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). Sensing and signalling in esponse o
oxygen dep i a ion in plan s and o he o ganisms. Ann. Bo . 96, 507–518.
doi: 10.1093/aob/mci206
Bak im, N., Eche a ia, C., C e in, C., A io-Dupon , M., Pie e, J. N., Vidal, J.,
e al. (1992). Regula o y phospho yla ion o So ghum lea phosphoenolpy u a e
ca boxylase. Iden i ica ion o he p o ein-se ine kinase and some elemen s o
he signal- ansduc ion cascade. Eu . J. Biochem. 204, 821–830. doi: 10.1111/j.
1432-1033.1992. b16701.x
Ba es, L. S., Wald en, R. P., and Tea e, I. D. (1973). Rapid de e mina ion o
ee p oline o wa e -s ess s udies. Plan Soil 39, 205–207. doi: 10.1007/
BF00018060
Baumel, A., Rousseau-Gueu in, M., Sapienza-Bianchi, C., Ga eil, A., Duong,
N., Rousseau, H., e al. (2016). Spa ina e sicolo Fab e: Ano he case o
Spa ina ans-A lan ic in oduc ion? Biol. In asions 18, 2123–2135. doi: 10.
1007/s10530-016-1128-z
Bax e , A., Mi le , R., and Suzuki, N. (2014). ROS as key playe s in plan s ess
signalling. J. Exp. Bo . 65, 1229–1240. doi: 10.1093/jxb/e 375
Be ness, M. D. (1991). Zona ion o Spa ina pa ens and Spa ina al e ni lo a
in New England Sal Ma sh. Ecology 72, 138–148. doi: 10.2307/19
38909
B ad o d, M. M. (1976). A apid and sensi i e me hod o he quan i a ion o
mic og am quan i ies o p o ein u ilizing he p inciple o p o ein-dye binding.
Anal. Biochem. 72, 248–254. doi: 10.1016/0003-2697(76)90527-3
B iens, M., and La he , F. (1982). Osmo egula ion in halophy ic highe
plan s: a compa a i e s udy o soluble ca bohyd a es, polyols, be aines and
ee p oline. Plan Cell En i on. 5, 287–292. doi: 10.1111/1365-3040.ep115
72682
B unne , I., B odbeck, S., and Wal he , L. (2002). Fine oo chemis y, s a ch
concen a ion, and “ i ali y” o subalpine coni e o es s in ela ion o soil pH.
Fo . Ecol. Manage 165, 75–84. doi: 10.1016/S0378-1127(01)00633-8
Bu dick, D. M. (1989). Roo ae enchyma de elopmen in Spa ina pa ens in
esponse o looding. Am. J. Bo . 76, 777–780. doi: 10.2307/2444425
Bu dick, D. M., and Mendelssohn, I. A. (1987). Wa e logging esponses in
dune, swale and ma sh popula ions o Spa ina pa ens unde ield condi ions.
Oecologia 74, 321–329. doi: 10.1007/BF00378924
Caçado , I., Ne o, J. M., Dua e, B., Ba oso, D. V., Pin o, M., and Ma ques, J. C.
(2013). De elopmen o an Angiospe m Quali y Assessmen Index (AQuA-
Index) o ecological quali y e alua ion o Po uguese wa e bodies - A
mul i-me ic app oach. Ecol. Indic. 25, 141–148. doi: 10.1016/j.ecolind.2012.
09.021
Ca mo-Sil a, A. E., Powe s, S. J., Keys, A. J., A abaça, M. C., and Pa y, M. A. J.
(2008). Pho o espi a ion in C4 g asses emains slow unde d ough condi ions.
Plan Cell En i on. 31, 925–940. doi: 10.1111/j.1365-3040.2008.01805.x
Ca mo-Sil a, A. E., Soa es, A. S., Ma ques, da Sil a, J., Be na des da Sil a, A.,
Keys, A. J., e al. (2007). Pho osyn he ic esponses o h ee C4 g asses o
di e en me abolic sub ypes o wa e de ici . Func . Plan Biol. 34, 204–213.
doi: 10.1071/FP06278
Cla ke, K. R., and Go ley, R. N. (2006). PRIMER 6: Use Manual/Tu o ial.
England: Plymou h.
Co eia, P. M. P., Sil a, A. B., Roi sch, T., Ca mo−Sil a, E., and Ma ques da Sil a, J.
(2020). Pho op o ec ion and op imiza ion o suc ose usage con ibu e o as e
eco e y o pho osyn hesis a e wa e de ici a high empe a u es in whea .
Physiol. Plan 2020:13227. doi: 10.1111/ppl.13227
Cousins, A. B., Adam, N. R., Wall, G. W., Kimball, B. A., Pin e , P. J., O man,
M. J., e al. (2002). Pho osys em II ene gy use, non-pho ochemical quenching
and he xan hophyll cycle in So ghum bicolo g own unde d ough and ee-
ai CO2 en ichmen (FACE) condi ions. Plan . Cell En i on. 25, 1551–1559.
doi: 10.1046/j.1365-3040.2002.00935.x
Cou o, T., Dua e, B., Ba oso, D., Caçado , I., and Ma ques, J. C. (2013).
Halophy es as sou ces o me als in es ua ine sys ems wi h low le els
o con amina ion. Func ion. Plan Biol. 2013, 931–939. doi: 10.1071/FP
12300
C uz de Ca alho, R., Feijão, E., Kle schkus, E., Ma ques, J. C., Reis-San os, P.,
Fonseca, V. F., e al. (2020a). Halophy e bio-op ical pheno yping: A mul i a ia e
F on ie s in Ma ine Science | www. on ie sin.o g 16 Augus 2021 | Volume 8 | A icle 699528

ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 17
C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens
pho ochemical p essu e index (Mul i-PPI) o classi y sal ma sh an h opogenic
p essu es le els. Ecol. Indic. 119:106816. doi: 10.1016/j.ecolind.2020.1
06816
C uz de Ca alho, R., Feijão, E., Ma os, A. R., Cab i a, M. T., No ais, S. C.,
Lemos, M. F. L., e al. (2020b). Glyphosa e-Based He bicide Toxicophenomics
in Ma ine Dia oms: Impac s on P ima y P oduc ion and Physiological Fi ness.
Appl. Sci. 10:10217391. doi: 10.3390/app10217391
Cu ado, G., Gallego-Té a , B., Figue oa, E., and Cas illo, J. M. (2020). E ec s o
emo al o alien Spa ina densi lo a and es o a ion o na i e S. ma i ima on
succession and zona ion in Eu opean sal ma shes. Es ua . Coas . Shel Sci.
244:105815. doi: 10.1016/j.ecss.2018.04.011
Demmig-Adams, B., and Adams, W. W. (1992). Pho op o ec ion and
o he esponses o plan s o high ligh s ess. A ailable online a :
www.annual e iews.o g (accessed Ap il 23, 2021).
Dua e, B., Bae a, A., Rousseau-Gueu in, M., Ainouche, M., Ma ques, J. C., and
Caçado , I. (2015). A ale o wo spa inas: Clima ic, pho obiological and
iso opic insigh s on he i ness o non-indigenous e sus na i e species. Es ua .
Coas . Shel Sci. 167, 178–190. doi: 10.1016/j.ecss.2015.06.015
Dua e, B., Ca ei as, J., and Caçado , I. (2021). Clima e Change Impac s on Sal
Ma sh Blue Ca bon, Ni ogen and Phospho ous S ocks and Ecosys em Se ices.
Appl. Sci. 11, 1–18. doi: 10.3390/app11041969
Dua e, B., Cou o, T., F ei as, J., Valen im, J., Sil a, H., Ma ques, J. C., e al. (2013).
Abio ic modula ion o Spa ina ma i ima pho obiology in di e en la i udinal
popula ions. Es ua . Coas . Shel Sci. 130, 127–137. doi: 10.1016/j.ecss.2013.
02.008
Dua e, B., Feijão, E., C uz de Ca alho, R., Dua e, I. A., Sil a, M., Ma os, A. R.,
e al. (2020). E ec s o P op anolol on G ow h, Lipids and Ene gy Me abolism
and Oxida i e S ess Response o Phaeodac ylum ico nu um. Biology 9:478.
doi: 10.3390/biology9120478
Dua e, B., Ma ques, J. C., and Caçado , I. (2016). Ecophysiological esponse
o na i e and in asi e Spa ina species o ex eme empe a u e e en s in
Medi e anean ma shes. Biol. In asions 18, 2189–2205. doi: 10.1007/s10530-
015-0958-4
Dua e, B., Ped o, S. S., Ma ques, J. C., Adão, H., and Caçado , I. (2017). Zos e a
nol ii de elopmen p obing using chlo ophyll a ansien analysis ({JIP}- es )
unde ield condi ions: In eg a ing physiological insigh s in o a pho ochemical
s ess index. Ecol. Indic. 76, 219–229. doi: 10.1016/j.ecolind.2017.
01.023
Dua e, C. M., Poine , I., and Gunn, J. (2018). Pe spec i es on a Global Obse ing
Sys em o Assess Ocean Heal h. F on . Ma . Sci. 5:265. doi: 10.3389/ ma s.2018.
00265
Edwa ds, E. A., Raws ho ne, S., and Mullineaux, P. M. (1990). Subcellula
dis ibu ion o mul iple o ms o glu a hione educ ase in lea es o pea (Pisum
sa i um L.). Plan a 180, 278–284. doi: 10.1007/BF00194008
EU (2020). Communica ion om he Commission o he Eu opean Pa liamen ,
he Council, he Eu opean Economic and Social Commi ee and he Commi ee
o he Regions - EU Biodi e si y S a egy o 2030. A ailable online a :
h ps://eu -lex.eu opa.eu/legal-con en /EN/TXT/HTML/?u i=CELEX:
52020DC0380& om=EN. (accessed Ap il 16, 2021).
Feijão, E., Gamei o, C., F anzi a, M., Dua e, B., Caçado , I., Cab i a, M. T., e al.
(2018). Hea wa e impac s on he model dia om Phaeodac ylum ico nu um:
Sea ching o pho ochemical and a y acid bioma ke s o he mal s ess. Ecol.
Indic. 95, 1026–1037. doi: 10.1016/j.ecolind.2017.07.058
Hea h, R. L., and Packe , L. (1968). Pho ope oxida ion in isola ed chlo oplas s. I.
Kine ics and s oichiome y o a y acid pe oxida ion. A ch. Biochem. Biophys.
125, 189–198. doi: 10.1016/0003-9861(68)90654-1
Hedge, P., K iwoken, L., and Pa en, K. (2003). A Re iew o Spa ina Managemen
in Washing on S a e. US. J. Aqua . Plan Manag. 41, 82–90.
Heywood, V. (1989). “Pa e ns, ex en s and modes o in asions by e es ial
plan s,” in Biological in asions: a global pe spec i e, eds M. D ake, J. A. Mooney,
H. A. di Cas i, F. G o es, R. H. K uge , F. J. Rejmanek, e al. (Chiches e : John
Wiley), 31–60.
Hul én, E. (1958). he amphi-a lan ic plan s and hei phy ogeog aphical connec ions.
S ockholm: Almquis & Wiksell.
Iqbal, N., Uma , S., and Naza , R. (2014). “Manipula ing Osmoly es o B eeding
Salini y-Tole an Plan s,” in Eme ging Technologies and Managemen o C op
S ess Tole ance, (Ams e dam: Else ie Inc.), 385–404. doi: 10.1016/B978-0-12-
800875-1.00016-8
Jolio , P., and Jolio , A. (2002). Cyclic elec on ans e in plan lea . P oc. Na l.
Acad. Sci. U. S. A. 99, 10209–10214. doi: 10.1073/pnas.102306999
Kalaji, H. M., Bosa, K., and Ko, J. (2011). E ec s o sal s ess on pho osys em II
e iciency and CO 2 assimila ion o wo Sy ian ba ley land aces. En i on. Exp.
Bo . 73, 64–72. doi: 10.1016/j.en expbo .2010.10.009
Ke , D. W., Hogle, I. B., O , B. S., and Tho n on, W. J. (2016). A e iew o 15
yea s o Spa ina managemen in he San F ancisco Es ua y. Biol. In asions 18,
2247–2266. doi: 10.1007/s10530-016-1178-2
Kobayashi, K., Endo, K., and Wada, H. (2016). “Roles o Lipids in Pho osyn hesis,”
in Lipids in Plan and Algae De elopmen , eds Y. Nakamu a and Y. Li-Beisson
(Cham: Sp inge In e na ional Publishing), 21–49. doi: 10.1007/978-3-319-
25979-6_2
Koy o, H.-W., Geissle , N., Hussin, S., and Huchze meye , B. (2006). Mechanisms
O Cash C op Halophy es To Main ain Yields And Reclaim Saline Soils In A id
A eas in Ecophysiology o High Salini y Tole an Plan s. Ne he lands: Sp inge ,
345–366. doi: 10.1007/1-4020-4018-0_22
Lich en hale , H. K., and Buschmann, C. (2001). Chlo ophylls and Ca o enoids:
Measu emen and Cha ac e iza ion by UV-VIS Spec oscopy. Cu . P o oc.
Food Anal. Chem. 1, 1–.4. doi: 10.1002/0471142913. a 0403s01
Majo , W., G ue, C., G assley, J., and Conques , L. (2003). Mechanical and
Chemical Con ol o Smoo h Co dg ass in Waillapa Bay, Washing on. J. Aqua .
Plan Manag. 41, 6–12.
Ma klund, S., and Ma klund, G. (1974). In ol emen o he Supe oxide Anion
Radical in he Au oxida ion o Py ogallol and a Con enien Assay o
Supe oxide Dismu ase. Eu . J. Biochem. 47, 469–474. doi: 10.1111/j.1432-1033.
1974. b03714.x
Ma ínez-Jau egui, M., Soliño, M., Ma ínez-Fe nández, J., and Touza, J. (2018).
Managing he Ea ly Wa ning Sys ems o In asi e Species o Plan s, Bi ds, and
Mammals in Na u al and Plan ed Pine Fo es s. Fo es s 9:9040170. doi: 10.3390/
9040170
Mika, A., and Lü hje, S. (2003). P ope ies o Guaiacol Pe oxidase Ac i i ies
Isola ed om Co n Roo Plasma Memb anes. Plan Physiol. 132, 1489L–1498.
doi: 10.1104/pp.103.020396
Pan, J., Sha i , R., Xu, X., and Chen, X. (2021). Mechanisms o Wa e logging
Tole ance in Plan s: Resea ch P og ess and P ospec s. F on . Plan Sci. 11:2319.
doi: 10.3389/ pls.2020.627331
Pa y, M. A. J., And alojc, P. J., Pa ma , S., Keys, A. J., Habash, D., Paul, M. J., e al.
(1997). Regula ion o Rubisco by inhibi o s in he ligh . Plan Cell En i on. 20,
528–534. doi: 10.1046/j.1365-3040.1997.d01-85.x
Pa en, K., O’Casey, C., and Me zge , C. (2017). La ge-Scale Chemical Con ol
o Smoo h Co dg ass (Spa ina al e ni lo a) in Willapa Bay, WA: Towa ds
E adica ion and Ecological Res o a ion. In asi e Plan Sci. Manag. 10, 284–292.
doi: 10.1017/inp.2017.25
Pé ez-Rome o, J. A., Idaszkin, Y. L., Dua e, B., Bae a, A., Ma ques, J. C., Redondo-
Gómez, S., e al. (2018). A mosphe ic CO2 en ichmen e ec on he Cu-
ole ance o he C4 co dg ass Spa ina densi lo a. J. Plan Physiol. 220, 155–166.
doi: 10.1016/j.jplph.2017.11.005
Puccia iello, C., Pa lan i, S., Ban i, V., No i, G., and Pe a a, P. (2012). Reac i e
oxygen species-d i en ansc ip ion in A abidopsis unde oxygen dep i a ion.
Plan Physiol. 159, 184–196. doi: 10.1104/pp.111.191122
Qiu, N., Lu, Q., and Lu, C. (2003). Pho osyn hesis, pho osys em II e iciency and he
xan hophyll cycle in he sal −adap ed halophy e A iplex cen alasia ica.New
Phy ol. 159, 479–486. doi: 10.1046/j.1469-8137.2003.00825.x
Repolho, T., Dua e, B., Dionísio, G., Paula, J. R., Lopes, A. R., Rosa, I. C.,
e al. (2017). Seag ass ecophysiological pe o mance unde ocean wa ming and
acidi ica ion. Sci. Rep. 7:41443. doi: 10.1038/s ep41443
Ru , M., and B unne , I. (2003). Vi ali y o ee ine oo s: ee alua ion o he
e azolium es . T ee Physiol. 23, 257–263. doi: 10.1093/ eephys/23.4.257
Sha ma, P., Jha, A. B., Dubey, R. S., and Pessa akli, M. (2012). Reac i e
Oxygen Species, Oxida i e Damage, and An ioxida i e De ense Mechanism in
Plan s unde S ess ul Condi ions. J. Bo . 2012:217037. doi: 10.1155/2012/21
7037
S asse , R. J., S i as a a, A., and Go indjee. (1995). Polyphasic Chlo ophyll a
Fluo escence T ansien in Plan s and Cyanobac e ia. Pho ochem. Pho obiol. 61,
32–42. doi: 10.1111/j.1751-1097.1995. b09240.x
Teixei a, A., Dua e, B., and Caçado , I. (2014). “Sal Ma shes and Biodi e si y,”
in Sabkha Ecosys ems: Volume IV: Cash C op Halophy e and Biodi e si y
Conse a ion, eds M. A. Khan, B. Böe , M. Öz ü k, T. Z. Al Abdessalaam, M.
F on ie s in Ma ine Science | www. on ie sin.o g 17 Augus 2021 | Volume 8 | A icle 699528
ma s-08-699528 Sep embe 1, 2021 Time: 9:1 # 18
C uz de Ca alho e al. Ecoenginee ing o Impai ing Spa ina pa ens
Clüsene -God , and B. Gul (Do d ech : Sp inge ), 283–298. doi: 10.1007/978-
94-007-7411-7_20
Te anishi, Y., Tanaka, A., Osumi, M., and Fukui, S. (1974). Ca alase ac i i ies o
hyd oca bon-u ilizing candida yeas s. Ag ic. Biol. Chem. 38, 1213–1220. doi:
10.1080/00021369.1974.10861301
Ti yakioglu, M., Eke , S., Ozku lu, F., Hus ed, S., and Cakmak, I. (2006).
An ioxidan de ense sys em and cadmium up ake in ba ley geno ypes di e ing
in cadmium ole ance. J. T ace Elem. Med. Biol. 20, 181–189. doi: 10.1016/j.
j emb.2005.12.004
☼UN (2020). UN Decade on Ecosys em Res o a ion. A ailable online a : h ps:
//www.decadeon es o a ion.o g/. (accessed Ap il 16, 2021).
on Caemme e , S. (2020). Rubisco ca boxylase/oxygenase: F om he enzyme o
he globe: A gas exchange pe spec i e. J. Plan Physiol. 252:153240. doi: 10.1016/
j.jplph.2020.153240
Zhang, R., Zhou, Y., Yue, Z., Chen, X., Cao, X., Ai, X., e al. (2019). The lea -ai
empe a u e di e ence e lec s he a ia ion in wa e s a us and pho osyn hesis
o so ghum unde wa e logged condi ions. PLoS One 14:e0219209. doi: 10.1371/
jou nal.pone.0219209
Con lic o In e es : The au ho s decla e ha he esea ch was conduc ed in he
absence o any comme cial o inancial ela ionships ha could be cons ued as a
po en ial con lic o in e es .
Publishe ’s No e: All claims exp essed in his a icle a e solely hose o he au ho s
and do no necessa ily ep esen hose o hei a ilia ed o ganiza ions, o hose o
he publishe , he edi o s and he e iewe s. Any p oduc ha may be e alua ed in
his a icle, o claim ha may be made by i s manu ac u e , is no gua an eed o
endo sed by he publishe .
Copy igh © 2021 C uz de Ca alho, Feijão, Dua e, Pin o, Sil a, Ma os, da
Sil a, Caçado , Reis-San os, Fonseca and Dua e. This is an open-access a icle
dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY).
The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he
o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal
publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice.
No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h
hese e ms.
F on ie s in Ma ine Science | www. on ie sin.o g 18 Augus 2021 | Volume 8 | A icle 699528