RESEARCH ARTICLE
Changes in he Me abolome o Picea
bal ou iana Emb yogenic Tissues Tha We e
Linked o Di e en Le els o 6-BAP by Gas
Ch oma og aphy-Mass Spec ome y
App oach
Q. F. Li
1,2
, J. H. Wang
1
*, P. Pulkkinen
3
, L. S. Kong
4
1S a e Key Labo a o y o Fo es Gene ics and T ee B eeding, Resea ch Ins i u e o Fo es y, Chinese
Academy o Fo es y, Beijing, China, 2Guangxi Key Labo a o y o Special Non-wood Fo es Cul i a ion &
U iliza ion, GuangXi Zhuang Au onomous Region Fo es y Resea ch Ins i u e, Nanning, China, 3Finnish
Fo es Resea ch Ins i u e, Haapas ensy jä, Finland, 4Uni Cen e o Fo es Biology, Depa men o Biology,
Uni e si y o Vic o ia, Vic o ia, Canada
*[email p o ec ed]
Abs ac
Emb yogenic cul u es o Picea bal ou iana, which is an impo an comme cial species o
e o es a ion in Sou he n China, easily lose hei emb yogenic abili y du ing long- e m cul-
u e. Emb yogenic issue ha p oli e a ed a lowe concen a ions (3.6 μM and 2.5 μM) o 6-
benzylaminopu ine (6-BAP) we e mo e p oduc i e, and gene a ed 113 ±6 and 89 ±3
ma u e emb yos pe 100 mg emb yogenic issue, espec i ely. A me abolomic app oach
was used o s udy he changes in me aboli es linked o emb yogenic compe ence ela ed o
h ee di e en 6-BAP concen a ions (2.5 μM, 3.6 μM, and 5 μM). A o al o 309 compounds
we e ob ained, among which 123 me aboli es mapped o Kyo o Encyclopedia o Genes and
genomes (KEGG) pa hways. The le els o 35 me aboli es we e signi ican ly di e en ially
egula ed among he h ee 6-BAP ea men s, and 32 me aboli es di e ed be ween he
2.5 μM and 5 μM ea men s. A o al o 17 me aboli es appea ed only once among he h ee
compa isons. The combina ion o a sco e plo and a loading plo showed ha in he samples
wi h highe emb yogenic abili y (3.6 μM and 2.5 μM), up- egula ed me aboli es we e mos ly
amino acids and down- egula ed me aboli es we e mos ly p ima y ca bohyd a es (espe-
cially suga s). These esul s sugges ed ha 6-BAP may in luence emb yogenic compe-
ence by ni ogen me abolism, which could cause an inc ease in amino acid le els and
highe amoun s o aspa a e, isoleucine, and leucine in issues wi h highe emb yogenic
abili y. Fu he mo e, we specula ed ha 6-BAP may a ec he amoun o yp ophan in is-
sues, which would change he indole-3-ace ic acid le els and in luence he emb yogenic
abili y.
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 1/13
OPEN ACCESS
Ci a ion: Li QF, Wang JH, Pulkkinen P, Kong LS
(2015) Changes in he Me abolome o Picea
bal ou iana Emb yogenic Tissues Tha We e Linked
o Di e en Le els o 6-BAP by Gas
Ch oma og aphy-Mass Spec ome y App oach.
PLoS ONE 10(10): e0141841. doi:10.1371/jou nal.
pone.0141841
Edi o : Mohana K ishna Reddy Mudiam, CSIR-
Indian Ins i u e o Toxicology Resea ch, INDIA
Recei ed: July 21, 2015
Accep ed: Oc obe 13, 2015
Published: Oc obe 30, 2015
Copy igh : © 2015 Li e al. 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, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any
medium, p o ided he o iginal au ho and sou ce a e
c edi ed.
Da a A ailabili y S a emen : Ou da a a e all
con ained wi hin he pape and Suppo ing
In o ma ion iles.
Funding: This esea ch was unded by he
Ad anced Gene a ion B eeding Technological
Resea ch and Demons a ion o No he n Coni e
Species (2012BAD01B01).
Compe ing In e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
In oduc ion
The p og essi ely diminishing emb yogenic abili y o emb yogenic issues has been well cha -
ac e ized in coni e ees. A p ima y example o such ecalci ance in sp uce species is he
inabili y o dec eased compe ence o es ablished emb yogenic issue o gene a e ea ly s age
emb yos in esponse o sui able ma u a ion condi ions and o de elop ully ma u e emb yos.
Al hough echnology o he ini ia ion and p oli e a ion o soma ic issues and subsequen gen-
e a ion o ma u e co yledona y emb yos in sp uces and o he coni e s has imp o ed [1–10],
some emb yogenic issues om many coni e species con inue o exhibi a high deg ee o a i-
abili y and o he s ha e los emb yogenic abili y.
Picea bal ou iana is an e e g een sp uce ee ha is dis ibu ed mos ly in he sou hwes and
no he n egions o he Tibe an pla eau. Because o he high quali y o he wood and i s as
g ow h, P.bal ou iana is a majo species o choice o a o es a ion. Howe e , he e a e se e al
d awbacks o using P.bal ou iana o a o es a ion, including he ac ha i ep oduces p i-
ma ily sexually, i s seedlings ini ially g ow slowly, and i se s seed la e [11]. In ou labo a o y
we ha e es ablished he whole soma ic emb yogenesis sys em o P.bal ou iana and applied o
a pa en [12]. We ound ha he emb yogenic abili y o he sys em was easily dec eased when
he amoun o 2,4-dichlo ophenoxyace ic acid (2,4-D) and 6-benzylaminopu ine (6-BAP) in
p oli e a ion s age we e main ained o emo ed, and ealized he e was an u gen need o
s udy he ea ly s age o soma ic emb yogenesis [13]. We ound ha i he le el o 2,4-D
was dec eased du ing p oli e a ion, he emb yogenic issue would ha dly gene a e soma ic
emb yos. The amoun o he cy okinin 6-BAP ha added in he p oli e a ion s age would in lu-
ence he inal yield o ma u e emb yos om issues, especially du ing long- e m cul u e. I is
well known ha cy okinins play impo an oles in he con ol o cell di ision in plan s, and
he cy okinin signaling pa hway has been s udied ecen ly [14,15]. Howe e , he mechanism o
ac ion o 6-BAP in plan soma ic emb yogenesis is ha dly known. In a p e ious s udy, we
ound ha he in luence o 6-BAP on emb yogenic capaci y was h ough ele an mRNAs and
p o eins [16] and, he e o e, we in e ed ha 6-BAP may a ec he co esponding me aboli es.
Me abolomics, he global analysis o cellula me aboli es, is a powe ul ool based on unc-
ional p o eomics ha can be applied o gain insigh s in o biological unc ions, which may be
an e ec i e app oach o he unc ional cha ac e iza ion o genes, and may help in he desc ip-
ion and elucida ion o physiological esponses in plan s unde di e en en i onmen al condi-
ions [17–27]. Gas ch oma og aphy-mass spec ome y (GC-MS) is gene ally pe o med using
elec on-impac quad upole o ime-o - ligh mass spec ome y [28] and is one o he mos
de eloped analy ical pla o ms o plan me aboli e p o iling [29]. Using GC-MS, i is possible
o p o ile se e al hund ed compounds belonging o di e se chemical classes, including suga s,
o ganic acids, amino acids, suga alcohols, a oma ic amines, and a y acids. Fo example, he
egula ion o de elopmen al e en s has been elucida ed a he me abolic le el using me abolic
p o iling [30,31] and di e en capabili ies o emb yogenic cell lines o Pinus aeda L. [32] and
Picea abies (L.) [33] had been explained by a model based on he combined da a o me abolic
p o iles. Fu he , he combina ion o GC-MS and OPLS-DA (o hogonal p ojec ions o la en
s uc u es disc iminan analysis) [34–37] has been used o isualize and disc imina e in e es -
ing me aboli es.
In he p esen s udy, we in es iga ed he me abolic p o iles o emb yonic issues using h ee
6-BAP concen a ions and iden i ied impo an me aboli es ha we e a ec ed by 6-BAP and
associa ed wi h emb yogenic compe ence. The objec i e was o use he GC-MS app oach o
in es iga e changes in he me abolome ha we e linked o emb yogenic abili y, which was
ela ed o di e en le els o 6-BAP.
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 2/13
Ma e ials and Me hods
Plan ma e ial and sampling
One selec ed emb yogenic cell line o P.bal ou iana (Fig 1A) was used in his s udy. This cell
line was es ablished in 2011 and was ini ia ed a he Resea ch Ins i u e o Fo es , Chinese Acad-
emy o Fo es y using seeds om eli e geno ype 4 ha we e induced on solidi ied hal -s eng h
LM medium [38] supplemen ed wi h 10 μM 2,4-D and 5 μM 6-BAP[12], 1% suc ose, 500 mg/L
glu amine, 1 g/L casein hyd olysa e, and 2% Gel i e a 24 ± 1°C in he da k. This cell line was
p oli e a ed on solidi ied hal -s eng h LM medium wi h h ee concen a ions o 6-BAP
(2.5 μM, 3.6 μM, and 5 μM) and wi h o he supplemen s kep unchanged a 24 ± 1°C in he
da k. This p oduced emb yogenic issues wi h di e en emb yogenic capabili ies a e 3
mon hs. The emb yogenic cul u es we e sub-cul u ed a 2-week in e als. This soma ic
emb yogenesis cul u e expe imen was pe o med wice. Bo h expe imen al se ies yielded simi-
la esul s wi h espec o emb yo de elopmen , and samples om one o he expe imen al
se ies we e selec ed o me abolomic p o iling.
Samples o emb yogenic cul u e we e collec ed a e subcul u ing o 7 days. Fo each ea -
men , six biological eplica es we e collec ed. The samples we e deno ed as 2.5 μM-, 3.6 μM-,
and 5 μM- ea ed samples. All samples we e ans e ed o c yo ubes, lash ozen in liquid
ni ogen, and s o ed a −80°C un il u he p ocessing o me aboli e ex ac ion.
Ea ly emb yo di e en ia ion om issues was s imula ed by ans e ing he cul u es o
hal -s eng h LM medium lacking plan g ow h egula o s o 1 week. P omo ion o la e
emb yo de elopmen and ma u a ion (Fig 1B) was pe o med by ans e ing cul u es o hal -
s eng h LM medium supplemen ed wi h 61 μM abscisic acid and 0.4% ac i e cha coal, 6%
suc ose, 500 mg/L glu amine, 1 g/L casein hyd olysa e, and 4% Gel i e, and cul u ing a
24 ± 1°C in he da k. The e we e 10 eplica es o each ea men . Then, soma ic emb yos (a
leas 3 mm long) gene a ed om 100 mg emb yogenic issue ha had 3–5 co yledons and
could ge mina e we e coun ed a e 8 weeks (Fig 1C). Twen y soma ic emb yos om each o
he h ee 6-BAP ea men s we e pu on one qua e -s eng h LM medium wi h 0.5% ac i e
cha coal, 2% suc ose, 500 mg/L glu amine, 500 mg/L casein hyd olysa e, and 3% Gel i e o
ge mina ion a 24 ± 1°C in he ligh (30 μEm
-2
s
-1
, 16 h pho ope iod). The e we e 10 eplica es
o each ea men . A e 6 weeks, he numbe s o ge mina ed soma ic emb yos wi h elonga ed
oo and hypoco yl we e coun ed.
Fig 1. Soma ic emb yogenesis o Picea bal ou iana. (A) Emb yogenic issues. (B) Soma ic emb yos. (C) Ge mina ion o soma ic emb yos.
doi:10.1371/jou nal.pone.0141841.g001
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 3/13
His ological analysis
Ano he cell line ha was induced om eli e geno ype 3 in 2013 and ea ed in he same way as
he es g oup was used as he con ol o alida e he in luence o 6-BAP. The numbe s o
ma u e soma ic emb yos and ge mina ed soma ic emb yos o he con ol line we e coun ed in
he same way as in he es g oup. His ological di e ences be ween he es and con ol lines
we e analyzed. P oli e a ed issues on medium supplemen ed wi h di e en le els o 6-BAP
we e ea ed and s ained o gene al ligh mic oscopy obse a ions acco ding o Gup a and
Holms om [39] wi h some modi ica ion. The emb yonal head cells we e s ained b igh ed
(ace oca mine) and suspenso cells we e s ained blue. These wo sec ions oge he we e consid-
e ed as he ea ly s age emb yos and he numbe s o ea ly s age emb yos o each 6-BAP concen-
a ion we e coun ed.
S a is ical analysis
To compa e he in luence o di e en le els o 6-BAP on he ma u a ion o issues and he ge -
mina ion o hei gene a ed soma ic emb yos, he numbe s o ea ly soma ic emb yos, he num-
be s o ma u e soma ic emb yos, and he ge mina ion a es we e subjec ed o analysis o
a iance (ANOVA) using SPSS20 so wa e(h p://www-01.ibm.com/so wa e/analy ics/spss/
downloads.h ml). The le el o signi icance was P <0.05.
Me aboli e ex ac ion and gas ch oma og aphy– ime-o - ligh mass
spec ome y (GC/TOF/MS) analysis
Me aboli es om emb yogenic issue (100-mg esh weigh ) we e ex ac ed acco ding o Lisec
e al. [40] wi h mino modi ica ions. B ie ly, emb yogenic issue s o ed a −80°C was g ound in
a mo a using liquid ni ogen, and ans e ed in o 2-mL cen i uge ubes. Ribi ol (60 μL) was
added and o exed o 10 s, ollowed by he addi ion o 0.35 mL 100% me hanol and o exing
o 10 s. The ubes we e placed in o an ul asound machine a 70°C o 10 min, and hen cen i-
uged o 10 min a 12,000 pm a 4°C. Nex , 0.35 mL supe na an was ans e ed in o new
Eppendo ubes and samples we e blow-d ied using mode a e ni ogen. Me hoxamine hyd o-
chlo ide (80 μL) was added, o exed o 30 s, and allowed o eac o 2 h a 37°C. Finally,
100 μL BSTFA eagen (con aining 1% TMCS, / ) was added o he mix u e and allowed o
eac o 1 h a 70°C.
The GC/TOF/MS analysis was pe o med using an Agilen 7890A gas ch oma og aph sys-
em coupled wi h a Pegasus ou -dimensional ime-o - ligh mass spec ome e (Agilen ,
USA). The sys em used a DB-5MS capilla y column coa ed wi h 5% diphenyl c oss-linked wi h
95% dime hylpolysiloxane (30 m × 250-μm inne diame e , 0.25 μm ilm hickness; J&W Sci-
en i ic, Folsom, CA, USA). Nex , a 1-μL aliquo o he analy e was injec ed in spli less mode.
Helium was used as he ca ie gas, he on inle pu ge low was 3 mL min
−1
, and he gas low
a e h ough he column was 1 mL min
−1
. The ini ial empe a u e was main ained a 90°C o
0.25 min, and hen aised o 240°C a a a e o 5°C min
−1
, and inally o 285°C a a a e o 20°C
min
−1
o 11.5 min. The injec ion, ans e line, and ion sou ce empe a u es we e 280°C,
250°C, and 220°C, espec i ely. The ene gy was −70 eV in elec on-impac mode. The MS da a
we e acqui ed in ull-scan mode wi h he m/z ange o 20–600 a a a e o 100 spec a pe sec-
ond a e a sol en delay o 492 s.
Mul i a ia e and s a is ical analyses o aw signals, da a baseline il e ing, and peak iden i i-
ca ion and in eg a ion we e pe o med using he Simca so wa e (h p://www.ume ics.com/
p oduc s/simca). The da a we e hen impo ed in o he TagFinde so wa e [41] wi h de aul
pa ame e s o co ec ion o e en ion ime o mass deb is, peak alignmen , and decon olu ion
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 4/13
analysis [42]. The o al mass o he signal in eg a ion a ea was no malized o each sample; ha
is, he o al in eg al a ea o each sample was se as 1000. Then, p incipal componen analysis
(PCA) o in e nal s anda d peak a eas was pe o med o p o ide sample weigh s and 1 sco es
o no malize he da a be o e mul i a ia e analysis. In addi ion, me aboli e da a we e mean cen-
e ed and UV scaled. To ob ain an o e iew o he me aboli e da a, a PCA model was calcu-
la ed ini ially on he X-ma ix [43]. In he PCA, a ew la en a iables we e calcula ed, which
desc ibed he la ges sys ema ic a ia ion in he X-ma ix. Thus, bo h he in luence o noise
and dimensionali y on he da a was g ea ly educed. Using he esul an PCA sco e sca e s,
clus e s and ou lie s wi hin samples can be iden i ied [44]. Finally, he no malized da a we e
impo ed in o SIMCA-P + 12.0.1 (Ume ics AB, Umeå, Sweden) using he OPLS-DA model
wi h he i s p incipal componen o VIP ( a iable impo ance in he p ojec ion) alues (VIP
>1) combined wi h he S uden ’s - es ( - es ) (P <0.05) o iden i y di e en ially exp essed
me aboli es and o sea ch o me aboli es in comme cial da abases such as hose p o ided by
he Na ional Ins i u e o S anda ds and Technology (NIST; h p://www.nis .go /index.h ml)
and he publicly a ailable KEGG (Kyo o Encyclopedia o Genes and Genomes) da abase
(h p://www.genome.jp/kegg/). To cha ac e ize he physiological mechanisms o ea ly soma ic
emb yogenesis unde lying he e ec s o he 6-BAP ea men s, we examined he me abolic
changes in emb yogenic issue a h ee 6-BAP concen a ions. Fo each ea men , h ee
compa isons we e made: 2.5 μM s 5μM, 3.6 μM s 2.5 μM, and 3.6 μM s 5μM. In each com-
pa ison, he sample wi h he lowe emb yo p oduc ion was always made he con ol g oup.
Me aboli es ha di e ed be ween samples we e iden i ied using OPLS-DA loading plo s and a
- es o he espec i e me aboli e peak a eas. In all cases, models we e judged o quali y using
goodness o i (R
2
X) and goodness o p edic ion pa ame e s.
Visualiza ion
I in e es ing me aboli es a e selec ed based solely on he co ela ion, a numbe o biochemical
compounds ha a e p esen in e y low concen a ions also will be selec ed, and he isk o
selec ing alse posi i es will be high. Po en ially biochemically in e es ing compounds can be
be e selec ed based on a combina ion o co a iance and co ela ion in o ma ion, which is he
pu pose o he sco e plo (S-plo ). The a he along he x-axis (co a iance), he g ea e he
con ibu ion o he a iance be ween he g oups, while he a he he y-axis (co ela ion), he
highe he eliabili y o he analy ical esul .
OPLS-DA oge he wi h he S-plo and loading plo allows complex da a o be mined o
me aboli es ha a e s a is ically and po en ially biochemically in e es ing compounds. The use
o app op ia e isualiza ion ools helps in he communica ion and in e p e a ion o scien i ic
da a. The me abolic da a we e analyzed as desc ibed by Wiklund e al.[36]. B ie ly, he wo
ec o s used in he S-plo a e calcula ed as
Co ð ;XiÞ¼ TXi
N1ð1Þ
Co ð ;XiÞ¼Co ð ;XiÞ
s sXi
ð2Þ
whe e is he sco e ec o in he OPLS-DA model, iis he cen e ed a iable in da a ma ix X,
and sis he es ima ed s anda d de ia ion. The e o e, Co ( ,X) and Co ( ,X) a e ec o s wi h
he same leng h as he numbe o a iables in he mode. These ec o s a e plo ed in a sca e
plo and a e S-shaped unless he a iable a iance is uni o m. The x-axis (Co ( ,X)) in he
S-plo is a isualiza ion o con ibu ion (co a iance), and he y-axis (Co ( ,X)) spans a
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 5/13

heo e ical minimum (−1) and maximum (+1), whe e 1 is he co ela ion ( eliabili y). The s a-
is ical S-plo was used o iden i y possible biochemically in e es ing compounds o bo h he
p edic i e and o hogonal a ia ions. A complemen a y ool o iden i ying in e es ing com-
pounds is he loading plo whe e he ec o Co ( p,X), which includes he co esponding jack-
kni ed confidence in e als, p o ides addi ional in o ma ion abou me aboli e a iabili y.
Resul s
De elopmen o P.bal ou iana soma ic emb yos
Du ing he ma u a ion s age, emb yogenic issue o he cell lines exhibi ed di e en emb yo-
genic abili y a e being ea ed wi h 2.5 μM, 3.6 μM, o 5 μM 6-BAP. Emb yogenic issue p o-
li e a ed in medium con aining 3.6 μM 6-BAP and yielded he mos ully ma u e soma ic
emb yos wi h a no mal se o co yledons (96±7/pe 100 mg issue), which had highe ge mina-
ion a es (48.47%±0.06)) han he ma u e soma ic emb yos om he o he wo 6-BAP ea -
men s. Medium con aining 2.5 μM 6-BAP yielded he second highes numbe o ma u e
emb yos (64±4/pe 100 mg issue) wi h ge mina ion a es o 28.39±0.04 and medium con ain-
ing 5 μM 6-BAP yielded he lowes numbe o ma u e soma ic emb yos (11±1/pe 100 mg is-
sue) wi h he lowes ge mina ion a es (9.42%±0.03).
ANOVA o he con ol line also e ealed signi ican di e ences among he samples ea ed
wi h he h ee concen a ions o 6-BAP (Table 1). In addi ion, a mic o-examina ion showed
ha he numbe s o ea ly soma ic emb yos in he di e en ea men s we e signi ican ly di e -
en (Fig 2); ha is, 31±3, 47±4, and 5±2 ea ly soma ic emb yos pe 50 mg issue in he 2.5 μM,
3.6 μM, and 5 μM g oups, espec i ely.
Me abolic changes in esponse o 6-BAP
Analysis o he h ee 6-BAP- ea ed cul u e samples yielded 309 compounds, among which
123 me aboli es we e assigned o KEGG pa hways. A PCA model wi h wo p incipal compo-
nen s explained 52% o he a ia ion ac oss he h ee samples (Fig 3A).
S-plo o he i s wo p incipal componen s o each wo samples (2.5 μM e sus 5 μM,
3.6 μM e sus 2.5 μM and 3.6 μM e sus 5 μM), explained 54%, 59% and 58% o he o al a ia-
ion, espec i ely, and was used o an o e iew o he da a. The S-plo showed ha each
6-BAP- ea ed sample was sepa a ed om he main clus e and dis ibu ed. OPLS-DA was
used o disc imina e be ween he wo samples, and he OPLS-DA showed ha he samples
we e sepa a ed acco ding o he 6-BAP concen a ion (Fig 3B, 3C and 3D). Me aboli es ha
dis inguished he sample classes a e p esen ed in S1 Table (2.5 μM e sus 5 μM), S2 Table
(3.6 μM e sus 2.5 μM) and S3 Table (3.6 μM e sus 5 μM), espec i ely.
2.5 μM e sus 5 μM 6-BAP- ea ed samples
Mos o he di e en ially egula ed me aboli es (32) we e p esen in he 2.5 μM g oup com-
pa ed wi h he 5 μM g oup, bo h o which had lowe emb yogenic compe ence han he 3.6 μM
Table 1. Numbe o ma u e co yledona y emb yos gene a ed om each ea men (pe 100 mg o
emb yogenic issue) and hei ge mina ion a es compa ed wi h he con ol line.
Sample Ma u e emb yos / pe 100 mg o emb yogenic issue Ge mina ion a e
2.5 μM64 ±4
b
28.39% ±0.04
b
3.6 μM96 ±7
a
48.47% ±0.06
a
5μM11 ±1
c
9.42% ±0.03
c
a, b, c
indica e he significance o di e ence (P 0.05).
doi:10.1371/jou nal.pone.0141841. 001
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 6/13
g oup. The majo i y o he egula ed me aboli es (14 up- egula ed, ou down- egula ed)
we e amino acids o hei de i a i es associa ed wi h a ious biosyn he ic pa hways. Among
he egula ed me aboli es ha we e ca bohyd a es, he numbe o up- and down- egula ed
me aboli es was app oxima ely equal. A o al o eigh o ganic acids ( i e up- egula ed, h ee
Fig 2. Ea ly s age emb yos in issues ea ed wi h 6-BAP by mic o-examina ion. The ba s in each o he panels indica e 500 μm. (A) Ea ly soma ic
emb yos in issue ea ed wi h 2.5 μM 6-BAP. (B) Ea ly soma ic emb yos in issue ea ed wi h 3.6 μM 6-BAP. (C) Ea ly and ma u e soma ic emb yos in issue
ea ed wi h 5 μM 6-BAP.
doi:10.1371/jou nal.pone.0141841.g002
Fig 3. Sco e plo s o he i s wo p incipal componen s by mul i a ia e analysis. (A) PCA 1/ 2 sco e sca e (N = 18, R
2
X[1] = 0.38, R
2
X[2] = 0.14,
and Q
2
Y [cum] = 0.36). OPLS-DA sco e sca e s. (B) 2.5 μM e sus 5 μM(R
2
X = 0.553, R
2
Y = 0.96 and Q
2
= 0.834). (C) 3.6 μM e sus 2.5 μM(R
2
X = 0.548,
R
2
Y = 0.95 and Q
2
= 0.742). (D) 3.6 μM e sus 5 μM (R2X = 0.573, R2Y = 0.89 and Q2 = 0.676). Samples a e colo ed acco ding o he 6-BAP
concen a ions.
doi:10.1371/jou nal.pone.0141841.g003
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 7/13
down- egula ed) we e iden i ied in he 2.5 μM g oup compa ed wi h he 5 μM g oup; no ably,
lyxose 1 was signi ican ly inc eased (19- old) in he 5 μM 6-BAP g oup.
The sepa a ion be ween he 32 di e en ially egula ed me aboli es be ween he 2.5 μM and
5μM g oups was highligh ed in he S-plo (Fig 4A). In he aw da a plo bo h g oups o e -
lapped, while only 11 me aboli es had a high co ela ion in he S-plo and a high eliabili y in
he loading plo (Fig 4D) and we e deemed eliable o sepa a ing he g oups.
3.6 μM e sus 2.5 μM 6-BAP- ea ed samples
Th ee di e en ially egula ed me aboli es we e up- egula ed and wo we e down- egula ed in
he 3.6 μM g oup compa ed wi h he 2.5 μM g oup, and h ee we e amino acids, o which one
was down- egula ed and wo we e up- egula ed in he 2.5 μM g oup compa ed wi h he con ol
cells. Among he egula ed me aboli es ha we e o ganic acids, he numbe o up- and down-
egula ed me aboli es was equal.
In he S-plo , 12 me aboli es showed a high co ela ion (Fig 4B); howe e , in he loading
plo , mos o hem showed low eliabili y because he con idence in e al did no suppo he
S-plo selec ion (Fig 4E). Finally, only yp ophan 2 and so bose 2 showed easonable eliabili y
we e selec ed o u he in es iga ion o hei biochemical signi icance.
3.6 μM e sus 5 μM 6-BAP- ea ed samples
Th ee o he di e en ially egula ed me aboli es we e ca bohyd a es; one was up- egula ed and
wo we e down- egula ed in 3.6 μM cul u es compa ed wi h 5 μM. A common pa e n was
obse ed o he o ganic acid and amino acid g oups, bo h o which comp ised ou
Fig 4. S a egy o iden i ica ion o in e es ing me aboli es. (A),(B), and (C) show selec ed signi ican me aboli es ela ed o he di e ences be ween
each pai wise compa ison (see Fig 3). (D),(E), and (F) show he loading plo s de i ed om each pai wise compa ison. The plo s mainly show he selec ed
me aboli es om he S-plo (see Fig 3).
doi:10.1371/jou nal.pone.0141841.g004
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 8/13
compounds wi h one down- egula ed and h ee up- egula ed in he 5 μM cul u e compa ed
wi h he con ol cells. The di e en ially egula ed me aboli es wi h he highes le els in he
3.6 μM g oup we e oxalic acid, galac ose and, leucine, among which he leucine le el was signi -
ican ly up- egula ed compa ed wi h he 5 μM g oup.
Signi ican di e ences be ween he 3.6 μM and 5 μM samples we e iden i ied using he mass
peak in ensi ies o all he de ec ed me aboli es, which we e exp essed in an S-plo , OPLS-DA
(Fig 4C, and loading plo (Fig 4F). Bo h up- egula ed and down- egula ed me aboli es we e
ound in he 3.6 μM samples compa ed wi h 5 μM samples, bu only 1,5-anhyd ogluci ol,
3-hyd oxyp opionic acid 1 and leucine we e selec ed as signi ican egula ed ma ke s based on
he s a is ical analyses.
Discussion
In he p esen s udy, we ollowed he me abolic e en s in one emb yogenic cell line o P.bal-
ou iana ha displayed di e en emb yogenic ac i i ies a e being ea ed wi h h ee 6-BAP
concen a ions. O he emb yogenic issues, hose ea ed wi h 3.6 μM 6-BAP gene a ed he
g ea es numbe o soma ic emb yos, which also had highe ge mina ion a es han he soma ic
emb yos gene a ed om emb yogenic issues p oli e a ed on medium wi h lowe (2.5 μM) o
highe (5 μM) le els o 6-BAP. Fu he mo e, mic o-examina ion showed ha he emb yonal
heads and suspenso s o ea ly soma ic emb yos in he 5 μM- ea ed cul u es we e bigge han
hose in he 2.5 μM- and 3.6 μM- ea ed cul u es. These esul s oge he wi h he ANOVA o
ma u e emb yos and his ological analysis o ea ly soma ic emb yos in he con ol cell line dem-
ons a ed ha 6-BAP in luenced he ma u a ion o issues and showed ha he in luence had
uni e sali y. Many possible mechanisms could be p oposed o explain hese esul s; o exam-
ple, 6-BAP may ha e in luenced o he plan g ow h egula o s o genes in ol ed in me aboli e
egula ion. In he p esen s udy, we in es iga ed his phenomenon using a me abolomics
app oach and ound ha me aboli e p o iles we e al e ed signi ican ly in esponse o di e en
concen a ions o 6-BAP. The p e e en ial di e en ial egula ion o me aboli es may igge
adap i e esponses du ing soma ic emb yogenesis.
Al hough he - es is widely used o selec ing in e es ing compounds, we ound ha some
o he egula ed me aboli es iden i ied using he - es may no be eliable [45–47]. Fo exam-
ple, lyxose1 was signi ican ly up- egula ed (19- old) in he 5 μM 6-BAP g oup compa ed wi h
he 2.5 μM g oup and was selec ed as a eliably egula ed me aboli e based on he loading plo ,
bu would no ha e been selec ed based on he - es . The majo dissen is ha he - es gi es
no conside a ion o a iable in ensi y, which is o en ela ed o me aboli e concen a ion [48].
In he p esen s udy, we used a loading plo because i educes he impac o a i ac s and noise
in he models.
Leucine was up- egula ed in emb yogenic issues p oli e a ed in he 3.6 μM cul u e com-
pa ed wi h he 5 μM cul u e, while so bose 2 was signi ican ly down- egula ed in he 3.6 μM
cul u e compa ed wi h he 2.5 μM cul u e. The aspa ic acid, alanine, aspa agine, se ine, gly-
cine, and phenylalanine amino acids we e up- egula ed in he 2.5 μM cul u e compa ed wi h
he 5 μM cul u e, which did no e icien ly gene a e emb yos. This inding is in good ag eemen
wi h he esul s o Richa d e al. [49]. B oeckling e al. [50] epo ed a nega i e co ela ion
be ween amino acid and suga le els in emb yogenic issues, which is in ag eemen wi h ou
esul s. We ound ha amino acid me aboli es we e mos ly up- egula ed, whe eas mos o he
suga s we e down- egula ed. All he me aboli es ha we e di e en ially egula ed by 6-BAP
esul ed in a di e en emb yogenic capaci y o he cul u es.
6-BAP may a ec wo channels in ol ed in ea ly soma ic emb yogenesis: ni ogen me abo-
lism and/o he IAA concen a ion in issues. In ni ogen me abolism, NH
4+
and NO
3
−
a e
Me aboli e Response o Picea bal ou iana Emb yogenic Tissues o 6-BAP
PLOS ONE | DOI:10.1371/jou nal.pone.0141841 Oc obe 30, 2015 9/13