Plan Physiology and Biochemis y 210 (2024) 108569
A ailable online 26 Ma ch 2024
0981-9428/© 2024 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
Ni a e modula es he physiological ole ance esponses o he halophy ic
species Sa coco nia u icosa o coppe excess
Ped o Valle-Rome o
a
, Eloy Manuel Cas ellanos
b
, Ca los J. Luque
b
, No is J. Flo es-Dua e
c
,
Elena Romano-Rod íguez
a
, Susana Redondo-G´
omez
a
, Ignacio D. Rod íguez-Llo en e
c
,
Eloísa Pajuelo
c
, En ique Ma eos-Na anjo
a
,
*
a
Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de Se illa, 1095, 41080 Se illa, Spain
b
Depa amen o de Ciencias In eg adas, Facul ad de Ciencias Expe imen ales, Uni e sidad de Huel a, 21071 Huel a, Spain
c
Depa amen o de Mic obiología y Pa asi ología, Facul ad de Fa macia, Uni e sidad de Se illa, 1095, 41080 Se illa, Spain
ARTICLE INFO
Handling Edi o : K Kees Venema
Keywo ds:
An ioxidan ac i i y
Coppe
Halophy e
Ni a e
Pho osyn hesis
Sa coco nia u icosa
S ess ole ance
ABSTRACT
Coexis ence impac o pollu an s o di e en na u e on halophy es ole ance o me al excess has no been
ho oughly examined, and plan unc ional esponses desc ibed so a do no ollow a clea pa e n. Using he Cu-
ole an halophy e Sa coco nia u icosa as a model species, we conduc ed a g eenhouse expe imen o e alua e
he impac o wo concen a ion o coppe (0 and 12 mM CuSO
4
) in combina ion wi h h ee ni a e le els (2, 14
and 50 mM KNO
3
) on plan g ow h, pho osyn he ic appa a us pe o mance and ROS-sca enging enzymes sys em.
The esul s e ealed ha S. u icosa was able o g ow adequa ely e en when exposed o high concen a ions o
coppe and ni a e. This esponse was linked o he plan capaci y o up ake and e ain a la ge amoun o coppe
in i s oo s (up o 1500 mg kg
−1
Cu), p e en ing i s anspo o ae ial pa s. This con ol o ansloca ion was
u he magni ied wi h ni a e concen a ion inc emen . Likewise, al hough Cu excess impai ed S. u icosa
ca bon assimila ion capaci y, he plan was able o down egula e i s ligh -ha es ing complexes unc ion, as
indica ed i s lowe s ETR alues, especially a 12 mM Cu +50 mM NO
3
. This down egula ion would con ibu e o
a oid excess ene gy abso p ion and ans o ma ion. In addi ion, his s a egy o a oiding excess ene gy was
accompanied by he up egula ion o all ROS-sca enging enzymes, a esponse ha was u he enhanced by he
inc ease in ni a e concen a ion. The e o e, we conclude ha he coexis ence o ni a e would a o S. u icosa
ole ance o coppe excess, and his e ec is media ed by he combined ac i a ion o se e al ole ance
mechanisms.
1. In oduc ion
Many o hea y me als, such as Cu, Mn, Ni, Zn, e c., a e essen ial o
plan de elopmen , being key elemen s in impo an biological p o-
cesses such as pho osyn he ic ca bon assimila ion, pho ochemical e i-
ciency o ligh cap u e and ans o ma ion, and espi a ion p ocess
(Chen e al., 2022; Meng e al., 2018; Mish a and Tanna, 2017; Shabala,
2013). Howe e , when p esen in excess, hey can d as ically a ec
hese i al me abolic p ocesses. This has led o inc eased a en ion om
he scien i ic communi y in ecen decades, ocusing on esea ch in o
me al ole ance in plan s. This esea ch aims o unde s and: (i) he
ole ance h esholds o a wide ange o plan species agains di e en
ypes o me als, in o de o comp ehend he eco oxicological impac o
hese pollu an s on species communi ies e olu ion and/o de e mina e
hei po en ial o en i onmen al emedia ion (Capa ´
os e al., 2022;
Ma eos-Na anjo e al., 2008a; Redondo-G´
omez e al., 2010; Singh e al.,
2023), and (ii) elucida e unde lying ole ance mechanisms ha would
allow hese species o espond o s ess induced by he p esence o
me als (Singh e al., 2023). In his concep ual amewo k, he scien i ic
communi y has iden i ied se e al halophy e species as me al- ole an
due o hei special mo phological, ana omical, physiological, and
biochemical ea u es ha allow hem o e ed hem o comple e hei li e
Abb e ia ions: A
N
, ne pho osyn he ic a e; CAT, ca alase; ETR, elec on anspo a e; GPX, guaiacol pe oxidase; F
/F
m
, maximum quan um e iciency o PSII
pho ochemis y; Φ
PSII
, ac ual pho ochemical e iciency o PSII; g
s
, s oma al conduc ance; LDMC, d y mass con en o b anches; RDMC, d y mass con en o he oo s;
RH, ela i e humidi y; ROS, eac i e oxygen species; SOD, supe oxide dismu ase;
i
WUE, in insic wa e use e iciency;
ε
, appa en ca boxyla ion e iciency.
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (E. Ma eos-Na anjo).
Con en s lis s a ailable a ScienceDi ec
Plan Physiology and Biochemis y
jou nal homepage: www.else ie .com/loca e/plaphy
h ps://doi.o g/10.1016/j.plaphy.2024.108569
Recei ed 11 Janua y 2024; Recei ed in e ised o m 12 Ma ch 2024; Accep ed 25 Ma ch 2024
Plan Physiology and Biochemis y 210 (2024) 108569
2
cycle in en i onmen s con amina ed wi h bo h salini y and hea y me als
(Calone e al., 2022; Lu s and Le `
e e, 2015; Manousaki and Kaloge -
akis, 2011; Redondo-G´
omez e al., 2006). These species ha e ga ne ed
a en ion as model o ganisms o he s udy o me al ole ance
mechanisms.
Gene ally, he majo i y o halophy es ole ance mechanisms agains
me al excess aim o a oid he buildup o excessi e le els o his elemen
in sensi i e issues whe e i has oxic e ec s (Kaba a-Pendias, 2001).
This is achie ed h ough hei abili y o p e en me al up ake, anspo ,
o seques a ion in issues o cellula compa men s ha a e sensi i e o
me als. This can be accomplished by me al binding o he cellula wall
and ex acellula oo exuda es o by educing me al in lux ac oss he
plasma memb ane (Singh e al., 2023). Pa icula ly, some halophy es
demons a e enhanced esis ance o eac i e oxygen species (ROS), such
as supe oxide adicals (O
2−
) and hyd ogen pe oxide (H
2
O
2
), caused by
hea y me als. This esis ance is achie ed h ough he syn hesis o an i-
oxidan s (S u hi e al., 2017) and he modula ion o ROS-sca enging
enzymes, as supe oxide dismu ase (SOD), ca alase (CAT), asco ba e
pe oxidase (APX), pe oxidase (POX), and glu a hione educ ase (GR),
esul ing in educed damage o lipid memb anes om ROS unde hea y
me al s ess (Liang e al., 2017). Addi ionally, coppe ole ance in hal-
ophy es has been also asc ibed o he main enance o pho osyn he ic
unc ions unde me al excess. This ci cums ance is ela ed o he mod-
ula ion o i s a o emen ioned an ioxidan appa a us and he ac ha
many, o e en mos , s eps in pho osyn he ic pa hway (ligh ha es ing
sys em and/o di usional and biochemical componen s o CO
2
assim-
ila ion) may be ole an o he up ake and accumula ion o high amoun s
o me al in plan issues (Mallick and Mohn, 2003; Singh e al., 2023).
Despi e he eno mous p og ess made in de e mining he ole ance
po en ial o halophy ic species and disen angling hei cons i u i e
ole ance mechanisms, many s udies we e mos ly con ined o assess he
ole ance agains speci ic pollu an s (Nikalje and Sup asanna, 2018;
Redondo-G´
omez e al., 2010), wi hou conside ing he coexis ence o
se e al hea y me als and/o con aminan s o di e en na u es, which
could ha e a di e en signi ican impac on hese plan ole ance
mechanisms. This impac would depend on pollu an cha ac e is ics
such as concen a ion, na u e, o hei combina ion (P´
e ez-Rome o
e al., 2022). In his con ex , ni a e (NO
3
−
) con amina ion esul ing om
e iliza ion p ocesses associa ed wi h in ensi e a ming p ac ices has
been iden i ied as a p ima y ac o con ibu ing o a ious ad e se e -
ec s on ecosys em conse a ion (Ba os e al., 2012; Ma ínez-Dalmau
e al., 2021). Gi en he widesp ead occu ence o ni a e con amina ion
in saline en i onmen s, like sal ma shes (Mohammadzadeh and Haji-
boland, 2022), i becomes impe a i e o unde s and he po en ial impac
o ni a e p esence on he ole ance mechanisms and me al up ake ca-
paci y o halophy ic plan s wi hin hese ecosys ems. The e o e, his
s udy was designed and conduc ed o ill his gap in knowledge.
Sa coco nia u icosa (L.) A. J. Sco was chosen as a model halophy e
species o s udy in de ail he impac o ni a e and me al in e ac ion on
plan pe o mance and ole ance mechanism. Speci ically, he s udy
ocused on he unc ionali y o he pho osyn he ic appa a us, modula-
ion o an ioxidan enzyme machine y, and me al up ake. This species
has been desc ibed as a na u al phy os abilize o se e al me als wi h
dis ibu ion in coas al sys ems ac oss sou he n and wes e n Eu ope (Said
e al., 2019; Sil a e al., 2021). Mo eo e , se e al au ho s ha e sug-
ges ed a link be ween hea y me al ole ance and ni a e accumula ion in
ce ain species o he Ama an haceae amily, o which his selec ed
species belongs (Hawkes o d e al., 2023; Mo ei a da Sil a e al., 2015).
Conside ing hese ac o s, we hypo hesize ha he coexis ence o ni a e
in he g owing medium would modula e he ole ance and me al accu-
mula ion capaci y o he halophy e S. u icosa, which would be linked o
he ac i a ion o speci ic halophy ic ole ance mechanisms a o ed by
he g ea e a ailabili y o ni ogen esou ces in he en i onmen . Taking
all his in o accoun , he main objec i e o his s udy was o in es iga e
he abili y o S. u icosa pho osyn hesis and ROS-sca enging enzyme
sys ems o cope wi h coppe excess unde di e en le els o ni a e in he
g owing medium, and examine he possible ole o coppe and ni ogen
concen a ion in plan issues in explaining plan esponses. Coppe was
selec ed as i a ains p io i y pollu an s a us acco ding o bo h he Eu-
opean Communi y and UNEP (T´
o h e al., 2016; UNEP, 2013) and due
o i s biological impo ance in assessing plan ole ance mechanisms in
his s udy.
2. Ma e ial and me hods
2.1. Plan ma e ial and expe imen al design
Sa coco nia u icosa plan s comple ely de eloped and plan ed in 5.5
cm diame e mul i-pe o a ed po s illed wi h ockwool as subs a e
we e supplied by he company ‘Hal´
o i as Onuba S.L.’. (Isla C is ina,
Huel a; Spain). These plan s we e placed in a clima e-con olled, PLC-
au oma ed, so wa e-con olled, enlo glass g eenhouse module (Uni-
e si y o Se ille, Cen e o Resea ch, Technology and Inno a ion,
CITIUS II; 37◦24
′
N, 6◦0
′
W; Sou hwes Spain) unde con olled condi-
ions: 21–25 ◦C empe a u e, 40–50% RH, na u al ligh was supple-
men ed by luo escen /incandescen lamps se o a pho ope iod o 16 h
o ligh (wi h a maximum pho on densi y lux a lea le el o 1000
μ
mol
m
−2
s
−1
) and 8 h o da kness, wi h adequa e i iga ion du ing 15 days
o an acclima ion pe iod un il expe imen onse .
A e acclima ion pe iod, plan s wi h 10 cm heigh we e selec ed and
di ided in o six ea men s o o y indi iduals pe g oup and a anged
in a andomized plo . Fo ha , he po s we e andomly subjec ed o wo
coppe concen a ion ea men s (0 and 12 mM CuSO
4
), in combina ion
wi h h ee le els o ni a e (2, 14 and 50 mM KNO
3
) o 60 days (n =
240; 40 plan s pe ea men ). The coppe concen a ion was chosen
conside ing he cu en coppe le els p e iously eco ded in he ma shes
o he sou hwes e n peninsula by Redondo-G´
omez e al. (2009) and
S´
anchez-Ga il´
an e al. (2021). Fo ni a e, h ee concen a ions we e
chosen o make ou da a ep esen a i e o low (2 mM), medium (14 mM)
and high (50 mM) le els o en i onmen al ni a e con amina ion
de i ed om chemical e ilize applica ion acco ding o Mohammad-
zadeh and Hajiboland (2022). Coppe and ni a e ea men s we e
es ablished by combining 20% Hoagland solu ion (Hoagland and A non,
1950) wi h he app op ia e concen a ion o CuSO
4
- 7H
2
O and KNO
3
,
espec i ely. In addi ion, all ea men s we e supplied wi h a solu ion o
171 mM NaCl, since his is he op imal salini y wi hin he ange o
na u al condi ions in which his species g ows na u ally (P´
e ez-Rome o
e al., 2023). Fo ha , a he beginning o he expe imen , 3 L o he
app op ia e solu ion was applied o he ays o a dep h o 1 cm. Solu ion
le els and pH we e moni o ed daily, and opped up wi h 20% Hoagland
solu ion and adjus ed o 6.5 ±7.0 wi hou he addi ional applica ion o
CuSO
4
- 7H
2
O and KNO
3
, o a oid a ia ions in pH and coppe and ni-
a e concen a ions due o e apo a ion o he solu ion. The con ol, 0
mM CuSO
4
ea men , had 0.0005 mM Cu, as he Hoagland solu ion
con ains a small amoun o Cu as an essen ial nu ien . All solu ions,
including CuSO
4
- 7H
2
O and KNO
3
, we e changed weekly.
2.2. E alua ion o plan g ow h and pho osyn he ic appa a us
pe o mance
A e 60 days o ea men , he plan s we e ha es ed and di ided
in o b anches and oo s. These biomass ac ions we e hen o en d ied a
60 ◦C o 48 h and weighed o ob ain es ima es o he d y mass con en o
he b anches (LDMC) and oo s (RDMC). These measu emen s we e
complemen ed wi h ins an aneous lea gas exchange and PSII pho o-
chemical e iciency measu emen s, ob ained wi h an open sys em
in a ed gas analyse , equipped wi h a luo escence measu emen
chambe (LI-6800-01, LICOR Inc., Lincoln, NE, USA) and a po able
modulable luo ome e (FMS-2; Hansa ech Ins umen s L d., King ‘s
Lynn, UK) 20 and 60 days a e expe imen al ea men s imposi ion.
Thus, ne pho osyn he ic a e (A
N
), s oma al conduc ance (g
s
), appa en
ca boxyla ion e iciency (
ε
: A
N
/C
i
), in insic wa e use wa e use
P. Valle-Rome o e al.
Plan Physiology and Biochemis y 210 (2024) 108569
3
e iciency (
i
WUE), ac ual pho ochemical e iciency o PSII (Φ
PSII
) and
elec on anspo a e (ETR) we e de e mined a 400 ppm CO
2
, 25 ±
0.5 ◦C, 50 ±1% RH and 1000
μ
mol pho on m
−2
s
−1
(n =7 measu e-
men s pe ea men ). All measu emen s we e made be ween 10:00 and
13:00 h and we e co ec ed by pho osyn he ic a ea acco ding o
(P´
e ez-Rome o e al., 2018). Finally, he maximum pho ochemical
quan um e iciency o PSII (F
/F
m
) was measu ed in 30 min o
da k-adap ed b anches (n =12 measu emen s pe ea men ), acco ding
o he p o ocol desc ibed by Ma eos-Na anjo e al. (2020).
2.3. E alua ion o plan an ioxidan enzymes s a us
A he end o he expe imen , 500 mg o esh ae ial issue samples
om each o expe imen al ea men s we e collec ed, g ound and ho-
mogenized in 8 mL o a 50 mM sodium phospha e bu e solu ion (pH
7.6) and 0.1 mM Na-EDTA, unde ice-cold condi ions acco ding o he
p o ocol desc ibed by Dua e e al. (2015). The homogena e was hen
subjec ed o cen i uga ion a 9000 pm o 20 min a 4 ◦C and he su-
pe na an liquid ob ained was used o se e al enzyma ic analyses.
Th ee samples pe ea men we e used and h ee measu emen s pe
sample and enzyme ac i i y we e egis e ed. The p o ein con en in he
ex ac s was ob ained acco ding o B ad o d (1976), using bo ine se um
albumin as s anda d.
Guaiacol pe oxidase EC 1.11.1.7 (GPX) was calcula ed as Be gmeye
(1974) indica ed, wi h a eac ion mix u e made o 50 mM sodium
phospha e bu e (pH 7.0), 2 mM H
2
O
2
, and 20 mM guaiacol. Fo all
hese enzyma ic ac i i ies, 100
μ
L o ege al ex ac was added o he
eac ion mix u e o s a he eac ion. Supe oxide dismu ase EC 1.15.1.1
(SOD) ac i i y was assayed by moni o ing he educ ion o py ogallol a
325 nm, ollowing Ma klund and Ma klund (1974) wo k. The eac ion
mix u e was 50 mM sodium phospha e bu e (pH 7.6), 0.1 mM
Na-EDTA, 3 mM py ogallol, Mili-Q wa e . The eac ion s a ed wi h he
addi ion o 10
μ
L o enzyme ex ac . Ca alase EC 1.11.1.6 (CAT) was
p epa ed ollowing he p o ocol o Te anishi e al. (1974) in a mix u e o
890 mL o sodium phospha e bu e (50 mM, pH 7.0), 100
μ
L lea ex ac
and 10
μ
L H
2
O
2
(15%). Subsequen ly, o de e mine he enzyme ac i i y,
he dec ease in abso bance a 240 nm was calcula ed, using he mola
ex inc ion coe icien o H
2
O
2
(39.4 mM
−1
cm
−1
). Finally, asco ba e
pe oxidase EC 1.11.1.11 (APX) was analyzed acco ding o Dua e e al.
(2015). The eac ion mix u e was composed o 50 mM o sodium
phospha e bu e (pH 7.0), 12 mM
μ
L o H
2
O
2
and 0.25 mM L-asco ba e,
in addi ion o he 100
μ
L o plan ex ac . Enzyme ac i i y was eco ded
by measu ing he dec ease in abso bance a 290 nm due o asco ba e
oxida ion and calcula ed using he mola ex inc ion coe icien o
ε
(2.8
mM
−1
cm
−1
). The au o-oxida ion o he subs a es was e alua ed by a
con ol sample wi h he eac ion mix u e bu wi hou he enzyme
ex ac .
2.4. E alua ion o coppe and ni ogen con en in plan issues
A he end o he expe imen , d ied b anch and oo samples we e
collec ed and c ushed o ob ain 0.5 g aliquo s in iplica e om each
expe imen al ea men . Subsequen ly, he samples we e diges ed wi h
6 mL o HNO
3
, 0.5 mL o HF and 1 mL H
2
O
2
; and he concen a ion o
coppe in he issues was quan i ied by induc i ely coupled plasma
spec oscopy (ICP; ARL-Fison 3410, USA). Finally, he undiges ed d y
samples unde wen o al ni ogen concen a ion analysis using he
Dumas me hod wi h an elemen al au oanalyze (Leco CHNS-932-
T umac, Spain). This echnique in ol es sample combus ion o ans-
o m ni ogen in o ni ogen oxides, ollowed by he p ecise quan i ica-
ion o he eleased ni ogen gas, ensu ing accu a e de e mina ion o
ni ogen con en (L´
opez-Calde ´
on e al., 2020).
2.5. S a is ical analysis
S a is ical analyses we e ca ied ou using RS udio s a is ical
so wa e (R De elopmen Co e Team, 2008). The analysis o he e ec o
coppe and ni a e ea men s on he di e en pa ame e s o S. u icosa
was ca ied ou using gene al linea models (GLM es ). Coppe and
ni a e concen a ions we e conside ed as g ouping a iables and he
di e en quan i ied pa ame e s as con as a iables. Those analyses
ha yielded signi ican esul s we e subjec ed o a pos hoc pai wise
con as analysis (LSD es ).
3. Resul s
3.1. G ow h and gas exchange measu emen s
The d y mass con en o he b anches and oo s did no di e be-
ween he expe imen al ea men s a e 60 days o ea men (GLM
Fig. 1. (A) D y mass con en o he b anches (LDMC) and (B) d y mass con en
o he oo s (RDMC) in Sa coco nia u icosa plan s a e 60 days o ea men ,
wi h h ee ni a e le els (2, 14 and 50 mM KNO
3
) in combina ion wi h wo
coppe concen a ions (0 and 12 mM CuSO
4
). Each alue ep esen s he mean
±s anda d e o o en eplica es. Cu, NO
3
o Cu x NO
3
in he uppe co ne o
he panel indica es signi ican main e ec s o in e ac ions (LSD es , *p <0.05,
**p <0.01).
P. Valle-Rome o e al.
Plan Physiology and Biochemis y 210 (2024) 108569
4
Model: p >0.05; Fig. 1A and B). Con a ily, ou esul s showed a sig-
ni ican e ec o coppe and ni a e ea men s on he ins an aneous gas
exchange cha ac e is ics o S. u icosa in he di e en sampling pe iods
(GLM Model: p <0.05; Fig. 2). O e all, appa en ca boxyla ion e i-
ciency was lowe wi h inc eased coppe concen a ion independen ly o
ni a e concen a ion in he g own medium a e 20 days o ea men ,
and his di e ence was mo e acu e 60 days a e me al s ess imposi ion
and mainly in plan s exposed o 50 mM ni a e (GLM Model: Cu and
NO
3
, p <0.01; Fig. 2A and B). A e y simila pa e n was obse ed o
A
N
alues a e 20 and 60 days o ea men (GLM Model: Cu and NO
3
, p
<0.01; Fig. 2C and D), while s oma al conduc ance (g
s
) showed mean
alues c. 90 mmol
−1
H
2
O m
−2
s
−1
in all ea men s a e 20 days
(Fig. 2E), and dec eased conside ably a e 60 days in p esence o coppe
ega dless o he ni a e concen a ion (GLM Model: Cu, p <0.01;
Fig. 2F). Rega ding in insic wa e use e iciency (
i
WUE), i was
obse ed ha , in gene al, alues we e lowe in he p esence o coppe a
20 days o ea men (GLM Model: Cu, p <0.05; Fig. 3A). Howe e , a 60
days, no signi ican di e ences in ea men s we e obse ed and all
ea men s had a simila e iciency (Fig. 3B).
3.2. Chlo ophyll a luo escence measu emen s
Ou esul s showed a signi ican e ec o coppe and ni a e ea -
men s on he PSII e iciency o S. u icosa in he di e en sampling
pe iods (GLM Model: p <0.05; Fig. 4). The e o e, he maximum
pho ochemical quan um e iciency o PSII (F
/F
m
), did no a y signi -
ican ly be ween expe imen al ea men s a e 20 days (Fig. 4A), while
he e was a nega i e syne gis ic in plan s g own a 12 mM Cu +50 mM
KNO
3
ha showed he lowes F
/F
m
alues compa ed o he o he
ea men s (GLM Model: Cu x NO
3
, p <0.01; Fig. 4B).
Howe e , he o e all ac ual pho ochemical e iciency o he PSII
(Φ
PSII
) and elec on anspo a e (ETR) alues was lowe in plan s
g own in he p esence o coppe a e 20 days (GLM Model: Cu, p <0.01;
Fig. 4C and E). In addi ion, bo h coppe and ni a e p esence had sig-
ni ican e ec s on plan s Φ
PSII
and ETR alues a e 60 days, ob aining
he lowes alues in he plan s subjec ed wi h 12 mM Cu +50 mM ni-
a e in he medium (GLM Model: Cu and NO
3
, p <0.01; Fig. 4D and F).
3.3. An ioxidan enzymes measu emen s
Ou esul s showed a signi ican e ec o coppe and ni a e ea -
men s on he an ioxidan enzymes s a us o S. u icosa a he end o he
expe imen (GLM Model: p <0.05: Fig. 5). Thus, o e all asco ba e
pe oxidase (APX) ac i i y was highe in plan s g own in he p esence o
coppe (GLM: Cu, p <0.05) and ended o inc ease wi h ni a e con-
cen a ion in he g ow h medium wi h and wi hou Cu applica ion (GLM
Model: NO
3
, p <0.01; Fig. 5A). Ve y simila pa e ns we e eco ded o
ca alase (CAT), supe oxide dismu ase (SOD), and guaiacol pe oxidase
(GPX), bu o hese enzyme ac i i ies i should be no ed ha he di -
e ences be ween bo h coppe ea men s we e e en mo e p onounced
(GLM Model: Cu, p <0.01; Fig. 5B and D), ob aining o e all he highes
alues in he plan s subjec ed o 12 mM Cu +50 mM ni a e in he
medium and specially o GPX ac i i y (GLM Model: Cu, NO
3
and Cu x
NO
3
, p <0.01; Fig. 5D).
3.4. Analysis o coppe and ni ogen con en in issues
The e we e signi ican e ec s o CuSO
4
and NO
3
concen a ion
ea men s on Cu and N concen a ions in issues (GLM Model: p <0.05;
Fig. 6). The e o e, he coppe concen a ion was highe in oo s han in
lea es, and wi hin he issues a conside able inc ease was obse ed
along he ex e nal exposu e o Cu ega dless o he concen a ion o
ni a e in bo h issues (GLM Model: Cu, p <0.01; Fig. 6A and B), excep
o he Cu concen a ion o lea es, which showed a educ ion in plan s
g own a 50 mM ni a e compa ed o he es o Cu ea ed plan s (GLM
Model: Cu x NO
3
, p <0.01; Fig. 6A). Howe e , ni ogen concen a ion
was highe in abo eg ound issues han in belowg ound issues and
ended o inc ease wi h he concen a ion o ni a e in he g own me-
dium; being his inc ease was e en magni ied in Cu-supplied plan s
g own a 50 mM ni a e, especially in lea es (GLM Model: Cu x NO
3
, p <
0.01; Fig. 6C).
4. Discussion
The abili y o halophy es o cope wi h induced me al s ess has been
highligh ed by many s udies, and signi ican e o s ha e been made o
un a el he mechanisms in ol ed in his esponse (Flowe s and Colme ,
2008; Meng e al., 2018). Howe e , ew s udies ha e explo ed in dep h
how his inna e ole ance could be modula ed by he coexis ence o
pollu an s o di e en na u e, such as ni a e en ichmen in he en i-
onmen . In ac , p e ious s udies ha e highligh ed he o e lapping e -
ec o dis inc s esso s on plan s ole ance capaci y, esul ing in he
gene a ion o in eg a ed mechanical and chemical signals ha play a
pi o al ole in elici ing he ole ance (Ma eos-Na anjo e al., 2021, 2024;
Roe and MacFa lane, 2022). In his sense, ou s udy showed how
S. u icosa ole ance esponses o coppe excess we e, o some ex en ,
modula ed by he a ia ion in ni a e concen a ion in he g owing
medium, demons a ing a di e en ial ac i a ion o speci ic ole ance
mechanisms in his plan species (i.e., me al up ake and anspo lim-
i a ion, pho osyn he ic appa a us pe o mance, and ROS-sca enging
enzyme sys em modula ion; Meng e al., 2018; Mish a and Tanna,
2017; Shabala, 2013).
Focusing on he ob ained esul s, we ound ha S. u icosa exhibi ed
high ole ance o coppe s ess, since plan s we e able o sus ain hei
g ow h a concen a ions highe han 12 mM Cu, measu ed as LDMC and
RDMC, compa ed o hose g own a 0 mM Cu. This is no ewo hy,
especially conside ing he esul s ob ained o o he halophy ic species
belonging o he Ama an haceae amily, whe e he de imen al impac
o coppe excess nega i ely a ec s hei g ow h (O ego e al., 2020).
The obse ed g ow h main enance in S. u icosa was a ibu ed o he
ac i a ion o di e en ole ance mechanisms, which could ac indi id-
ually o syne gis ically, in esponse o Cu excess (Capa ´
os e al., 2022;
Liang e al., 2017). In his con ex , ou esul s e ealed a subs an ial
coppe up ake and e en ion plan capaci y in he plan , pa icula ly in
i s oo s, wi h mean alues a ound 1500 mg kg
−1
Cu o plan s g own a
12 mM Cu, ega dless o he ni a e le el. Consis en wi h his esul ,
nume ous s udies ha e co obo a ed he capaci y o S. u icosa o e ain
me als in he hizosphe e ( oo s and hizosedimen ), he eby p e en ing
hei ansloca ion o ae ial pa s (Cae ano e al., 2008; Mo ei a da Sil a
e al., 2015; Sil a e al., 2021). This Cu accumula ion pa e n could be
explained by well-desc ibed p ocess in halophy es, such as Cu binding o
oo cell walls, chela ion, and/o compa men aliza ion in acuoles o
non- oxic s o age, a oiding ion in e ac ion wi h essen ial cellula
me abolic p ocesses in mo e sensi i e o gans (Capa ´
os e al., 2022;
Sheo an e al., 2010). Addi ionally, ou esul s indica e ha he coppe
accumula ion pa e n was al e ed o a ce ain deg ee by he p esence o
ni a e. In his ega d, se e al au ho s ha e indica ed ha Cu accumu-
la ion pa e n may a y depending on plan species o eco ypes, de el-
opmen al s age, and en i onmen al ac o s such as ni ogen supply
(Y uela, 2009). Thus, ou esul s sugges ed ha o e all NO
3
supply led
o a highe ni ogen con en in plan issues. This e ec was e en mo e
p onounced in plan s subjec ed o 12 mM Cu +50 mM NO
3
, ollowed by
a concomi an educ ion in he concen a ion o coppe in hei lea es
compa ed wi h hei Cu-supplied coun e pa s, which would con ibu e
o explaining plan ole ance agains excess coppe . Acco ding o ou
esul s, Cui e al. (2022) indica ed ha Cu and N supply al e ed he
up ake pa e ns o bo h elemen s h ough a di e en ial egula ion o he
exp ession le els o NO
3
and Cu anspo e s in ice plan s. Hence, i is
possible ha some hing simila occu ed in S. u icosa, and his a ea
dese es u he esea ch as i could con ibu e o explaining i s high Cu
ole ance.
Despi e he ela i ely high ole ance demons a ed by S. u icosa in
P. Valle-Rome o e al.
Plan Physiology and Biochemis y 210 (2024) 108569
5
Fig. 2. (A, B) Appa en ca boxyla ion e iciency (
ε
), (C, D) ne pho osyn he ic a e (A
N
) and (E, F) s oma al conduc ance (g
s
) in andomly selec ed Sa coco nia
u icosa b anches o plan s subjec ed o wo coppe concen a ions (0 and 12 mM CuSO
4
) in combina ion wi h h ee ni a e le els (2, 14 and 50 mM KNO
3
) a e 20
and 60 days o ea men . Each alue ep esen s he mean ±s anda d e o o h ee eplica es. Cu, NO
3
o Cu x NO
3
in he uppe co ne o he panel indica es
signi ican main e ec s o in e ac ions (LSD es , *p <0.05, **p <0.01). In case o in e ac ion, Cu x NO
3
, means ha a e signi ican ly di e en om each o he
wi hin he same sampling pe iod a e indica ed by di e en le e s (GLM, p <0.05).
P. Valle-Rome o e al.
Plan Physiology and Biochemis y 210 (2024) 108569
6
e ms o g ow h, ou pho osyn he ic pe o mance analysis indica ed a
g ea e sensi i i y o his me abolic p ocess in e ms o ca boxyla ion
capaci y and pho ochemical e iciency unde ele a ed concen a ions
bo h Cu and NO
3
(i.e., 12 mM Cu and 50 mM NO
3
). I is no ewo hy ha
he pho osyn he ic appa a us o plan s, including halophy es, is o en
highly ulne able o hea y me al oxici y (Nikalje and Sup asanna,
2018). The e ec s o Cu on A
N
o S. u icosa we e clea ly disce nible
ega dless o ni a e concen a ion, wi h his e ec becoming mo e
p onounced a e 60 days o ea men . In addi ion, we ound ha
al hough se e al s udies ha e indica ed ha exogenous applied ni a e
could a o pho osyn he ic ac i i y (Agniho i and Se h, 2016; Wei
e al., 2024) as showed plan s g own a 14 mM NO
3
and wi hou Cu
addi ion, when i is in excess, i can damage pho osyn he ic ac i i y, as
indica ed by he educ ion o A
N
a 50 mM NO
3
(Han e al., 2023). These
endencies o Cu and NO
3
impac s on he pe o mance o he S. u icosa
pho osyn he ic appa a us could be pa ially explained by a di e en ial
modula ion e ec o expe imen al ac o s on he down- egula ion o key
s eps in he plan pho osyn he ic appa a us igge ed by each s ess
ac o in e ms o appa a us in eg i y and unc ioning; i.e., CO
2
di usion
limi a ion, RuBisCO ac i i y, and ligh -ha es ing an enna e iciency
(Flexas e al., 2012; Ma eos-Na anjo e al., 2013, 2015). Ou esul s
indica ed ha he limi a ion o A
N
in S. u icosa was media ed by bo h
di usional and biochemical limi a ions (Ama i e al., 2014; Mi e al.,
2021), as indica ed by he dec ease in g
s
and he inc eased in C
i
, which
gene a ed a consequen d op in pho osyn he ic appa en ca boxyla ion
e iciency (
ε
) wi h inc easing Cu concen a ion and in plan s g own a
50 mM NO
3
wi h and wi hou Cu supply. The educ ion in ins an aneous
ca boxyla ion e iciency would also sugges a me abolic impai men in
S. u icosa, p ima ily in biochemical ca boxyla ion, which would be
associa ed wi h a dec ease in RuBisCO ca boxylase ac i i y (Mi e al.,
2021). In his sense, se e al au ho s ha e indica ed ha enzyme ac i i y
down- egula ion is eally common in he p esence o hea y me als
(Siedlecka and K upa, 2004) and would be a ibu ed o he abili y o Cu
o compe e wi h Mg o he o ma ion o he RuBisCO–CO
2
–me al
2+
ca aly ic complex, he eby al e ing he a ini y o CO
2
and po en ially
impai ing he chlo ophyll syn hesis sys em (Ma eos-Na anjo e al.,
2008b; Mi e al., 2021). Howe e , i should be no ed ha he educ ion
o g
s
led o
i
WUE alues main enance, which would con ibu e o p e-
se ing he ade-o be ween CO
2
acquisi ion o pho osyn he ic p ocess
and wa e losses in S. u icosa in he p esence o bo h con aminan s, as
o he halophy es ha e shown in esponse o coppe excess
(P´
e ez-Rome o e al., 2018). This ci cums ance would a o plan wa e
ela ions, upholding a g ea e cell u go and wa e po en ial g adien ,
and consequen ly a la ge lea a ea, compensa ing in pa o he decline
in ca bon assimila ion capaci y pe uni a ea and ensu ing con inued
g ow h unde ad e se condi ions (Mohammadzadeh and Hajiboland,
2022), as has been add essed in o he halophy es (Redondo-G´
omez
e al., 2010).
On he o he hand, S. u icosa impai men in ca bon assimila ion
capaci y a ising om he indi idual o combined p esence o coppe and
high ni a e concen a ion was associa ed wi h di e en ial pollu an
e ec s on he pho ochemical appa a us unc ionali y. Thus, we obse ed
a dec ease in Φ
PSII
in he p esence o coppe , a ibu able o he pol-
lu an s nega i e impac on he PSII eac ion cen e h ough he
des uc ion o an ennae pigmen s (Anjum e al., 2016; Khan e al.,
2022). In his sense, i has been desc ibed ha coppe in e e es wi h he
up ake and assimila ion o essen ial ions such as Mg, Zn, Fe, and Ni,
leading o chlo oplas ul as uc u al al e a ions (Shabbi e al., 2020).
The e o e, i a ec s he plan capaci y o ene gy ans e om he
collec o an ennae o eac ion cen e s (Li e al., 2012). Addi ionally, ou
esul s indica ed ha he e was a nega i e syne gis ic e ec o coppe
supply in plan s g own a 50 mM KNO
3
, as hey p esen ed he lowes
F
/F
m
alues compa ed wi h he es o ea men s. This esponse sug-
ges s ha he combina ion o bo h pollu an s es ed would inc ease
pho oinhibi ion induced by ligh s ess (We ne e al., 2002). Likewise,
he nega i e impac o hese pollu an ac o s on S. u icosa pho o-
chemical appa a us was a ibu ed o he down- egula ion o i s elec on
anspo chain unc ionali y, as indica ed by he lowe ETR alues
eco ded in plan s g own a 12 mM Cu, ega dless o NO
3
concen a ion
in he g own medium, and especially in hose subjec ed o 12 mM Cu +
50 mM NO
3
a e 60 days o ea men . This esponse indica es ha he
p esence o Cu dis up s ene gy luxes pe lea c oss-sec ion (Anjum e al.,
2016; Ma eos-Na anjo e al., 2008a, 2013; Nikalje and Sup asanna,
2018), being his impac mo e p onounced due o he syne gis ic e ec
o he p esence o a high ni a e concen a ion. This elec on anspo
down egula ion would be an a oidance s a egy used by S. u icosa o
p o ec i s pho osyn he ic appa a us and o he chlo oplas s uc u es
agains pho oinhibi ion damage induce by coppe excess (Meng e al.,
Fig. 3. (A, B) In insic wa e use e iciency (
i
WUE) in Sa coco nia u icosa plan s a e 20 and 60 days o ea men , wi h h ee ni a e le els (2, 14 and 50 mM KNO
3
)
in andomly selec ed Sa coco nia u icosa b anches o plan s subjec ed o wo coppe concen a ions (0 and 12 mM CuSO
4
) in combina ion wi h h ee ni a e le els
(2, 14 and 50 mM KNO
3
) a e 20 and 60 days o ea men . Each alue ep esen s he mean ±s anda d e o o h ee eplica es. Cu, NO
3
o Cu x NO
3
in he uppe
co ne o he panel indica es signi ican main e ec s o in e ac ions (LSD es , *p <0.05, **p <0.01). In case o in e ac ion, Cu x NO
3
, means ha a e signi ican ly
di e en om each o he wi hin he same sampling pe iod a e indica ed by di e en le e s (GLM, p <0.05).
P. Valle-Rome o e al.
Plan Physiology and Biochemis y 210 (2024) 108569
7
Fig. 4. (A, B) Maximum pho ochemical quan um e iciency o PSII (F
/F
m
), (C, D) ac ual pho ochemical e iciency o PSII (Φ
PSII
) and (E, F) elec on anspo a e
(ETR) in andomly selec ed Sa coco nia u icosa b anches o plan s subjec ed o wo coppe concen a ions (0 and 12 mM CuSO
4
) in combina ion wi h h ee ni a e
le els (2, 14 and 50 mM KNO
3
) a e 20 and 60 days o ea men . Each alue ep esen s he mean ±s anda d e o o h ee eplica es. Cu, NO
3
o Cu x NO
3
in he
uppe co ne o he panel indica es signi ican main e ec s o in e ac ions (LSD es , *p <0.05, **p <0.01). In case o in e ac ion, Cu x NO
3
, means ha a e
signi ican ly di e en om each o he wi hin he same sampling pe iod a e indica ed by di e en le e s (GLM, p <0.05).
P. Valle-Rome o e al.
Plan Physiology and Biochemis y 210 (2024) 108569
8
2018). I educes he numbe o elec ons being abso bed bu a e no
ixed by ca boxyla ion ac i i y; he e o e, hey a e ee o c ea e ROS
(P´
e ez-Rome o e al., 2020; Salaza -Pa a e al., 2012). Cong uen ly, he
men ioned minimiza ion o ligh -ha es ing complexes has been
add essed in o he halophy es subjec ed o en i onmen al s ess (Koy o
e al., 2013).
In con as , ou esul s e ealed ha S. u icosa was able o modula e
i s an ioxidan sys em o acclima e o excess coppe , as do o he halo-
phy es acing luc ua ing s ess en i onmen (Bash i and P asad, 2015;
Mesnoua e al., 2016; Redondo-G´
omez e al., 2011; S u hi e al., 2017).
In his sense, ROS-sca enging enzymes such as APX, CAT, SOD and GPX
play an essen ial ole in egula ing ROS le els in plan s (Dazy e al.,
2009; Pa lak and Yilmaz, 2013). In pa icula , SOD con e s supe oxide
adicals o H
2
O
2
, while APX, GPX and CAT ca alyze he decomposi ion
Fig. 5. (A) Asco ba e pe oxidase (APX), (B) ca alase (CAT), (C) supe oxide dismu ase (SOD) and (D) guaiacol pe oxidase (GPX) in andomly selec ed Sa coco nia
u icosa plan s subjec ed o wo coppe concen a ions (0 and 12 mM CuSO
4
) in combina ion wi h h ee ni a e le els (2, 14 and 50 mM KNO
3
) a e 60 days o
ea men . Each alue ep esen s he mean ±s anda d e o o h ee eplica es. Cu, NO
3
o Cu x NO
3
in he uppe co ne o he panel indica es signi ican main e ec s
o in e ac ions (LSD es , *p <0.05, **p <0.01). In case o in e ac ion, Cu x NO
3
, means ha a e signi ican ly di e en om each o he a e indica ed by di e en
le e s (GLM, p <0.05).
Fig. 6. (A, B) Coppe con en (Cu) and (C, D) ni ogenous con en (N) in abo eg ound and belowg ound issues in andomly selec ed Sa coco nia u icosa plan s
subjec ed o wo coppe concen a ions (0 and 12 mM CuSO
4
) in combina ion wi h h ee ni a e le els (2, 14 and 50 mM KNO
3
) a e 60 days o ea men . Each alue
ep esen s he mean ±s anda d e o o h ee eplica es. Cu, NO
3
o Cu x NO
3
in he uppe co ne o he panel indica es signi ican main e ec s o in e ac ions (LSD
es , *p <0.05, **p <0.01). In case o in e ac ion, Cu x NO
3
, means ha a e signi ican ly di e en om each o he a e indica ed by di e en le e s (GLM, p <0.05).
P. Valle-Rome o e al.
Plan Physiology and Biochemis y 210 (2024) 108569
9
o H
2
O
2
in o H
2
O (Pi as eh-Anosheh e al., 2023). The inc eased o all
an ioxidan enzyme ac i i ies obse ed in his s udy is ela ed o an
inc ease in he a e o ROS p oduc ion due o Cu inc emen , allowing he
plan o cope wi h his s ess ul condi ion and explaining he high
ole ance demons a ed by S. u icosa. Howe e , beyond his end, he
mos ema kable aspec was ha ni a e applica ion inc eased he ac-
i i y o an ioxidan enzymes, being his e ec mo e p onounced a 50
mM NO
3
, which was consis en wi h he esul s ob ained by se e al
au ho s in plan s subjec ed o a ious en i onmen al ac o s (De Souza
e al., 2021; Moghaddam e al., 2023; Wei e al., 2015). The e o e, his
up egula ion ROS-sca enging enzymes, along wi h he a oidance o
excessi e ligh ene gy abso p ion and ans o ma ion in S. u icosa
pho osys ems a o emen ioned, would con ibu e o explaining he high
ole ance demons a ed by his species unde ele a ed concen a ions o
bo h Cu and NO
3
.
These physiological and biochemical indings a e, by hemsel es,
ex emely impo an in e ms o unde s anding he ole ance mecha-
nisms unde lying me al exposu e in halophy es unde he coexis ence o
pollu an s o di e en na u es. Ne e heless, om an ecological and
applica ion pe spec i e, ou indings a e also ele an . Thus, in an
ecological con ex , ou esul s sugges ha he de elopmen and main-
enance o na u al popula ions o S. u icosa would no be ad e sely
a ec ed by occasional episodes o di use pollu ion by ni a es de i ed
om e iliza ion p ac ices in ag icul u al a ms close o ma shes sys-
ems domina ed by his halophy ic species, which a e also subjec o
me al con amina ion (Redondo-G´
omez e al., 2009), ensu ing he
main enance o i s ecosys em se ices. The iden i ica ion o plan di -
e en ial ole ance/ esis ance and me al up ake beha io s unde he
coexis ence o di e en concen a ions o coppe and ni a e also p o-
ides a mo e ealis ic iew o he species po en ial o i s implemen a-
ion as bio- ools in pollu ed es ua ine ecosys ems. In his sense, al hough
nume ous s udies subs an ia e he ema kable phy o emedia ion capa-
bili ies exhibi ed by halophy es (Calone e al., 2022; Liang e al., 2017;
Nikalje and Sup asanna, 2018; Roe and MacFa lane, 2022), i has been
demons a ed ha he up ake o me als, such as coppe , when imp op-
e ly managed, comp omise plan g ow h and, consequen ly, he success
o phy o emedia ion p ojec s (Gibilisco e al., 2022; Ma eos-Na anjo
e al., 2008a; O ego e al., 2020; P´
e ez-Rome o e al., 2018). Rega ding
his conce n, ou esul s highligh ha S. u icosa, wi h i s p o icien
g ow h main enance and e ec i e seques a ion o coppe and ni a e
om he medium, eme ges as an adequa e species o phy o emedia ion
applica ions in soils ain ed wi h hese speci ic pollu an s.
5. Conclusion
Sa coco nia u icosa shows high ole ance o Cu-induced s ess and
ni a e p esence in he medium, as indica ed by he capaci y o main ain
i s g ow h a 12 mM Cu, e en wi h ni a e concen a ion as high as 50
mM NO
3
. This g ow h main enance was linked o he ac i a ion o
di e en ole ance mechanisms, such as he plan abili y o up ake and
e ain a la ge amoun o coppe in i s oo s, p e en ing i s anspo o
he mos sensi i e ae ial pa s. This ansloca ion limi a ion was u he
magni ied wi h he inc emen ni a e concen a ion. Likewise, main-
aining wa e balance, down- egula ion pho osys ems unc ion o a oid
excess ene gy abso p ion and ans o ma ion, along wi h an up egula-
ion o ROS-sca enging enzymes, con ibu ed o explain he high
ole ance demons a ed by his species unde ele a ed concen a ion o
bo h Cu and NO
3
. Finally, om a conse a ional pe spec i e, hese e-
sul s sugges he esilience o S. u icosa and i s po en ial o phy o -
emedia ion applica ions.
CRediT au ho ship con ibu ion s a emen
Ped o Valle-Rome o: W i ing – e iew & edi ing, W i ing – o iginal
d a , In es iga ion, Fo mal analysis, Da a cu a ion. Eloy Manuel Cas-
ellanos: W i ing – e iew & edi ing, Resou ces, P ojec adminis a ion,
Me hodology, In es iga ion, Funding acquisi ion, Concep ualiza ion.
Ca los J. Luque: W i ing – e iew & edi ing, Resou ces, Me hodology,
In es iga ion, Funding acquisi ion, Concep ualiza ion. No is J. Flo es-
Dua e: W i ing – e iew & edi ing, Me hodology, In es iga ion. Elena
Romano-Rod íguez: Me hodology, In es iga ion. Susana Redondo-
G´
omez: W i ing – e iew & edi ing, Supe ision, Resou ces, Me hod-
ology, In es iga ion, Concep ualiza ion. Ignacio D. Rod íguez-Llo -
en e: W i ing – e iew & edi ing, Supe ision, Resou ces, Me hodology.
Eloísa Pajuelo: W i ing – e iew & edi ing, Supe ision, Resou ces,
Me hodology. En ique Ma eos-Na anjo: W i ing – e iew & edi ing,
W i ing – o iginal d a , Supe ision, So wa e, Resou ces, Me hodol-
ogy, In es iga ion, Fo mal analysis, Da a cu a ion, Concep ualiza ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgemen s
This wo k has been inanced by he Campus o In e na ional Excel-
lence o he Sea (CEIMAR) wi hin he amewo k o he “Plan P opio
2021”, by he company “Hal´
o i as Onuba S.L” and suppo ed by he
g an p ojec PID2021-124750NB-I00 inanciado po MICIU/AEI/
10.13039/501100011033 y po FEDER, UE, and TED2021-
131605B–I00, MICIU/AEI/10.13039/501100011033 y po la Uni´
on
Eu opea Nex Gene a ionEU/ PRTR. We also like o hank he Uni e si y
o Se ille G eenhouse Resea ch Gene al Se ice (SGI, CITIUS) o
p o iding acili ies and equipmen . We also app ecia e he aluable
eedback om e iewe s inso a as i has helped us imp o e and en ich
ou wo k.
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