Jou nal o Expe imen al Bo any, Vol. 72, No. 14 pp. 5246–5261, 2021
doi:10.1093/jxb/e ab143 Ad ance Access Publica ion 30 Ma ch 2021
Abb e ia ions:ABA, abscisic acid; AN, ne pho osyn he ic a e; Cl−, chlo ide; CTR, con ol; DAS, days a e sowing; gm, mesophyll di usion conduc ance o CO2;
gs, s oma al conduc ance; NO3−, ni a e; NUE, ni ogen-use e iciency;PO43−, phospha e; Pp, pa ch ou pu p essu e; Qy, PSII quan um yield; RWC, ela i e wa e
con en ; SLA, speci ic lea a ea; SO42−, sul a e; WD, wa e de ici ; WUE, wa e -use e iciency;WUEi, in insic wa e -use e iciency; Ψ
π, osmo ic po en ial; Ψ
p, u go
po en ial; Ψ
W, wa e po en ial.
© The Au ho (s) 2021. Published by Ox o d Uni e si y P ess on behal o he Socie y o Expe imen al Biology.
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 (h p://c ea i ecommons.o g/licenses/by/4.0/),
which pe mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
RESEARCH PAPER
Chlo ide nu i ion imp o es d ough esis ance by enhancing
wa e de ici a oidance and ole ance mechanisms
JuanD.F anco-Na a o1,, PabloDíaz-Rueda1,, Ca losM.Ri e o-Núñez1, Ja ie B umós2,
Al edoE.Rubio-Casal3,, Al onsodeCi es3, JoséM.Colmene o-Flo es1,4,*, and MiguelA.Rosales1,4,*,
1 G oup o Plan Ion and Wa e Regula ion, Ins i u o de Recu sos Na u ales y Ag obiología, Consejo Supe io de In es igaciones
Cien í icas (CSIC), 41012 Se ille, Spain
2 Ins i u o Valenciano de In es igaciones Ag a ias, Cen o de Genómica, Moncada, 46113 Valencia, Spain
3 Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de Se illa, 41012 Se ille, Spain
4 Labo a o y o Plan Molecula Ecophysiology, Ins i u o de Recu sos Na u ales y Ag obiología, Consejo Supe io de In es igaciones
Cien í icas (CSIC), 41012 Se ille, Spain
* Co espondence: m [email p o ec ed] o chemac @i nase.csic.es
Recei ed 30 Sep embe 2020; Edi o ial decision 24 Ma ch 2021; Accep ed 25 Ma ch 2021
Edi o : Ian Dodd, Lancas e Uni e si y,UK
Abs ac
Chlo ide (Cl−), adi ionally conside ed ha m ul o ag icul u e, has ecen ly been de ined as a bene icial mac onu ien
wi h speci ic oles ha esul in mo e e icien use o wa e (WUE), ni ogen (NUE), and CO2 in well-wa e ed plan s.
When supplied in a bene icial ange o 1–5mM, Cl− inc eases lea cell size, imp o es lea osmo egula ion, and educes
wa e consump ion wi hou impai ing pho osyn he ic e iciency, esul ing in o e all highe WUE. Thus, adequa e man-
agemen o Cl− nu i ion a ises as a po en ial s a egy o inc ease he abili y o plan s o wi hs and wa e de ici . To
s udy he ela ionship be ween Cl− nu i ion and d ough esis ance, obacco plan s ea ed wi h 0.5–5mM Cl− sal s
we e subjec ed o sus ained wa e de ici (WD; 60% ield capaci y) and wa e dep i a ion/ ehyd a ion ea men s, in
compa ison wi h plan s ea ed wi h equi alen concen a ions o ni a e, sul a e, and phospha e sal s. The esul s
showed ha Cl− applica ion educed s ess symp oms and imp o ed plan g ow h du ing wa e de ici . D ough esis -
ance p omo ed by Cl− nu i ion esul ed om he simul aneous occu ence o wa e de ici a oidance and ole ance
mechanisms, which imp o ed lea u go , wa e balance, pho osyn hesis pe o mance, and WUE. Thus, i is p oposed
ha bene icial Cl− le els inc ease he abili y o c ops o wi hs and d ough , p omo ing a mo e sus ainable and esilien
ag icul u e.
Keywo ds: Bene icial mac onu ien , chlo ide, d ough esis ance, pho osyn hesis, u go , wa e de ici , wa e ela ions, WUE.
applypa as yle " ig//cap ion/p[1]" pa as yle "FigCap "
This pape is a ailable online ee o all access cha ges (see h ps://academic.oup.com/jxb/pages/openaccess o u he de ails)
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Chlo ide nu i ion imp o es d ough ole ance | 5247
In oduc ion
In he con ex o he clima e change and s ong wa e demand
om in ensi e ag icul u e, d ough is ce ainly he abio ic
s ess ha mos se e ely a ec s c op p oduc i i y (Comas e al.,
2013; FAO, 2016). Unde s anding how plan s espond o wa e
a ailabili y and how wa e is used o op imal biomass p oduc-
ion and yield has gained eno mous impo ance in ag icul u e
(Da ies and Benne , 2015; Mau el and Nac y, 2020). In gen-
e al, lowe a ailabili y o soil wa e du ing d ough leads o a
dec ease o lea ela i e wa e con en (RWC) and lea wa e
po en ial (Ψ
w) ha causes abscisic acid (ABA) biosyn hesis
(McAdam and B od ibb, 2016, 2018; Sack e al., 2018), ig-
ge ing complex plan acclima iza ion esponses a molecula ,
cellula , and physiological le els. These esponses include wa e
de ici (WD) a oidance and ole ance mechanisms, acco ding
o he nomencla u e o Le i (1972). A oidance esponses in-
clude mechanisms ha main ain plan wa e con en and Ψw
close o uns essed le els, mainly by inc easing wa e up ake o
limi ing wa e loss. Induc ion o s oma al closu e educes wa e
loss h ough anspi a ion (Rosales e al., 2012; Koe oe s e al.,
2016; Buckley, 2019), bu leads o a educ ion o CO2 a ail-
abili y and pho osyn hesis and, consequen ly, o he dec ease o
ege a i e g ow h and yield (Galmés e al., 2007; Cha es e al.,
2009; Fe guson e al., 2018). In addi ion, mechanisms ha im-
p o e soil wa e up ake a e also s imula ed (Rosales e al., 2019;
Scha wies and Dinneny, 2019). When WD a oidance mech-
anisms a e o e come and plan issues expe ience cellula de-
hyd a ion, ole ance mechanisms mus ensu e cell su i al and
he plan abili y o esume g ow h, including he induc ion o
cell osmo ic adjus men and he biosyn hesis o p o ec i e sol-
u es and p o eins (Ve slues e al., 2006).
Conside ing ha 80% o he a ailable eshwa e esou ces
a e cu en ly consumed by ag icul u e, he imp o emen o
wa e -use e iciency (WUE), de ined as he amoun o ca bon
ixed in pho osyn hesis pe uni o wa e anspi ed, emains es-
sen ial o es ablishing a balance be ween ag icul u e and wa e
esou ces (Condon e al., 2004; Flexas e al., 2016). Because o
he u gen need o imp o e he wo ld’s c op p oduc ion, WUE
is conside ed an essen ial ai o minimize he loss o wa e
in plan s. As a consequence, conside able e o s ha e been
made o elucida e physiological and gene ic ac o s associa ed
wi h his ai (Condon e al., 2004; Blum, 2009; Hessini e al.,
2009; Med ano e al., 2015). Se e al s a egies ha e ocused on
ob aining new c op a ie ies wi h highe WUE and on be e
managemen o wa e esou ces, such as: (i) imp o ing he i i-
ga ion p ocesses and educing he wa e loss h ough soil e ap-
o a ion o leakage; (ii) inc easing he e iciency o ixing ca bon
in ela ion o wa e anspi ed; and (iii) pa i ioning mo e o
he achie ed biomass in o he ha es ed p oduc ( e iewed in
Condon e al., 2004). Howe e , due o he complexi y o hese
ai s, simple and mo e speci ic aspec s o WUE a e equi ed
o iden i y single a ge s o manipula ion (Flexas e al., 2016).
Chlo ide (Cl−) has been well cha ac e ized as a mic onu-
ien , playing an essen ial ole as a co ac o o PSII and egu-
la ing he ac i i y o some enzymes (B oadley e al., 2012). In
addi ion, Cl− is a majo osmo ically ac i e solu e in he acuole
(Flowe s, 1988). As a coun e anion, Cl− plays ele an oles
in egula ing he elec ical po en ial o di e en memb anes,
he o ganella pH g adien s, and he elec ical exci abili y o
plan cells (Whi e and B oadley, 2001). Howe e , Cl− has been
adi ionally conside ed ha m ul o ag icul u e, o wo main
easons: (i) he oxici y esul ing om excessi e Cl− accumu-
la ion in sensi i e c ops unde sal s ess condi ions (Li e al.,
2017; Geil us, 2018); and (ii) he gene alized belie ha Cl− an-
agonizes ni a e (NO3−) homeos asis, impai ing he abili y o
c ops o anspo and accumula e NO3− (Ka ka i e al., 1982;
Siddiqi e al., 1990; Xu e al., 2000; Wege e al., 2017). Howe e ,
Cl− nu i ion o ypical mac onu ien le els has been ecen ly
unco e ed as bene icial o plan g ow h unde well-wa e ed
condi ions, wi h new biological unc ions ha imp o e cell
wa e balance, whole-plan wa e ela ions, pho osyn hesis pe -
o mance, WUE, and ni ogen-use e iciency (NUE; i.e. he
ege a i e o ep oduc i e biomass yield pe uni o ni ogen
a ailable in he soil) in plan s (F anco-Na a o e  al., 2016,
2019; Rosales e al., 2020). Thus, Cl− has been p oposed as a
bene icial mac onu ien (F anco-Na a o e al., 2016), a de -
ini ion u he suppo ed by o he s (Ra en, 2017; Wege e al.,
2017; Geil us, 2018; O ieux e al., 2018; Bazihizina e al., 2019;
Ra en, 2020). Fi s ly, when supplied abo e he mic onu ien
equi emen and below he oxici y h eshold (e.g. 1–5mM
Cl−), Cl− plays speci ic oles in he egula ion o cell osmola i y
and u go , s imula ing lea cell size and lea wa e balance. The
esul ing enla gemen o lea cell size educes he s oma al
densi y, which in u n lowe s s oma al conduc ance (gs) and
wa e consump ion. Secondly, Cl− also inc eases mesophyll
di usion conduc ance o CO2 (gm), which makes i possible
o main ain he plan pho osyn he ic capaci y despi e he e-
duc ion o gs, esul ing in o e all highe WUE in well-wa e ed
plan s (F anco-Na a o e al., 2019). The e o e, adequa e man-
agemen o Cl− nu i ion o imp o e c op yield while also e-
ducing wa e consump ion is pa icula ly challenging in C3
plan s (Ma on e al., 2019).
Cl− luxes a e also ele an o adequa e egula ion o s o-
ma al closu e (Nie es-Co dones e al., 2019) and speci ically
equi ed o cell osmo ic adjus men in esponse o osmo ic
s ess (Shabala and Lew, 2002). The e o e, h ough i s ole in
he egula ion o cell osmola i y, wa e balance, and WUE
unde well-wa e ed condi ions, Cl− homeos asis a ises as a
po en ial adap i e mechanism ha migh inc ease he abili y
o plan s o wi hs and d ough s ess. So a , all p e iously
epo ed unc ions o Cl− nu i ion as a bene icial mac o-
nu ien ha e been expe imen ally pe o med unde well-
wa e ed condi ions. No di ec ela ionship be ween Cl− and
d ough esis ance in glycophy e plan s has been es ablished
o da e. The e o e, he aim o his wo k is o elucida e his
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5248 | F anco-Na a o e al.
ques ion by: (i) quan i ying he deg ee o WD esis ance
o Cl−- ea ed plan s compa ed wi h plan s ea ed wi h
equi alen concen a ions o anionic mac onu ien s such
us NO3−, phospha e, and sul a e; and (ii) iden i ying ele an
physiological mechanisms egula ed by Cl− nu i ion ha
imp o e WD esis ance in plan s.
Ma e ials andme hods
Plan cul i a ion and expe imen aldesign
Tobacco (Nico iana abacum L. a . Ha ana) plan s we e g own unde
g eenhouse expe imen al condi ions ( empe a u e o 25/17±2 °C
day/nigh , ela i e humidi y o 60±10%, and a 16 h/8h pho ope iod
wi h a pho osyn he ic pho on lux densi y o 300–350μmol m−2 s−1).
Plan s we e g own in 7.5 li e po s (20×17×25cm) con aining a mix
o pe li e: e miculi e (4:6), and wa e ed wi h a basal nu ien solu ion
supplemen ed wi h h ee nu i ional ea men s: 5mM Cl− sal s (CL),
5mM NO3− sal s (N), and a mix o sul a e+phospha e (SO42−+PO43−)
sal s (SP), as p e iously epo ed in F anco-Na a o e al. (2016). The CL
ea men was pe o med wi h he applica ion o 5mM Cl−: 2.5mM
KCl, 0.625mM MgCl2, and 0.625mM CaCl2. To e alua e he speci i-
ci y o Cl− in he s udied phenomena, wo addi ional ea men s we e
used: N ea men con aining 2.5mM KNO3, 0.625mM Mg(NO3)2, and
0.625mM Ca(NO3)2; and SP ea men con aining 1.25mM KH2PO4,
0.625mM K2SO4, 0.625mM MgSO4, and 0.625mM CaSO4. All ea -
men s (CL, N, and SP) con ained he same ca ionic balance as shown in
F anco-Na a o e al. (2016). Nu ien s p esen in he basal nu ien so-
lu ion we e as ollows: 1.25mM KNO3, 0.625mM KH2PO4, 0.053mM
K2HPO4, 2 mM Ca(NO3)2, 1 mM MgSO4, 0.1 mM FeNa-EDTA,
0.1 mM H3BO3, 0.1 mM MnSO4, 29 μM ZnSO4, 0.11 μM CoCl2,
53μM KCl, 0.1μM CuSO4, 1μM Na2MoO4, and 5μM KI. All expe i-
men al solu ions we e adjus ed o pH 5.7 wi hKOH.
A e 30 d (45days a e sowing; DAS), in addi ion o he h ee nu-
i ional ea men s, plan s we e subjec ed o wo i iga ion ea men s:
op imal i iga ion (con ol; CTR), in which po s con aining obacco
plan s we e i iga ed up o 100% ield capaci y (3.5 ml g−1 subs a e)
h oughou he expe imen , and WD, wi h po s i iga ed e e y 2–3 d up
o 60% o ield capaci y (2.1ml g−1 subs a e) o 20 d (65 DAS). Du ing
he WD ea men , he esul ing a e age soil wa e con en anged be-
ween 60% and 10% o ield capaci y (Supplemen a y Fig. S1A).
Ano he se o expe imen s wi h inc easing concen a ions o Cl− and
SO42−+PO43− sal s, in combina ion wi h CTR and WD egimes (100%
and 60% o ield capaci y, espec i ely) as explained abo e, was pe o med
o 26 d unde simila expe imen al condi ions o hose p e iously de-
sc ibed (Supplemen a y Fig. S1B). Fo CL ea men s, 0.5, 2, and 5mM
Cl− sal s we e applied o he basal solu ion, whe eas he equi alen SP
ea men s we e also added o ensu e he same ca ionic balance as in di -
e en CL ea men s (as desc ibed in F anco-Na a o e al., 2016).
Plan sampling and de e mina ion o biomass and lea
pa ame e s
Samplings we e pe o med om each combina ion o nu i ional and
i iga ion ea men s a e 20 d o 26 d o wa e es ic ion, in which all
plan s we e non-senescen and a he ea ly ep oduc i e s age. Di e en
plan issues we e ha es ed sepa a ely and lea a ea was measu ed as ex-
plained below. Subsequen ly, FW alues om di e en plan issues we e
ob ained, and samples we e d ied in a o ced-ai o en a 75°C o 48h
o ob ain he DW alues, bo h pa ame e s eco ded as g ams pe plan .
A e ob aining FW alues, de ached lea es o each obacco plan
we e pho og aphed and hei lea a ea was measu ed h ough pixel
quan i ica ion wi h ImageJ2 So wa e wi h a high p ecision o 99.95–
100% (Rasband, 1997; Rueden e al., 2017). Da a we e ob ained in cm2.
Speci ic lea a ea (SLA) was calcula ed as ollows (Ma celis e al., 1998):
SLA=( o al lea a ea)( o al lea DW)−1.
Nu ien con en de e mina ion
O en-d ied lea issue was g ound o powde using a homogenize
(Tau us, 25790, Ba celona, Spain) and he concen a ion o Cl−, NO3−,
SO42−, and PO43− was de e mined as p e iously epo ed (F anco-
Na a o e al., 2016).
Wa e pa ame e s
Lea wa e con en , RWC, succulence, lea osmo ic po en ial (Ψ
π), lea
Ψ
w, and lea u go (o p essu e) po en ial (Ψ
p) we e de e mined as p e-
iously desc ibed in F anco-Na a o e al. (2016).
Wa e consump ion was quan i ied g a ime ically by eco ding he
weigh loss o each po , equi alen o he olume o solu ion consumed
and los by e apo anspi a ion by each plan . In WD- ea ed plan s, wa e
consump ion was quan i ied as he olume o wa e needed o main ain
ield capaci y up o 60%. In eg a ed WUE (WUEi) was calcula ed as he
inc ease o plan DW o e ime ela ed o he accumula ed wa e con-
sump ion (g DW ml−1 H2O), as well as he DW ob ained h oughou he
expe imen and a e ha es ing ela ed o o al wa e consump ion (g
DW ml−1 H2O) (Abba e e al., 2004).
Wa e dep i a ion and ehyd a ion assay: quan um yield and
p essu ep obes
Six obacco plan s o each nu i ional ea men (SP, CL, and N) we e
main ained unde CTR condi ions up o 73 DAS, when wa e dep i -
a ion was applied o 4 d, and, a 77 DAS, plan s we e ehyd a ed a 100%
o ield capaci y and moni o ed un il 80 DAS. Th ee plan s om each nu-
i ional ea men we e moni o ed e e y day by g a ime ic me hods o
e i y he wa e con en in he soil, and PSII quan um yield (Qy) meas-
u emen s we e pe o med. Fo Qy de e mina ion, chlo ophyll luo es-
cence in ligh -adap ed plan s was measu ed using a po able luo ome e
(Fluo Pen FP-100; Pho on Sys em Ins umen s, B no, Czech Republic),
as desc ibed in F anco-Na a o e al. (2016). Fo each ea men , 3–5
pho osyn he ically ac i e and ully expanded in e media e lea es om six
plan s we e used. Qy measu emen s we e conduc ed e e y day be ween
10 h and 12h om he beginning o he wa e es ic ion ea men
(46–64DAS).
Fo he o he h ee plan s, each plan was moni o ed wi h 2–3
LPCP p obes (so-called ZIM p obes; ZIM Plan Technology GmbH,
Hennigsdo , Ge many), a non-in asi e echnique ha eco ds lea
u go p essu e in eal- ime (desc ibed in de ail in Zimme mann e al.,
2008, 2010). The lea pa ch ou pu p essu e (Pp) is eco ded in a lea
ha is pa ched be ween a me allic sensing chip and a magne ic pad.
Pp is in e sely co ela ed wi h he lea u go p essu e (Eh enbe ge
e al., 2012). Signals a e sen wi elessly by ansmi e s o a con olle
ha ans e s he da a o a GPRS modem linked o an In e ne se e .
P obes and he In e ne -based da a ans e sys em we e pu chased
om ZIM Plan Technology GmbH. P obes we e clamped on 2–3
pho osyn he ically ac i e and ully expanded in e media e lea es
( i h–six h lea es om he op o he plan , a ~0.80 m abo e he
g ound), be ween he cen al ascula bundle and he edge o he
lea es (~3cm away om he edge), and in he middle pa o hose
lea es, in o de o es ablish a uni o m con ac wi h he lea issue
a oiding ne es (Fe nández e al., 2011). The clamping was pe o med
p e-dawn a maximum u gidi y as ecommended by Zimme mann
e al. (2008, 2010, 2013). P essu e signals we e app op ia ely adjus ed
be ween 10 kPa and 25 kPa, changing he dis ance be ween he wo
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Chlo ide nu i ion imp o es d ough ole ance | 5249
magne s. The p essu e senso magne was placed on he abaxial side o
he lea es. When p essu e p obe eco dings became s able a 68 DAS,
a ia ion in he diu nal ampli ude o Pp was ound because o possible
di e ences in he ini ial clamp p essu e, lea hickness, o comp ess-
ibili y a ia ions as epo ed in Zimme mann e al. (2008).
Lea gas exchange pa ame e s
Ne pho osyn he ic a e (AN) and s oma al conduc ance (gs) we e meas-
u ed be ween 12.00h and 14.00h using an open gas exchange sys em
(LI-6400, LI-COR, Lincoln, NE, USA) equipped wi h a 2×3cm LED
chambe (LI-6400-02B) as desc ibed in F anco-Na a o e  al. (2016).
The WUEi was calcula ed as he a io be ween he a e o pho osyn hesis
and s oma al conduc ance (AN/gs).
S a is ical analyses
S a is ical analyses we e pe o med using STATGRAPHICS Cen u ion
XVI so wa e (S a Poin Technologies, Wa en on, VA, USA). Shapi o–
Wilk (W) es was used o e i y he no mali y o he da a se s. One-way
ANOVA and mul i a ia e analysis o a iance (MANOVA) we e pe -
o med o de e mine signi ican di e ences be ween g oups o samples,
and le els o signi icance we e desc ibed by as e isks: *P≤0.05; **P≤0.01;
***P≤0.001. Non-signi ican (ns) di e ences we e indica ed when P
was >0.05. Mul iple compa isons o means we e de e mined by he
Tukey’s HSD (hones ly signi ican di e ence) and MRT (mul iple ange
es ) s a is ical es s included in he men ioned so wa e. Analysis o co-
a iance (ANCOVA) was pe o med wi h R so wa e (h ps://www. -
p ojec .o g/) o compa e he slopes o he ela ionship o o al biomass
wi h wa e consump ion be ween CL and SP ea men s. Values ep e-
sen he mean o a leas six obacco plan s in each ea men , which we e
ep oduced in a leas h ee independen expe imen s (Supplemen a y
Table S1).
Resul s
The e ec o Cl− on plan g ow h du ing wa e de ici
To s udy whe he Cl− nu i ion pa icipa es in plan adap-
i e esponses o d ough s ess, g eenhouse expe imen s we e
pe o med unde wo i iga ion egimes: op imal i iga ion
(CTR) and sus ained wa e de ici (WD). Fo he WD ea -
men , plan s we e wa e ed e e y 2–3 d wi h he h ee nu i-
ional ea men s (CL, N, and SP) un il he subs a e eached
2.1 ml g−1 (60% o ield capaci y). The WD ea men was
main ained o 20 d, whe eas wa e ing up o 100% o ield
capaci y was es ablished o he CTR ea men (3.5ml g−1).
The ime cou se o he subs a e wa e loss h oughou a ep e-
sen a i e expe imen is p esen ed in Supplemen a y Fig. S1A.
Fi s , we e i ied whe he he e ec s o he 5mM Cl− ea -
men (CL) on plan g ow h we e consis en wi h hose p e i-
ously ob ained in F anco-Na a o e al. (2016, 2019). Wi h his
aim, we conduc ed new se s o expe imen s (Supplemen a y
Table S1) and compa ed di e en nu i ional and physiological
e ec s o he CL ea men wi h hose o plan s subjec ed o
low Cl− (SP and N ea men s). Consis en ly, lea anion con-
en s (Cl−, NO3−, SO42−, and PO43−) we e di e en ially ac-
cumula ed in plan s acco ding o he espec i e nu i ional
ea men s (CL, N, and SP) unde bo h i iga ion egimes
(Table 1; Supplemen a y Table S2). Nu i ional and i iga ion
ea men s and hei in e ac ion signi ican ly a ec ed Cl− and
NO3− con en s in obacco lea es (Table 1; Supplemen a y
Table S2). Unde con ol condi ions, he Cl− concen a ion
in CL- ea ed lea es was 106.5mM (i.e. 55.7mg g−1 DW),
eaching ypical mac onu ien le els. In SP and N plan s,
Cl− con en was 100 imes lowe , al hough a exceeding he
c i ical le els o de iciency equi ed o ul il essen ial mic onu-
ien unc ions (B oadley e al., 2012; Colmene o-Flo es e al.,
2019). In e es ingly, he Cl‒ con en signi ican ly inc eased in
d ough -s essed CL and SP plan s (1.12 and 2.5 imes, espec -
i ely), whe eas no changes we e obse ed in N plan s. In add-
i ion, he NO3− concen a ion was s ongly dec eased by WD
in SP and N plan s (2.1 and 3.3 imes, espec i ely), whe eas no
ele an changes in SO42− and PO43− con en s we e obse ed
(Table 1).
Table 1. Anion concen a ion in lea es subjec ed o di e en nu i ional and i iga ion ea men s
Cl− (mM) NO3− (mM) PO43− (mM) SO42− (mM)
CTR WD PCTR WD PCTR WD PCTR WD P
SP 1.02±0.08 b 2.56±0.61 b * 6.71±1.18 b 3.20±0.78 b * 15.7±1.51 a 13.8±1.62 a ns 33.5±1.80 a 30.8±4.06 a ns
CL 106.5±3.85 a 118.9±2.50 a * 2.48±0.28 b 2.00±0.42 b ns 6.97±0.58 b 8.76±0.29 b ** 12.0±2.55 b 11.1±0.38 b ns
N1.01±0.16 b 1.05±0.10 b ns 46.9±7.22 a 14.2±1.19 a ** 9.02±0.28 b 9.01±0.24 b ns 15.5±3.56 b 17.2±1.20 b ns
P*** *** *** *** *** *** *** ***
I** *** ns ns
NT *** *** *** ***
I×NT ** *** ns ns
Nu i ional ea men (NT) consis ed o a basal nu ien solu ion supplemen ed wi h 5mM chlo ide (CL), 5mM ni a e (N), o he sul a e+phospha e (SP)
sal mix u e con aining he same ca ionic balance as in he CL and N ea men s. I iga ion ea men (I) consis ed o a con ol ea men o well-wa e ed
plan s (CTR; 100% ield capaci y) and sus ained wa e de ici (WD; 60% ield capaci y) ea men s. Mean alues ±SE, n=6. Le els o signi icance:
***P≤0.001,**P≤0.01, *P≤0.05, and P >0.05 (‘ns’, non-signi ican ). ‘Homogeneous g oup’ s a is ics we e calcula ed h ough ANOVA and MANOVA es s,
whe e mean alues wi h di e en le e s a e signi ican ly di e en acco ding o Tukey’s es .
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5250 | F anco-Na a o e al.
As demons a ed in F anco-Na a o e al. (2016), he appli-
ca ion o 5mM Cl− unde con ol condi ions p omo ed plan
g ow h when compa ed wi h SP plan s (Supplemen a y Figs
S2, S3A–C), mainly due o highe lea expansion and shoo
g ow h (Supplemen a y Fig. S3D), which was in u n a con-
sequence o he s imula o y e ec o Cl− on cell expansion
(F anco-Na a o e al., 2016). On he o he hand, he N ea -
men s ongly s imula ed plan g ow h and lea expansion as a
esul o a highe a e o bo h cell di ision and me abolic ac-
i i y gi en he impo an ole o ni ogen in plan me abolism,
g ow h, and de elopmen (Hawkes o d e  al., 2012; F anco-
Na a o e  al., 2016, 2019; Supplemen a y Figs S2, S3A–C).
When e alua ing g ow h esponses o d ough , we ound ha
plan s subjec ed o WD showed educed o al, lea , and oo
biomass unde all nu i ional ea men s, wi h signi ican in e -
ac ions be ween i iga ion and nu i ional ea men s (Fig. 1A;
Supplemen a y Figs S2, S3A–C). Howe e , he Cl− applica ion
caused lowe educ ion o plan g ow h (35–45% educ ions o
o al and o gans biomass) han SP and N ea men s (45–55%
and 50–60% educ ions, espec i ely) du ing WD (Fig. 1A).
To u he explo e he ole o Cl− in plan acclima iza ion o
WD, di e en mo phological pa ame e s widely used as key
lea ai s we e measu ed: lea a ea, numbe o lea es, and SLA
(i.e. he lea a ea pe uni o biomass in es ed). Unde con ol
condi ions, N plan s showed he signi ican ly highes lea a ea
due o he occu ence o la ge and mo e nume ous lea es,
while CL plan s p esen ed highe lea a ea han SP plan s
(Supplemen a y Fig. S3D, E). Howe e , non-signi ican di e -
ences in SLA be ween he h ee nu i ional ea men s we e
obse ed (Supplemen a y Fig. S3F). The WD ea men caused
a s ong educ ion in bo h he a ea and numbe o lea es in SP
and N plan s, which was mo e signi ican in N- ea ed plan s
(Fig. 1B; Supplemen a y Fig. S3D, E). In e es ingly, WD caused
no changes in he numbe o lea es in CL plan s, exhibi ing
a smalle educ ion o lea a ea in compa ison wi h he SP
and N ea men s (Fig. 1B). Fu he mo e, whe eas SP and N
plan s showed a simila SLA educ ion unde WD, Cl− appli-
ca ion signi ican ly inc eased i (Fig. 1B; Supplemen a y Fig.
S3F). Taken oge he , ou esul s alida e he bene icial e ec
o Cl− nu i ion on plan g ow h unde bo h well-wa e ed and
WD condi ions in obacco plan s, whe eas N- ea ed plan s
exhibi ed he highes sensi i i y oWD.
The e ec o Cl− nu i ion on whole-plan wa e -use
e iciency and wa e balance du ing wa e de ici
Conside ing ha Cl− nu i ion imp o es whole-plan WUE
and wa e balance in well-wa e ed plan s (F anco-Na a o
e  al., 2016, 2019), and alle ia es de imen al e ec s o WD
on plan g ow h (Fig. 1), we wonde ed whe he Cl− nu i ion
induces plan physiological esponses linked o wa e ela ions
du ing WD. Measu emen o he o al plan weigh ela i e o
accumula ed wa e consumed showed highe in eg a ed WUE
alues in Cl‒- ea ed plan s du ing bo h CTR (Fig. 2A) and
WD (Fig. 2B) ea men s. In e es ingly, when compa ed wi h
well-wa e ed plan s, we obse ed ha WUE alues exhibi ed
a >2- old inc ease du ing WD, emaining highe always in
CL plan s (Fig. 2B). To be e compa e di e ences be ween
Fig. 1. E ec o Cl− nu i ion and sus ained wa e de ici on plan g ow h.
Plan s we e al e na i ely ea ed wi h (i) h ee nu i ional ea men s:
5mM chlo ide sal s (CL), 5mM ni a e sal s (N), and a mix u e o
sul a e+phospha e sal s (SP) con aining he same ca ionic balance as
in he CL and N ea men s; and (ii) wo i iga ion ea men s: 100%
ield capaci y (CTR, con ol) and 60% ield capaci y (WD, wa e de ici ).
(A) E ec on o al, lea , and oo DW (%) in WD plan s no malized o
CTR plan s. (B) E ec on lea a ea and numbe and speci ic lea a ea
(SLA) in WD plan s no malized o CTR plan s. Absolu e alues o CTR
ea men s we e as ollows: o al DW (g), SP=23.2±1.19, CL=27.9±0.58,
N=43.8±1.66; lea DW (g), SP=7.60±0.34, CL=9.43±0.40, N=16.4±0.63;
oo DW (g), SP=2.47±0.16, CL=2.96±0.13, N=4.82±0.25; lea a ea
(cm2), SP=2156±44.5, CL=2575±49.7, N=4198±75.1; numbe o
lea es, SP=17.8±0.47, CL=14.3±0.43, N=26.5±0.73; SLA (cm2 g-1 DW),
SP=273.5±6.72, CL=269.4±1.53, N=263.1±4.45. Mean alues ±SE. n=6.
‘Homogeneous g oup’ s a is ics we e calcula ed h ough ANOVA, whe e
mean alues wi h di e en le e s a e signi ican ly di e en acco ding o
Tukey’s es a P≤0.05. Le els o signi icance: ***P≤0.001.
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Chlo ide nu i ion imp o es d ough ole ance | 5251
high and low Cl− ea men s, biomass e sus wa e use ela-
ionships we e plo ed in Fig. 2C (CTR) and Fig. 2D (WD).
Gi en ha he biomass o N plan s di e s g ea ly om ha
o he o he ea men s (Supplemen a y Fig. S3), only he CL
e sus SP ionic ea men s we e compa ed. Using ANCOVA
(as epo ed in Pué olas e al., 2017), signi ican di e ences
be ween CL and SP ea men s we e obse ed in bo h CTR
and WD ea men s, showing ha Cl−- ea ed plan s ha e a
g ea e capaci y o p oduce biomass in ela ion o he amoun
o wa e consumed (Fig. 2C, D). I is no ewo hy ha unde
con ol condi ions, CL plan s main ained highe g ow h wi h
less wa e consumed han SP plan s (Fig. 2C). Howe e , unde
WD condi ions, CL plan s main ained highe WUE (Fig. 2B)
despi e consuming mo e wa e , due o highe biomass p o-
duc ion (Fig. 2D). When con ol and WD alues we e plo ed
oge he (Supplemen a y Fig. S4), he ANCOVA showed ha
he slopes o he ela ionship a ied be ween CL and SP ea -
men s, u he suppo ing WUE di e ences be ween ionic
ea men s. When we del ed in o lea -le el esponses o WD,
ou esul s showed ha WD educed lea wa e con en , RWC,
and succulence in plan s subjec ed o all nu i ional ea -
men s (Fig. 3). Howe e , hese wa e pa ame e s exhibi ed a
signi ican Cl−-dependen s imula ion in compa ison wi h
SP and N ea men s unde bo h con ol and WD condi ions,
showing ha Cl− alle ia es nega i e e ec s o WD on plan
wa e balance.
To u he in es iga e he ole o Cl− in egula ing whole-
plan wa e ela ions and, pa icula ly, u go main enance
du ing wa e dep i a ion/ ehyd a ion, we moni o ed he
u go p essu e changes o obacco lea es by using magne ic
lea pa ch-clamp p essu e p obes (ZIM-p obe; Zimme mann
e al., 2008). This non-in asi e echnique allows he eal- ime
moni o ing o he u go p essu e o in ac lea es wi h high
p ecision (Fig. 4A). The measu ed lea pa ch ou pu p essu e Pp
is in e sely p opo ional o he lea u go . Be o e wa e dep i -
a ion, Pp alues eco ded in he h ee ea men s (SP, CL, and N)
g adually inc eased du ing he day, indica ing u go loss a e
sun ise, and ab up ly dec eased du ing sunse , indica ing lea
Fig. 2. E ec o Cl− nu i ion and sus ained wa e de ici on in eg a ed wa e -use e iciency. Plan s we e al e na i ely ea ed wi h (i) h ee nu i ional
ea men s (NT): 5mM chlo ide sal s (CL); 5mM ni a e sal s (N); and a mix u e o sul a e+phospha e sal s (SP) con aining he same ca ionic balance as
in he CL and N ea men s; and (ii) wo i iga ion ea men s we e also applied: 100% ield capaci y (CTR, con ol) and 60% ield capaci y (WD, wa e
de ici ). E ec on in eg a ed wa e -use e iciency (WUE) in plan s subjec ed o CTR (A) and WD (B) ea men s. Rela ionship be ween o al biomass
and accumula ed wa e consump ion in plan s du ing CTR (C) and WD (D) ea men s. Mean alues ±SE, n=6. ‘Homogeneous g oup’ s a is ics we e
calcula ed h ough ANOVA and MANOVA, whe e mean alues wi h di e en le e s a e signi ican ly di e en acco ding o Tukey’s es a P≤0.05. The
eg ession line o each SP and CL pool is shown in bo h panels (C and D), whe e P- alues and ANCOVA o compa e eg ession slopes a e shown.
Le els o signi icance: ***P≤0.001.
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5252 | F anco-Na a o e al.
u go eco e y du ing he nigh . Al hough some di e ences
in ampli ude we e ound be ween ea men s, he kine ics o
he Pp cu es om di e en plan s showed he same ci cadian
ends. I iga ion wi h he h ee nu i ional ea men s (SP,
CL, and N) was wi hheld o 4 d un il he wa e con en o
d ough -s essed po s eached be ween 10% and 20% o he
wa e con en measu ed in well-wa e ed po s. Subsequen ly,
i iga ion was es o ed o con ol wa e le els. A e WD im-
posi ion, s ong loss o u go (inc ease in Pp alues) was ob-
se ed in plan s subjec ed o SP and N ea men s. Howe e ,
u go alues we e no signi ican ly al e ed by WD in CL
plan s, which main ained a Pp pa e n simila o ha o well-
wa e ed plan s (Fig. 4A). To quan i y cell damage p oduced by
he esul ing lea issue dehyd a ion, he pho osyn he ic e i-
ciency o PSII was measu ed wi h a chlo ophyll luo ome e
in a da k-adap ed s a e. The CL ea men de e mined much
g ea e p o ec ion o he pho osyn he ic machine y unde se-
e e WD, wi h signi ican ly highe Qy alues han hose o SP
and N ea men s (Fig. 4A). A e ehyd a ion, CL plan s, bu
no SP and N plan s, ully eco e ed Pp and Qy alues o hose
o con ol condi ions.
To de e mine whe he imp o ed wa e balance pa am-
e e s o CL plan s we e associa ed wi h he Cl− osmo egu-
la o y p ope ies and he esul ing s imula ion o lea u go
obse ed in well-wa e ed plan s (F anco-Na a o e al., 2016;
Colmene o-Flo es e al., 2019), Ψ
π, Ψ
w, and Ψ
p we e meas-
u ed in lea es o obacco plan s. Cl–- ea ed plan s showed
mo e nega i e alues o Ψ
π unde bo h con ol and WD con-
di ions (Fig. 4B), indica ing g ea e osmo egula o y capaci y
due o highe accumula ion o osmo ically ac i e solu es in
hei lea issues. This in u n led o signi ican ly highe Ψ
p
alues in CL plan s (Fig. 4B) and, consequen ly, o a be e
ole ance o WD. Highe u go o CL plan s was also a conse-
quence o less nega i e Ψ
w alues unde bo h con ol and WD
condi ions (Fig. 4B), caused by he highe lea wa e con en
o Cl−- ea ed plan s (Fig. 3A). In e es ingly, he mo e posi i e
lea Ψ
w o CL plan s, in compa ison wi h SP and N plan s, in-
dica es ha o he e en s a ec ing plan wa e ela ions migh
be egula ed by Cl−, as desc ibed below.
The e ec o Cl− nu i ion on gas exchange and
pho osyn he ic wa e -use e iciency du ing wa e de ici
To be e unde s and he ole o Cl− on he egula ion o plan
wa e ela ions, gs was quan i ied unde CTR and WD con-
di ions (Fig. 5A). As p e iously shown (F anco-Na a o e al.,
Fig. 3. E ec o Cl− nu i ion and sus ained wa e de ici on wa e
pa ame e s. Plan s we e al e na i ely ea ed wi h (i) h ee nu i ional
ea men s (NT): 5mM chlo ide sal s (CL), 5mM ni a e sal s (N), and
a mix u e o sul a e+phospha e sal s (SP) con aining he same ca ionic
balance as in he CL and N ea men s; and (ii) wo i iga ion ea men s
(I): 100% ield capaci y (CTR, con ol) and 60% ield capaci y (WD, wa e
de ici ). E ec on lea wa e con en (A), lea ela i e wa e con en (RWC)
(B), and lea succulence (C) in CTR and WD ea men s. Mean alues ±SE,
n=6. ‘Homogeneous g oup’ s a is ics we e calcula ed h ough ANOVA,
whe e mean alues wi h di e en le e s a e signi ican ly di e en acco ding
o Tukey’s es a P≤0.05. Le els o signi icance: ***P≤0.001, **P≤0.01,
and ‘ns’ P>0.05.
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Chlo ide nu i ion imp o es d ough ole ance | 5253
2016), he CL ea men ga e ise o lowe gs in well-wa e ed
plan s due o he lowe s oma al densi y. The lowe gs did no
impai he ne pho osyn he ic a e when compa ed wi h SP
plan s (AN; Fig. 5B) as a consequence o he posi i e e ec o
Cl− on he gm (F anco-Na a o e al., 2019), leading o highe
pho osyn he ic o in insic WUEi (AN/gs; Fig. 5C). As a esul ,
Fig. 4. E ec o Cl− nu i ion on wa e s a us du ing wa e dep i a ion and ehyd a ion ea men s. Plan s we e al e na i ely ea ed wi h h ee nu i ional
ea men s (NT): 5mM chlo ide sal s (CL); 5mM ni a e sal s (N); and a mix u e o sul a e+phospha e sal s (SP) con aining he same ca ionic balance
as in he CL and N ea men s. Tobacco plan s o each NT (SP, CL, and N) we e main ained unde op imal i iga ion (CTR) up o 73 DAS, hen wa e
dep i a ion was applied o 4 d and, a 77 DAS, plan s we e ehyd a ed up o 100% o ield capaci y and u he moni o ed un il 80 DAS. (A) E ec
on ield capaci y, e iciency o PSII, and eal- ime measu emen o lea u go using he non-in asi e magne ic lea pa ch-clamp p essu e p obes
(Zimme mann e al., 2008). Pa ch p essu e (Pp) is in e sely co ela ed wi h lea u go p essu e and posi i ely co ela ed wi h lea wa e po en ial and plan
anspi a ion (Zimme mann e al., 2008, 2010). (B) E ec o Cl− nu i ion and sus ained wa e de ici on lea osmo ic po en ial (Ψ
π), lea wa e po en ial
(Ψ
w), and lea u go (o p essu e) po en ial (Ψ
p) in CL, N, and SP plan s, which we e ea ed o 20 d wi h wo i iga ion egimes (I): 100% ield capaci y
(CTR, con ol) and 60% ield capaci y (WD, wa e de ici ). Mean alues ±SE, n=6. ‘Homogeneous g oup’ s a is ics we e calcula ed h ough ANOVA
and MANOVA, whe e mean alues wi h di e en le e s a e signi ican ly di e en acco ding o Tukey’s es a P≤0.05. Le els o signi icance: ***P≤0.001;
**P≤0.01; and *P≤0.05.
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5254 | F anco-Na a o e al.
he be e wa e balance (Fig. 4) and WUE o Cl−- ea ed plan s
inc eased hei ole ance o WD, as e idenced by he lowe cell
damage su e ed in pho osyn he ic issues (Fig. 5D). The e o e,
SP and N plan s, wi h mo e dehyd a ed and less u gid lea es
(Fig. 4B), became mo e s essed by he WD ea men (Fig.
5D), leading o s onge gs educ ion (Fig. 5A) and g ea e loss
Fig. 5. E ec o Cl− nu i ion and sus ained wa e de ici on gas exchange pa ame e s, wa e -use e iciency, and s abili y o PSII. Plan s we e al e na i ely
ea ed wi h (i) h ee nu i ional ea men s (NT): 5mM chlo ide sal s (CL); 5mM ni a e sal s (N); and a mix u e o sul a e+phospha e sal s (SP) con aining
he same ca ionic balance as in he CL and N ea men s; and (ii) wo i iga ion ea men s (I) we e also applied: 100% ield capaci y (CTR, con ol) and
60% ield capaci y (WD, wa e de ici ). E ec on (A) s oma al conduc ance (gs), (B) ne pho osyn he ic a e (AN), and (C) pho osyn he ic o ins an aneous
wa e -use e iciency (WUEi) measu ed in ully expanded pho osyn he ically ac i e lea es om plan s be ween 51 and 65days a e sowing (DAS). (D)
E ec on he highly sensi i e physiological s ess ma ke quan um yield (Qy; s abili y o PSII) measu ed in ully expanded pho osyn he ically ac i e lea es
om plan s be ween 46 and 65 DAS. Mean alues ±SE. n=6. ‘Homogeneous g oup’ s a is ics we e calcula ed h ough ANOVA and MANOVA, whe e
mean alues wi h di e en le e s a e signi ican ly di e en acco ding o Tukey’s es a P≤0.05. Le els o signi icance: ***P≤0.001; **P≤0.01; *P≤0.05; and
‘ns’ P>0.05.
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