Jou nal o Expe imen al Bo any, Vol. 66, No. 19 pp. 5769–5781, 2015
doi:10.1093/jxb/e 279 Ad ance Access publica ion 2 July 2015
This pape is a ailable online ee o all access cha ges (see h p://jxb.ox o djou nals.o g/open_access.h ml o u he de ails)
RESEARCH PAPER
Mala e as a key ca bon sou ce o lea da k- espi ed CO2
ac oss di e en en i onmen al condi ions in po a oplan s
Ma co M.Lehmann1,2,*, Ka ja T.Rinne1, Ca olaBlessing2, Rol T. W.Siegwol 1, NinaBuchmann2 and
Roland A.We ne 2
1 Labo a o y o A mosphe ic Chemis y, Paul Sche e Ins i u e (PSI), CH-5232 Villigen, Swi ze land
2 Ins i u e o Ag icul u al Sciences, ETH Zu ich, Uni e si ae ss . 2, CH-8092 Zu ich, Swi ze land
* To whom co espondence should be add essed. E-mail: ma [email p o ec ed]
Recei ed 28 Janua y 2015; Re ised 30 Ap il 2015; Accep ed 6 May 2015
Edi o : Howa d G i i hs
Abs ac
Dissimila ion o ca bon sou ces du ing plan espi a ion in suppo o me abolic p ocesses esul s in he con inu-
ous elease o CO2. The ca bon iso opic composi ion o lea da k- espi ed CO2 (i.e. δ13CR) shows daily en ichmen s
up o 14.8‰ unde di e en en i onmen al condi ions. Howe e , he easons o his 13C en ichmen in lea da k-
espi ed CO2 a e no ully unde s ood, since daily changes in δ13C o pu a i e lea espi a o y ca bon sou ces (δ13CRS)
a e no ye clea . Thus, we exposed po a o plan s (Solanum ube osum) o di e en empe a u e and soil mois u e
ea men s. We de e mined δ13CR wi h an in- ube incuba ion echnique and δ13CRS wi h compound-speci ic iso ope
analysis du ing a daily cycle. The highes δ13CRS alues we e ound in he o ganic acid mala e unde di e en en i on-
men al condi ions, showing less nega i e alues compa ed o δ13CR (up o 5.2‰) and compa ed o δ13CRS o soluble
ca bohyd a es, ci a e and s a ch (up o 8.8‰). Mo eo e , linea ela ionships be ween δ13CR and δ13CRS among di -
e en pu a i e ca bon sou ces we e s onges o mala e du ing day ime ( 2=0.69, P≤0.001) and nigh ime ( 2=0.36,
P≤0.001) unde all en i onmen al condi ions. Amul iple linea eg ession analysis e ealed δ13CRS o mala e as he
mos impo an ca bon sou ce in luencing δ13CR. Thus, ou esul s s ongly indica e mala e as a key ca bon sou ce o
13C en iched da k- espi ed CO2 in po a o plan s, p obably d i en by an anapleu o ic lux eplenishing in e media es
o he K ebs cycle.
Key wo ds: Compound-speci ic iso ope analysis (CSIA), d ough , o ganic acids, plan espi a ion, s able ca bon iso opes,
suga s, empe a u e, ica boxylic acid (TCA) cycle.
In oduc ion
The in es iga ion o plan espi a ion as a majo p ocess in
plan biochemis y has expanded ou unde s anding o ca -
bon cycling in au o ophic o ganisms. Plan s dissimila e ca -
bon sou ces o he p oduc ion o in e media es and educing
equi alen s in suppo o me abolic p ocesses, he eby con in-
uously eleasing CO2 ia plan espi a ion (Hopkins, 2006).
Lea - espi ed CO2 is mainly de i ed om oxida i e deca box-
yla ion eac ions ca alysed by enzymes om he K ebs cycle
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/3.0/), 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.
© The Au ho 2015. Published by Ox o d Uni e si y P ess on behal o he Socie y o Expe imen al Biology.
Abb e ia ions: δ13CR, ca bon iso opic composi ion o lea da k- espi ed CO2; δ13CRS, ca bon iso opic composi ion o pu a i e lea espi a o y ca bon sou ces;
An, ne assimila ion a e; Ci, in e cellula CO2 concen a ion; CSIA, compound-speci ic iso ope analysis; gs, s oma al conduc ance; HPLC, high pe o mance liquid
ch oma og aphy; KC, K ebs cycle; LEDR, ligh -enhanced da k espi a ion; ME, malic enzyme; OAA, oxaloace a e; PDH, py u a e dehyd ogenase; PEPC,
phosphoenolpy u a e ca boxylase; SPS, suc ose phospha e syn hase; SWC, olume ic soil wa e con en .
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5770 | Lehmann e al.
(KC) and om in e ac ing anabolic and ca abolic eac ions
(Voe and Voe , 2011).
Using s able iso opes, he pa hway o ca bon can be aced
om pho osyn he ic ca bon ixa ion o espi a o y ca bon
loss. On he one hand, C3 plan s disc imina e hea ily agains
13C due o pho osyn he ic iso ope ac iona ion, leading
o gene al 13C deple ion in plan biomass o abou 20‰ in
compa ison o a mosphe ic CO2 (Fa quha e al., 1989). The
exac magni ude o pho osyn he ic ca bon iso ope disc imi-
na ion depends on he in e cellula CO2 concen a ion (Ci) in
he subs oma al ca i y, which is egula ed by o he physiolog-
ical pa ame e s such as ne assimila ion a e (An) and s oma-
al conduc ance (gs). En i onmen al condi ions such as ligh ,
empe a u e, soil mois u e, and ai humidi y will in luence
hese pa ame e s and wi h hem he pho osyn he ic ca bon
iso ope disc imina ion. On he o he hand, he ca bon iso-
opic composi ion o lea da k- espi ed CO2 (i.e. δ13CR) has
clea ly been shown o be less nega i e han lea me aboli es in
se e al plan species (Ghashghaie e al., 2003; Bowling e al.,
2008; We ne and Gessle , 2011; Ghashghaie and Badeck,
2014). In a daily cycle, lea da k- espi ed CO2 ollows a p o-
g essi e 13C en ichmen du ing he day and a g adual 13C
deple ion du ing he cou se o he nigh (Hymus e al., 2005;
P a e e al., 2006), esul ing in a s ong empo al a iabili y
o up o 14.8‰ (Ba bou e  al., 2007; We ne e  al., 2009;
Wegene e al., 2010), which di e s among unc ional g oups
(P iaul e al., 2009; We ne e al., 2009).
δ13CR is he eby linked o he ca bon iso opic composi-
ion o pu a i e lea espi a o y ca bon sou ces (i.e. δ13CRS)
such as ca bohyd a es (soluble mono- and di-saccha ides,
and s a ch) and o ganic acids. P e ious s udies showed ha
en i onmen al d i e s such as empe a u e and soil mois u e
in luence δ13CR and δ13CRS. Mo e nega i e δ13CR alues wi h
inc easing empe a u e ha e been obse ed wi h sho - e m
changes in lea empe a u e du ing da kness in Phaseolus
ulga is (Tche kez e  al., 2003), while long- e m e ec s o
highe empe a u es on δ13CR and δ13CRS ha e no ye been
in es iga ed unde con olled condi ions. O he s udies ha e
demons a ed less nega i e δ13CR and δ13CRS alues unde
d y condi ions compa ed o hose unde we condi ions
(Du anceau e  al., 1999; Ghashghaie e  al., 2001). Simila
obse a ions we e made in ield expe imen s (Sun e al., 2009;
Dubbe e al., 2012). Con e sely, mo e nega i e δ13CR al-
ues ha e been ound unde d y condi ions o Medi e anean
ees and he bs such as Que cus ilex and Tube a ia gu a a
compa ed o hose unde we condi ions (Unge e al., 2010),
which ha e been explained wi h accompanied inc eases in
empe a u es and apou p essu e de ici . Ne e heless, he
combined e ec s o empe a u e and soil mois u e on δ13CR
and δ13CRS unde con olled condi ions ha e ye o be es ed.
Mo eo e , δ13CR is de e mined by a ious pos -pho o-
syn he ic ca bon iso ope ac iona ion p ocesses a pi o al
b anching poin s in espi a o y pa hways, ca bon iso ope
e ec s on enzyma ic eac ions, and changes in espi a o y
subs a es ( o a de ailed e iew see We ne and Gessle ,
2011). The 13C en ichmen in lea da k- espi ed CO2 i sel
is hough o be a esul o agmen a ion ac iona ion p o-
cesses based on he e ogeneous in amolecula ca bon iso ope
dis ibu ion in espi a o y ca bon sou ces (Tche kez e  al.,
2004). Fo ins ance, C-3 and C-4 posi ions o glucose a e
known o be en iched in 13C compa ed o he o he mole-
cule posi ions due o an iso ope e ec o he aldolase eac-
ion (Rossmann e  al., 1991; Gleixne and Schmid , 1997).
B eakdown o glucose du ing glycolysis p oduces py u a e
wi h a 13C en iched C-1 posi ion ( o me C-3 and C-4 posi-
ions o glucose). The ea e , he py u a e dehyd ogenase
eac ion (PDH) eleases he C-1 posi ion as 13C en iched CO2,
whe eas he mo e 13C deple ed ace yl-CoA esidue is used
in he KC (P iaul e  al., 2009; We ne and Gessle , 2011).
Thus, a PDH domina ed espi a o y pa hway may lead o 13C
en ichmen in lea da k- espi ed CO2.
Howe e , he knowledge abou δ13CR is o en based on
ligh -acclima ed lea es, which ha e been ans e ed in o
da kness o allow espi a o y measu emen s. This app oach
holds an unp e en able bias known as ‘ligh -enhanced da k
espi a ion’ (LEDR), which needs o be aken in o accoun
when in e p e ing day ime δ13CR alues. LEDR is a sho -
e m ligh -da k ansi ion pe iod, desc ibing an inc ease in
he amoun o lea da k- espi ed CO2 sho ly upon da k-
ening o abou 20 min, which depends on ligh in ensi y
(A kin e al., 1998). On he one hand, LEDR may be in lu-
enced by eassembly o he KC, which is hough o be only
pa ially ac i e unde ligh condi ions (Tche kez e al., 2005;
Swee lo e e  al., 2010; We ne and Gessle , 2011; We ne
e al., 2011). On he o he hand, LEDR may be d i en by
a b eakdown o a ligh -accumula ed mala e pool, causing
13C-en iched lea da k- espi ed CO2 (Ba bou e al., 2007;
Gessle e al., 2009; We ne e al., 2009; Ba bou e al., 2011;
We ne and Gessle , 2011). Mala e i sel is also known o
be 13C en iched compa ed o o he ca bon sou ces (Gleixne
e al., 1998; Ghashghaie e  al., 2001). The 13C en ichmen
in mala e was a ibu ed o an anapleu o ic lux ia he
phosphoenolpy u a e ca boxylase eac ion (PEPC), which
ixes 13C-en iched hyd ogen ca bona e and eplenishes KC
in e media es (Melze and O’Lea y, 1987; Sa idge and Blai ,
2004). Thus, a possible b eakdown o mala e by he mi o-
chond ial malic enzyme eac ion, o wi hin he KC, may
in luence δ13CR (Ba bou e al., 2007; We ne e al., 2011).
In addi ion, plan s may also use o a ce ain ex en mo e
complex ca bon sou ces such as lipids and p o eins unde
se e e en i onmen al condi ions o unde p olonged da k-
ness (Tche kez e  al., 2003; Usadel e  al., 2008). Howe e ,
he d i ing p ocesses, he espi a o y ca bon sou ces, and he
mechanisms causing changes in δ13CR du ing day and nigh
a e no ully esol ed hus a .
Hence, wi h his s udy we in end o assess wo majo
esea ch ques ions. Wha causes he high daily a ia ions in
δ13CR? How a e δ13CR and δ13CRS in luenced by empe a u e
and soil mois u e condi ions? Ou main objec i es we e (i) o
analyse he ela ionship be ween δ13CR and δ13CRS alues and
(ii) o de e mine changes in δ13CR and δ13CRS alues, as well as
in concen a ions o he pu a i e ca bon sou ces unde di e -
en en i onmen al condi ions. The e o e, we exposed po a o
plan s o di e en con olled empe a u e and soil mois u e
condi ions and measu ed δ13CR wi h an in- ube incuba ion
echnique, as well as δ13CRS and concen a ions o soluble
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Mala e as a key ca bon sou ce o lea da k- espi ed CO2 in po a o | 5771
ca bohyd a es, o ganic acids and s a ch om lea es wi h
compound speci ic iso ope analysis (CSIA) on a daily basis.
Ma e ials and me hods
Plan ma e ial
Po a o plan s (Solanum ube osum L. c . Annabell) we e g own
om ube s o he same size in 5 l po s illed wi h ba k humus soil
(Ökohum, He enho , Swi ze land) in a g eenhouse, wi h a e age
empe a u es o 20/16°C and apou p essu e de ici s (VPD) o abou
0.9/0.4 kPa (day/nigh ). The plan s we e exposed o a 16 h dayligh
pe iod supplemen ed by 400 W sodium-lamps (Powe one Son-T
Plus, Philips, Ams e dam, Ne he lands). Fo y days a e plan ing,
plan s we e ans e ed in o walk-in clima e chambe s o acclima i-
za ion o 2 weeks. The 16 h dayligh in he clima e chambe s had an
a e aged pho osyn he ic pho on lux densi y o ~400µmol m-2 s-1 a
lea le el, hus plan s we e no ully ligh -sa u a ed. Be o e he ea -
men pe iod, soil wa e s a us was op imal o a leas 3 d a e wa e -
ing, while an indi idual plan consumed abou 300 ml wa e pe day.
50 ml o a 0.4% e ilize solu ion ( / , Gesal, Zü ich, Swi ze land)
was applied wice o all plan s du ing he whole expe imen o 70d.
T ea men s we e applied du ing he las 15 d o he expe imen .
Plan s we e exposed o high empe a u e (Thigh) o 28/23°C (day/
nigh ) and low empe a u e condi ions (Tlow) o 22/17°C, a a VPD
o abou 0.9/0.35 kPa o bo h empe a u e ea men s. Th ee cli-
ma e chambe s we e used o eplica ion o each empe a u e ea -
men . Wi hin each clima e chambe he e we e wo soil-mois u e
ea men s wi h nine plan s each. D y soil mois u e condi ions we e
kep cons an ly a 50–60% o he daily wa e consump ion o each
indi idual plan , de e mined by weighing he en i e po s. Plan s
unde we condi ions we e kep a 100%.
The inal sampling pe iod las ed 32 h du ing he las 2 d o he
expe imen , when d y soil condi ions we e es ablished o bo h em-
pe a u e ea men s. Sampling was done on a daily basis e e y 2 h
(nigh ime) o 4 h (day ime). Du ing sampling, indi idual plan s had
3–6 anks, wi h abou ou ully de eloped lea es pe ank. Always
he hi d-las ully de eloped lea pe ank was sampled a all poin s
in ime, bu wi hin 24 h only one sample was aken om each indi id-
ual plan o a oid any s ess esponse induced by sampling. Sampled
lea ma e ial was immedia ely ozen in liquid ni ogen and s o ed a
−80°C. Subsequen ly, he lea ma e ial was eeze-d ied and milled
o powde by a s eel ball mill (MM200, Re sch, Haan, Ge many)
o all u he iso opic and biochemical analyses. In addi ion o lea
sampling, ai CO2 samples om all six clima e chambe s we e col-
lec ed a he same poin s in ime du ing he sampling pe iod, show-
ing a mean δ13C alue o −12.2‰ and ypical daily a ia ions o SD
≤1.4‰; no di e ences be ween empe a u e ea men s (P≥0.05) and
poin s in ime (P≥0.05; linea mixed e ec s model) we e obse ed
du ing he daily cycle.
Physiological measu emen s and biomass de e mina ion
Se e al lea physiological pa ame e s we e de e mined wi h an
in a ed gas analyse (LI-6400, LI-COR, Lincoln, Neb aska, USA),
including ne assimila ion a e (An), in e cellula CO2 concen a ion
(Ci), and s oma al conduc ance (gs). All measu emen s we e aken in
he las 4 h o he dayligh phase. To moni o olume ic soil wa e
con en (SWC), up o h ee soil mois u e senso s (EC-5 and log-
ge Em5b, Decagon De ices, Pullman, USA) we e ins alled o each
ea men . Sho ly a e he sampling pe iod, o al plan biomass
was ha es ed, o en-d ied (a 60°C), and weighed. The esh ube
weigh and ube coun (numbe o po a oes) we e de e mined.
Ca bon iso ope and concen a ion analyses
δ13C alues a e exp essed as desc ibed by C aig (1957) and modi ied
by Coplen (2011):
δ
13
sample s anda d
C RR
1
‰/
()
=−
whe e Rsample is he 13C/12C a io o he sample ma e ial and Rs anda d
is ha o he in e na ional s anda d VPDB (Vienna Pee Dee
Belemni e).
De e mina ion o δ13CR
The in- ube incuba ion echnique was used o he collec ion o lea
da k- espi ed CO2 du ing day ime and nigh ime (We ne e  al.,
2007). A lea was placed in a 12 ml gas- igh exe aine (Labco,
Lampe e , UK), which was immedia ely da kened wi h a ligh p oo
casing o igge lea da k espi a ion. The ube was hen lushed
o 1 min wi h syn he ic ai un il a CO2- ee a mosphe e was es ab-
lished, which was moni o ed wi h an in a ed gas analyse (LI-6262,
LI-COR, Lincoln, Neb aska, USA). A e an incuba ion ime o
3 min in da kness, an aliquo o da k- espi ed CO2 was ans e ed
wi h a gas- igh sy inge in o a new exe aine illed wi h d y N2. δ13CR
alues we e de e mined wi h an IRMS, using a modi ied Gasbench
II (The mo Fishe , B emen, Ge many) connec ed o a Del aplusXP-
IRMS, simila o Zeeman e al. (2008). The ans e o he CO2 sam-
ple in o a new exe aine , as well as he IRMS measu ing p ocedu e,
we e bo h es ed wi h ai o known δ13C o CO2 o ensu e no iso-
ope ac iona ion had occu ed. Measu emen p ecision o a qual-
i y con ol s anda d ( h ee s anda ds pe 24 samples) was SD≤0.1‰.
De e mina ion o δ13C in bulk lea es and lea s a ch
Ex ac ion o lea s a ch was pe o med as desc ibed in p e ious s ud-
ies (Wanek e al., 2001; Goe liche e al., 2006; Rich e e al., 2009).
Lea s a ch was isola ed om 50 mg lea ma e ial wi h me hanol/
chlo o o m/wa e (MCW, 12:5:3, / / ) a 70°C o 30 min. Samples
we e cen i uged (10 000 ×g, 2 min) and supe na an s emo ed,
while he lea -s a ch-con aining pelle s we e washed wi h MCW and
deionized wa e and d ied a oom empe a u e (RT). Pelle s we e
hen e-suspended in wa e and boiled a 99°C o 15 min o acili-
a e s a ch gela iniza ion. Subsequen ly, lea s a ch was enzyma i-
cally diges ed wi h α-amylase (EC 3.2.1.1, Sigma-Ald ich, Buchs,
Swi ze land) a 85°C o 2 h, and cleaned wi h cen i uga ion il e s
o emo e enzymes (Vi aspin, Sa o ius, Gö ingen, Ge many). To
de e mine δ13C o bulk lea es (δ13Clea ) and s a ch, an elemen al ana-
lyse (Flash EA 1112 Se ies) coupled o a Del aplusXP-IRMS was
used (bo h The mo Fishe , B emen, Ge many; We ne e al., 1999).
Measu emen s o samples, blanks, and e e ence ma e ial ollowed
he iden ical ea men p inciple desc ibed by We ne and B and
(2001). The long- e m p ecision o a quali y con ol s anda d o all
sequences was SD≤0.12‰.
Iso opic and concen a ion analysis o soluble ca bohyd a es
and o ganicacids
Wa e -soluble compounds we e ex ac ed om 100 mg lea ma e-
ial wi h wa e a 85°C o 30 min, simila o S ei e  al. (2013).
Subsequen ly, soluble ca bohyd a es and o ganic acids we e
sepa a ed by ion-exchange ch oma og aphy (Wanek e  al., 2001;
Goe liche e al., 2006; Rich e e al., 2009), using Dowex 50WX8
in H+- o m and Dowex 1X8 in NaCOO-- o m (bo h 100–200 mesh,
Sigma-Ald ich, Buchs, Swi ze land). To a oid clogging o he HPLC
column by polyphenols, all samples designa ed o ca bohyd a e
analyses we e il e ed wi h 100 mg Sep-Pak C18 Vac RC Ca idges
(Wa e s AG, Mil o d, Massachuse s, USA). Finally, all ca bohy-
d a e and o ganic acid samples we e cleaned wi h 0.45µm PTFE
sy inge il e (In och oma AG, Zug, Swi ze land) p io o HPLC
measu emen s.
To de e mine δ13CRS alues and he concen a ions o soluble ca bo-
hyd a es and o ganic acids, a HPLC-IRMS sys em consis ing o a high
pe o mance liquid ch oma og aph coupled o a Del a V Ad an age
IRMS by a LC IsoLink (all The mo Fishe , B emen, Ge many)
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5772 | Lehmann e al.
was used acco ding o K ummen e al. (2004). Ca bohyd a es we e
sepa a ed on a 3 × 150 mm anion-exchange column Ca boPac PA20
(Dionex, Ol en, Swi ze land) using 2 mM NaOH as he mobile phase
and a low speed o 250µl min-1 (Boschke e al., 2008; Rinne e al.,
2012). Low column empe a u e o 20°C was used o p e en isome i-
za ion o hexoses (Rinne e al., 2012). This enabled ch oma og aphic
sepa a ion o suc ose and glucose, bu uc ose δ13CRS and concen-
a ion measu emen s we e a ec ed by pa ial co-elu ion o uc ose
wi h o he compounds. To co ec δ13CRS alues and o calcula e con-
cen a ions om he peak a eas, in e spe sed s anda d solu ions in a
concen a ion ange o 20–180 ng C µl-1 we e measu ed wi hin each
sequence. The measu emen p ecision o δ13CRS alues in all ca bohy-
d a e s anda ds was SD<0.5‰. Below a concen a ion o 60 ng C µl-1,
he p ecision o uc ose s anda ds was lowe o ce ain ba ches, and
he e o e hese esul s we e excluded.
O ganic acids we e sepa a ed on a 4.6 × 300 mm Allu e O ganic
Acids column (Res ek, Belle on e, USA) a 5–10°C. The mobile
phase was a 100 mM monopo assium phospha e bu e (pH 3)wi h a
low speed o 500μl min-1 (He mann e al., 2005). The measu emen
p ecision o δ13C in o ganic acid s anda ds was SD<0.4‰. Low ci -
a e concen a ions om Tlow samples (<45 ng C µl-1) impeded he
analy ical accu acy o he δ13CRS alues, he e o e hese samples
we e no aken in o accoun .
All pu i ica ion s eps we e e i ied o each ba ch o 24 samples
using 2.5 mg s anda d solu ions o known δ13C (by EA-IRMS) o all
ca bohyd a es and o ganic acids measu ed in his s udy. Di e ences
be ween δ13C alues be o e and a e pu i ica ion we e gene ally
≤0.2‰, indica ing no signi ican iso ope ac iona ion o any s and-
a d. Mean eco e y was 101 ± 6% o uc ose, 96 ± 6% o glucose,
89 ± 3% o suc ose, 91 ± 3% o mala e, and 86 ± 3% o ci a e.
De e mina ion o s a ch concen a ion
Fo he ex ac ion o lea s a ch o concen a ion analyses we used
a modi ied me hod o C i chley e al. (2001). Lea s a ch was iso-
la ed wi h 1.12 M pe chlo ic acid om 50 mg lea ma e ial a RT
o 15 min and cen i uged (10 min, 3000×g, 4°C). The supe na an
was emo ed and he lea -s a ch-con aining pelle was washed ee
om pigmen s wi h deionized wa e and e hanol. Pelle s we e hen
d ied a RT, esuspended in wa e , and gela inized. Subsequen ly,
s a ch samples we e enzyma ically hyd olysed o glucose o 2 h a
37°C wi h a solu ion mix o α-amylase (EC 3.2.1.1, Sigma-Ald ich,
Buchs, Swi ze land) and α-amyloglucosidase (EC 3.2.1.3, Roche,
Ro k euz, Swi ze land) in 220 mM sodium ace a e bu e (pH 4.8).
The glucose concen a ion was de e mined a 340 nm wi h a 96-well
mic opla e eade (EL×800, BioTek, Luze n, Swi ze land) using a
coupled enzyma ic eac ion (Hoch e al., 2002). Po a o s a ch was
used as a s anda d. Glucose concen a ions a e exp essed in mola -
i y o s a ch monome s.
Da a analysis
R e sion 3.0.2 (R Co e Team, 2013) was used o (mul iple) lin-
ea eg ession analyses and linea mixed e ec s models (R package
nlme). Models included ixed e ec s ( empe a u e, soil mois u e,
sampling ime) and andom e ec s (clima e chambe s, indi idual
plan s). I applicable, δ13C alues and concen a ions we e loga i h-
mically ans o med o ensu e no mal dis ibu ion. Fo he bes - i
combina ion o he mul iple linea eg ession analysis, a iables
we e excluded i P≥0.05.
Resul s
Physiological pa ame e s and biomass
Physiological pa ame e s (An, Ci, gs, and SWC) o po a o plan s
exposed o ou di e en ea men s we e moni o ed du ing
he ea men pe iod o 15 d (Fig.1). The ne assimila ion a e
declined du ing he ea men pe iod unde all ou ea men s
(Fig.1A). Du ing he sampling pe iod (Fig.1A, day 15), An
was signi ican ly in luenced by soil mois u e (P=0.02, Table1),
wi h lowes alues (1.9µmol m-2 s-1) unde Thigh and d y condi-
ions, and highes alues (5.4µmol m-2 s-1) unde Tlow and we
condi ions, whe eas he empe a u e in luence on An was no
signi ican (P=0.07, Table1) bu ended o cause lowe An al-
ues unde Thigh han unde Tlow unde bo h soil mois u e con-
di ions. The in e cellula CO2 concen a ion inc eased du ing
he ea men pe iod o all ou ea men s (Fig.1B). Du ing
he sampling pe iod (Fig.1B, day 15), Ci was independen ly
in luenced by empe a u e (P=0.012, Table1) and soil mois u e
(P=0.01, Table1), wi h lowes Ci (247.5µmol mol-1) unde Tlow
and d y condi ions and highes Ci (332.8µmol mol-1) unde Thigh
and we condi ions. S oma al conduc ance du ing he ea -
men pe iod was lowe unde d y ea men s compa ed o hose
unde we ea men s (Fig.1C). Du ing he sampling pe iod
(Fig.1C, day 15), gs was signi ican ly in luenced by soil mois-
u e (P≤0.001, Table1), wi h lowes gs (abou 0.06 mol m-2 s-1)
in plan s o bo h d y ea men s and highes gs (0.22 mol m-2
s-1) in plan s unde Thigh and we condi ions, whe eas he em-
pe a u e in luence unde we condi ions ended o cause highe
gs alues unde Thigh han unde Tlow. The olume ic soil wa e
con en was lowe unde d y condi ions (~7–14%) compa ed
o we condi ions (23–27.5%) o he las 9 d o he ea men
pe iod (Fig.1D), including he sampling pe iod (Fig.1D, day
15), whe e SWC was signi ican ly a ec ed only by soil mois u e
ea men s (P=0.002, Table1). Gene ally, no signi ican in e -
ac ions be ween empe a u e and soil mois u e we e obse ed
o any pa ame e (Table1). In addi ion, only soil mois u e
ea men s a ec ed plan biomass (P=0.008, Table1) and ube
weigh (P=0.023, Table 1) aken sho ly a e he sampling
pe iod, independen o empe a u e ea men s. Highes alues
ended o be unde Tlow and we condi ions and lowes alues
unde Thigh and d y condi ions (Tables 1, 2), indica ing di e en
s ess le els c ea ed by he ou ea men s.
Ca bon iso opes in po a olea es
Daily cycles o δ13CR and δ13Clea
δ13C alues o lea da k- espi ed CO2 (δ13CR) a ied signi i-
can ly o e ime (P≤0.001, Table3) wi h alues in he ange
o −21.9‰ and −32‰, declining s ongly du ing nigh ime
and inc easing again du ing he day ime o all ou ea -
men s (Fig.2A). An in e ac ion be ween empe a u e and ime
showed ha he in luence o empe a u e di e ed wi h ime
(P=0.014, Table3). Day ime δ13CR alues unde Thigh we e up o
4.7‰ mo e nega i e compa ed o hose unde Tlow, independ-
en o soil mois u e condi ions, whe eas nigh ime δ13CR alues
o bo h empe a u e ea men s we e e y simila , pa icula ly
in he second nigh . D y soil mois u e condi ions caused less
nega i e δ13CR alues compa ed o hose unde we condi ions
du ing he daily cycle (P=0.013, Table3), wi h a maximum di -
e ence o 2.7‰, independen o empe a u e ea men s. On
a e age, he di e ence be ween day ime and nigh ime δ13CR
alues was highes unde Tlow and we condi ions, a 5.7‰, and
lowes unde Thigh and d y condi ions, a 2.5‰.
a Na u al Resou ces Ins i u e Finland (Luke) on No embe 29, 2016h p://jxb.ox o djou nals.o g/Downloaded om
Mala e as a key ca bon sou ce o lea da k- espi ed CO2 in po a o | 5773
The bulk lea ma e ial e lec s all en i onmen al condi ions
expe ienced du ing he whole g ow h pe iod. δ13Clea o all
ea men s showed no changes du ing he sampling pe iod
and no in e ac ions be ween ea men s and ime (Fig.2B;
Table3). Unde Thigh, δ13Clea alues we e up o 2.2‰ mo e
nega i e compa ed o hose unde Tlow, esul ing in a signi i-
can empe a u e e ec independen o soil mois u e condi-
ions (P=0.022, Table3). Simila ly, soil mois u e showed a
signi ican e ec on δ13Clea (P=0.005, Table 3), independ-
en o empe a u e ea men s, wi h alues up o 1.1‰ less
nega i e unde d y han unde we condi ions mainly du ing
nigh ime.
δ13CRS o soluble ca bohyd a es, o ganic acids, ands a ch
Highes δ13C alues in pu a i e lea espi a o y ca bon
sou ces (δ13CRS) we e ound in he o ganic acid mala e, while
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●Tlow We
Tlow D y
Thigh We
Thigh D y
(D)
SWC (m3 m−3)
T ea men pe iod (d)
Fig.1. Physiological pa ame e s unde di e en en i onmen al condi ions
du ing he ea men pe iod: (A) ne assimila ion a e (An, µmol m-2 s-1), (B)
in e cellula CO2 concen a ion (Ci, µmol mol-1), (C) s oma al conduc ance
(gs, mol m-2 s-1), (D) olume ic soil wa e con en (SWC, m3/m-3). Po a o
plan s we e ea ed wi h a combina ion o Tlow (low empe a u e; closed
symbols), Thigh (high empe a u e; open symbols), and we (ci cles) o d y
( iangles) condi ions. Boxed a eas indica e he sampling pe iod. Means
±SE a e gi en (n=3).
Table1. En i onmen al in luences on physiological pa ame e s
Resul s o linea mixed e ec s models es ing he e ec s o empe a u e (low, high) and soil mois u e (we , d y) on physiological pa ame e s (An, ne
assimila ion a e; Ci, in e cellula CO2 concen a ion; gs, s oma al conduc ance; SWC, olume ic soil wa e con en ), o al plan biomass, ube weigh ,
and ube coun du ing he sampling pe iod. P- alues a e gi en o ea men s and hei in e ac ion. Signi ican di e ences a e gi en in bold (P≤0.05).
Pa ame e AnCigsSWC Plan biomass Tube weigh Tube coun
Tempe a u e 0.070 0.012 0.127 0.863 0.978 0.359 0.400
Soil mois u e 0.020 0.010 0.001 0.002 0.008 0.023 0.233
Temp.:mois u e 0.522 0.110 0.174 0.845 0.565 0.892 0.486
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δ13CR (‰)
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048121620242
83
2
−34
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−30
−28
−26
−24
−22
−20 (B)
Sampling pe iod (h)
δ13Clea (‰)
●Tlow We
Tlow D y
Thigh We
Thigh D y
Fig.2. Daily cycles o he ca bon iso opic composi ion o (A) lea
da k- espi ed CO2 (δ13CR) and (B) bulk lea es (δ13Clea ) unde di e en
en i onmen al condi ions du ing he sampling pe iod. Po a o plan s
we e ea ed wi h a combina ion o Tlow (low empe a u e; closed symbols),
Thigh (high empe a u e; open symbols), and we (ci cles) o d y ( iangles)
condi ions. G ey a eas indica e nigh ime. Means ±SE a e gi en
(n=3).
a Na u al Resou ces Ins i u e Finland (Luke) on No embe 29, 2016h p://jxb.ox o djou nals.o g/Downloaded om

5774 | Lehmann e al.
soluble ca bohyd a es ( uc ose, glucose and suc ose) exhib-
i ed gene ally lowes δ13CRS alues (Fig.3). δ13CRS o soluble
ca bohyd a es o all ea men s we e in he ange o −27.2‰
and −36.6‰. Mo e nega i e δ13CRS alues o glucose and
suc ose unde Thigh compa ed o hose unde Tlow we e ound,
independen o soil mois u e condi ions, while less nega-
i e δ13CRS alues unde d y condi ions compa ed o hose
unde we condi ions we e obse ed, independen o em-
pe a u e ea men s (Fig.3B, C; Table3). Signi ican in e -
ac ions be ween empe a u e and ime o δ13CRS o glucose
(P=0.008, Table3) and suc ose (P=0.003, Table3) showed
ha daily cycles di e ed be ween empe a u es. Addi ionally,
soil mois u e condi ions caused signi ican empo al a ia-
ions du ing he daily cycle in δ13CRS o suc ose (P=0.002,
Table3).
We obse ed signi ican linea ela ionships be ween
uc ose and glucose o δ13CRS ( 2=0.74, P≤0.001) and
concen a ion alues ( 2=0.8, P≤0.001), while ela ionships
be ween he o he δ13CRS alues and concen a ions o di e -
en ca bon sou ces we e weake (da a no shown). Howe e ,
he de ian esul s o δ13CRS o uc ose in compa ison o he
o he suga s a e assumed o e lec peak o e lap issues o his
suga (Tables 3, 4). This is clea ly e lec ed also in he concen-
a ion esul s (Fig.4A). Consequen ly, he uc ose esul s
will no be discussed u he in de ail.
δ13CRS o mala e (Fig. 3D) in he ange o −24‰ and
−29.3‰ and δ13CRS o ci a e (Fig.3E) in he ange o −29.6‰
and −32.1‰ showed no empo al a ia ions (P=0.198
and P=0.052 o mala e and ci a e, espec i ely, Table 3).
Signi ican in e ac ions be ween empe a u e and soil mois-
u e ea men s we e obse ed o δ13CRS o mala e (P=0.017;
Table3), esul ing in la ge di e ences be ween δ13CRS al-
ues o soil mois u e condi ions unde Thigh han unde Tlow
(Fig.3D). Ci a e showed less nega i e δ13CRS alues unde
d y condi ions han unde we condi ions (P=0.009; Table3).
δ13CRS o s a ch o all ea men s (Fig.3F), anging om
−25.2‰ and −32.1‰, was in luenced by soil mois u e con-
di ions (P=0.046, Table 3), independen o empe a u e
ea men s, while empe a u e showed no signi ican e ec
(P=0.107, Table 3). In addi ion, soil mois u e condi ions
caused signi ican empo al a ia ions du ing he daily cycle
in δ13CRS o s a ch (P=0.032, Table3).
Concen a ions o soluble ca bohyd a es, o ganic
acids, ands a ch
Concen a ions o glucose o all ea men s (Fig.4B), ang-
ing om 27 o 95µmol g DW-1, showed no empo al a ia-
ions (P=0.927, Table3). In con as , concen a ions o suc ose
Table2. Biomass and ube analyses a e sampling pe iod
To al plan biomass (d y weigh ), ube weigh ( esh weigh ), and ube
coun (numbe o po a oes) a e he sampling pe iod. Po a o plan s
we e ea ed wi h a combina ion o Tlow (low empe a u e), Thigh (high
empe a u e), and we o d y condi ions. Means ±SE a e gi en (n=3).
Re e o Table 1 o s a is ical analysis.
T ea men s
Pa ame e Tlow we Tlow d y Thigh we Thigh d y
To al biomass (g) 10.6 ± 1.4 7.8 ± 1.2 10.3 ± 1 8.1 ± 0.5
Tube weigh (g) 513.9 ± 18.4 458.3 ± 15 481.4 ± 15.9 430 ± 24.2
Tube coun (no.) 21.3 ± 2.7 20.2 ± 1.7 19.3 ± 1.2 19 ± 2.3
Table3. En i onmen al in luences on lea da k- espi ed CO2 and espi a o y ca bon sou ces
Resul s o linea mixed e ec s models es ing he e ec s o empe a u e (low, high) and soil mois u e (we , d y) on δ13C alues in di e en
pu a i e lea espi a o y ca bon sou ces, bulk lea es (δ13Clea ), and in lea da k- espi ed CO2 (δ13CR), as well as on concen a ions o di e en
ca bon sou ces du ing he sampling pe iod. Resul s o uc ose a e a ec ed by co-elu ion wi h o he compounds. P- alues a e gi en o
ea men s, ime, and hei in e ac ions. Signi ican di e ences a e gi en in bold (P≤0.05).
δ13C
Pa ame e F uc ose Glucose Suc ose Mala e Ci a e S a ch δ13CLea δ13CR
Tempe a u e 0.019 0.004 0.028 0.015 n.a. 0.107 0.022 0.044
Soil mois u e 0.001 0.001 0.001 0.049 0.009 0.046 0.005 0.013
Time 0.001 0.195 0.081 0.198 0.052 0.001 0.066 0.001
Temp.:mois u e 0.035 0.063 0.543 0.017 n.a. 0.270 0.165 0.875
Temp.: ime 0.256 0.008 0.003 0.807 n.a. 0.113 0.812 0.014
Mois u e: ime 0.061 0.291 0.002 0.060 0.411 0.032 0.596 0.883
Concen a ion
Pa ame e F uc ose Glucose Suc ose Mala e Ci a e S a ch
Tempe a u e 0.663 0.352 0.142 0.011 n.a. 0.002
Soil mois u e 0.001 0.001 0.031 0.999 0.052 0.001
Time 0.016 0.927 0.001 0.035 0.110 0.001
Temp:mois u e 0.475 0.705 0.462 0.796 n.a. 0.001
Temp.: ime 0.901 0.847 0.113 0.387 n.a. 0.324
Mois u e: ime 0.831 0.629 0.063 0.889 0.895 0.071
n.a., no a ailable
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Mala e as a key ca bon sou ce o lea da k- espi ed CO2 in po a o | 5775
(Fig.4C) in he ange o 23 o 159µmol g DW-1 showed clea
daily a ia ions (P≤0.001, Table3), wi h highes concen a ions
o all ea men s by he end o he day, excep o Thigh and d y
condi ions. Glucose concen a ions we e signi ican ly highe
unde d y han unde we condi ions (P≤0.001, Table3), while
con e se esul s we e obse ed o suc ose (P=0.031, Table3).
Gene ally, no e ec o empe a u e on he concen a ion o
any soluble ca bohyd a e was obse ed.
Mala e concen a ions o all ea men s (Fig.4D), anging
om 23 o 163µmol g DW-1, showed a daily pa e n wi h
declining concen a ions in he beginning o he nigh and an
inc ease a e 2–4 h in he da k (P=0.035, Table3). In con as
o soluble ca bohyd a es, mala e concen a ions we e signi i-
can ly highe unde Thigh han unde Tlow (P=0.011, Table3),
bu we e no a ec ed by soil mois u e ea men s (P=0.999,
Table3). Ci a e concen a ions unde Thigh o ~15µmol g
DW-1 we e he lowes o all measu ed pu a i e ca bon sou ces
a ailable o lea da k espi a ion and showed no changes due
o soil mois u e ea men s and ime (Fig.4E; Table3).
S a ch concen a ions (Fig. 4F), anging om 67 o
282 µmol g DW-1, showed signi ican empo al a ia ions
(P≤0.001, Table3), independen o any ea men . The a e age
s a ch concen a ion o 243µmol g DW-1 unde Tlow and we
condi ions was clea ly highe (~2.5 imes) compa ed o hose
unde o he ea men s. In addi ion, in e ac ions be ween
empe a u e and soil mois u e ea men s led o smalle di -
e ences be ween he alues o we and d y condi ions unde
Thigh compa ed o hose unde Tlow (P≤ 0.001, Table3).
Linea ela ionships be ween δ13CR and δ13CRS
Linea eg ession analyses we e pe o med o unde s and
he biochemical link be ween δ13CR and δ13CRS ac oss
all ea men s (Table 4; Supplemen a y Fig. S1). δ13CRS
o mala e explained mos o he daily a ia ion o δ13CR
( 2=0.26, P≤ 0.001), while he explana o y powe o uc-
ose, glucose, and ci a e was lowe . The lowes linea ela-
ionships du ing he daily cycle we e ound be ween δ13CR
and δ13CRS o suc ose and s a ch. Due o he high daily
a ia ions in δ13CR we ca ied ou he same analysis sepa-
a ely o day ime and nigh ime. Day ime linea ela ion-
ships we e gene ally s onge han du ing nigh ime, wi h
δ13CR s ongly ela ed o δ13CRS o mala e, ci a e, and
δ13Clea ( 2>0.6, P≤0.001), bu lowe ela ed o δ13CRS o sol-
uble ca bohyd a es and s a ch. Du ing nigh ime, δ13CRS o
mala e explained 36% o he a ia ion in δ13CR, bu δ13CRS
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δ
13
CRS (‰)
Sampling pe iod (h)
Fig.3. Daily cycles o he ca bon iso opic composi ion o di e en lea espi a o y ca bon sou ces (δ13CRS) unde di e en en i onmen al condi ions
du ing he sampling pe iod: (A) uc ose, (B) glucose, (C) suc ose, (D) mala e, (E) ci a e, and (F) s a ch. Po a o plan s we e ea ed wi h a combina ion
o Tlow (low empe a u e; closed symbols), Thigh (high empe a u e; open symbols), and we (ci cles) o d y ( iangles) condi ions. Resul s o uc ose a e
a ec ed by co-elu ion wi h o he compounds. G ey a eas indica e nigh ime. Means ± SE a e gi en (n=2–3).
Table4. Rela ionships be ween δ13C o lea da k- espi ed CO2
and δ13C o espi a o y ca bon sou ces
Linea eg ession analyses ela ing δ13C o lea da k- espi ed CO2
o δ13C o pu a i e espi a o y ca bon sou ces and o δ13C o bulk
lea es (δ13Clea ) ac oss all en i onmen al condi ions o day ime
(0 h, 16 h, 24 h), o nigh ime (2 h, 4 h, 8 h, 26 h, 28 h, 32 h), and o
he o al daily cycle (sampling pe iod o e 32 h). Resul s o uc ose
a e a ec ed by co-elu ion wi h o he compounds. Gene ic eg ession
equa ion y=mx+b was used. 2 alues a e gi en, s a s indica e
P- alues. All co ela ion coe icien s we e posi i e.
2
Pu a i e ca bon sou ces Day ime Nigh ime Daily
F uc ose 0.35*** 0.34*** 0.12***
Glucose 0.54*** 0.34*** 0.13***
Suc ose 0.59*** 0.20*** 0.04*
Mala e 0.69*** 0.36*** 0.26***
Ci a e 0.67*** 0.28** 0.17**
S a ch 0.48*** 0.16** 0.06*
δ13CLea 0.63*** 0.33*** 0.20***
*, P≤0.05; **, P≤0.01; ***, P≤0.001
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5776 | Lehmann e al.
o uc ose and glucose, as well as δ13Clea , showed simila ly
high explana o ypowe .
In luence o en i onmen al d i e s and ca bon sou ces
on δ13CR
Fu he mo e, a s epwise (backwa d) mul iple linea eg ession
analysis was pe o med o iden i y en i onmen al d i e s and
ca bon sou ces in luencing δ13CR (Table5). Day ime/nigh ime
showed he s onges posi i e e ec on δ13CR (β=0.73, P≤0.001),
while δ13CRS o mala e was he ca bon sou ce ha a ec ed δ13CR
mos (β=0.4, P≤0.001). By compa ison, he in luence o δ13CRS
o s a ch and soil mois u e condi ions on δ13CR alues wasmino .
Discussion
This s udy clea ly demons a es ha di e en empe a u e and
soil mois u e condi ions in luence δ13C o lea da k- espi ed
CO2 (δ13CR), δ13C o di e en pu a i e lea espi a o y ca -
bon sou ces (δ13CRS), and concen a ions o ca bon sou ces
du ing a daily cycle in po a o lea es. Fu he mo e, ou ind-
ings s ongly indica e mala e as a key ca bon sou ce o day-
ime and nigh ime δ13CR ac oss di e en en i onmen al
condi ions.
In luence o empe a u e and soil mois u e on iso opic
composi ions
A e 2 weeks o ea men , we al eady ound a clea empe a-
u e e ec on δ13Clea , wi h up o 2.2‰ mo e nega i e δ13Clea
alues unde Thigh condi ions compa ed o hose unde Tlow
condi ions (Fig.2B). This is in ag eemen wi h a s udy show-
ing mo e nega i e δ13C alues wi h inc easing empe a u e o
bulk lea es o Xan hium species (Smi h e al., 1976). Simila
o Tche kez e al. (2003) unde sho - e m empe a u e ea -
men s, we obse ed mo e nega i e δ13CR alue wi h inc easing
empe a u e (Fig.2A), bu due o ou long- e m ea men we
ound also mo e nega i e δ13CRS alues (Fig.3). On he o he
hand, d y condi ions in bo h o he empe a u e ea men s
caused less nega i e δ13Clea , δ13CR, and δ13CRS alues com-
pa ed o hose unde we condi ions, which is consis en wi h
p e ious s udies unde con olled condi ions (Du anceau
e al., 1999; Ghashghaie e al., 2001).
The iso opic esul s unde he di e en en i onmen al con-
di ions can be di ec ly linked o he lea gas exchange obse ed
du ing he 32 h sampling pe iod (day 15 o he ea men
pe iod). Inc easing empe a u e caused lowe An alues unde
bo h soil mois u e condi ions (Fig.1A; Table1), indica ing ha
plan s unde Thigh we e beyond he pho osyn he ic op imum.
This esul is in ag eemen wi h ea lie s udies, showing ha
cold-adap ed po a o plan s ha e educed a es o pho osyn he-
sis wi h empe a u es abo e 20°C (Le y and Veilleux, 2007).
Addi ionally, An migh be also in luenced by lea ageing, since
Table5. En i onmen al d i e s and ca bon sou ces in luencing
δ13C o lea da k- espi ed CO2
Resul o s epwise (backwa d) mul iple linea eg ession analysis
showing he bes - i combina ion o independen en i onmen al
d i e s ( empe a u e, soil mois u e, day ime/nigh ime), ime, and
δ13C o glucose, suc ose, mala e, and s a ch as a iables in luencing
δ13C o lea da k- espi ed CO2 (δ13CR) du ing he sampling pe iod in
po a o lea es. S anda dized β-coe icien s and P- alues a e gi en.
D i e s and ca bon sou ces
in luencing δ13CR
S anda dized
β-coe icien
P- alue
Day ime/nigh ime 0.73 <0.001
Mala e 0.40 <0.001
S a ch 0.11 0.019
Soil mois u e 0.14 0.013
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Tlow D y
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(F) S a ch
Sampling pe iod (h)
Concen a ion (µmol g DW
−
1)
Fig.4. Daily cycles o he concen a ion o di e en lea espi a o y ca bon sou ces unde di e en en i onmen al condi ions du ing he sampling pe iod:
(A) uc ose, (B) glucose, (C) suc ose, (D) mala e, (E) ci a e, and (F) s a ch. Po a o plan s we e ea ed wi h a combina ion o Tlow (low empe a u e; closed
symbols), Thigh (high empe a u e; open symbols), and we (ci cles) o d y ( iangles) condi ions. G ey a eas indica e nigh ime. Resul s o uc ose a e
a ec ed by co-elu ion wi h o he compounds. To acili a e compa ison wi h o he me aboli es, suc ose concen a ions we e mul iplied by 2 o coun o
hexose uni s, while s a ch concen a ions a e gi en in mola i y o s a ch monome s. No e di e en y-axis scale in (F). Means ±SE a e gi en (n=2–3).
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Mala e as a key ca bon sou ce o lea da k- espi ed CO2 in po a o | 5777
An dec eased unde all ea men s du ing he ea men pe iod.
On he o he hand, gs ended o highe alues wi h inc easing
empe a u e, bu only unde we condi ions (Fig.1C; Table1).
An inc ease o gs unde Thigh migh be igge ed by inc easing
anspi a ion a es, which could be a physiological esponse o
compensa e educed a es o An by cooling he lea empe a-
u e unde Thigh condi ions. Howe e , his was only obse ed
in plan s unde Thigh and well-wa e ed condi ions, when SWC
was high. Subsequen ly, lowe ca bon ixa ion and highe CO2
di usion in o he s oma al ca i ies unde Thigh, in compa ison
o Tlow, caused an inc ease o Ci (Fig.1B) and mo e nega i e
δ13CR and δ13CRS alues (Table6). Fu he mo e, d y soil mois-
u e condi ions caused educed a es o An and gs compa ed
o hose unde we condi ions (Fig.1A, C; Table1), independ-
en o empe a u e ea men s. This can be explained wi h he
se e e d ough s ess, e lec ing low SWC alues (Fig. 1D).
Consequen ly, plan s unde d y condi ions expe ienced
educed CO2 di usion in o he s oma al ca i ies, leading o
lowe Ci and less nega i e δ13C alues (Table6).
Plan s unde Thigh and d y condi ions showed he low-
es pe o mance du ing he sampling pe iod compa ed o
plan s unde o he ea men s, which is e lec ed in low An
alues (Fig.1A), plan biomass, ube weigh and ube coun
(Table2). δ13CR and δ13CRS in hese plan s we e expec ed o
be he mos posi i e compa ed o o he ea men s due o a
se e e d ough caused by he double e ec o high empe a-
u e and d y soil mois u e. Ins ead, δ13CR and δ13CRS o he
plan s unde he highes s ess le el (Thigh and d y condi ions)
we e a he simila o hose unde lowes s ess le el (Tlow and
we condi ions). This was pa icula ly obse ed o δ13CRS o
soluble ca bohyd a es and s a ch (Fig.3). Again, his is an
indica o o low An unde Thigh and d y condi ions, esul ing in
a mode a e educ ion o Ci, while a he same ime gs s ongly
educes CO2 di usion in o he s oma al ca i ies, causing an
inc ease o Ci. Consequen ly, his led o in e media e δ13CR
and δ13CRS alues unde Thigh and d y condi ions (Table6). In
summa y ou indings indica e ha combined e ec s o em-
pe a u e and soil mois u e condi ions on δ13CR and δ13CRS
could cancel ou he indi idual e ec o each d i e .
En i onmen al in luences on concen a ions o pu a i e
ca bon sou ces
Soil mois u e and empe a u e a ec ed concen a ions o
pu a i e lea espi a o y ca bon sou ces di e en ly. Suc ose
concen a ion dec eased unde d y condi ions (Fig. 4C;
Table3), which is in con as o he ecen s udy by Lemoine
e  al. (2013). This may be explained by educed a es o
suc ose syn hesis due o lowe ing o he suc ose phospha e
syn hase eac ion (SPS) (Vu e  al., 1998). The dec ease in
he enzyme ac i i y is p obably igge ed by limi ed a es
o phloem suga anspo obse ed unde d ough (Rueh
e al., 2009). This in u n could be an explana ion o lowe
plan biomass and ube weigh /coun in esponse o highe
empe a u es and d y condi ions (Tables 1, 2). Subsequen ly,
he inc ease o uc ose and glucose concen a ions unde
d ough may also be a consequence o lowe SPS ac i i y
(Fig. 4A, B; Table 3), since he demand o bo h hexoses
o suc ose syn hesis was educed. Addi ionally, inc easing
uc ose and glucose concen a ions unde d ough migh
ha e osmo ic unc ionali y, main aining me abolic ac i i y
(Lemoine e al., 2013).
On he o he hand, mala e concen a ions inc eased wi h
empe a u e (Fig.4D; Table3), which is mos likely a conse-
quence o highe PEPC ac i i y (Chin hapalli e al., 2003).
Highe mala e concen a ions may also suppo espi a-
o y p ocesses in he KC o egula ion o s oma al opening
(Finkemeie and Swee lo e, 2009). Mo eo e , dec eased
s a ch concen a ions in lea es unde ea men s wi h highe
en i onmen al s ess han Tlow and we condi ions (Fig.4F)
we e simila o p e ious indings (Lemoine e al., 2013). The
esul also suppo s he assump ion ha educed amoun s o
assimila ed ca bon due o lowe An unde Thigh o d y con-
di ions we e used o main enance o biochemical p ocesses
a he han o ca bon s o age. Addi ionally, his indica es
ha plan s unde Thigh o d y condi ions we e unde se e e
en i onmen al s ess.
Mala e as a key espi a o y ca bon sou ce o day ime
and nigh ime δ13CR
The daily cycle o δ13CR was highly a iable, showing less
nega i e day ime and mo e nega i e nigh ime alues, while
δ13CRS alues gene ally showed lowe changes du ing he
same pe iod (Figs 2A, 3; Table3). δ13CRS alues o all ea -
men s compa ed o δ13CR alues we e mo e nega i e o sol-
uble ca bohyd a es (up o 9.3‰) and ci a e (up o 4.1‰),
bu also less nega i e o s a ch (up o 4‰) and mala e (up
o 5.2‰) du ing he daily cycle (Figs 2A, 3). In pa icula ,
mala e was s ongly en iched in 13C, by up o 8.8‰, compa ed
o all o he pu a i e ca bon sou ces (Fig.3). This was simila
o a p e ious s udy in es iga ing me aboli es in po a o lea es
(Gleixne e al., 1998) and indica es a possible biochemical
link be ween 13C en iched lea da k- espi ed CO2 and 13C
en iched mala e.
Fo a be e unde s anding o he o e all biochemical con-
nec ions be ween δ13CR and di e en pu a i e ca bon sou ces,
we ca ied ou linea eg ession analyses, independen o
en i onmen al condi ions (Table4; Supplemen a y Fig. S1).
Table6. Cohe ence be ween lea physiological pa ame e s and
δ13C alues. Lea physiological pa ame e s and δ13C alues du ing he
sampling pe iod in po a o plan s unde di e en ea men s compa ed
o hose in po a o plan s g owing unde Tlow and we condi ions
The ollowing a iables we e conside ed: An, ne assimila ion a e; Ci,
in e cellula CO2 concen a ion; gs, s oma al conduc ance; δ13CR, δ13C
o lea da k- espi ed CO2; δ13CRS, δ13C o di e en pu a i e espi a o y
ca bon sou ces ( uc ose, glucose, suc ose, s a ch, and mala e).
A ows indica e s ong (↑, ↓), in e media e (↗, ↘), o no changes (→)
due o he in luence o ea men combina ions (Tlow, low empe a u e;
Thigh, high empe a u e; and we o d y condi ions).
T ea men s AngsCiδ13CRδ13CRS
Tlow d y ↘ ↓ ↓ ↑ ↑
Thigh we ↘ ↑ ↑ ↓ ↓
Thigh d y ↓ ↓ ↗ → →
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