Antioxidant Response and Calcium-Dependent Protein Kinases Involvement in Canola (Brassica napus L.) Tolerance to Drought

  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 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 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 .