF] Larras | Using bioassays and species sensitivity 614 distributions to assess herbicide toxicity towards benthic diatoms[END_REF]. Indeed it was argued that heterotrophic 426 diatoms were more tolerant than autotrophic diatoms species to PSII inhibitors [START_REF] Pérès | 718 Effects of the phenylurea herbicide isoproturon on periphytic diatom communities in freshwater 719 indoor microcosm[END_REF][START_REF] Bérard | Characterisation of phytoplankton from 517 two limnic systems contaminated by a herbicidal photosynthetic inhibitor The PICT method 518 (pollution-induced community tolerance): application and significance[END_REF][START_REF] Debenest | Sensitivity of 555 freshwater periphytic diatoms to agricultural herbicides[END_REF]. For this assumption, authors explained that 428 conversely to autotrophic species, facultative or obligatory heterotrophic species (eventually 429 the case of P. mannii in this study) could metabolize other substrata to decrease their 430 dependence on photosynthesis [START_REF] Larras | Using bioassays and species sensitivity 614 distributions to assess herbicide toxicity towards benthic diatoms[END_REF]) and thus to prevent effects of PSII inhibitor 431 herbicides. Heterotrophy has been clearly evidenced for several species of the genus Pseudo-432 nitzschia (Mengelt and Prezelin, 2005[START_REF] Loureiro | The significance of organic 638 nutrients to Pseudo-nitzschia delicatissima (Bacillariophyceae) nutrition[END_REF][START_REF] Ben Garali | Effects of nitrogen supply on Pseudo-nitzschia calliantha and Pseudo-nitzschia cf seriata: 671 field and laboratory experiments[END_REF].

433

In our experiment, the growth of P. mannii was inhibited by C. decipiens which 434 dominated the co-culture, substantially on days 6 and 9. The Herbicide induced increase of 435 Chl a cell content seemed then to be likely related to C. decipiens (Fig. 2). This suggest that the 436 competitive advantage of C. decipiens might not affect its Chl a response to herbicides.

437

Increased Chl a content in response to herbicides has been noted for several microalgae, 438 including C. gracilis, contaminated by PSII inhibitors such as diuron [START_REF] Magnusson | Comparative effects of herbicides on photosynthesis and 650 growth of tropical estuarine microalgae[END_REF]439 Stachowski-Haberkorn et al. 2013), atrazine [START_REF] Adler | Flow cytometry as a tool to study phytotoxic 498 modes of action[END_REF], bentazon [START_REF] Mine | Mode of action of bentazon: Effect on photosynthesis[END_REF]440 Matsunaka, 1975), isoproturon andterbutryn (Rioboo et al. 2002). This reaction, also known as 441 "greening", is widely described as an increase in pigment in antennae in order to maximize light 442 harvesting as a strategy to counterbalance the herbicide action [START_REF] Magnusson | Comparative effects of herbicides on photosynthesis and 650 growth of tropical estuarine microalgae[END_REF]Ricart 443 et al. 2009;[START_REF] Mansano | 656 Effects of diuron and carbofuran and their mixtures on the microalgae Raphidocelis subcapitata[END_REF]) and hence maintain an efficient conversion of light energy 444 into chemical energy [START_REF] Coquillé | Use of diatom motility features as endpoints of 543 metolachlor toxicity[END_REF]. This would allow an adaptation of photosynthetic 445 metabolism to herbicide effects. decipiens (Fig. 3), which has by far dominated the diatom co-culture. So, as for Chl a, the 451 competitive advantage of C. decipiens on co-culture did not also influence its photosynthesis

118(126

  photosynthetic efficiency, and antioxidant enzyme activities, to better assess the herbicide

  130

  in 125 mL flasks 131 incubated at 20 o C and with a photon flux density of 100 μE m -2 s -1 , provided by cool-white 132 fluorescent tubes, with a 12:12 h light:dark cycle in temperature-controlled chamber 133 (Aqualytic, Dortmund, Germany). Diatom cultures were acclimated to these experimental 134 conditions for a minimum of 4-5 generations before dilution with fresh media and the start of 135 the experiments. For this, early exponentially growing cells were transferred on new f/2 media, 136 every 8 days of incubation. Inoculations were carried out in a laminar flow hood and using 137 sterile laboratory material to avoid contamination. All Flasks were swirled by hand at least 138 twice daily, and their positions on the incubator shelves were changed arbitrarily at least once 139 each day to moderate any discontinuities in irradiance or temperature within the incubator. two diatom isolates up to the genus level was made by light 142 microscopy under a Zeiss AxioCam HRc light microscope (Zeiss, Oberkochen, Germany).

  143

  159sterilized Milli-Q water) were used to prepare an herbicide mixture (diuron, DIA, alachlor and 160 simazine) containing ~ 1 mg l -1 of each herbicide. 161 Experimental design 162 Herbicide contamination experiments were carried out on single-species bioassays 163 (monoculture of Pseudo-nitzschia mannii or Chaetoceros decipiens), as well as on a co-culture 164 of both species. Bioassays were conducted in triplicate in 2 L glass Erlenmeyers flasks, 165

  169 treatments were carried out: control without any herbicide (treatment C) and treatment 170 contaminated with the herbicide blend solution to have the final concentration of ~1 µg L -1 of 171 each herbicide (treatment Herb). The concentration of total herbicides in contaminated flasks 172 was 4.64 µg L -1 . The chosen herbicide concentration represents ~5 times the maximum level 173 observed in Bizerte Lagoon watershed environment (1.246 µg L -1 , Mhadhbi et al. 2019).

  ) used to determine cell density were immediately fixed with 3% acid 185 Lugol's solution. Counts were done in triplicata on 30 µl aliquots under an inverted microscope 186 at magnification of x 40. The instantaneous specific growth rate (μinst, d -1 ) was determined by 187 linear regression of the natural logarithm (ln) of cell count vs time over the sampling days. 188 Measurements of photosynthetic parameters 189 Samples (10 ml) were dark-adapted for 15 min prior to fluorescence measurements. The 190 AquaPen AP-P100 (Photon Systems Instruments, Czech Republic) was used to measure the 191 rapid polyphasic Chl a fluorescence rise during 3 sec by using a LED blue emitter light intensity 192 of 3000 µE m -2 s -1 . 193 Fluorescence yield (F) at investigated fluorescence transients was determined according 194 to Strasser and Strasser (1995). The fluorescence yield at 50 µs was considered as O transient 195 (F50µs) when all reaction centers of PSII are open. The maximal fluorescence yield (FM) was 196 determined at P transient and the variable fluorescence yields related to J and I transients were 197 determined at 2 ms and 30 ms, respectively. Photosynthetic parameters indicating PSII activity 198 (ABS0/RC: absorption of photons per active reaction center (RC); TR0/RC: trapped energy flux 199 per RC; ET0/RC: electron transport flux per RC and DI0/RC: non-photochemical energy 200 dissipation per active reaction centre) were evaluated following to Strasser et al. (1999). 201 Detection of antioxidant enzyme activities 202

  deviation values were calculated for each treatment from three 219 independent replicate cultures and graphed. A two-way ANOVA was used the test the effect of 220 herbicide mixture and culture mode on the instantaneous growth rate of P. mannii and C. 221 decipiens maintained in monoculture and co-culture. The normality of data distribution 222 (Kolmogorov-Smirnov test) and homogeneity of variance (Bartlett-Box test) were respected 223 and differences were accepted as significant when p < 0.05. 224 For each experiment (P. mannii monoculture, C. decipiens monoculture and P. mannii + 225 C. decipiens co-culture), Student's t-tests were performed to detect significant differences in 226 Chl a cell content, photosynthetic parameters and antioxidant enzyme activities between control and herbicide treatments and for comparisons between the different cultures (with a p value 228 <0.05 considered as statistically significant) after having checked the homoscedasticity with 229 Fisher's F-test).

255

  content for P. mannii monoculture (Student's t-test, P < 0.05) and values decreased by 55-89% 256 compared to the control after 2-3 days. Conversely, the addition of herbicides did not provoke 257 any significant effect on the specific biomass of C. decipiens in monoculture (Student's t-test, 258 P > 0.05, Fig. 2b) whose mean values varied in a range from 31.76 ± 7.77 to 53.61 ± 7.90 pg 259 cell -1 . 260

  that the herbicide mixture did not significantly affect the 316 instantaneous growth rate of P. mannii and C. decipiens grown as monocultures (P > 0.05, Fig.3171a, b). Literature data on the effect of herbicides on diatom growth are highly contrasted.318Indeed, it seems difficult to distinguish a general response of diatoms to herbicide exposure. 319 Previously, authors have reported a decrease in growth for freshwater diatoms in response to 320 herbicides, such as atrazine (1-1000 μg L -1 : Larras et al. 2013; 500 μg l -1 : Wood et al. 2014), 321 metolachor (5-30 µg L -1 : Roubeix et al. 2011), isoproturon (5-20 µg L -1 : Pérès et al. 1996) and 322 diuron (10 µg L -1 :

  338 per-cell Chl a content would decrease under herbicide contamination probably due to inhibition 339 of pigment synthesis or pigment destruction. In fact, herbicides, particularly the PSII inhibitors 340 (e.g. DIA, diuron and simazine), are known to act by binding to the exchangeable quinone site 341 in the PSII reaction center, thus blocking electron transfer (Gomes and Juneau, 2017; Thomas 342 et al. 2020). The excitation of the PSII complex is therefore directly transferred to O2, resulting 343 in the formation of singlet oxygen causing uncontrolled oxidation of membranes components 344 in particular chlorophyll pigments, including Chl a (Thomas et al. 2020). Furthermore, PSII 345 inhibiting herbicides would be responsible for the accumulation of compounds such as 346 polyenes, phytils and phytophloenes that inhibit chlorophyll synthesis in microalgae (Thomas 347 et al. 2020). Conversely to P. mannii, herbicides did not cause any effect on Chl a cell content 348 of C. decipiens monoculture (P > 0.05, Fig.

354

  Photosynthetic response and antioxidant enzymes activity

  369 fluxes per RC were affected by herbicides for C. decipiens monoculture (P > 0.05, Fig 3), 370

  ; Thomas 381 et al. 2020). For C. decipiens, the eventual oxidative damage could be likely induced by the 382 Very-Long-Chain Fatty Acid (VLCFA) inhibitor (alachlor) as the PSII inhibiting herbicides has 383 not impacted its photosynthetic performance (P > 0.05, Fig 3). It is widely known that the 384 toxicity of chloroacetamides like alachlor is linked to the oxidation of cell components with the 385 increase of ROS and lipid peroxidation

  387

  4), probably indicating an oxidative short-term stress in both species under herbicide exposure,388as it has been observed with other chemical toxic compounds(Manimaran et al. 2012, Nguyen 389 Deroche et al. 2012). In summary, it seems that the antioxidant enzymatic response to 390 herbicides could provide an efficient protection to C. decipiens, probably resulting in the 391 maintenance of its photosynthetic apparatus integrity and Chl a per-cell content. On the other 392 hand, the antioxidant defense system of P. mannii was only triggered at the short time scale 393 which might result in significant effects on the photosynthetic apparatus integrity. 394 Herbicide toxicity towards diatoms in co-culture 395 Growth and Chl a cell content 396

446

  Photosynthetic response and antioxidant enzymes activity

  450

  Figure 1 1 2

Figure

  Figure

Figure 2 Figure 3 Figure 4 Table 1

 2341 Figure 2

  , respectively. The herbicides used in this study are representative of two different modes of action and 158 have different solubility Kow values (Table 1). Stock solutions of herbicides (all dissolved in

	156
	157	Chemicals preparation

b, c Mode of action/Site of action a

					Growth inhibitor/ Very-Long-Chain Fatty Acid (VLCFA)
	Alachlor	Chloroacetamide	3.52	210 ± 38.62	Inhibitors
	DIA	Triazine	1.15	3200	Photosynthesis inhibitor/ PSII inhibitor
	Diuron	Phenylurea	2.68	47.30 ± 30	Photosynthesis inhibitor/ PSII inhibitor
	Simazine	Triazine	2.18	6.43 ± 4.29	Photosynthesis inhibitor/ PSII inhibitor

Table Table 2

 Table F ratios of two-way ANOVA analysis testing for effects of treatment (control/herbicide) and culture mode (monoculture/co-culture) on instantaneous growth rates (IGR) of P. mannii and C. decipiens

	Treatment	Culture mode	Treatment x Culture

mode F statistics Prob value F statistics Prob value F statistics Prob

	IGR P. mannii	1.943	0.170	152.611	0.000	1.769	value 0.152
	IGR C. decipiens	1.196	0.281	11.687	0.001	19.078	0.000

to herbicide resulted in a significant increase in catalase activity (Student's t-test, P < 0.05, response to the herbicide mixture. As observed for P. mannii or C. decipiens monocultures, the 453 GPX activity was stimulated only after one day of contamination in co-culture (Fig. 4), 454 suggesting that this enzyme could be considered as the antioxidant defense at early exposure to 455 herbicides. There was a significant increase of CAT activity by herbicides in co-culture (P < 456 0.05, Fig. 4), especially at late herbicide exposure, similarly to that observed for C. decipiens 457 monoculture, providing additional support that the physiological response of the co-culture to 458 herbicides was likely attributed to C. decipiens. However, CAT activity levels were 459 significantly higher than those measured for C. decipiens monoculture (Student's t-test, P < 460 0.05, Fig. 4) and values at days 3 and 6 were 1.2 to 2 folds higher than in C. decipiens 461 monoculture, probably suggesting a higher CAT response due to the competitive stress. These 462 results provided further evidence that GPX and CAT enzymes were triggered by the herbicide-463 induced oxidative stress, at early and tardy exposure, in order to repair damage and keep the 464 diatom in good physiological state during most period of herbicide contamination.

465

CONCLUSION

466

The study found that response to herbicides differ between the two marine diatoms and 467 between the culture conditions. The diatom C. decipiens was more sensitive to herbicides in co-468 culture than in the single species-pollutant experiment, which suggest that herbicide pollution 469 in natural environments might considerably alter the diatom populations and phytoplankton 470 communities. We link altered growth of C. decipiens to a modified physiological profile under 471 herbicide conditions, thereby suggesting that chemical pollutants effects are not only reflected 472 by changes in cell densities but also by changes in the physiology of the cells. In order to attain Click here to access/download Suppl Mtrls (not cover letter -place cover letter in "comments") Figure captions (Sahraoui et al.).docx


Hae Min Lee

Dong Wook Choi

Seahyun Kim

Aro Lee

Yeon Minseo Kim

Jin Roh

Young Ho

Hwa Yeon Cho

Ho-Jae Lee

Seung-Rock Lee

Lionel Tarrago

Vadim N Gladyshev

Ji Hyung Kim

Byung Cheon Lee
email: cheonii@korea.ac.kr

Byung Cheon Lee

Hae Min Lee

Yeong Ho

Biosensor-linked immunosorbent assay for quantification of methionine oxidation in target proteins

Keywords: methionine oxidation, biosensor, methionine sulfoxide reductase B, immunosorbent assay, redox lecular Devices, CA, USA

Methionine oxidation is involved in regulating protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor "tpMetROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidations in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay could prove to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.

Peroxide, superoxide, and hydroxyl radical are highly reactive oxygen species (ROS). [START_REF] Muthukumar | Cadmium-induced oxidative stress in Saccharomyces cerevisiae[END_REF] In aerobic organisms, ROS are generated as by-products of the mitochondrial respiratory chain reaction, but they can also be produced during specific immune responses. [START_REF] Zhao | Pivotal role of reactive oxygen species in differential regulation of lipopolysaccha-ride-induced prostaglandins production in macrophages[END_REF] Accumulated ROS in cells can exert both beneficial or detrimental influence on numerous cellular events by altering functions of various macromolecules, including proteins, lipids, and nucleic acids. [START_REF] Nita | The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults[END_REF][START_REF] Tabner | Protein aggregation, metals and oxidative stress in neurodegenerative diseases[END_REF][START_REF] Catala | Lipid peroxidation of membrane phospholipids generates hydroxy-alkenals and oxidized phospholipids active in physiological and/or pathological conditions[END_REF] In particular, several amino acid residues in proteins can be readily oxidized by ROS. Sulfur-containing amino acids, including methionine (Met), are especially easily oxidized by ROS. Oxidation of sulfur in methionine produces either methionine-S-sulfoxide (MetSO) or methionine-R-sulfoxide (MetRO). [START_REF] Boschi-Muller | The enzymology and biochemistry of methionine sulfoxide reductases[END_REF] MetSO and MetRO are reduced by MsrA and MsrB, respectively, which are conserved in animals, including mammals.

Emerging evidence suggests that reversible oxidation/reduction of methionine residues is an adaptation for functional regulation of proteins. [START_REF] Kaya | Regulation of protein function by reversible methionine oxidation and the role of selenoprotein MsrB1[END_REF][START_REF] Henry | Redox controls RecA protein activity via reversible oxidation of its methionine residues[END_REF][START_REF] Nicklow | Activity of the yeast cytoplasmic Hsp70 nucleotide-exchange factor Fes1 is regulated by reversible methionine oxidation[END_REF][START_REF] Wang | CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution[END_REF] Oxidation of methionine residues 281 and 282 within the regulatory region of calcium (Ca 2+ )/calmodulin-dependent protein kinase II (CaMKII) increases its enzymatic activity even in the absence of to Ca 2+ /Calmodulin. [START_REF] Erickson | A dynamic pathway for calciumindependent activation of CaMKII by methionine oxidation[END_REF] Oxidation of methionine residue in hypochlorite-responsive transcription factor (HypT), a protein expressed in Escherichia. coli, induces the expression of various genes required for escaping cell death due to the oxidative stress mediated by neutrophils during an innate immune response. [START_REF] Drazic | Methionine oxidation activates a transcription factor in response to oxidative stress[END_REF] In F-actin, the stereoselective oxidation of methio-nine 46 and 49 catalyzed by molecule interacting with CasL (MICAL) family of proteins results in actin depolymerization and the generation of G-actin monomers. [START_REF] Dalle-Donne | Methionine oxidation as a major cause of the functional impairment of oxidized actin[END_REF][START_REF] Grintsevich | F-actin dismantling through a redoxdriven synergy between Mical and cofilin[END_REF] These oxidized methionine residues involved in altering protein functions were shown to be reduced by either MsrA or MsrB, which brings the proteins back to the original reduced state. For example, G-actin monomers disassembled by oxidation of the two conserved methionine residues are reassembled to F-actin polymer upon treatment with MsrB. [START_REF] Lee | Selenoprotein MsrB1 promotes anti-inflammatory cytokine gene expression in macrophages and controls immune response in vivo[END_REF][START_REF] Lee | MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation[END_REF] Additionally, the oxidation of specific methionine residues in proteins may alter protein structure, thereby diminishing activity. [START_REF] Mccarthy | Impact of methionine oxidation on calmodulin structural dynamics[END_REF][START_REF] Moen | Structural and functional impact of site-directed methionine oxidation in myosin[END_REF][START_REF] Shao | Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I[END_REF] Therefore, it can be surmised that global methionine oxidation may confer differential effects on individual protein function. It is critical to identify proteins where methionine oxidation levels regulate protein function. However, detecting methionine oxidation in a specific protein has been challenged due to the spontaneous oxidation that occurs during sample preparation. To eliminate this oxidation source, Sun and his colleagues replaced the ammonium persulfate (APS) with a Flavin mononucleotide/diphenyliodonium chloride/sodium toluenesulfinic acid system and UV light. [START_REF] Sun | Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate-gel electrophoresis[END_REF] Also artificial oxidation could be distinguished by using the use of isotopes through LC-MS/MS. [START_REF] Shipman | Method for Quantifying Oxidized Methionines and Application to HIV-1 Env[END_REF][START_REF] Bettinger | Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome[END_REF] But high cost and incomplete specificity still remains a challenge to be overcome. [START_REF] Ghesquiere | Proteomics methods to study methionine oxidation[END_REF][START_REF] Liu | Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis[END_REF] In recent, an advanced approach was developed by Tarrago and his colleagues. It is a ratiometric fluorescent sensor that detects a level of global methionine oxidation in vitro and in vivo. [START_REF] Tarrago | Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors[END_REF] However, there is still a need for measuring the degree of methionine oxidation of specific protein target. Here, we introduce a new method for measuring methionine oxidation in a target-specific manner based on the strategy developed from the sensor devised by Tarrago and his colleagues. Using the ability of protein G to bind immunoglobulins, we made a recombinant protein composed of yeast MsrB, cpYFP, and yeast Trx3, followed by protein G with a short amino acid linker. The biosensor was then applied in immunosorbent assays to measure methionine oxidation of specific purified proteins from cell lysates and organ extracts.

MATERIALS AND METHODS

Construction of the target protein MetRO protein G (tpMetROG) biosensor. All polymerase chain reactions (PCR) and site-directed mutagenesis were performed using the KOD Plus polymerase (TOYOBO, Osaka, Japan). MsrB and Trx3 were amplified from the S. cerevisiae genomic DNA using PCR with a specific pairs of primers (listed in Supplementary Table S1) containing overlapping extensions for cpYFP. cpYFP was amplified from the H 2 O 2 sensor, HyPer [START_REF] Belousov | Genetically encoded fluorescent indicator for intracellular hydrogen peroxide[END_REF] using PCR with a specific pairs of primers (listed in Supplementary Table S1). Flexible linker and protein G were de novo synthesized (listed in Supplementary Table S2) from Cosmogenetech (South Korea). MsrB and Trx3 amplicon were subsequently fused to cpYFP by PCR. Sequentially flexible linker and protein G were fused to C terminal of Trx3 by PCR with a specific pairs of primers (listed in Supplementary Table S1). The fused MsrB/cpYFP/Trx3/flexible linker/protein G was purified using a PCR/Gel purification kit (Bioneer, Daejeon, Republic of Korea). For the construction of the MsrB/cpYFP/Trx3/flexible linker/protein G expression plasmid, purified fragments were proportionally mixed with the pET 21a cloning vector (Addgene, MA, USA) plasmid that was previously digested by BamHI and XhoI restriction enzyme (Thermo fisher scientific, MA, USA) and further ligated using T4 DNA ligase (Invitrogen, MA, USA). Then the ligation mixture was transformed into Escherichia coli (E. coli) DH5α (Invitro-gen, MA, USA) for selection and amplification for the correct recombinant plasmid.

Expression and purification of tpMetROG and methionine-rich proteins (MRPs). All constructed plasmids were introduced into E. coli Rosetta2 (DE3) (Novagen, WI, USA) grown in Luria-Bertani medium (Formedium, Norfolk, England) containing ampicillin (50 μg/ml) at 37 °C. When absorption at 600 nm (A 600 ) reached ~0.6, synthesis of the recombinant protein was induced by adding 100 μM isopropyl β-D-1thiogalactopyranoside (IPTG) (Gold biotechnology, MO, USA) at 18 ℃. After 18-24 h incubation, the cells were collected by centrifugation at 3000 g for 30 min. Pellets were resuspended in buffer A (50 mM Tris-HCl, 150 mM NaCl, 5 mM 2mercaptoethanol, pH 8.0) and subsequently sonicated. Proteins were purified using the HisTrap HP column (GE Healthcare, IL, USA) and eluted with buffer B (buffer A supplement 500 mM imidazole). Protein solutions were concentrated using Amicon Ultra-15 30 kDa centrifugal filters (Millipore, MA, USA). Initial protein concentrations were measured using the Pierce bicinchoninic acid (BCA) protein kit (Thermo Scientific, MA, USA). The recombinant proteins were aliquoted and stored at -80 °C.

For the analysis of MRPs, we selected four proteins, including IDLO from Idiomarina loihiensis L2TR, Sacde from Saccharophagus degradans 2-40, LegP from Legionella pneumophila (LegP), calmodulin from Mus musculus. [START_REF] Liang | Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins[END_REF] All proteins were prepared as His-tag forms using a pET-21a (+) cloning vector (Novagen, WI, USA). All constructed plasmids were introduced into E. coli Rosetta2 (DE3) (Novagen, WI, USA) grown in Luria-Bertani medium containing ampicillin (50 μg/ml) at 37 °C. The purification was carried out similarly as described above.

Spectroscopic characterization of tpMetROG biosensors with oxidized MRPs. For assessing the tpMetROG biosensor, purified proteins (10-20 μM) were reduced using 10 mM dithiothreitol (DTT) at room temperature for 30 min. After reduction, the proteins were desalted using HiTrap Desalting columns (GE Healthcare, IL, USA) in 50 mM Tris-HCl, pH 7.5, and were diluted to 1-5 μM in the same buffer for recording fluorescence. Purified MRPs (calmodulin, IDLO, LegP, Sacde) were oxidized using 40 mM hydrogen peroxide (H 2 O 2 ) at room temperature for 2 h. Residual H 2 O 2 was removed by centrifugation (14,000 g, 10 minutes, 4 °C) using Amicon Ultra-0.5 centrifugal filter with 10 K cutoffs. The centrifugation step was repeated five times. The protein concentrations were measured using the Pierce bicinchoninic acid (BCA) protein kit (Thermo Scientific, MA, USA). Fully reduced tpMetROG (1 μM) in working buffer (50mM Tris-HCl, 150mM NaCl, pH 7.5) were incubated with MRP (1-2 μM), partially oxidized MRP (1-2 μM) at room temperature for 30 min. Fully oxidized tpMetROG (1 μM) were prepared by incubating fully reduced tpMetROG with N-acetyl methionine sulfoxide (100 μM) at room temperature for 30 min. Methionine (Met) and methionine sulfoxide (MetO) (Sigma-Aldrich, MO, USA) were prepared in 1 mM in 50 mM Tris-HCl (pH 8.0). Fully reduced tpMetROG were incubated with Met (100 μM) or MetO (100 μM) at room temperature for 30 min.

The fluorescence spectrum is recorded in Costar black clearbottom 96 well microplates using SpectraMax I3 (Molecular Devices, CA, USA). Absorbance spectra were recorded from 360 to 800 nm in a 1 nm step increment. Excitation spectra were recorded from 380 nm to 510 nm with 535 nm emission and a 530-nm cutoff in a 1 nm step increment. The device was typically set to 10 reads per well with the photomultiplier tube (PMT) set on 'auto.' Emission spectrum were recorded from 500 nm to 750 nm with an excitation wavelength of 410 nm, in 1 nm step increment. The normalized fluorescence intensity (NFI) was obtained by dividing all spectrum values by the maximum value recorded at 535 nm emission wavelength. The percentage of oxidized tpMetROG was represented as relative fluorescence intensity (RFI), and RFI was determined using the following equation (equation A).

RFI of tpMetROG = (R Reduced -R) / (R Reduced -R Oxidized ) (equation A) Where R is measured F 500 nm /F 415 nm ratio of the excitation spectrum. R Reduced is the F 500 nm /F 415 nm ratio of fully reduced tpMetROG, and ROxidized is the F 500 nm /F 415 nm ratio of fully oxidized tpMetROG. [START_REF] Peterfi | Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide[END_REF] pH calibration and kinetic measurement. For the analyzing the pH influence, following buffers were used: 50 mM Phosphate, 150 mM NaCl (pH 5.8, 6.4 and 7.0); 50 mM Tris-HCl, 150 mM NaCl (pH 7.5, 8.0, 8.3 and 9.0). Fully reduced tpMetROG (1 μM) or inactive tpMetROG (1 μM) was incubated with IDLO (2 μM) or oxidized IDLO (2 μM) in a Costar black clear-bottom 96 well microplate for 30 min at 37 ℃. The Fluorescence spectrum is recorded using SpectraMax I3 (Mo- First, the plate is coated with the protein or a sample containing a protein to measure the oxidation state of methionine. After adding the primary antibody for a target protein, the unbound antibody is washed away, and tpMetROG is added at low temperature. The unbound tpMetROG is washed using the working buffer. After incubation at 37 ℃, the tpMetROG fluorescence spectrum was measured. at 500 nm and 415 nm for the excitation spectrum with an emission wavelength of 535 nm.

For measuring kinetic reactivity of tpMetROG with various substrates, the reduced biosensor (1 μM) was incubated for 60 min with buffer A (50 mM Tris-HCl, 150 mM NaCl, pH 7.5), DTT (1 mM), oxidized calmodulin (2 μM) or reduced calmodulin (2 μM) as control. The examination of the excitation spectrum and calculating F 500 nm /F 415 nm was performed as described above. The F 500 nm /F 415 nm (R) were normalized to the value at t = 0 (R 0 ) Application of tpMetROG using purified MRPs in antibody-based immunoassays. tpMetROG was applied in antibody-based immunoassays. Purified MRPs were oxidized with 10-40 mM hydrogen peroxide (H 2 O 2 ) at room temperature for 2 h. Residual H 2 O 2 was removed by repeating five cycles of washing-centrifugation (14,000 g, 10 minutes, 4 °C) using Amicon Ultra-0.5 centrifugal filter with 10 K cutoffs. Protein concentrations were measured using the Pierce bicinchoninic acid (BCA) protein kit (Thermo Scientific, MA, USA). MRPs were prepared at 2 μM in 200 μl buffer A. Black immunoplates (SPL Life sciences, Korea) were coated with MRPs for 2 hours at room temperature and then blocked with SuperBlock (Thermo Scientific, MA, USA). Dilutions of antiidlo (1:1000; Convance), anti-sacde (1:1000; Convance), antilegp (1:1000; Convance), or anti-calmodulin (1:1000; Convance) antibodies were added to the plates for 2 h at room temperature. After washing with a wash buffer (100 mM phosphate, 150 mM sodium chloride, 0.05% tween 20, pH 7.2), the plates were incubated with the fully reduced tpMetROG (1 μM) in 200 μl working buffer (50mM Tris, 150mM NaCl, pH 7.5) for 2 h at 18 ℃. After the plate were washed again with working buffer, 200 μl working buffer was added and incubat-ed at 37 ℃ for 30 min. Then, fluorescence was recorded using SpectraMax I3. Excitation spectrum was recorded from 380 nm to 510 nm with 535 nm emission and a 530-nm cutoff in 1 nm increments. The percentage of oxidized tpMetROG is represented as RFI.

Application of tpMetROG using whole cell lysates. tpMetROG was used to analyze the oxidation of calmodulin obtained from the lysates of Raw264.7 and U87MG cells. Raw264.7 cells were cultured in HyClone RPMI 1640 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, penicillin 100 U/ml, and 100 μg/ml streptomycin. U87MG cells were cultured in HyClone DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, penicillin 100 U/ml, and 100 μg/ml streptomycin. Cells were oxidized with 15, 30, and 45 mM H 2 O 2 for 40 minutes at 37 ℃ or 5, 10, and 15 mM NaOCl for 15 min at 37 ℃ or with 100 ng/ml LPS for 6 h -16 h. After the treatment, cells were washed three times with cold PBS, and the harvested cells were recovered by centrifugation. Cells were lysed in RIPA buffer (30 mM HEPES, pH 7.3, containing 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100 and 1% (w/v) Nadeoxycholate), and the lysates were centrifuged at 16000 g for 10 min at 4 ℃. The protein concentrations of the supernatant were measured using the Pierce BCA protein kit. The same amount of total protein (1.6 mg/ml, 200 μl) was coated on Black immunoplates, incubated for 2 h at room temperature, and blocked with SuperBlock. A dilution of anti-calmodulin antibodies (1:1000; Convance) was applied for 2 h at room temperature. After washing with a wash buffer (100 mM phosphate, 150 mM sodium chloride, 0.05% tween 20, pH 7.2), the plates were incubated with the fully reduced tpMetROG (1 μM) in 200 μl working buffer (50mM Tris, 150mM NaCl, pH 7.5) for 2 h at 18 ℃. After the plate were washed again with working buffer, 200 μl working buffer was added and incubated at 37 ℃ for 30 min. Then, fluorescence was recorded in same manner as described above.

Quantification of oxidized methionine from mouse tissues using tpMetROG. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea University (KUIACUC-2019-0107). 10-12 weeks old MsrB1 knock-out, [START_REF] Fomenko | MsrB1 (Methionine-R-sulfoxide Reductase 1) Knock-out Mice ROLES OF MsrB1 IN REDOX REGULATION AND IDENTIFICATION OF A NOVEL SELENOPROTEIN FORM[END_REF] and age-and sex-matched C57BL/6J (Orientbio) mice were used in this study. Mice were anesthetized by intraperitoneal injection of sterile avertin (tribromoethanol: 200 mg/10 ml/kg) and euthanized by cervical dislocation. The small intestine, large intestine, brain, heart, liver, kidneys, and lungs were dissected under sterile conditions. Each organ was immediately rinsed with PBS and weighed. Each organ was placed in a 15 ml polystyrene tube containing 2 ml of 0.2 % Tween 20/TBS and homogenized on ice with a Polytron PT1200E homogenizer (Kinematica AG, Luzern, Switzerland). The homogenates were centrifuged at 16000 g for 10 min at 4 ℃. The protein concentration of the supernatants was measured using the Pierce BCA protein kit. Same amount of total protein (3.2 mg/ml, 200 μl) was coated into Black immunoplates and incubated for 2 h at room temperature and then blocked with SuperBlock. Anti-calmodulin (1:1000; Convance) antibodies were applied for 2 h at room temperature. After washing with a wash buffer (100 mM phosphate, 150 mM sodium chloride, 0.05% tween 20, pH 7.2), the plates were incubated with the fully reduced tpMetROG (1 μM) in 200 μl working buffer (50mM Tris, 150mM NaCl, pH 7.5) for 2 h at 18 ℃. After the plate were washed again with working buffer, 200 μl working buffer was added and incubated at 37℃ for 30 min. Then, fluorescence was recorded in same manner as described above.

Analysis of stereospecific oxidation in F-actin using tpMetROG. Recombinant mouse Mical1 and oxidized form of F-actin were prepared as described previously. [START_REF] Kim | Structural and kinetic insights into flavin-containing monooxygenase and calponinhomology domains in human MICAL3[END_REF] Mical1oxidized F-actin and actin were coated on Black immunoplates and incubated for 2 h at room temperature and then blockedwith SuperBlock. A proper dilution of mouse monoclonal anti-β actin antibody (1:1000) (Santa Cruz Biotechnology, TX, USA) was applied for 2 h at room temperature. After washing with a wash buffer (100 mM phosphate, 150 mM sodium chloride, 0.05% tween 20, pH 7.2), the plates were incubated with the fully reduced tpMetROG (1 μM) in 200 μl working buffer (50mM Tris, 150mM NaCl, pH 7.5) for 2 h at 18 ℃. After the plate were washed again with working buffer, 200 μl working buffer was added and incubated at 37 ℃ for 30 min. Then, fluorescence was recorded in same manner as described above.

Statistical analysis. Data are presented as mean ± standard error. To compare two groups, the two-tailed unpaired t-test, Mann-Whitney, or Wilcoxon test was used with or without the assumption of normality. The number of asterisks indicates the degree of statistical significance according to the p-value. All statistical analyses were calculated using Prism 8 software (GraphPad Software, CA, USA). of methionine residues in target proteins. We prepared a fusion protein named tpMetROG, consisting of MsrB, circularly permuted yellow fluorescent protein (cpYFP), and thioredoxin3 (Trx3), peptide linker, and protein G in succession (Fig 1A). Protein G is a cell surface protein from Streptococci, and its B1 domain binds to the Fc region of immunoglobulin G (IgG). [START_REF] Tarrago | Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors[END_REF][START_REF] Sasaki | Recombinant protein (EGFP-Protein G)-coated PbS quantum dots for in vitro and in vivo dual fluorescence (visible and second-NIR) imaging of breast tumors[END_REF] Once the biosensor reacts with MetRO, a stable disulfide bond is formed between MsrB and Trx3, causing changes in the spectral properties of cpYFP; (1) Reduction of the methionine R-sulfoxide residue of a target protein leads to the formation of sulfenic acid on catalytic cysteine (Cys129) of MsrB, (2) the resolving cysteine (Cys69) in proximity attacks the sulfenic acid intermediate to form an intramolecular disulfide, reducing the sulfenic acid, (3) the disulfide bond between two cysteines is reduced by Trx3 by a similar mechanism as in (2), resulting in the formation of a transient intermolecular bond between Cys69 of MsrB and Cys417 of Trx3. In our recombinant Trx3, the resolving cysteine (Cys422) has been substituted to serine, which makes the transient disulfide bond between MsrB and Trx3 stable, thereby affecting the structure of cpYFP permanently. [START_REF] Tarrago | Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors[END_REF][START_REF] Peterfi | Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide[END_REF][START_REF] Choi | Development of a novel fluorescent biosensor for dynamic monitoring of metabolic methionine redox status in cells and tissues[END_REF] Importantly, this tpMetROG biosensor is designed to bind to the Fc region via the Protein G, allowing it to detect methionine sulfoxide in a protein-specific manner using antibodies (Fig. 1B). Using the sensing ability of tpMetROG, we designed a method named biosensor-linked immunosorbent assay (BLISA), similar to the indirect ELISA (Fig 1B ); as follows:

RESULT Construction of a biosensor detecting the oxidation state

(1) Coat proteins from the tissue lysates on the plate. (2) Add primary antibody for a target protein. (3) Wash away the unbound antibody and then add tpMetROG at low temperature. (4) Wash away the unbound tpMetROG and then add the working buffer with increasing temperature. (5) Measure the change of fluorescence intensity in response to the reduction of methionine sulfoxides of a target protein.

Optical properties of tpMetROG. When purified tpMetROG was analyzed for absorbance spectra, two peaks appeared at 410 nm and 500 nm (Fig. 2A). The purified tpMetROG showed a single peak at 515 nm in the emission spectrum (Fig. 2B) and two peaks at 415 nm and 500 nm in the excitation spectrum (Fig. 2C). To characterize the optical properties of tpMetROG and the changes upon Met oxidation, the excitation spectrum was analyzed by incubating tpMetROG with or without partially oxidized, fully oxidized, or untreated IDLO. For readability, we used the normalized fluorescence intensity (NFI) of the excitation spectrum (Fig. 2D), calculated by dividing all excitation spectrum values by the maximum value of the excitation spectrum. Indeed, the NFIs of tpMetROG at 500 nm decreased after reacting with the fully or partially oxidized IDLOs, but the NFIs at 415 nm were not changed. The NFIs at 500 nm showed the greatest difference for all tested samples, and the NFIs at 415 nm were constant (Fig. 2D). Based on this observation, the NFI at both wavelengths were selected to calculate a ratio of fluorescence intensity (RFI) by dividing the 500 nm value by the 415 nm value to quantify methionine oxidation (See the materials and methods). The RFI increased quantitatively after incubation with the oxidized IDLO, reflecting the degree of protein methionine oxidation (Fig. 2E).

Characterization of tpMetROG with various oxidized MRPs. To examine whether tpMetROG can be used as a biosensor for various proteins, the purified tpMetROG was incubated with free MetRO and oxidized MRPs. The spectrum of the NFI obtained by incubating tpMetROG with Met was similar to that obtained after incubating tpMetROG without any substrate. However, a slight reduction was observed at 500 nm in the NFI when the biosensor was incubated with free MetRO (Fig. 3A). Accordingly, the RFI also showed a slight increase, suggesting that tpMetROG senses free MetRO with low efficiency (Fig. 3A). Next, a significant reduction in theNFIs at 500 nm and increase in the RFIs were observed when tpMetROG was incubated with several oxidized MRPs such as mouse calmodulin, hypothetical protein (accession no. YP_155605) from Idiomarina loihiensis L2TR (IDLO), hypothetical protein (accession no. YP_095088) from Legionella (Fig. 3B-E). Finally, there were no changes in the NFI at 500 nm and the RFI when mutated tpMetROGC129S (cysteine in the catalytic part of MsrB replaced by serine) was incubated with the oxidized Sacde (Fig. 3F). Collectively, these results indicate that tpMetROG can quantitatively measure the methionine oxidation in proteins. Next, we measured the kinetic change in the biosensor to determine the optimal incubation time with the substrate. tpMetROG was incubated with calmodulin (2 μM), oxidized calmodulin (2 μM), or dithiothreitol (DTT) (1 mM) for 1500 s. Then, the RFIs after every 30 s (R) were normalized to the RFI at t=0 (R 0 ) and are presented as R/R 0 , (Figure S1A). A rapid decrease and increase of R/R 0 were observed up to 150 s in the reaction with oxidized calmodulin and DTT. After 150 seconds, R/R 0 reaches a stable plateau until the end of the reaction, indicating that incubation for 150 seconds could be sufficient to obtain a stable RFI value (Figure S1A). We examined the effect of pH on tpMetROG activity. The fluorescence ratio was recorded while tpMetROG or tpMetROG C129S was incubated with reduced or oxidized IDLO in the buffers with different pH values (5.8 to 9.0)(Figure S1B). The fluorescence intensity (FI, F 500 nm /F 415 nm ) of all used sensors increases to the same level with increase of pH. In particular, tpMetROG C129S with reduced IDLO, tpMetROG C129S with oxidized IDLO, and tpMetROG with reduced IDLO show the same value and increase rate with increase of pH. Accordingly, this data supports the idea that tpMetROG C129S can be used as a control dead mutant to determine pH dependency of tpMetROG under variable pH conditions. However, in vitro BLISA assay are performed at controlled pH by using the working buffer (50mM Tris, 150mM NaCl, pH 7.5)(see the materials and methods). Thus, the control dead mutant does not need to be applied for BLISA to determine pH dependency. Nevertheless, in vivo application of tpMetROG needs to consider pH variation and so this control dead mutant will be a useful tool to determine pH dependency of fluorescence intensity.

Application of tpMetROG in the immunosorbent assay for purified proteins. Indirect ELISA intends to quantify specific protein antigen, which requires the primary and enzyme-labeled secondary antibody. The tpMetROG biosensor was applied to develop a novel method, BLISA, a modified version of indirect ELISA. The biosensor (instead of specific protein antigen) that has an affinity for the constant region of the primary antibody replaces the enzyme-labeled secondary antibody to quantify the methionine oxidation of specific protein. Before applying it to various protein substrates, we performed control experiments to examine non-specific interactions (Fig. 4A). For the oxidized Sacde, the biosensor was pneumophila (LegP), and hypothetical protein (accession no.YP_527410) from Saccharophagus degradans (Sacde) incubated with anti-Sacde antibody in the oxidized Sacdecoated plate. In the control A experiment, the biosensor was incubated with an anti-Sacde antibody in a non-coated plate, and in the control B experiment, the biosensor was incubated with oxidized Sacde without anti-Sacde antibody. The experiments with tpMetROG Δprotein G indicate that the sensor lacking protein G was incubated with an anti-Sacde antibody in the oxidized Sacde-coated plate (Fig. 4A). The NFI, including the value at 500 nm, of control A, control B, and tpMetROG Δprotein G were almost similar as a basal level, whereas the NFI of the oxidized Sacde showed a dramatic increase, indicating that the tpMetROG biosensor could successfully detect the methionine oxidation in a specific protein during BLISA. Therefore, it shows a successful development of functional BLISA to analyze methionine oxidation of a specific protein without any experimental noise.

Next, we applied BLISA to various protein substrates with various oxidation levels; calmodulin, IDLO, LegP, and Sacde were oxidized by short-term exposure to H 2 O 2 (10 mM, 20 mM, 30 mM, and 40 mM) and used to coat plates. The RFI for all proteins (calmodulin (Fig. 4B), IDLO (Fig. 4C), LegP (Fig. 4D), and Sacde (Fig. 4E)) gradually increased as the concentration of H 2 O 2 used to oxidize proteins increased. Finally, we applied BLISA to the active MICAL1-treated F-actin. Active MICAL1 contains flavin-containing monooxygenase (FMO) and calponin-homology domain (CH), which stereoselectively oxidize the two conserved methionine residues of F-actin to MetRO. [START_REF] Lee | MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation[END_REF][START_REF] Kim | Structural and kinetic insights into flavin-containing monooxygenase and calponinhomology domains in human MICAL3[END_REF] As a result, the active MICAL1-treated F-actin shows increased RFI compared to the untreated F-actin (Fig. 4F). Taken together, these results prove that BLISA using tpMetROG can be applied to detect methionine oxidation in a protein-specific manner.

Analysis of Methionine oxidation of calmodulin in cells using BLISA. We examined methionine oxidation of calmodulin in cells using BLISA after the induction of oxidative stress. Human glioblastoma U87MG cells (Fig 5A -B Lipopolysaccharides (LPS) trigger the production of ROS in the macrophages by binding with toll-like receptor 4. [START_REF] Zhao | Pivotal Role of Reactive Oxygen Species in Differential Regulation of Lipopolysaccharide-Induced Prostaglandins Production in Macrophages[END_REF] BLISA was applied to quantify the methionine oxidation of calmodulin in Raw264.7 cells stimulated with LPS for 6 and 16 h. The RFIs of calmodulin dramatically increased in LPS-stimulated cells for 6 and 16 hours (Fig. 5E). Therefore, these results demonstrate that the tpMetROG biosensor is able to quantify protein-specific methionine oxidation occurring in the cells.

Analysis of methionine oxidation in calmodulin obtained from various tissues of MsrB1 knock-out mice using BLISA. Methionine oxidation of calmodulin in animal tissues was analyzed using BLISA and tpMetROG. For this tissue samples of the small intestine, large intestine, brain, heart, liver, kidney, and lung were collected from MsrB1 knock-out and wild-type (WT) mice. Interestingly, the RFIs for calmodulin were much higher in all organ tissues from MsrB1 knock-out mice than those from WT mice (Fig. 6). Consequently, this result demonstrates that the ablation of MsrB1 leads to severe methionine oxidation of calmodulin, thereby supporting the idea that MsrB1 is essential for maintaining the reduced state of methionine in proteins.

DISCUSSION

Oxidation of methionine residues often leads to structural abnormalities in protein and their malfunction. Therefore, it is critical to quantitatively measure the methionine oxidation status of proteins under various physiological conditions. Recently, two strategies were used to quantify the amount of methionine sulfoxide residues in proteins in vivo; the LC-MS using an isotope labeling with [START_REF] Moen | Structural and functional impact of site-directed methionine oxidation in myosin[END_REF] O 34 and the fluorescencebased biosensor using cpYFP. [START_REF] Tarrago | Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors[END_REF] However, these methods have limitations in terms of complexity and the lack of specificity. In particular, considering that the oxidation of specific methionine residues in proteins, and not all methionines, is associated with the changes in protein functions, it is essential to quantify oxidation of a specific methionine residue within a protein. In this regard, the tpMetROG biosensor is an advanced tool that employs a fluorescent biosensor and antibodies against specific proteins, allowing for quantifying methionine oxidation of target proteins; it could be applied for any proteins if specific antibodies are available. Accurate analysis of methionine sulfoxide residues in specific proteins using LC-MS/MS has been challenging due to a high degree of spontaneous methionine oxidation during sample preparation. However, the immunosorbent assay using the tpMetROG adopted the sampling process that does not require incubation with buffers containing methanol or heating; thereby, preventing the spontaneous methionine oxidation.

Calmodulin is a multifunctional calcium-binding protein expressed in all eukaryotic cells. [START_REF] Chin | Calmodulin: a prototypical calcium sensor[END_REF] Upon calcium binding, calmodulin undergoes conformational changes, which facilitate its interaction with various target proteins such as the kinases, phosphatases, phosphodiesterases, and ion channels. [START_REF] Chin | Calmodulin: a prototypical calcium sensor[END_REF][START_REF] Vougier | Essential role of methionine residues in calmodulin binding to Bordetella pertussis adenylate cyclase, as probed by selective oxidation and repair by the peptide methionine sulfoxide reductases[END_REF] However, oxidation of the methionine residues of calmodulin also leads to conformational changes that allow interaction with various target proteins without binding calcium. [START_REF] Heiby | Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk[END_REF][START_REF] Bartlett | Oxidation of Met(144) and Met(145) in calmodulin blocks calmodulin dependent activation of the plasma membrane Ca-ATPase[END_REF][START_REF] Mccarthy | Impact of methionine oxidation on calmodulin structural dynamics[END_REF][START_REF] Marimoutou | Oxidation of Methionine 77 in Calmodulin Alters Mouse Growth and Behavior[END_REF] The tpMetROG biosensor was able to quantify the oxidation of methionine residues in calmodulin under oxidative stress. Therefore, this biosensor can potentially be used to predict the changes in calmodulin functions associated with methionine oxidation. Many proteins, such as nitrogen assimilation transcription factor nirA (NirA), nuclear factor of kappa B inhibitor alpha (IkBα), HypT, CaMKII, actin, and, calcineurin are functionally altered by reversible oxidation and reduction of methionine residues causing structural changes or altered interactions. [START_REF] Aledo | Methionine in proteins: The Cinderella of the proteinogenic amino acids[END_REF] In particular, the two conserved methio-nine residues of actin are stereoselectively oxidized to methionine-R-sulfoxide by MICALs, which results in actin depolymerization. The tpMetROG biosensor allows understanding the functional role of oxidation and reduction of methionine residues and conducting research on methionine residues in protein as the potential regulators of cellular function under various physiological conditions.

However, this novel biosensor still has some limitations. Currently, its application is limited to in vitro analysis. In vivo analysis such as live-cell imaging and immunohistochemistry should be developed to expand its use and contribution to various biological studies for better understanding association of methionine oxidation and functional regulation within a specific protein. Also, this sensor is devised for quantifying relative change of methionine oxidation in a specific protein, whereas the absolute value of methionine oxidation in a single protein cannot be analyzed in the present way. To make this possible, analysis of the number of methionine oxidation in a specific protein by using LC-MS/MS can be applied to examine correlation of the number of methionine oxidation and fluorescence intensity. Future study will be conducted to overcome this hurdle of tpMetROG sensors in order to expand its application.

CONCLUSION

We developed the recombinant fluorescence-based sensor that includes MsrB, cpYFP, Trx3, and protein G. This sensor could successfully replace the secondary antibody to measure the oxidation of methionine residues of specific proteins in an immunosorbent assay, BLISA. In particular, methionine oxidation of calmodulin upon oxidative stress could be quantified in cells and tissues. For future research, this advanced tool would be useful for studying mechanisms linking methionine oxidation to changes in the function of various proteins. We need to consider the extended application of this biosensor, beyond the immunosorbent assay, as a versatile tool.

Figure 1 .

 1 Figure 1. Schematic representation of tpMetROG biosensor and its application in an immunosorbent assay. (A) Schematic representation of the tpMetROG biosensor construct encoding cpYFP flanked by MsrB1 and Trx3 at the N-and C-terminus, and protein G linked to the C-terminus of Trx3 by a peptide linker. (B) Schematic representation of the biosensor-linked immunosorbent assay using tpMetROG.First, the plate is coated with the protein or a sample containing a protein to measure the oxidation state of methionine. After adding the primary antibody for a target protein, the unbound antibody is washed away, and tpMetROG is added at low temperature. The unbound tpMetROG is washed using the working buffer. After incubation at 37 ℃, the tpMetROG fluorescence spectrum was measured.

Figure 2 .

 2 Figure 2. Optical spectral analysis of the tpMetROG sensor. (A) Absorption spectrum of reduced tpMetROG. (B) The emission spectrum of reduced tpMetROG (1 μM) when the maximum absorbance wavelength (410 nm) was used as the excitation wavelength. (C) Excitation spectrum and (D) normalized fluorescence intensity (NFI) of fully reduced tpMetROG (1 μM) incubated with or without IDLO (2 μM), partially oxidized or N-acetyl methionine sulfoxide (100 μM) when emission wavelength was fixed at 535 nm. The dotted vertical lines indicate the 415 and 500 nm. (E) A relative fluorescence intensity (RFI) of tpMetROG (1 μM) incubated with IDLO (2 μM) or partially oxidized IDLO (2 μM). Data are presented as means ± SD and are representative of 3 replicates. *** p < 0.001, as determined by two-tailed paired t-test.working buffer, 200 μl working buffer was added and incubated at 37 ℃ for 30 min. Then, fluorescence was recorded in same manner as described above.Quantification of oxidized methionine from mouse tissues using tpMetROG. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea University (KUIACUC-2019-0107). 10-12 weeks old MsrB1 knock-out,[START_REF] Fomenko | MsrB1 (Methionine-R-sulfoxide Reductase 1) Knock-out Mice ROLES OF MsrB1 IN REDOX REGULATION AND IDENTIFICATION OF A NOVEL SELENOPROTEIN FORM[END_REF] and age-and sex-matched C57BL/6J (Orientbio) mice were used in this study. Mice were anesthetized by intraperitoneal injection of sterile avertin (tribromoethanol: 200 mg/10 ml/kg) and euthanized by cervical dislocation. The small intestine, large intestine, brain, heart, liver, kidneys, and lungs were dissected under sterile conditions. Each organ was immediately rinsed with PBS and weighed. Each organ was placed in a 15 ml polystyrene tube containing 2 ml of 0.2 % Tween 20/TBS and homogenized on ice with a Polytron PT1200E homogenizer (Kinematica AG, Luzern, Switzerland). The homogenates were centrifuged at 16000 g for 10 min at 4 ℃. The protein concentration of the supernatants was measured using the Pierce BCA protein kit. Same amount of total protein (3.2 mg/ml, 200 μl) was coated into Black immunoplates and incubated for 2 h at room temperature and then blocked with SuperBlock. Anti-calmodulin (1:1000; Convance) antibodies were applied for 2 h at room temperature. After washing with a wash buffer (100 mM phosphate, 150 mM sodium chloride, 0.05% tween 20, pH 7.2), the plates were incubated with the fully reduced tpMetROG (1 μM) in 200 μl working buffer (50mM Tris, 150mM NaCl, pH 7.5) for 2 h at 18 ℃. After the plate were washed again with

Figure 3 .

 3 Figure 3. Analysis of various MRPs using tpMetROG. (A) Normalized fluorescence intensity (NFI) and relative fluorescence intensity (RFI) of reduced tpMetROG (1 μM) incubated with methionine (Met, 10 μM) or methionine sulfoxide (MetO, 10 μM) when emission wavelength was fixed at 535 nm. (B) NFI and RFI of reduced tpMetROG (1 μM) incubated with (B) calmodulin (1 μM) or partially oxidized calmodulin (1 μM), (C) IDLO (1 μM) or partially oxidized IDLO (1 μM), (D) LegP (1 μM) or partially oxidized LegP (1 μM), (E) Sacde (1 μM) or partially oxidized Sacde (1 μM) when emission wavelength was fixed at 535 nm. (F) NFI and RFI of inactive mutant tpMetROG C129S incubated with Sacde (1 μM) or partially oxidized Sacde (1 μM) when emission wavelength was fixed at 535 nm. The dotted vertical lines indicate the 415 and 500 nm. Data are presented as means ± SD and are representative of 3 replicates. ** p < 0.01, *** p < 0.001; ns, not significant, as determined by two-tailed paired t-test.

Figure 4 .

 4 Figure 4. Characterization of tpMetROG used in immunosorbent assays. (A) Fluorescence excitation intensity of tpMetROG was measured after tpMetROG was incubated with antibodies in the Sacde coated plate. Control A: tpMetROG incubated with anti-sacde antibody in a non-coated plate. Control B: tpMetROG incubated in sacde coated plate without antibody. tpMetROG △ protein G ; tpMetROG lacking protein G incubated with antibodies in the Sacde coated plate. (B-F) Relative fluorescence intensity (RFI) of tpMetROG after tpMetROG was incubated with antibodies in a plate coated with purified protein. RFIs were obtained from the excitation spectrum recorded with a 535 nm emission wavelength, according to the oxidation state of calmodulin, IDLO, LegP, or Sacde. (F) Detection of stereospecific oxidation states of MICAL1-oxidized actin using tpMetROG. Static measurements of fluorescence in MICAL1-oxidized actin using tpMetROG. Actin oxidized by MICAL1 (0.2 M) for 2 h was coated on a plate, incubated with mouse monoclonal anti-β actin antibody, and the RFI of tpMetROG in the immunoassay after oxidation was measured. Data are presented as means ± SD and are representative of 3 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant, as determined by two-tailed paired t-test.

Figure 5 .

 5 Figure 5. Quantitative analysis of calmodulin methionine oxidation in cell lysates. Relative fluorescence intensity (RFI) of tpMetROG after tpMetROG was incubated with antibodies in the plate coated with U87MG and Raw264.7 cell lysates. (A-D) RFI of tpMetROG in the immunoassay with oxidized cell lysates. U87MG or Raw264.7 cells were treated with H 2 O 2 (0 mM, 15 mM, 30 mM, 45 mM, A, B) or NaOCl (0 mM, 5 mM, 10 mM, 15 mM, C, D) for 45 min (H 2 O 2 ) or 15 min (NaOCl). Cell lysates were coated in a plate, incubated with an anti-calmodulin (1:1000; Covance) antibody, followed by sensing using tpMetROG. (E) RFI of tpMetROG in the immunoassay with LPS treated Raw264.7 cell lysates. Lysates from Raw264.7 cells treated with 100 ng/ml LPS for 0, 6 h, or 16 h were used to quantify methionine oxidation of calmodulin. Data are presented as means ± SD and representative of 3 replicates. ** p < 0.01, *** p < 0.001; ns, not significant, as determined by two-tailed paired t-test.

Figure 6 .

 6 Figure 6. Quantitative analysis of calmodulin methionine oxidation in tissue extracts. Lysates of the small intestine, large intestine, large intestine, brain, heart, liver, kidney and lung isolated from WT mice and MsrB1 knockout mice were used to compare the state of methionine oxidation of calmodulin. Organ extracts were coated in a plate, incubated with an anti-calmodulin (1:1000; Covance) antibody, following by sensing using tpMetROG. (A) RFI of tpMetROG in immunoassay according to MsrB1 knock-out and WT mice organ extracts. Expression level of calmodulin in each organ was analyzed by western blot in MsrB1 knock-out and WT mice. Data are presented as means ± SD and are representative of 3 replicates (A). ** p < 0.01, *** p < 0.001; ns, not significant, as determined by two-tailed paired ttest.

  ) and mouse macrophage Raw264.7 cells (Fig 5C-D) were incubated with various concentrations of H 2 O 2 (15 mM, 30 mM, and 45 mM) or NaOCl (5 mM, 10 mM, and 15 mM) for 30 min, and the cell lysates were used to coat plates. After treating with anti-calmodulin antibody and tpMetROG, the RFIs were analyzed (Fig 5A-D). As expected, the RFIs for calmodulin gradually increased as the concentration of H 2 O 2 or NaOCl used increased in all tested cell lines.

ACKNOWLEDGMENT

This work was supported by National Research Foundation of Korea (NRF) grants (2021R1A2C4086540) funded by the Korean government (Ministry of Science, ICT & Future Planning) to B.C.L.

ASSOCIATED CONTENT

Notes

The authors have filed a patent application relating to the technology.

ABBREVIATIONS

MsrB, methionine sulfoxide reductase B; cpYFP, circularly permuted yellow fluorescent protein; ROS, Reactive oxygen species; Met, methionine; MetSO, methionine-S-sulfoxide; MetRO, methionine-R-sulfoxide; CaMKII, calcium/calmodulin-dependent protein kinase II; HypT, hypochlorite-responsive transcription factor; MICAL, molecule interacting with CasL; NFI, normailized fluorescence intensity; RFI, relative fluorescence intensity; MRP, methionine rich protein; IgG, immunoglobulin G; BLISA, biosensor-linked immunosorbent assay; NirA, nitrogen assimilation transcription factor nirA; IkBα, nuclear factor of kappa B inhibitor alpha.

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