

Ci a ion: Ahmadi, H.; Abbasi, A.;
Taleei, A.; Mohammadi, V.; Pueyo, J.J.
An ioxidan Response and
Calcium-Dependen P o ein Kinases
In ol emen in Canola (B assica
napus L.) Tole ance o D ough .
Ag onomy 2022,12, 125. h ps://
doi.o g/10.3390/ag onomy12010125
Academic Edi o s: Monica Boscaiu
and Ana Fi a
Recei ed: 29 No embe 2021
Accep ed: 30 Decembe 2021
Published: 5 Janua y 2022
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Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license (h ps://
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ag onomy
A icle
An ioxidan Response and Calcium-Dependen P o ein Kinases
In ol emen in Canola (B assica napus L.) Tole ance o D ough
Hossein Ahmadi 1,2, Ali eza Abbasi 1,*, Ali eza Taleei 1, Valiollah Mohammadi 1and JoséJ. Pueyo 2,*
1Depa men o Ag onomy and Plan B eeding, Uni e si y o Teh an, Ka aj 77871-31587, I an;
[email p o ec ed] (H.A.); [email p o ec ed] (A.T.); [email p o ec ed] (V.M.)
2Ins i u e o Ag icul u al Sciences, ICA-CSIC, 28006 Mad id, Spain
*Co espondence: [email p o ec ed] (A.A.); [email p o ec ed] (J.J.P.)
Abs ac :
Canola is an impo an empe a e oil c op ha can be se e ely a ec ed by d ough . Unde -
s anding he physiological and molecula mechanisms in ol ed in canola ole ance o wa e de ici
is essen ial o ob ain d ough - ole an p oduc i e cul i a s. To in es iga e he ole o an ioxidan
esponse and he possible in ol emen o calcium-dependen p o ein kinases (CDPKs) in canola
ole ance o d ough , we analyzed ou geno ypes wi h di e en sensi i i y o wa e s ess. Lea
ela i e wa e con en , canopy empe a u e, PSII e iciency, elec oly e leakage index and lipid pe -
oxida ion we e used as indica o s o classi y he cul i a s as d ough - ole an o d ough -sensi i e.
An ioxidan enzymes supe oxide dismu ase, guaiacol pe oxidase and ca alase displayed signi ican ly
highe ac i i ies in d ough - ole an han in d ough -sensi i e cul i a s subjec ed o wa e de ici ,
sugges ing ha he e iciency o he an ioxidan esponse is essen ial in canola d ough ole ance.
The inc eased exp ession o genes BnaCDPK6 and BnaCDPK14 unde d ough condi ions, hei
di e en ial exp ession in d ough - ole an and d ough -sensi i e geno ypes, and he p esence o
mul iple cis-ac ing s ess- ela ed elemen s in hei p omo e egions sugges ha CDPKs a e pa o
he signaling pa hways ha egula e d ough esponse in canola. We p opose he BnaCDPK genes
and hei egula o elemen s as po en ial molecula a ge s o ob ain d ough - ole an p oduc i e
canola cul i a s h ough b eeding o gene ic ans o ma ion.
Keywo ds:
B assica napus; canola; apeseed; d ough ; calcium-dependen p o ein kinases; CDPK;
an ioxidan enzymes
1. In oduc ion
Canola (B assica napus L.) is one o he mos widely g own oil c ops wo ldwide. Canola
oil has a high amoun o polyunsa u a ed a y acids, and i is ex ensi ely used in he ood
indus y [
1
]. Al hough canola ole an cul i a s ha e been iden i ied, as a empe a e c op,
canola is gene ally conside ed sensi i e o d ough , which has a subs an ial nega i e e ec
on his c op’s g ow h and de elopmen [
2
]. In gene al, abio ic s ess ad e sely a ec s he
majo biological p ocesses, esul ing in al e a ions a he physiological, mo phological,
biochemical, molecula and cellula le els. D ough s ess o sca ci y o wa e is a c i ical
en i onmen al ac o in ag icul u e wo ldwide. I nega i ely a ec s c op yield and, in he
con ex o clima e change, d ough is expec ed o ha e a p o ound impac on sus ainable
c op p oduc ion in a id and semi-a id soils. In canola, d ough s ess causes a dec ease in
plan biomass and induces changes in physiological and biochemical pa ame e s, including
dec eases in ela i e wa e con en , educed chlo ophyll con en due o loss o pigmen s
and damaged hylakoid memb anes, educed pho osyn he ic a e, s oma al conduc ance,
cell expansion, g ow h, damage o cellula memb anes and a ious gene ic esponses
[3–5]
.
Pho osyn hesis is one o he key physiological p ocesses ha bo h d ough and hea s ess
comp omise by educing pho osyn he ic e iciency and/o inc easing he dissipa ion o
exci a ion ene gy as hea , leading o al e a ions in luo escence pa ame e s. Unde d ough ,
Ag onomy 2022,12, 125. h ps://doi.o g/10.3390/ag onomy12010125 h ps://www.mdpi.com/jou nal/ag onomy
Ag onomy 2022,12, 125 2 o 15
plan s educe wa e loss by educing he s oma al opening. Howe e , his also es ic s CO
2
en y in o he lea , which may lead o he dec ease o he pho osyn hesis a e [
6
–
8
]. Ele a ed
CO
2
has been epo ed o alle ia e he nega i e e ec s o bo h hea s ess and d ough in
whea [
9
,
10
] Wa e s ess induces he p oduc ion o eac i e oxygen species (ROS) in plan
cells, which can ac as signaling molecules, media ing many key physiological p ocesses, bu
high ROS p oduc ion is oxic o plan s, and ROS can a ack lipids, p o eins, ca bohyd a es,
nucleic acids and, ul ima ely, lead o cell dea h [
11
,
12
]. Plan s ha e some mechanisms,
such as physiological, biochemical and me abolic esponses h ough he ac i a ion o many
s ess- esponsi e genes and he syn hesis o p o eins, o manage en i onmen al s ess [
13
].
Plan esponse mechanisms include enzyma ic and non-enzyma ic an ioxida i e sys ems,
such as supe oxide dismu ase (SOD, EC 1.15.1.1), which is he i s de ense enzyme ha
con e s supe oxide o H
2
O
2
, which can be sca enged by ca alase (CAT, EC 1.11.1.6) and
di e en classes o pe oxidases, including guaiacol pe oxidase (GPOX, EC. 1.11.1.7) [
14
].
Se e al an ioxidan and ca bohyd a e-me abolic enzymes ha e been epo ed o co ela e
wi h d ough e ec s on whea g ain yield and ha e been p oposed as bio-signa u es o he
b eeding and selec ion o d ough - ole an geno ypes [
15
]. The i s s ep in abio ic s ess
esponse is sensing o pe cei ing s ess, which is a complex p ocess in ol ing calcium
signals and phospho yla ion cascades ha ac i a e an ioxida i e sys ems [
16
]. One o he
mos common esponses o s ess pe cep ion is he inc eased accumula ion o ee calcium
(Ca
2+
) in he cy osol leading o he al e ed exp ession o s ess- esponsi e genes h ough
Ca
2+
senso s, such as calcium-dependen p o ein kinases (CDPKs/CPKs), calcineu in B-like
p o eins (CBLs), calmodulins (CaMs) and calmodulin-like p o eins (CMLs) [
17
,
18
]. Among
Ca
2+
senso s, CDPKs play a key ole in he esponse o abio ic and bio ic s imuli [
19
]. They
con ain a single polypep ide chain, a calmodulin domain wi h ou EF-hand Ca
2+
binding
mo i s and a kinase domain; hey pe cei e Ca
2+
a ia ions and quickly ansla e hem
in o a phospho yla ion signal [
20
]. P e ious s udies on CDPK genes in A abidopsis ha e
shown ha hey play a key ole in esponse o abio ic and bio ic s imuli and ha hey
a e speci ically in ol ed in d ough esis ance [
21
–
23
]. Canola and A abidopsis belong
o he same plan amily (B assicaceae) and, he e o e, sha e some gene ic simila i ies. A
leas
25 CDPK genes
ha e been iden i ied in he canola genome, some o which ha e been
u he analyzed unde a ious abio ic s esses [24].
The aims o he p esen s udy we e o in es iga e di e en ial esponses o wa e
de ici in d ough -sensi i e and d ough - ole an canola geno ypes. Lea ela i e wa e
con en , lea canopy empe a u e, PSII e iciency, lea elec oly e leakage index and lipid
pe oxida ion we e measu ed o es whe he hey could cons i u e alid indica o s o
d ough ole ance in canola. We hypo hesized ha he an ioxidan esponse was likely
o ha e an impo an ole in canola d ough ole ance, and we de e mined supe oxide
dismu ase, guaiacol pe oxidase and ca alase enzyma ic ac i i ies o check whe he he e
we e di e ences be ween sensi i e and ole an cul i a s. Based on he epo ed ole o
CDPKs in A abidopsis, we heo ized hey migh be in ol ed in canola esponse o d ough .
To es his hypo hesis, we quan i ied he exp ession o h ee BnaCDPK genes in sensi i e
and ole an canola cul i a s subjec ed o wa e de ici , and we examined he BnaCDPK
p omo e egions o iden i y mo i s ha could indica e ha hei exp ession migh be
egula ed in esponse o d ough s ess.
2. Ma e ials and Me hods
2.1. Plan Ma e ial, Expe imen al Design, and D ough S ess T ea men s
Fou cul i a s o canola (B assica napus L.), namely Za am and Slm046 ( ole an
geno ypes) and Okapi and Sa igol (sensi i e geno ypes), we e used in his s udy [
25
,
26
].
A comple ely andomized ac o ial expe imen wi h h ee eplica ions was ca ied ou in
a g ow h chambe unde con olled condi ions (16 h/8 h day/nigh pho ope iod, 22
◦
C).
Seeds we e su ace-s e ilized and g own in plas ic po s illed wi h 5 kg clay loam soil o
3 weeks. D ough s ess ea men s we e applied a e 3 weeks. I iga ion egimes we e
based on po weigh and soil wa e con en . Con ol plan s we e kep in good i iga ion a
Ag onomy 2022,12, 125 3 o 15
100% ield capaci y (FC), and wo d ough ea men s, 70% FC and 40% FC, we e applied
o 3 mo e weeks. Lea es we e hen collec ed, ozen in liquid ni ogen and s o ed a
−
80
◦
C.
2.2. Rela i e Wa e Con en , Lea Tempe a u e, Chlo ophyll Fluo escence (F /Fm) and Elec oly e
Leakage Index
Rela i e wa e con en (RWC) is a physiological measu e o cellula hyd a ion. A com-
ple ely de eloped young lea om each plan was used. F esh weigh (FW) was measu ed;
sa u a ed weigh (SW) was de e mined a e keeping he lea es in wa e o 24 h a 4
◦
C in
he da k, and d y weigh (DW) was measu ed a e 72 h in an o en a 45
◦
C. Then, RWC was
es ima ed using he Wea he ley o mula [
27
]:
RWC (%) = [(FW −DW)/(SW −DW)] ×100
.
Lea empe a u e (LT) is a de e mining ac o in plan p oduc i i y. A po able in a ed
he mome e (Model OS1327D, OMEGA, No walk, CT, USA) was used o measu e LT (
◦
C).
In o de o a oid he e ec o soil empe a u e, da a we e aken on high lea co e age a eas.
Two o ou measu emen s pe po we e aken.
Chlo ophyll luo escence was measu ed wi h a po able luo escence spec ome e
(Handy PEA, Handsa ech Ins umen s L d., King’s Lynn, UK) [
28
]. Lea clips we e applied
o 30 min o da k adap a ion, hen minimal luo escence (F
0
) and maximum luo escence
(F
m
) we e measu ed unde a weak ed ligh (<0.1
µ
mol m
−2
s
−1
) and a 0.8 s sa u a ing
pulse (8000
µ
mol m
−2
s
−1
). The a iable luo escence (F
) and he maximum quan um
yield o PSII (F /Fm) we e calcula ed using he measu ed pa ame e s F0and Fm.
Lea elec oly e leakage index (ELI) was used as a unc ion o cellula inju y om
osmo ic s ess ha was de e mined o be ELI (%) = (EC
1
/EC
2
)
×
100 [
29
]. Plan ma e ial
(0.8 g lea ) was imme sed in s e ile wa e in 20 mL ubes and incuba ed a 25
◦
C o
24 h
;
a e incuba ion, he elec ical conduc i i y o he solu ion (EC
1
) was measu ed; hen,
samples we e au ocla ed a 120
◦
C o 20 min, and he inal elec ical conduc i i y (EC
2
)
was measu ed a e cooling o 25 ◦C.
2.3. Lipid Pe oxida ion
The amoun o malondialdehyde (MDA) is indica i e o he le el o damage o mem-
b ane lipid o lipid pe oxida ion. Lea samples (0.05 g) we e pul e ized in liquid ni ogen,
homogenized wi h 0.1% ichlo oace ic acid (TCA) and cen i uged a 10,000
×
g o 15 min
a 4
◦
C, hen he supe na an (0.6 mL) was mixed wi h 0.6 mL o 20% (w/ ) ichlo oace ic
acid (TCA) con aining 0.5% (w/ ) TBA; he mix u e was hea ed a 95
◦
C o 30 min and
hen quickly cooled down in an ice ba h. A e cen i uga ion a 10,000
×
g o 10 min, he
abso bance and nonspeci ic abso p ion o he supe na an was measu ed a 532 and
600 nm
.
The ex inc ion coe icien (155 mM
−1
cm
−1
) was used o calcula ing he amoun o MDA
as µmol MDA mg−1 esh weigh [30].
2.4. Enzyme Ex ac ion, P o ein De e mina ion and An ioxidan Enzyme Ac i i y
Lea samples we e g ound by liquid ni ogen and ex ac ion bu e con aining 0.1 M
T is-HCl; 5% (w/ ) suc ose and 0.1% 2-me cap oe hanol, a pH 7.5, was added (3:1 bu e
olume:lea FW). Then he homogena e was cen i uged (10,000
×
g) o 20 min a 4
◦
C,
and he supe na an was used o enzyma ic ac i i ies and p o ein de e mina ions. The
B ad o d me hod [31] was used o de e mine he p o ein concen a ion.
Supe oxide dismu ase ac i i y (SOD) was de e mined as he abili y o inhibi he
pho ochemical educ ion o ni o blue e azolium [
32
]. The eac ion mix u e, 50 mM
po assium phospha e bu e (pH 7.8), 13 mM me hionine, 75
µ
M NBT, 0.1
µ
M EDTA, 4
µ
M
ibo la in and he equi ed amoun o enzyme ex ac ( o al olume 1 mL) was illumina ed
wi h wo 15 W luo escen lamps o 15 min. A con ol sample con ained he eac ion
mix u e wi hou he enzyme. One SOD uni ep esen s enough enzyme o cause 50%
inhibi ion in he educ ion o NBT as obse ed a 560 nm. Guaiacol pe oxidase (GPOX)
ac i i y is based on he abili y o con e guaiacol o e a guaiacol (
ε
= 26.6 mM
−1
cm
−1
)
as moni o ed a 436 nm [
33
]. The eac ion mix u e con ained 100 mM po assium phospha e
bu e (pH 7.0), 20 mM guaiacol, 10 mM H
2
O
2
and enzyme ex ac ( o al olume 1 mL).
Ag onomy 2022,12, 125 4 o 15
Abso bance was measu ed a 436 nm o 5 min. One GPOX uni is de ined as 1
µ
M oxidized
guaiacol pe min pe mg p o ein (uni mg
−1
p o ein). Ca alase (CAT) ac i i y assay is
based on he disappea ance o H
2
O
2
a 240 nm (
ε
= 40 mM
−1
cm
−1
) [
34
]. The eac ion
mix u e con ained 50 mM po assium phospha e bu e (pH 7.0), 33 mM H
2
O
2
and enzyme
ex ac ( o al olume 1 mL). Abso bance was measu ed a 240 nm o 180 s. CAT ac i i y is
exp essed as uni s (µM H2O2pe min) pe mg o p o ein.
2.5. RNA Isola ion and Gene Exp ession
To al RNA was isola ed and pu i ied om canola lea es using he RNeasy plan mini
ki (Qiagen, Valencia, CA, USA). The quali y o he RNA samples was assessed in an
aga ose gel 2%, and i s concen a ion was de e mined wi h a NanoD op ND-1000 (The mo
Fishe Scien i ic, Wal ham, MA, USA). cDNA was syn hesized om RNA (1
µ
g) using he
Quan iTec Re e se T ansc ip ion Syn hesis ki (Qiagen. Valencia, CA, USA). Real- ime
PCR (RT-PCR) was pe o med using he luo escen in e cala ing dye SYBRG een wi h
he S ep One Plus Real-Time PCR Sys em (Applied Biosys ems, Wal ham, MA, USA),
acco ding o he manu ac u e ’s ins uc ions. Gene-speci ic p ime s we e designed using
he P ime 3 So wa e (h p://p ime 3.u .ee/, accessed on 20 June 2019) (Table 1). The cycle
h eshold (C ) was measu ed o h ee biological samples wi h h ee echnical eplica es
pe sample. Fo no malizing he le el o a ge gene exp ession, BnaAc in7 was used as a
con ol cons i u i e gene. Analysis o he ela i e gene exp ession was ca ied ou using
he 2−∆∆C o mula [35].
Table 1. P ime s used in his s udy.
Gene Accession No. Gene Locus P ime Sequence
BnaCDPK6 XM_013788076.2 LOC106348352 Fo 50TCAAGGACAAAGTCTACGAGGGTAA 30
Re 50TCTTTTACAGGAACGGGATTGTTG 30
BnaCDPK10 XM_013860191.2 LOC106419410 Fo 50AGTCAGATCGAGATTGAAGCAGTT 30
Re 50TTGAGTTCCGGGCAAGTTATTCTA 30
BnaCDPK14 XM_013896624 LOC106454505 Fo 50CGGATTGCGTAAACTAGGAATTGTTG 30
Re 50CTGCCCATCTTTCTGATGTGTACC 30
BnaAc in7 XM_013858992 LOC106418315 Fo 50TGGGTTTGCTGGTGACGAT 30
Re 50TGCCTAGGACGACCAACAATACT 30
2.6. S a is ical Analyses
Da a we e analyzed using SAS ( . 9.4) by wo-way ANOVA, conside ing d ough and
geno ype as ixed ac o s, and he signi icance o he ixed ac o s and hei in e ac ion
was e alua ed o all da a (Supplemen a y Table S1). Pos -hoc compa isons o means we e
pe o med using he Duncan’s mul iple ange es (DMRT), p= 0.05.
3. Resul s
3.1. E ec o D ough S ess on Physiological T ai s
The lea ela i e lea wa e con en (RWC), lea empe a u e (LT) and maximum quan-
um yield o PSII (F
/F
m
) we e measu ed in he lea es o canola d ough - ole an cul i a s
(Za am and Slm046) and d ough -sensi i e cul i a s (Okapi and Se igold) unde con ol
and d ough -s ess condi ions. The e we e no signi ican di e ences among cul i a s unde
well-wa e ed condi ions o any o he measu ed pa ame e s.
The ela i e lea wa e con en dec eased signi ican ly as a esul o he d ough
ea men s. Bo h mode a e (70% FC) and se e e d ough (40% FC) signi ican ly educed
RWC in compa ison o well-wa e ed (100% FC) condi ions. Mo eo e , in e ac ion be ween
s ess and geno ypes was signi ican , and d ough -sensi i e geno ypes we e signi ican ly
mo e a ec ed han ole an geno ypes unde bo h d ough egimes (Figu e 1).
Ag onomy 2022,12, 125 5 o 15
Ag onomy 2022, 12, x FOR PEER REVIEW 5 o 16
con ol and d ough -s ess condi ions. The e we e no signi ican di e ences among cul i-
a s unde well-wa e ed condi ions o any o he measu ed pa ame e s.
The ela i e lea wa e con en dec eased signi ican ly as a esul o he d ough ea -
men s. Bo h mode a e (70% FC) and se e e d ough (40% FC) signi ican ly educed RWC
in compa ison o well-wa e ed (100% FC) condi ions. Mo eo e , in e ac ion be ween
s ess and geno ypes was signi ican , and d ough -sensi i e geno ypes we e signi ican ly
mo e a ec ed han ole an geno ypes unde bo h d ough egimes (Figu e 1).
Figu e 1. In e ac ion e ec o d ough s ess and geno ype on he ela i e wa e con en (RWC) o
canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among
ea men s acco ding o he Duncan es .
Lea empe a u e inc eased wi h inc easing d ough . In e ac ion be ween s ess and
geno ypes was signi ican , and he inc ease in LT was signi ican ly lowe in d ough - ol-
e an geno ypes han in d ough -sensi i e geno ypes unde he se e e d ough ea men
(Figu e 2).
Figu e 2. In e ac ion e ec o d ough s ess and geno ype on he lea empe a u e (LT) o canola
plan s. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among ea men s
acco ding o he Duncan es .
aaaa
bbccd
cd dee
0
20
40
60
80
100
Za am Slm046 Okapi Sa igol
RWC (%)
Geno ype
100% FC
70% FC
40% FC
dddd
ccbc b
bbaa
0
10
20
30
40
50
Za am Slm046 Okapi Sa igol
LT (°C)
Geno ype
100% FC
70% FC
40% FC
Figu e 1.
In e ac ion e ec o d ough s ess and geno ype on he ela i e wa e con en (RWC) o
canola lea es. Da a a e means (n= 3)
±
SE. Di e en le e s indica e di e ences (p< 0.05) among
ea men s acco ding o he Duncan es .
Lea empe a u e inc eased wi h inc easing d ough . In e ac ion be ween s ess and
geno ypes was signi ican , and he inc ease in LT was signi ican ly lowe in d ough -
ole an geno ypes han in d ough -sensi i e geno ypes unde he se e e d ough ea men
(Figu e 2).
Ag onomy 2022, 12, x FOR PEER REVIEW 5 o 16
con ol and d ough -s ess condi ions. The e we e no signi ican di e ences among cul i-
a s unde well-wa e ed condi ions o any o he measu ed pa ame e s.
The ela i e lea wa e con en dec eased signi ican ly as a esul o he d ough ea -
men s. Bo h mode a e (70% FC) and se e e d ough (40% FC) signi ican ly educed RWC
in compa ison o well-wa e ed (100% FC) condi ions. Mo eo e , in e ac ion be ween
s ess and geno ypes was signi ican , and d ough -sensi i e geno ypes we e signi ican ly
mo e a ec ed han ole an geno ypes unde bo h d ough egimes (Figu e 1).
Figu e 1. In e ac ion e ec o d ough s ess and geno ype on he ela i e wa e con en (RWC) o
canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among
ea men s acco ding o he Duncan es .
Lea empe a u e inc eased wi h inc easing d ough . In e ac ion be ween s ess and
geno ypes was signi ican , and he inc ease in LT was signi ican ly lowe in d ough - ol-
e an geno ypes han in d ough -sensi i e geno ypes unde he se e e d ough ea men
(Figu e 2).
Figu e 2. In e ac ion e ec o d ough s ess and geno ype on he lea empe a u e (LT) o canola
plan s. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among ea men s
acco ding o he Duncan es .
aaaa
bbccd
cd dee
0
20
40
60
80
100
Za am Slm046 Okapi Sa igol
RWC (%)
Geno ype
100% FC
70% FC
40% FC
dddd
ccbc b
bbaa
0
10
20
30
40
50
Za am Slm046 Okapi Sa igol
LT (°C)
Geno ype
100% FC
70% FC
40% FC
Figu e 2.
In e ac ion e ec o d ough s ess and geno ype on he lea empe a u e (LT) o canola
plan s. Da a a e means (n= 3)
±
SE. Di e en le e s indica e di e ences (p< 0.05) among ea men s
acco ding o he Duncan es .
The maximum quan um yield o PSII (F
/F
m
) dec eased unde d ough condi ions,
and di e en ial esponses be ween d ough - ole an and d ough -sensi i e geno ypes
we e measu ed in bo h s ess ea men s. In e ac ion be ween s ess and geno ypes was
signi ican . As compa ed o o he geno ypes, unde se e e d ough , Za am main ained a
signi ican ly highe F /Fm alue. (Figu e 3).
Analysis o elec oly e leakage index (ELI) showed inc eases o all geno ypes unde
bo h d ough ea men s, and he e we e signi ican di e ences be ween ole an and sensi-
i e geno ypes unde bo h d ough egimes. In e ac ion be ween ac o s was signi ican .
Unde se e e d ough , be ween he wo sensi i e cul i a s, Sa igol was mo e a ec ed han
Okapi (Figu e 4).

Ag onomy 2022,12, 125 6 o 15
Ag onomy 2022, 12, x FOR PEER REVIEW 6 o 16
The maximum quan um yield o PSII (F /Fm) dec eased unde d ough condi ions,
and di e en ial esponses be ween d ough - ole an and d ough -sensi i e geno ypes
we e measu ed in bo h s ess ea men s. In e ac ion be ween s ess and geno ypes was
signi ican . As compa ed o o he geno ypes, unde se e e d ough , Za am main ained a
signi ican ly highe F /Fm alue. (Figu e 3).
Figu e 3. In e ac ion e ec o d ough s ess and geno ype on he maximum quan um yield (F /Fm)
o canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among
ea men s acco ding o he Duncan es .
Analysis o elec oly e leakage index (ELI) showed inc eases o all geno ypes unde
bo h d ough ea men s, and he e we e signi ican di e ences be ween ole an and sen-
si i e geno ypes unde bo h d ough egimes. In e ac ion be ween ac o s was signi ican .
Unde se e e d ough , be ween he wo sensi i e cul i a s, Sa igol was mo e a ec ed han
Okapi (Figu e 4).
Figu e 4. In e ac ion e ec o d ough s ess and geno ype on he elec oly e leakage index (ELI) o
canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among
ea men s acco ding o he Duncan es .
aaaa
bcde
de gg
0.72
0.76
0.80
0.84
0.88
Za am Slm046 Okapi Sa igol
F /Fm
Geno ype
100% FC
70% FC
40% FC
eedd
ccba
0
10
20
30
40
50
60
Za am Slm046 Okapi Sa igol
ELI (%)
Geno ype
100% FC
70% FC
40% FC
Figu e 3.
In e ac ion e ec o d ough s ess and geno ype on he maximum quan um yield (F
/F
m
)
o canola lea es. Da a a e means (n= 3)
±
SE. Di e en le e s indica e di e ences (p< 0.05) among
ea men s acco ding o he Duncan es .
Ag onomy 2022, 12, x FOR PEER REVIEW 6 o 16
The maximum quan um yield o PSII (F /Fm) dec eased unde d ough condi ions,
and di e en ial esponses be ween d ough - ole an and d ough -sensi i e geno ypes
we e measu ed in bo h s ess ea men s. In e ac ion be ween s ess and geno ypes was
signi ican . As compa ed o o he geno ypes, unde se e e d ough , Za am main ained a
signi ican ly highe F /Fm alue. (Figu e 3).
Figu e 3. In e ac ion e ec o d ough s ess and geno ype on he maximum quan um yield (F /Fm)
o canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among
ea men s acco ding o he Duncan es .
Analysis o elec oly e leakage index (ELI) showed inc eases o all geno ypes unde
bo h d ough ea men s, and he e we e signi ican di e ences be ween ole an and sen-
si i e geno ypes unde bo h d ough egimes. In e ac ion be ween ac o s was signi ican .
Unde se e e d ough , be ween he wo sensi i e cul i a s, Sa igol was mo e a ec ed han
Okapi (Figu e 4).
Figu e 4. In e ac ion e ec o d ough s ess and geno ype on he elec oly e leakage index (ELI) o
canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among
ea men s acco ding o he Duncan es .
aaaa
bcde
de gg
0.72
0.76
0.80
0.84
0.88
Za am Slm046 Okapi Sa igol
F /Fm
Geno ype
100% FC
70% FC
40% FC
eedd
ccba
0
10
20
30
40
50
60
Za am Slm046 Okapi Sa igol
ELI (%)
Geno ype
100% FC
70% FC
40% FC
Figu e 4.
In e ac ion e ec o d ough s ess and geno ype on he elec oly e leakage index (ELI) o
canola lea es. Da a a e means (n= 3)
±
SE. Di e en le e s indica e di e ences (p< 0.05) among
ea men s acco ding o he Duncan es .
3.2. Lipid Pe oxida ion
The malondialdehyde (MDA) con en is indica i e o lipid pe oxida ion. MDA con en
inc eased wi h d ough o all geno ypes. The e we e signi ican di e ences be ween
ole an and sensi i e geno ypes unde se e e d ough . (Figu e 5). In e ac ion be ween
s ess and geno ypes was signi ican . As compa ed o o he geno ypes, unde se e e
d ough , Za am main ained a signi ican ly lowe MDA con en , and his cul i a was less
a ec ed han Slm046, which is also d ough - ole an (Figu e 5).
Ag onomy 2022,12, 125 7 o 15
Ag onomy 2022, 12, x FOR PEER REVIEW 7 o 16
3.2. Lipid Pe oxida ion
The malondialdehyde (MDA) con en is indica i e o lipid pe oxida ion. MDA con-
en inc eased wi h d ough o all geno ypes. The e we e signi ican di e ences be ween
ole an and sensi i e geno ypes unde se e e d ough . (Figu e 5). In e ac ion be ween
s ess and geno ypes was signi ican . As compa ed o o he geno ypes, unde se e e
d ough , Za am main ained a signi ican ly lowe MDA con en , and his cul i a was less
a ec ed han Slm046, which is also d ough - ole an (Figu e 5).
Figu e 5. In e ac ion e ec o d ough s ess and geno ype on he malondialdehyde (MDA) con en
o canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e ences (p < 0.05) among
ea men s acco ding o he Duncan es .
3.3. An ioxidan Enzyme Ac i i ies
The e we e no signi ican di e ences in supe oxide dismu ase (SOD) ac i i y be-
ween d ough - ole an and d ough -sensi i e geno ypes unde con ol condi ions. SOD
ac i i y signi ican ly inc eased in all cul i a s when mode a e d ough (70% FC) was ap-
plied, bu s ill no signi ican di e ences among cul i a s we e de ec ed (Figu e 6A). Unde
mo e se e e d ough condi ions (40% FC), SOD ac i i y inc eased in he ole an cul i a s
and dec eased in he sensi i e ones wi h signi ican di e ences be ween ole an and sen-
si i e geno ypes. The same beha io was obse ed o guaiacol pe oxidase (GPOX) (Fig-
u e 6B). The e we e no signi ican di e ences in ca alase (CAT) ac i i y among cul i a s
unde con ol condi ions. CAT ac i i y inc eased signi ican ly wi h d ough se e i y in
he ole an cul i a s, while i dec eased in he sensi i e ones (Figu e 6C). Di e ences be-
ween ole an and sensi i e cul i a s we e signi ican unde bo h d ough egimes. In e -
ac ion be ween ac o s geno ype and s ess was signi ican o all h ee enzyma ic ac i i-
ies.
gggg
e dde
cb
aa
0.0
0.5
1.0
1.5
2.0
Za am Slm046 Okapi Sa igol
MDA (µmol·mg-1 FW)
Geno ype
100% FC
70% FC
40% FC
Figu e 5.
In e ac ion e ec o d ough s ess and geno ype on he malondialdehyde (MDA) con en
o canola lea es. Da a a e means (n= 3)
±
SE. Di e en le e s indica e di e ences (p< 0.05) among
ea men s acco ding o he Duncan es .
3.3. An ioxidan Enzyme Ac i i ies
The e we e no signi ican di e ences in supe oxide dismu ase (SOD) ac i i y be ween
d ough - ole an and d ough -sensi i e geno ypes unde con ol condi ions. SOD ac i i y
signi ican ly inc eased in all cul i a s when mode a e d ough (70% FC) was applied, bu
s ill no signi ican di e ences among cul i a s we e de ec ed (Figu e 6A). Unde mo e
se e e d ough condi ions (40% FC), SOD ac i i y inc eased in he ole an cul i a s and
dec eased in he sensi i e ones wi h signi ican di e ences be ween ole an and sensi i e
geno ypes. The same beha io was obse ed o guaiacol pe oxidase (GPOX) (Figu e 6B).
The e we e no signi ican di e ences in ca alase (CAT) ac i i y among cul i a s unde
con ol condi ions. CAT ac i i y inc eased signi ican ly wi h d ough se e i y in he ole an
cul i a s, while i dec eased in he sensi i e ones (Figu e 6C). Di e ences be ween ole an
and sensi i e cul i a s we e signi ican unde bo h d ough egimes. In e ac ion be ween
ac o s geno ype and s ess was signi ican o all h ee enzyma ic ac i i ies.
Ag onomy 2022, 12, x FOR PEER REVIEW 8 o 16
(A)
(B)
(C)
ede ee
bbbbc
aa
cde bcd
0.0
2.0
4.0
6.0
8.0
Za am Slm046 Okapi Sa igol
SOD (Uni s·mg-1 p o ein)
Geno ype
100% FC
70% FC
40% FC
de cd de e
c
bccd
aa
0.0
2.0
4.0
6.0
Za am Slm046 Okapi Sa igol
GPOX (Uni s·mg-1 p o ein)
Geno ype
cd cde ccd
bb
cde cde
aa
de e
0.0
0.5
1.0
1.5
2.0
2.5
Za am Slm046 Okapi Sa igol
CAT (Uni s·mg-1 p o ein)
Geno ype
Figu e 6. Con .
Ag onomy 2022,12, 125 8 o 15
Ag onomy 2022, 12, x FOR PEER REVIEW 8 o 16
(A)
(B)
(C)
ede ee
bbbbc
aa
cde bcd
0.0
2.0
4.0
6.0
8.0
Za am Slm046 Okapi Sa igol
SOD (Uni s·mg-1 p o ein)
Geno ype
100% FC
70% FC
40% FC
de cd de e
c
bccd
aa
0.0
2.0
4.0
6.0
Za am Slm046 Okapi Sa igol
GPOX (Uni s·mg-1 p o ein)
Geno ype
cd cde ccd
bb
cde cde
aa
de e
0.0
0.5
1.0
1.5
2.0
2.5
Za am Slm046 Okapi Sa igol
CAT (Uni s·mg-1 p o ein)
Geno ype
Figu e 6.
In e ac ion e ec o d ough s ess and geno ype on SOD (
A
), GPOX (
B
) and CAT (
C
)
enzyma ic ac i i ies in canola lea es. Da a a e means (n= 3)
±
SE. Di e en le e s indica e di e ences
(p< 0.05) among ea men s acco ding o he Duncan es .
3.4. Calcium-Dependen P o ein Kinases (CDPK) Gene Exp ession in Canola Lea es and
P omo e Analysis
The e we e signi ican di e ences in ela i e gene exp ession due o geno ype and
d ough egime o he genes BnaCDPK6 and BnaCDPK14. Signi ican di e ences in
BnaCDPK10 exp ession we e a esul o he d ough ea men only (
Supplemen a y Table S1
).
BnaCDPK6 and BnaCDPK14 exp ession inc eased wi h d ough se e i y in all cul i a s
(
Figu e 7A,B
). BnaCDPK10 exp ession inc eased when mild d ough was applied and
sligh ly dec eased unde he mo e se e e condi ions (Figu e 7C).
A de ailed analysis using he PLACE da abase [
36
] o BnaCDPK6, BnaCDPK10 and
BnaCDPK14 p omo e sequences, lying 2000 bp ups eam o hei coding sequences, iden-
i ied se e al s ess- esponsi e egula o y elemen s ha a e known o con ibu e o he
exp ession o s ess- ela ed genes a a ansc ip ional le el (Table 2).
Ag onomy 2022,12, 125 9 o 15
Ag onomy 2022, 12, x FOR PEER REVIEW 9 o 16
Figu e 6. In e ac ion e ec o d ough s ess and geno ype on SOD (A), GPOX (B) and CAT (C)
enzyma ic ac i i ies in canola lea es. Da a a e means (n = 3) ± SE. Di e en le e s indica e di e -
ences (p < 0.05) among ea men s acco ding o he Duncan es .
3.4. Calcium-Dependen P o ein Kinases (CDPK) Gene Exp ession in Canola Lea es and
P omo e Analysis
The e we e signi ican di e ences in ela i e gene exp ession due o geno ype and
d ough egime o he genes BnaCDPK6 and BnaCDPK14. Signi ican di e ences in BnaC-
DPK10 exp ession we e a esul o he d ough ea men only (Supplemen a y Table S1).
BnaCDPK6 and BnaCDPK14 exp ession inc eased wi h d ough se e i y in all cul i a s
(Figu e 7A,B). BnaCDPK10 exp ession inc eased when mild d ough was applied and
sligh ly dec eased unde he mo e se e e condi ions (Figu e 7C).
(A)
(B)
eeee
bc ab
de cd
aa
bc bc
0
0.5
1
1.5
2
Za am Slm046 Okapi Sa igol
BnaCDPK6
Geno ype
FC100 FC70 FC40
dddd
bc bc cc
aa
bbc
0
0.5
1
1.5
2
2.5
Za am Slm046 Okapi Sa igol
BnaCDPK14
Geno ype
Ag onomy 2022, 12, x FOR PEER REVIEW 10 o 16
(C)
Figu e 7. E ec s o d ough s ess and geno ype on BnaCDPK6 (A), BnaCDPK14 (B) and
BnaCDPK10 (C) ansc ip accumula ion in canola lea es. Da a a e means (n = 3) ± SE.
Di e en le e s indica e di e ences (p < 0.05) among ea men s acco ding o he Dun-
can es .
A de ailed analysis using he PLACE da abase [36] o BnaCDPK6, BnaCDPK10 and
BnaCDPK14 p omo e sequences, lying 2000 bp ups eam o hei coding sequences, iden-
i ied se e al s ess- esponsi e egula o y elemen s ha a e known o con ibu e o he
exp ession o s ess- ela ed genes a a ansc ip ional le el (Table 2).
Table 2. S ess- esponsi e cis elemen s in he 2000 bp ups eam egion o BnaCDPK6, BnaCDPK10
and BnaCDPK14 coding sequences.
Elemen Name
Sequence
Numbe o Elemen s
BnaCDPK6
BnaCDPK10
BnaCDPK14
ABRERATCAL
MACGYGB
1
1
-
ABRELATERD1
ACGTG
1
1
1
CGCGBOXAT
VCGCGB
8
2
-
WBOXNTCHN48
CTGACY
1
2
2
WBOXNTERF3
TGACY
3
5
3
WBOXHVISO1
TGACT
2
3
2
WBOXATNPR1
TTGAC
3
5
-
WBBOXPCWRKY1
TTTGACY
1
-
-
RAV1AAT
CAACA
3
5
2
MYBGAHV
TAACAAA
2
2
-
MYBCORE
CNGTTR
5
2
7
MYB1AT
WAACCA
6
2
-
MYBST1
GGATA
5
2
3
MYBPLANT
MACCWAMC
2
-
2
MYBCOREATCYCB1
AACGG
1
-
6
MYBPZM
CCWACC
1
1
1
MYB1LEPR
GTTAGTT
-
1
-
4. Discussion
This esea ch was ca ied ou o gain deepe insigh s in o he physiological, biochem-
ical and molecula esponse mechanisms induced by di e en le els o d ough in canola
plan s. Se e al physiological and biochemical ai s we e sco ed unde con ol and
c
a
b
0
0.3
0.6
0.9
1.2
1.5
1.8
FC100 FC70 FC40
BnaCDPK10
D ough s ess
Figu e 7.
E ec s o d ough s ess and geno ype on BnaCDPK6 (
A
), BnaCDPK14 (
B
) and BnaCDPK10
(
C
) ansc ip accumula ion in canola lea es. Da a a e means (n= 3)
±
SE. Di e en le e s indica e
di e ences (p< 0.05) among ea men s acco ding o he Duncan es .