Ecoengineering Solutions for the Impairment of Spreading and Growth of Invasive Spartina patens in Mediterranean Salt Marshes

ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 1 ORIGINAL RESEARCH published: 30 Augus 2021 doi: 10.3389/ ma s.2021.699528 Edi ed by: S elios Ka sane akis, Uni e si y o he Aegean, G eece Re iewed by: Césa Cos a, Fede al Uni e si y o Rio G ande, B azil Jenneke Visse , Uni e si y o Louisiana a La aye e, Uni ed S a es *Co espondence: Rica do C uz de Ca alho [email p o ec ed] Special y sec ion: This a icle was submi ed o Ma ine Ecosys em Ecology, a sec ion o he jou nal F on ie s in Ma ine Science Recei ed: 23 Ap il 2021 Accep ed: 09 Augus 2021 Published: 30 Augus 2021 Ci a ion: C uz de Ca alho R, Feijão E, Dua e I, Pin o V, Sil a M, Ma os AR, da Sil a AB, Caçado I, Reis-San os P, Fonseca VF and Dua e B (2021) Ecoenginee ing Solu ions o he Impai men o Sp eading and G ow h o In asi e Spa ina pa ens in Medi e anean Sal Ma shes. F on . Ma . Sci. 8:699528. doi: 10.3389/ ma s.2021.699528 Ecoenginee ing Solu ions o he Impai men o Sp eading and G ow h o In asi e Spa ina pa ens in Medi e anean Sal Ma shes Rica do C uz de Ca alho1,2*, Edua do Feijão1, I ina Dua e1, Vanessa Pin o1, Ma isa Sil a1, Ana Ri a Ma os3,4, Anabela Be na des da Sil a3,4, Isabel Caçado 1,3, Pa ick Reis-San os1,5, Vanessa F. Fonseca1,6 and Be na do Dua e1,3 1MARE – Ma ine and En i onmen al Sciences Cen e, Faculdade de Ciências da Uni e sidade de Lisboa, Lisbon, Po ugal, 2cE3c – Cen e o Ecology, E olu ion and En i onmen al Changes, Faculdade de Ciências da Uni e sidade de Lisboa, Lisbon, Po ugal, 3Depa amen o de Biologia Vege al, Faculdade de Ciências, Uni e sidade de Lisboa, Lisbon, Po ugal, 4Biosys ems and Applied Sciences Ins i u e, Faculdade de Ciências da Uni e sidade de Lisboa, Lisbon, Po ugal, 5Sou he n Seas Ecology Labo a o ies, School o Biological Sciences, The Uni e si y o Adelaide, Adelaide, SA, Aus alia, 6Depa amen o de Biologia Animal da Faculdade de Ciências da Uni e sidade de Lisboa, Lisbon, Po ugal The in asion o na u al communi ies by non-indigenous species ep esen s one o he mos se ious h ea s o biodi e si y. Unde s anding he ecophysiology o in asi e species can p o ide insigh s in o po en ial physiological handicaps ela i e o na i e species. By doing so, we can le e age he de elopmen o ecoenginee ing solu ions o he emo al o non-indigenous species, p e e ably using non-chemical me hods. Spa ina pa ens is a known in asi e species o co dg ass agg essi ely p oli e a ing in Medi e anean sal ma shes, p oducing impene able monospeci ic s ands. As i s occu ence is delimi ed by he uppe high ide wa e le el, we hypo hesized ha S. pa ens is in ole an o wa e logging. The e o e, we de eloped a ield expe imen whe e s ands o S. pa ens we e kep wa e logged o e he en i e idal cycle o 30 days. A he end o he expe imen al pe iod, plan s in he ial plo s exhibi ed se e e s ess symp oms a di e en physiological le els compa ed wi h con ol plo s (no in e en ion). A he pho obiological le el, in e ened plan s exhibi ed lowe e iciency in p oducing chemical ene gy om ligh , whils a he biochemical le el wa e logging impai ed he an ioxidan sys em and inc eased lipid pe oxida ion p oduc s. Fu he mo e, he applica ion o chlo ophyll apulse ampli ude modula ed (PAM) luo ome y, a non-in asi e echnique, allowed us o e alua e he e ec i eness o he implemen ed measu es, being he ool ha p o ided he bes sepa a ion be ween he con ol and in e ened popula ion. Conside ing he physiological ai s obse ed he e, ecoenginee ing solu ions F on ie s in Ma ine Science | www. on ie sin.o g 1Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 2 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens based on inc eased wa e logging o S. pa ens s ands, can be a low-cos and e icien measu e o educe he sp eading and g ow h o his in asi e species in he Medi e anean and o he sal ma shes wo ldwide wi h li le dis u bance. Keywo ds: ecological es o a ion, in asi e species, sal ma sh, emo e sensing, halophy es INTRODUCTION Sal ma shes p o ide a wide ange o ecological se ices, including nu se y habi a s o many animals, p o ec ion agains coas al e osion, wa e pu i ica ion, ha ing he conside able capaci y o s o e and seques e ca bon, and a e key playe s in he ecosys em na u al emedia ion capaci y (Cou o e al., 2013; Teixei a e al., 2014;Dua e e al., 2018, 2021), being hese se ices alua ed in se e al millions o eu os pe yea (Dua e e al., 2021). Being p e e ed loca ions o human se lemen and a p o usion o an h opogenic ac i i ies, coas al, and ansi ional a eas ha e been se e ely impac ed in hei heal h and unc ioning wo ldwide. Habi a loss and deg ada ion, clima e change and he in oduc ion o in asi e species a e amongs he majo h ea s o sal ma sh ecosys ems (Dua e e al., 2015, 2018;Repolho e al., 2017;Pé ez-Rome o e al., 2018). The e o e, in he con ex o inc easing deg ada ion a es, he need o es o e sal ma sh ecosys ems has been ecognized as a p io i y by manage s, scien is s, and gene al socie y. This is well-emphasized in he EU Biodi e si y S a egy o 2030 and i s EU Na u e Res o a ion Plan (EU, 2020) and ein o ced by he UN Decade on Ecosys em Res o a ion (UN, 2020). Sal ma shes ha e been widely a ec ed by non-indigenous species (NIS) being a se ious h ea o we land biodi e si y (Heywood, 1989). Al hough many NIS plan s we e in oduced long ago (mo e han a cen u y), ecen a i als a e o much conce n (Aguia and Fe ei a, 2013;Ainouche and G ay, 2016; Ma ínez-Jau egui e al., 2018). The Spa ina genus is highly success ul amongs he halophy e plan g oup, being widesp ead ac oss he globe. These plan s ha e C4- ype pho osyn hesis, in which a CO2concen a ion mechanism a ibulose-1,5- bisphospha e ca boxylase-oxygenase (Rubisco) le el, in ol ing he ixa ion o a mosphe ic CO2by phosphoenol-py u a e ca boxylase (PEPC), inc eases Rubisco ca boxylase ac i i y and allows a as e g ow h a e ( on Caemme e , 2020). Spa ina pa ens (Ai .) Muhl. (G amineae) is a pe ennial g ass dis ibu ed along a wide ange o coas al habi a s, agg essi ely compe ing wi h na i e species (Dua e e al., 2015). I was in oduced in he Medi e anean Sea p obably due o ship a ic om Ame ica, being used as packing ma e ial in ships boxes and Abb e ia ions: APx, asco ba e pe oxidase; CAP, canonical analysis o p incipal coo dina es; CAT, ca alase; DBI, double bond index; DTT, di hio h ei ol; EDTA, e hylenediamine e aace ic acid; ETC, elec on anspo chain; FAME, a y acids me hyl es e s; GOPx, guaiacol pe oxidase; GR, glu a hione educ ase; LC- PUFA, polyunsa u a ed a y acids; MDA, malondialdehyde; NIS, non-indigenous species; PAM, pulse ampli ude modula ed; PCO, p incipal coo dina es analysis; PEP, phosphoenol-py u a e; PEPC, phosphoenol-py u a e ca boxylase; PMSF, phenylme hylsul onyl luo ide; PS I, pho osys em I; PS II, pho osys em II; PVC, poly inyl chlo ide; PVP, poly inylpy olidone; RC, eac ion cen e s; ROS, eac i e oxygen species; Rubisco, ibulose-1,5-bisphospha e ca boxylase-oxygenase; SOD, supe oxide dismu ase; TBA, hioba bi u ic acid; TCA, ichlo oace ic acid; TPF, iphenyl o mazan; TTC, iphenyl- e azolium chlo ide. c a es (Hul én, 1958). The e o e, al hough being p esen o some ime along he Wes e n Medi e anean coas s, i had no been eco ded on he Eas e n Ibe ian coas un il ecen ly (Baumel e al., 2016). Con olling in asi e species is ele an o es o a ion e o s, and se e al app oaches ha e been de eloped o add ess his issue, anging om he bicide applica ion o mowing and physical emo al (Ke e al., 2016). While he i s appea s as a simple and cos -e ec i e me hod (Majo e al., 2003;Pa en e al., 2017), he long- e m e ec s o he bicides may ha e se ious impac s on he ecosys em (C uz de Ca alho e al., 2020b). As o he o he wo me hods, hey will only p e en u he sp ead needing o be cons an ly applied and being e y ime- and cos -consuming (Hedge e al., 2003). The e o e, we conside ed an al e na i e me hod unde pinned on he physiology o he species, in pa icula he ac ha S. pa ens has a low ole ance o wa e logging, due o poo ae a ion o he hizosphe e which consequen ly impai s i s g ow h (Bu dick, 1989;Be ness, 1991; Cu ado e al., 2020), and na u ally limi s his species dis ibu ion o he high ide bo de o sal ma shes. Thus, in he p esen wo k, we es ed he applica ion o a physical ba ie ha would p olong wa e logging a ound he plan s and e alua ed he impac on i s physiology. MATERIALS AND METHODS Plan Ma e ial and Collec ion Si e Sampling occu ed be o e he s a o he g owing season (Feb ua y 2020) in he Ho as sal ma sh (Alcoche e; 38◦ 45.661’ N, 8◦56.116’ W), loca ed in he middle es ua y, adjacen o he Tagus Es ua y Na u al Rese e (Figu e 1). This sal ma sh is looded wice pe day, being domina ed by he halophy e species Spa ina ma i ima in he lowe ma sh (ci ca 12% co e age), Halimione po ulacoides in he mid-uppe ma sh (ci ca 35% co e age), and Sa coco nia u icosa in he uppe ma sh (ci ca 20% co e age) (Caçado e al., 2013). Mo e ecen ly, he halophy e in asi e species S. pa ens has managed o es ablish i sel in he ma gins, jus i ying he impo ance o he cu en s udy. Applied T ea men s Spa ina pa ens plan s we e subjec ed o wo ea men s: (1) con ol, whe e he plan s did no unde go any in e en ion, and (2) he wa e logging ea men (wa e logged) whe e plan u s illed he inside o a 50 cm long and 10 cm diame e PVC ube bu ied down in o he sedimen s, lea ing an 8 cm ma gin ou side, which allowed he idal wa e o en e he ube and emain longe inside and, hus, inc ease he wa e logging o he plan s (n= 5 o each ea men ). The expe imen las ed o 30 days. Se e al F on ie s in Ma ine Science | www. on ie sin.o g 2Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 3 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens FIGURE 1 | Map o he loca ion o he sampling si e in he Ho as sal ma sh (Alcoche e) in he Tagus es ua y. ield measu emen s we e made, namely, in i o chlo ophyll a pulse ampli ude modula ed (PAM) luo ome y measu emen s. Mo eo e , 20 lea es om each ea men we e also collec ed di ec ly in o indi idual ubes wi h liquid ni ogen (see sec ions below). Finally, whole plan s o S. pa ens om bo h ea men s we e also collec ed, anspo ed o he lab, washed, emo ed om excess wa e , weigh ed ( esh weigh ), and o en-d ied a 60◦C o cons an weigh (d y weigh ). Chlo ophyll aPulse Ampli ude Modula ed Fluo ome y Ten lea es om plan s om each ea men we e da k-adap ed o 15 min and PAM measu emen s we e pe o med using a Fluo oPen FP100 (Pho o Sys em Ins umen s, Czechia). Fo he analysis o chlo ophyll ansien ligh cu es (Kau sky plo ), and he de i ed luo ome ic pa ame e s (Table 1), he JIP- es was used (Dua e e al., 2017). Lea In a ed The mog aphy The mal images we e ob ained wi h a FLIR E50bx in a ed came a (FLIR Sys ems, Inc., Wilson ille, OR, Uni ed S a es) p oducing images o 320 ×240 esolu ion wi h an accu acy o ±0.045◦C. Ten lea es we e andomly selec ed om plan s o bo h ea men s, ha ing a wa e bo le a ambien empe a u e nea he lea es as e e ence. The a e age empe a u e o each lea was calcula ed on each image. All image p ocessing and analysis we e pe o med in FLIR Tools so wa e ( e sion 6.4.18039.1003, FLIR Sys ems, Inc.). Roo Respi a ion F esh ine oo s o each ea men weighing app oxima ely 100 mg we e ans e ed o 10-mL eac ion ubes, and 6 mL o TTC-solu ion [0.6% (w/ ) iphenyl- e azolium chlo ide in 0.06 M Na2HPO4–KH2PO4and 0.05% ( / ) Tween 20] was added o each ube (n= 5) (B unne e al., 2002). In duplica e ubes, 0.15 mM KCN was added o de e mine he inhibi ed espi a ion. The samples we e hen incuba ed o 24 h a 25◦C. A e incuba ion, he TTC solu ion was decan ed and iphenyl o mazan (TPF) ex ac ion was made by adding 2 mL o e hanol and boiling a 80◦C o 15 min (Ru and B unne , 2003). A e collec ing he supe na an in new ubes, he abso bance o 1 mL was measu ed a 520 nm wi h a spec opho ome e (UV500 UV- Visible Spec ome e , Unicam, Wal ham, MA, Uni ed S a es). The oo esidues in he es ubes we e d ied a 80◦C o 72 h and weighed. Reduc ion o TTC was calcula ed as µg o TPF p oduced pe hou pe g d y weigh (DW). P oline Quan i ica ion P oline con en was de e mined acco ding o Ba es e al. (1973). Fo each ea men , plan lea es (n= 5) we e homogenized in 3% aqueous sul osalicylic acid and he homogena e cen i uged a 9,000 g o 15 min a 0◦C (Sigma F on ie s in Ma ine Science | www. on ie sin.o g 3Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 4 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens TABLE 1 | Fluo ome ic analysis pa ame e s and hei desc ip ion. JIP- es A ea Co esponds o he oxidized quinone pool size a ailable o educ ion and is a unc ion o he a ea abo e he Kau sky plo N Reac ion cen e u no e a e SMCo esponds o he ene gy needed o close all eac ion cen e s M0Ne a e o PS II RC closu e γRC P obabili y ha a PS II chlo ophyll molecule unc ion as a RC 9Eo P obabili y ha an abso bed pho on will mo e an elec on in o he ETC ϕ0P obabili y ha a apped exci a ion mo es an elec on in o he ETC beyond QA δRo E iciency o he ans e o an elec on om PQH2 o inal PS I accep o s RE0/RC Flux o elec ons ans e ed om PQH2 o inal PSI accep o s pe ac i e PS II ABS/CS Abso bed ene gy lux pe c oss-sec ion TR0/CS T apped ene gy lux pe c oss-sec ion ET0/CS Elec on anspo ene gy lux pe c oss-sec ion DI0/CS Dissipa ed ene gy lux pe c oss-sec ion RC/CS Numbe o a ailable eac ion cen e s pe c oss-sec ion PGG ouping p obabili y o he connec i i y be ween he wo PS II uni s δRo/(1-δRo) Con ibu ion o PSI, educing i s end accep o s 90/(1- 90) Con ibu ion o he da k eac ions om QA− o PC 9Eo/(1- 9Eo) Equilib ium cons an o he edox eac ions be ween PS II and PS I RC/ABS Reac ion cen e II densi y wi hin he an enna chlo ophyll bed o PS II TR0/DI0Con ibu ion o pa ial pe o mance due o he ligh eac ions o p ima y pho ochemis y SFI S uc u e unc ional index o pho osyn hesis SFI (NO) Non-pho osyn he ic o dissipa ion s uc u e unc ional index 2-16K, SIGMA Labo zen i ugen GmbH, Os e ode am Ha z, Ge many). The supe na an was collec ed, and he eac ion consis ed o 2 mL o ex ac combined wi h 2 mL o glacial ace ic acid and 2 mL o acid ninhyd in. The eac ion occu ed o 1 h a 100◦C, a e which he eac ion was s opped in an ice ba h. The eac ion mix u e was ex ac ed wi h 4 mL o oluene and i s abso bance ead a 520 nm wi h a spec opho ome e (UV500 UV-Visible Spec ome e , Unicam, Wal ham, MA, Uni ed S a es) and compa ed wi h a s anda d cu e o p oline, exp essed in µmol g−1 esh weigh (FW). C4-Pho osyn he ic Ca boxyla ing Enzymes Ac i i y and Pigmen Analysis Ca boxyla ing enzymes, PEPC and Rubisco, we e ex ac ed om ozen lea samples acco ding o Ca mo-Sil a e al. (2008), excep ha 50 mM HEPES-KOH pH 7.3 was used and 0.5% ( / ) T i on X-100 added. B ie ly, app oxima ely 50 mg FW we e ex ac ed in a cold mo a con aining qua z sand, 1% (w/ ) insoluble poly inylpy olidone (PVP) and 1 mL o ice-cold ex ac ion medium [50 mM HEPES-KOH pH 7.3, 1 mM EDTA, 5% (w/ ) PVP25000, 6% (w/ ) polye hylene glycol (PEG4000), 10 mM di hio h ei ol (DTT), 1% ( / ) p o ease cock ail inhibi o (Sigma, S Louis, MO, Uni ed S a es) and 0.5% ( / ) T i on X-100]. A e aking aliquo s o pigmen analysis, he homogena e was cen i uged o 3 min a 16,800 g a 4◦C (Sigma 2-16K, SIGMA Labo zen i ugen GmbH, Os e ode am Ha z, Ge many) and he supe na an (c ude ex ac ) was kep a 4◦C and immedia ely used o measu ing he ac i i ies o Rubisco (EC 4.1.1.39) and PEPC (EC 4.1.1.31). The ac i i ies o Rubisco we e assayed a 25◦C by 14CO2 inco po a ion in o acid-s able p oduc s acco ding o Pa y e al. (1997) o wi h modi ica ions (Co eia e al., 2020). The assay medium (1 mL pe sample) con ained 50 mM Bicine-KOH pH 8.2, 40 mM MgCl2, 10 mM NaH14CO3(7.4 kBq µM−1) and 0.4 mM ibulose-1,5-bisphospha e (RuBP). To measu e Rubisco ini ial ac i i y (Vi), 25 µL o c ude ex ac was added o assay medium, and he eac ion s opped a e 1 min wi h he addi ion o 100 µL o 1 M HCl. To de e mine Rubisco o al ac i i y (V ), 25 µL o c ude ex ac was added o assay medium wi hou RuBP o 3 min, o allow he ca bamyla ion o enzyme ca aly ic si es. Rubisco V eac ion was s a ed by adding RuBP and s opped a e 1 min wi h he addi ion o 100 µL o 1 M HCl. The mix u e was comple ely d ied a 60◦C a e which he esidue was esuspended in 0.5 mL o dis illed wa e and mixed wi h 5 mL o scin illa ion liquid (BioSa e LS Cock ail, Beckman, Uni ed S a es). Radioac i i y o he 14C inco po a ed in he acid- s able p oduc s was measu ed by scin illa ion coun ing (LS 7800 spec opho ome e , Beckmann Ins umen s Inc., Fulle on, CA, Uni ed S a es). Rubisco ac i a ion s a e (%) was de e mined by he Vi/V a io. PEPC physiological (Vphysiol) and maximum (Vmax) ac i i ies we e measu ed in a con inuous assay a 340 nm and 25◦C (UV500 UV-Visible spec opho ome e , Unicam, Camb idge, Uni ed Kingdom) acco ding o Bak im e al. (1992) wi h some modi ica ions (Ca mo-Sil a e al., 2007). The eac ion mix u e o Vphysiol (1 mL) consis ed o 50 mM HEPES-KOH pH 7.2, 10 mM MgCl2, 10 mM NaHCO3, 2.5 mM PEP (Sigma), 12 uni s o MDH (Sigma) and 20 µL o c ude ex ac . Fo Vmax, he eac ion mix u e consis ed o 50 mM HEPES-KOH pH 8.0, 10 mM MgCl2, 10 mM NaHCO3, 10 mM PEP (Sigma), 12 uni s o MDH (Sigma) and 20 µL o ex ac . In bo h cases, he eac ion was s a ed by he addi ion o 0.2 mM ( inal concen a ion) NADH (Sigma). Each measu ed ac i i y is he mean o h ee eplica e on he same ex ac . PEPC ac i a ion s a e (%) was calcula ed as Vphysiol/Vmax a io. Fo pigmen analysis, each 20 µL aliquo p e iously e ie ed om he lea ex ac was dilu ed in 980 µL o me hanol. A e mixing in he o ex, he samples we e le in he da k a 4◦C o e nigh . A e cen i uging o 1 min a 13,000 g a 4◦C (Sigma 2-16K, SIGMA Labo zen i ugen GmbH, Os e ode am Ha z, Ge many), he abso bance a 470, 652.4, 665.2, and 700 nm we e measu ed in an EpochTM 2 Mic opla e Spec opho ome e (BioTek, Winooski, VT, Uni ed S a es). Pigmen concen a ions we e de e mined acco ding o he equa ions in Lich en hale and Buschmann (2001). An ioxidan Enzyme Assays To ex ac he soluble p o ein ac ion, lea samples we e g inded in a cooled mo a wi h 0.5 mL o 50 mM sodium/po assium phospha e ex ac ion bu e (wi h 0.1 mM Na-EDTA, 2 mM PVP, 10 mM DTT, 0.1 mM PMSF, and 24 µM NADP, pH 7.6). F on ie s in Ma ine Science | www. on ie sin.o g 4Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 5 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens The homogena e was cen i uged a 13,000 g o 10 min a 4◦C (Sigma 2-16K, SIGMA Labo zen i ugen GmbH, Os e ode am Ha z, Ge many) and he supe na an was collec ed o a new ube. P o ein concen a ion was de e mined acco ding o B ad o d (1976) in an EpochTM 2 Mic opla e Spec opho ome e (BioTek, Winooski, VT, Uni ed S a es). Ca alase (CAT; EC 1.11.1.6) ac i i y was measu ed acco ding o Te anishi e al. (1974), h ough H2O2consump ion moni o ing and he dec ease in abso bance a 240 nm (ε= 39.4 mM−1cm−1). The eac ion mix u e con ained 50 mM o sodium/po assium phospha e bu e (pH 7.0), and 30 mM o H2O2wi h he eac ion being s a ed by he addi ion o 5 µL o ex ac . Asco ba e pe oxidase (APx; EC 1.11.1.11) was assayed acco ding o Ti yakioglu e al. (2006). The eac ion mix u e con ained 50 mM o sodium/po assium phospha e bu e (pH 7.0), 0.1 mM o H2O2, and 0.25 mM L-asco ba e, and he eac ion was also ini ia ed wi h he addi ion o 5 µL o he ex ac . The ac i i y was eco ded as he dec ease in abso bance a 290 nm and he amoun o asco ba e oxidized calcula ed om he mola ex inc ion coe icien (ε= 2.8 mM−1cm−1). Guaiacol pe oxidase (GOPx; EC 1.11.1.7) ac i i y was assayed acco ding o Mika and Lü hje (2003) h ough he moni o iza ion o guaiacol oxida ion a 470 nm (ε= 26.6 mM−1cm−1). The eac ion mix u e con ained 50 mM o sodium/po assium phospha e bu e (pH 7.0), 10 mM o H2O2, and 8 mM guaiacol, and he eac ion was ini ia ed wi h he addi ion o 5 µL o he ex ac . Supe oxide dismu ase (SOD; EC 1.15.1.1) ac i i y was assayed acco ding o Ma klund and Ma klund (1974) by measu ing he educ ion o py ogallol a 325 nm. The eac ion mix u e con ained 30 mM o sodium/po assium phospha e bu e (pH 7.0) and 0.24 mM o py ogallol, wi h he eac ion being s a ed by he addi ion o 5 µL o ex ac . Glu a hione educ ase (GR; EC 1.8.1.7) ac i i y was assayed acco ding o Edwa ds e al. (1990) by measu ing he all in abso bance a 340 nm as NADPH was oxidized (ε= 6.22 mM−1cm−1). The eac ion mix u e con ained 25 mM o sodium/po assium phospha e ex ac ion bu e (pH 7.6), 0.5 mM o oxidized glu a hione and 0.2 mM NADPH, s a ing he eac ion by he addi ion o 5 µL o ex ac . Con ol assays we e done in he absence o subs a e o e alua e he au oxida ion o he subs a es. All assays we e pe o med in a o al olume o 200 µL pe well a 25◦C in an EpochTM 2 Mic opla e Spec opho ome e (BioTek, Winooski, VT, Uni ed S a es). Lipid Pe oxida ion Analysis Lipid pe oxida ion p oduc s we e de e mined as p e iously desc ibed (Hea h and Packe , 1968). Lea es we e homogenized b ie ly in 1.5 mL o 10% ( / ) ichlo oace ic acid (TCA), con aining 0.4% (w/ ) hioba bi u ic acid (TBA). The eac ion was conduc ed a 100◦C o 30 min, being hal ed h ough placemen in ice. A e cen i uga ion a 15,000 g o 10 min a 4◦C (Sigma 2-16K, SIGMA Labo zen i ugen GmbH, Os e ode am Ha z, Ge many), 1 mL o he supe na an was collec ed and mixed wi h 1 mL o 0.4% TBA and incuba ed again unde he same condi ions. The abso bance o he supe na an was eco ded a 532 and 600 nm by spec opho ome y (UV500 UV- Visible Spec ome e , Unicam, Wal ham, MA, Uni ed S a es). The concen a ion o malondialdehyde (MDA) was de e mined using he mola ex inc ion coe icien (ε= 155 mM−1cm−1). Fa y Acid P o iles The analysis o a y acid was pe o med by di ec ans- es e i ica ion o lea samples, in eshly p epa ed me hanol sulphu ic acid (97.5:2.5, / ), a 70◦C o 60 min, using he in e nal s anda d pen adecanoic acid (C15:0) (Feijão e al., 2018). Fa y acid me hyl es e s (FAME) we e eco e ed using pe oleum e he , d ied wi h an N2 low, and e-suspended in an adequa e amoun o hexane. The FAME solu ion was analyzed h ough gas ch oma og aphy (Va ian 430-GC gas ch oma og aph equipped wi h a hyd ogen lame ioniza ion de ec o se a 300◦C, Middelbu g, Ne he lands), by addi ion o 1 µL, se ing he injec o empe a u e o 270◦C, wi h a spli a io o 50. The used-silica capilla y column (50 m ×0.25 mm; WCOT Fused Silica, CP-Sil 88 o FAME; Va ian, Middelbu g, Ne he lands) was main ained a a cons an ni ogen low o 2.0 mL min−1and he o en se o 190◦C. Fa y acids iden i ica ion was pe o med by compa ison o e en ion imes wi h s anda ds (Sigma-Ald ich) and ch oma og ams we e analyzed by he peak su ace me hod, using he Galaxy so wa e. The double bond index (DBI) was calcula ed, o de e mine he memb ane sa u a ion le els, as p e iously desc ibed (Feijão e al., 2018): DBI =2×% monoenes +2×% dienes +3×% ienes 100 (1) S a is ical Analysis Since he da a lacked no mali y and homogenei y, he s a is ical analysis was based on Mann-Whi ney non-pa ame ic es s (G aphPad P ism 8.4.2 o Windows, G aphPad So wa e, San Diego, CA, Uni ed S a es). Mul i a ia e s a is ical analyses [SIMPER and Canonical Analysis o P incipal Coo dina es (CAP)] we e pe o med using P ime 6 so wa e (Cla ke and Go ley, 2006). The da a ob ained om he Kau sky plo s, he mog aphy da a, oxida i e s ess and a y acids we e used FIGURE 2 | Chlo ophyll ansien kine ics (Kau sky plo s) in lea es o con ol plan s (da k g ay) and wa e logged plan s (ligh g ay) o Spa ina pa ens a e 30 days (mean ±s.d., n= 10). F on ie s in Ma ine Science | www. on ie sin.o g 5Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 6 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens FIGURE 3 | Boxplo s o lea ene gy luxes [A, abso bed (ABS/CS); B, apped (TR0/CS); C, anspo ed (ET0/CS); D, dissipa ed (DI0/CS)] and (E) he numbe o a ailable eac ion cen e s pe c oss-sec ion (RC/CS) in lea es o con ol plan s (whi e boxes) and wa e logged plan s (g ay boxes) o Spa ina pa ens a e 30 days (n= 10, di e en le e s indica e signi ican di e ences a p<0.05). as he basis o he cons uc ion o he espec i e esemblance ma ixes based on he Euclidean dis ances be ween samples. To e alua e he di e en me abolic da ase s ob ained as a whole (in opposi ion o uni a ia e analysis), s a is ical mul i a ia e models based on Kau sky plo s, he mog aphy da a, oxida i e s ess and a y acids we e gene a ed using P incipal Coo dina es Analysis (PCO) (Cla ke and Go ley, 2006). RESULTS Chlo ophyll aPAM Analysis Obse ing he Kau sky plo s esul an om he in i o PAM luo ome ic analysis, lowe luo escence alues could be obse ed in plan s subjec ed o he ea men when compa ed wi h con ol plan s (Figu e 2). F on ie s in Ma ine Science | www. on ie sin.o g 6Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 7 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens FIGURE 4 | Boxplo s o he pho osys em II and ETC ela ed pho ochemical ai s [A, oxidized quinone pool; B, eac ion cen e u no e a e (N); C, he ene gy needed o close all eac ion cen e s (SM); D, he p obabili y ha a PSII chlo ophyll molecule unc ion as a RC (γRC); E, ne a e o PS II RC closu e (M0)], in lea es o con ol plan s (whi e boxes) and wa e logged plan s (g ay boxes) o Spa ina pa ens a e 30 days (n= 10, di e en le e s indica e signi ican di e ences a p<0.05). The ou ene gy luxes [Figu e 3: A, ene gy abso bed by he pho osys em II (PS II) an ennae (ABS/CS); B, ene gy apped inside he PS II (TR0/CS); C, ene gy anspo ed wi hin he elec on anspo chain (ETC) (ET0/CS); and D, he ene gy dissipa ion lux (DI0/CS)] showed he same pa e n p esen ing lowe alues in he F on ie s in Ma ine Science | www. on ie sin.o g 7Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 8 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens FIGURE 5 | Boxplo s o he pho osys ems I (PS I) and II (PS II) pho ochemical ai s. (A) Ac i e oxygen-e ol ing complexes (OECs); (B) g ouping p obabili y be ween he wo PS II uni s (PG); (C) he con ibu ion o he da k eac ions om quinone A o plas oquinone [ψ0/(1 - ψ0)]; (D) he equilib ium cons an o he edox eac ions be ween PS II and PS I [ψE0/(1 - ψE0)]; (E) elec on anspo om PQH2 o he educ ion o PS I end elec on accep o s (RE0/RC); (F) he con ibu ion o PS I educing i s end accep o s [δR0/(1- δR0)]; (G) eac ion cen e II densi y wi hin he an enna chlo ophyll bed o PS II (RC/ABS); (H) con ibu ion o pa ial pe o mance due o he ligh eac ions o p ima y pho ochemis y (TR0/DI0), in lea es o con ol plan s (whi e boxes) and wa e logged plan s (g ay boxes) o Spa ina pa ens a e 30 days (n= 10, di e en le e s indica e signi ican di e ences a p<0.05). F on ie s in Ma ine Science | www. on ie sin.o g 8Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 9 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens FIGURE 6 | Boxplo s o he s uc u e unc ional indexes o pho osyn hesis (A, SFI) and non-pho osyn he ic o dissipa ion p ocesses [B, SFI (NO)] in lea es o con ol plan s (whi e boxes) and wa e logged plan s (g ay boxes) o Spa ina pa ens a e 30 days (n= 10, di e en le e s indica e signi ican di e ences a p<0.05). FIGURE 7 | Boxplo s o he lea su ace empe a u e measu ed h ough in a ed he mog aphy in lea es o con ol plan s (whi e box) and wa e logged plan s (g ay box) o Spa ina pa ens a e 30 days (n= 10, di e en le e s indica e signi ican di e ences a p<0.05). wa e logged plan s, al hough ha dec ease was only s a is ically signi ican o TR0/CS and ET0/CS. I was also a simila educ ion in he numbe o oxidized PS II eac ion cen e s (RC/CS) (Figu e 3E). Fu he analysis o he unc ioning o di e en componen s o he pho osys ems and ETC (Figu e 4) showed a dec ease in he oxidized quinone pool size in he wa e logged plan s, ollowed by an enhancemen in he numbe o QA edox u no e s un il maximum luo escence was eached (N). Al hough no signi ican changes we e obse ed in he ene gy needed o close all RCs (SM), he e was a dec ease in he p obabili y o a PS II chlo ophyll molecule unc ioning as a RC (γRC) in he ea ed plan s. Howe e , no signi ican di e ences we e obse ed in he QA educ ion a e (M0). Al hough he ac i e oxygen-e ol ing complexes (OEC) showed no di e ences be ween con ol and wa e logged plan s (Figu e 5A), he PG, he g ouping p obabili y ha co ela es wi h he disconnec ion be ween he wo PS II uni s, inc eased in he la e plan g oup (Figu e 5B). Rega ding PS II and PS I, wa e logged plan s p esen ed a signi ican dec ease in pho ochemical p ocesses, bo h in he con ibu ion o ligh (TR0/DI0;Figu e 5H) and da k [ψ0/(1 -ψ0); Figu e 5C] eac ions o he pho ochemical cycle. On he o he hand, a he PS I le el he e was a signi ican inc ease in he ac i i y o his pho osys em [δR0/(1 - δR0); Figu e 5F] in he in e ened plan s, al hough he e was a dec ease in he equilib ium cons an o he edox eac ion be ween bo h pho osys ems owa d he PS II [ψE0/(1 - ψE0); Figu e 5D]. Ne e heless, he e we e no signi ican changes in he eac ion cen e densi y wi hin he PS II an enna chlo ophyll bed (RC/ABS; Figu e 5G) o in he elec on anspo om PQH2 o he educ ion o he PS I end accep o s (RE0/RC; Figu e 5E). In summa y and obse ing he s uc u e unc ional indexes, he e was a dec ease o he pho ochemical p ocesses (Figu e 6A) and an inc ease o he non-pho ochemical o dissipa i e p ocesses (Figu e 6B) in he wa e logged plan s. Lea The mog aphy Rega ding lea su ace empe a u e measu ed h ough in a ed he mog aphy, he e was a s a is ically signi ican inc ease in empe a u e in he ea ed plan s (11.06◦C) ela i ely o con ol ones (10.77◦C) (Figu e 7). F on ie s in Ma ine Science | www. on ie sin.o g 9Augus 2021 | Volume 8 | A icle 699528 ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 16 C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens S. pa ens. Al hough long- e m s udies need o be pe o med, his ecoenginee ing solu ion has he po en ial o con ol and elimina e his species om sal ma shes and o he in e idal sys ems in u u e ecosys em es o a ion p og ams. Since S. pa ens u s a e easily iden i ied, he upscaling o his echnique could in ol e he applica ion o ubes wi h di e en diame e s acco ding o plan u s densi y, allowing wa e logging o be p olonged in space and ime. Fu he mo e, he applica ion o a simple bio-op ical ool will allow he s akeholde s o easily ollow he p ocess o supp essing he species wi hou he in e e ence o he p ocess and allowing epea ed measu es o e he in e en ion pe iod. DATA AVAILABILITY STATEMENT The aw da a suppo ing he conclusions o his a icle will be made a ailable by he au ho s, wi hou undue ese a ion. AUTHOR CONTRIBUTIONS BD, VF, and PR-S: concep ualiza ion. BD: me hodology, supe ision, p ojec adminis a ion, and unding acquisi ion. RC and BD: o mal analysis. RC, EF, ID, VP, MS, AS, and AM: in es iga ion. RC: da a cu a ion and w i ing—o iginal d a p epa a ion. EF, ID, VP, MS, AM, AS, IC, PR-S, VF, and BD: w i ing— e iew and edi ing. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . FUNDING BD and VF we e suppo ed by in es iga ion con ac s (CEECIND/00511/2017 and DL57/2016/CP1479/CT0024). PR-S was suppo ed by FCT h ough a pos doc o al g an (SFRH/BPD/95784/2013). We would like o hank he Fundação pa a a Ciência e a Tecnologia (FCT) o unding he esea ch ia p ojec g an s PTDC/CTA-AMB/30056/2017 (OPTOX), UID/MAR/04292/2019, and UIDB/04046/2020. We would also like o hank he MAR2020 p og am h ough he p ojec RESTAURA2020 (16-01-04-FMP-0014). SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h ps://www. on ie sin.o g/a icles/10.3389/ ma s. 2021.699528/ ull#supplemen a y-ma e ial REFERENCES Aguia , F. C. F., and Fe ei a, M. T. (2013). Plan in asions in he i e s o he Ibe ian Peninsula, sou h-wes e n Eu ope: A e iew. Plan Biol. 147, 1107–1119. doi: 10.1080/11263504.2013.861539 Ainouche, M., and G ay, A. (2016). In asi e Spa ina: lessons and challenges. Biol. In as. 18, 2119–2122. doi: 10.1007/s10530-016-1201-7 Aziz, I., Gul, B., Gulza , S., and Ajmal Khan, M. (2011). Seasonal a ia ions in plan wa e s a us o ou dese halophy es om semi-a id egion o Ka achi. Pak. J. Bo . 43, 587–594. Bailey-Se es, J., and Chang, R. (2005). 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