Transcriptomic Analysis During Olive Fruit Development and Expression Profiling of Fatty Acid Desaturase Genes

Ci a ion: Se ano, A.; Ga cía-Ma ín, J.; Mo e , M.; Ma ínez-Ri as, J.M.; Luque, F. T ansc ip omic Analysis Du ing Oli e F ui De elopmen and Exp ession P o iling o Fa y Acid Desa u ase Genes. In . J. Mol. Sci. 2024,25, 11150. h ps://doi.o g/ 10.3390/ijms252011150 Academic Edi o : Malgo za a Kloc Recei ed: 1 Oc obe 2024 Re ised: 14 Oc obe 2024 Accep ed: 15 Oc obe 2024 Published: 17 Oc obe 2024 Copy igh : © 2024 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/). In e na ional Jou nal o Molecula Sciences A icle T ansc ip omic Analysis Du ing Oli e F ui De elopmen and Exp ession P o iling o Fa y Acid Desa u ase Genes Alicia Se ano 1,* , Judi h Ga cía-Ma ín1, Ma ín Mo e 1, JoséManuel Ma ínez-Ri as 2 and F ancisco Luque 1,* 1The Uni e si y Ins i u e o Resea ch in Oli e G o e and Oli e Oils (INUO), Uni e si y o Jaén, 23071 Jaén, Spain; [email p o ec ed] (J.G.-M.); mmo [email p o ec ed] (M.M.) 2Ins i u o de la G asa, CSIC (Consejo Supe io de In es igaciones Cien í icas), 41013 Se ille, Spain; [email p o ec ed] *Co espondence: [email p o ec ed] (A.S.); [email p o ec ed] (F.L.) Abs ac : The oli e ui is a d upe whose de elopmen and ipening akes se e al mon hs om lowe ing o ull ma u a ion. Du ing his pe iod, se e al biochemical and physiological changes occu ha a ec he skin colo , ex u e, composi ion, and size o he mesoca p. The inal esul is a ui ich in a y acids, phenolic compounds, ocophe ols, pigmen s, s e ols, e penoids, and o he compounds o nu i ional in e es . In his wo k, a ansc ip omic analysis was pe o med using lowe s (T0) and mesoca p issue a se en di e en s ages du ing oli e ui de elopmen and ipening (T1–T7) o he ‘Picual’ cul i a . A o al o 1755 genes o e exp essed a any ime wi h espec o he lowe ing s age we e u he analyzed. These genes we e g ouped in o eigh clus e s based on hei exp ession p o ile. The gene en ichmen analysis e ealed he mos ele an biological p ocess o e e y clus e . Highligh ing he impo an ole o ho mones a e y ea ly s ages o ui de elopmen (T1, Clus e 1), whe eas genes in ol ed in a y acid biosyn hesis we e ele an h oughou he ui de elopmen al p ocess. Hence, genes coding o di e en a y acid desa u ase (SAD, FAD2, FAD3, FAD4, FAD5, FAD6, and FAD7) enzymes ecei ed special a en ion. In pa icula , 26 genes coding o di e en a y acid desa u ase enzymes we e iden i ied in he ‘Picual’ genome, con ibu ing o he imp o emen o he genome anno a ion. The exp ession pa e n o hese genes du ing ui de elopmen co obo a ed hei ole in de e mining a y acid composi ion. Keywo ds: Olea eu opaea L.; oli e ui ; ipening p ocess; RNAseq; a y acid desa u ase 1. In oduc ion The oli e ee (Olea eu opaea L.) is cul i a ed o he nu i ional alue o i s ui , a d upe consis ing o a leshy mesoca p and a woody endoca p. The de elopmen and ipening o he oli e ui is a long p ocess las ing se e al mon hs, which could be di ided in o i e phases [ 1 ]. The de elopmen p ocess s a s immedia ely a e he pollina ion and ui se , ollowing a double sigmoid g ow h cu e pa e n [ 2 ]. The i s phase is cha ac e ized by an exponen ial g ow h o he emb yo due o a apid cell di ision ha usually las s a ound hal a mon h a e lowe ing [ 3 ]. Du ing he ollowing 30 days, a phase o cell di ision and expansion akes place, leading o he mesoca p g ow h and endoca p de elopmen . Pi ha dening concu s wi h a ecession o cell di ision and a educ ion in ui g ow h. Subsequen ly, he g een ui con inues g owing un il i eaches he inal size by cell expansion a he yellowish s age [ 4 ]. Finally, he ipening phase leads o changes o he pu ple colo o he skin, om he u ning s age o e aison o comple ely pu ple o black. The leng h o he ui de elopmen and ipening p ocess exhibi s a ia ions among cul i a s [ 4 ], and he inal composi ion can di e signi ican ly, e en du ing ui de el- opmen [ 2 ]. In any case, oli e ui has unique cha ac e is ics due o i s composi ion, In . J. Mol. Sci. 2024,25, 11150. h ps://doi.o g/10.3390/ijms252011150 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2024,25, 11150 2 o 21 including a y acids, phenolic compounds, ocophe ols, pigmen s, s e ols, and e penoids. These compounds a e ans e ed o i gin oli e oil, gi ing i a unique la o and heal hy p ope ies [ 5 ]. The biosyn hesis o hese compounds in ol es se e al biochemical and physiological changes du ing oli e ui de elopmen [ 1 ]. Despi e he e o s made o unde s and he molecula mechanisms unde lying hese me abolic p ocesses, nume ous genes in ol ed in hei biosyn he ic pa hways a e s ill unknown [6]. T ansc ip omic s udies ocused on a y acid biosyn hesis ha e been epo ed in he cul i a s ‘syl es is’ [ 7 ], ‘Leccino’ [ 8 ], ‘A bequina’, ‘F an oio’, and ‘Niki skii’ [ 9 ], whe eas in he ‘Picual’ cul i a a e mainly ela ed o ui abscission [ 10 – 12 ], ea ly ui de elopmen [ 3 ], and ho monal con ol [13]. Rega ding he oil con en , i accumula es mainly in he mesoca p a e pi ha dening and eaches i s maximum du ing e aison, ep esen ing up o 30% o he esh weigh o he ipe ui [ 1 , 14 ]. The mos abundan a y acid is oleic acid (C18:1), which ep esen s abou 75% o o al a y acids, ollowed by linoleic (C18:2), palmi ic (C16:0), s ea ic (C18:0), and linolenic (C18:3) acids. The pa e n o a y acid biosyn hesis and desa u a ion a ies du ing ui de elopmen and be ween cul i a s, wi h a highe p opo ion o oleic han linoleic acid in he inal con en being desi able [ 15 ]. The e o e, a ho ough unde s anding o he a y acid biosyn hesis p ocess du ing ui de elopmen could con ibu e o p oducing highe -quali y oils. Fou genes encoding o s ea oyl–ACP desa u ase (SAD) enzyme ha e been desc ibed in oli e in ol ed in oleic acid o ma ion: OeSAD1 [ 16 ], OeSAD2,OeSAD3 [ 17 ], and Oe- SAD4 [ 18 ]. Mo eo e , i e genes ha e been cha ac e ized as encoding o mic osomal olea e desa u ases (OeFAD2-1,OeFAD2-2,OeFAD2-3,OeFAD2-4,OeFAD2-5) [ 19 – 22 ], which a e esponsible o linoleic acid con en oge he wi h he plas idial iso o m OeFAD6 [ 19 , 23 ]. Simila ly, genes in ol ed in he biosyn hesis o linolenic acid ha e been s udied in oli e, including ou linolea e desa u ases. Two a e loca ed in he endoplasmic e iculum: Oe- FAD3A [ 24 ] and OeFAD3B [ 25 ]. Ano he wo possess plas idial localiza ion: OeFAD7-1 [ 26 ] and OeFAD7-2 [ 25 ]. In addi ion, genes co esponding o FAD4 [ 27 ] and FAD5 [ 28 ] ha e no been s udied in oli e. The exp ession o genes coding o mos o hese enzymes and iso o ms has been u he analyzed in se e al cul i a s wi h low and high linoleic acid con en [ 15 ], bu , up o da e, he e a e no ansc ip omic s udies encompassing all hese genes du ing ui de elopmen . This wo k ep esen s an o e iew o he genes o e exp essed h oughou ui de el- opmen , om lowe o ipe ui mesoca p. We analyze hei exp ession p o iles and he biological p ocesses in which hey a e in ol ed in o de o iden i y ele an genes o he biosyn hesis o oli e ui compounds. Mo eo e , in he ‘Picual’ genome, his wo k aims o iden i y genes coding o a y acid desa u ases, which could con ibu e o imp o ing he genome anno a ion, and hei exp ession was analyzed h oughou ui de elopmen om lowe o ipened ui . In sho , his wo k aims o p o ide a be e unde s anding o he molecula mechanism in ol ed in ui de elopmen , which could be c ucial o molecula ma ke de elopmen . 2. Resul s 2.1. Gene Exp ession Du ing F ui De elopmen The de elopmen o he oli e ui is a p ocess ha akes a ound 6 mon hs om ull bloom o ui ipening (Figu e 1A). In his wo k, di e en gene exp essions we e obse ed a each sampling ime h ough- ou ui de elopmen . The lowe ing s age (T0) clea ly di e ed om he es o he ui s ages, and samples collec ed a 0.5, 1, and 2 mon hs a e ull bloom (AFB) (T1, T2, and T3, espec i ely) and co esponding o ea ly de elopmen al s ages o he whole ui a e signi i- can ly dis inc om he o he mesoca p samples om ui collec ed a 3, 4, 5, and 6 mon hs AFB (T4, T5, T6, and T7, espec i ely) (Figu e 1B). The pe cen ages o ead mapping o he e e ence genome anged om 74.3% o 78.7% (Table S1). In . J. Mol. Sci. 2024,25, 11150 3 o 21 In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 o 23 Figu e 1. (A) De elopmen al s ages collec ed o RNAseq analysis. (B) PCA plo showing he ex- p ession diffe ences among oli e ui de eloping samples. Samples collec ed in iplica e: T0: low- e s a ull bloom, T1: ui s a 15 days a e ull blooming (AFB), T2: ui s a 1 mon h AFB, T3: ui s a 2 mon hs AFB, T4: ui s a 3 mon hs AFB, T5: ui s a 4 mon hs AFB, T6: ui s a 5 mon hs AFB, and T7: ui s a 6 mon hs AFB. Thus, he lowe ing s age (T0) was conside ed he e e ence ime o he analysis o he diffe en ial gene exp ession h oughou he ui de elopmen om 15 days o 6 mon hs AFB (T1–T7) (Figu e 2). The esul s poin ed ou a p edominance o ep essed genes (Down) along he en i e oli e ui de elopmen p ocess, which means ha mo e genes a e o e exp essed in he lowe ing s age. Likewise, he numbe o ep essed genes in ui s inc eases h oughou ui de elopmen , showing he highes alue when he ui is ully ma u e a T7 (6 mon hs AFB). The numbe o o e exp essed genes in ui anged om 539 a 15 days a e lowe ing (T1) o 932 one mon h a e lowe ing (T2). O e all, 13,486 diffe en ially exp essed genes we e iden i ied wi h a p-adj alue < 0.01 and a old change highe han 4. O hose, 2,160 genes we e o e exp essed a any ime du ing ui de elopmen compa ed o he lowe ing s age. The exp ession p o ile o hose genes o e - exp essed a some ime wi h espec o he lowe ing s age and wi h an exp ession alue (log 2 CPM) highe han 0 was analyzed wi h DPGP 0.1 so wa e [29]. Genes o e exp essed a any s age o ui de elopmen ecei ed special a en ion be- cause o hei po en ial ole in in e es ing biological p ocesses in oli e ui . In o al, 1755 genes we e ga he ed in o 37 clus e s acco ding o hei exp ession end along he ui de elopmen (Figu e S1). Howe e , clus e s showing simila ends we e eo ganized in o eigh clus e s (A–H), excluding hose clus e s showing a andom exp ession pa e n du - ing ui de elopmen (clus e s 12, 33, 36, and 37 om DPGP). Genes included in each clus e , along wi h hei anno a ion, a e a ailable in he supplemen a y in o ma ion (Table S2). The eigh clus e s e ealed se e al GO e ms ela ed o biological p ocesses cha ac- e is ic o ui de elopmen (Table S3). In he ollowing clus e desc ip ion, only he 20 mos ep esen a i e p ocesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.8 will be p esen ed. Figu e 1. (A) De elopmen al s ages collec ed o RNAseq analysis. (B) PCA plo showing he exp ession di e ences among oli e ui de eloping samples. Samples collec ed in iplica e: T0: lowe s a ull bloom, T1: ui s a 15 days a e ull blooming (AFB), T2: ui s a 1 mon h AFB, T3: ui s a 2 mon hs AFB, T4: ui s a 3 mon hs AFB, T5: ui s a 4 mon hs AFB, T6: ui s a 5 mon hs AFB, and T7: ui s a 6 mon hs AFB. Thus, he lowe ing s age (T0) was conside ed he e e ence ime o he analysis o he di e en ial gene exp ession h oughou he ui de elopmen om 15 days o 6 mon hs AFB (T1–T7) (Figu e 2). The esul s poin ed ou a p edominance o ep essed genes (Down) along he en i e oli e ui de elopmen p ocess, which means ha mo e genes a e o e exp essed in he lowe ing s age. Likewise, he numbe o ep essed genes in ui s inc eases h oughou ui de elopmen , showing he highes alue when he ui is ully ma u e a T7 (6 mon hs AFB). The numbe o o e exp essed genes in ui anged om 539 a 15 days a e lowe ing (T1) o 932 one mon h a e lowe ing (T2). O e all, 13,486 di e en ially exp essed genes we e iden i ied wi h a p-adj alue < 0.01 and a old change highe han 4. O hose, 2,160 genes we e o e exp essed a any ime du ing ui de elopmen compa ed o he lowe ing s age. The exp ession p o ile o hose genes o e exp essed a some ime wi h espec o he lowe ing s age and wi h an exp ession alue (log2CPM) highe han 0 was analyzed wi h DPGP 0.1 so wa e [29]. Genes o e exp essed a any s age o ui de elopmen ecei ed special a en ion because o hei po en ial ole in in e es ing biological p ocesses in oli e ui . In o al, 1755 genes we e ga he ed in o 37 clus e s acco ding o hei exp ession end along he ui de elopmen (Figu e S1). Howe e , clus e s showing simila ends we e eo ganized in o eigh clus e s (A–H), excluding hose clus e s showing a andom exp ession pa e n du ing ui de elopmen (clus e s 12, 33, 36, and 37 om DPGP). Genes included in each clus e , along wi h hei anno a ion, a e a ailable in he Supplemen a y In o ma ion (Table S2). The eigh clus e s e ealed se e al GO e ms ela ed o biological p ocesses cha ac e is ic o ui de elopmen (Table S3). In he ollowing clus e desc ip ion, only he 20 mos ep esen a i e p ocesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.8 will be p esen ed. Clus e A (Figu e 3) co esponds o clus e 35 ob ained in DPGP and includes 15 o e - exp essed genes a T1, i.e., 15 days AFB. In e es ingly, he mos en iched e m in his clus e is ela ed o he syn hesis o pacli axel (GO:0042617), a di e penoid no ed o i s an icance p ope ies [ 30 ]. This compound has no been p e iously desc ibed in oli e since e penes ha e been s udied in ad anced s ages o ui de elopmen , ep esen ing a mino i y pe cen age among he ola ile compounds, and hei con en dec eases as he ui ipens [ 31 – 33 ]. Howe e , he e penoids s udied in oli e ui a e hose coming om In . J. Mol. Sci. 2024,25, 11150 4 o 21 he phenylp opanoid pa hway, which ac s o he de imen o he pacli axel syn hesis pa hway [30]. In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 o 23 Figu e 2. Diffe en ially exp essed genes h oughou ui de elopmen owing o he lowe ing s age. T0: lowe s a ull bloom, T1: 15 days a e ull blooming (AFB), T2: 1 mon h AFB, T3: 2 mon hs AFB, T4: 3 mon hs AFB, T5: 4 mon hs AFB, T6: 5 mon hs AFB, and T7: 6 mon hs AFB. Clus e A (Figu e 3) co esponds o clus e 35 ob ained in DPGP and includes 15 o e exp essed genes a T1, i.e., 15 days AFB. In e es ingly, he mos en iched e m in his clus e is ela ed o he syn hesis o pacli axel (GO:0042617), a di e penoid no ed o i s an icance p ope ies [30]. This compound has no been p e iously desc ibed in oli e since e penes ha e been s udied in ad anced s ages o ui de elopmen , ep esen ing a mino i y pe cen age among he ola ile compounds, and hei con en dec eases as he ui ipens [31–33]. Howe e , he e penoids s udied in oli e ui a e hose coming om he phenylp opanoid pa hway, which ac s o he de imen o he pacli axel syn hesis pa hway [30]. The unc ional en ichmen analysis also e ealed genes ela ed o he ho mone e- sponse, such as auxin, gibbe ellin, jasmonic acid, e hylene, and salicylic acid, which a e e y impo an in plan g ow h and de elopmen . Two GO e ms ela ed o he syn hesis o indole-3-ace ic acid (GO:0103075, GO:0010279), he main auxin in plan s, and wo GO e ms ela ed o gibbe ellin (GO:0045544, GO:0009739) we e de ec ed. Bo h ho mones play a c ucial ole in he ui se and in cell di ision in he ea ly s ages o ui de elopmen [3,34]. Fo ins ance, in oma o and melon, an o e exp ession o auxin syn hesis- ela ed genes has been obse ed in he ea ly s ages o ui de elopmen , coinciding wi h an ac- cumula ion o indole-3-ace ic acid, dec easing as he ipening p ocess p og esses [35,36]. Likewise, N,N-dime hylaniline monooxygenase (GO:0004499) is a la in monooxygenase, as well as indole-3-py u a e monooxygenase (GO:0103075). Bo h compounds in luence he na u al syn hesis o gibbe ellin and auxins [36]. Among o he s, en iched e ms ela ed o mic onu ien s equi ed o ui de elopmen , such as i on and manganese, also ap- pea ed in clus e A [37]. Figu e 2. Di e en ially exp essed genes h oughou ui de elopmen owing o he lowe ing s age. T0: lowe s a ull bloom, T1: 15 days a e ull blooming (AFB), T2: 1 mon h AFB, T3: 2 mon hs AFB, T4: 3 mon hs AFB, T5: 4 mon hs AFB, T6: 5 mon hs AFB, and T7: 6 mon hs AFB. In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 5 o 23 Figu e 3. Clus e A. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p o- cesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. The second clus e (Figu e 4) was he esul o me ging clus e s 20 and 34 gene a ed by he DPGP algo i hm (Figu e S1), including a o al o 118 o e exp essed genes a T2 (1 mon h AFB). En ichmen analysis e ealed GO e ms ela ed o acyl–CoA biosyn hesis (GO:0046949) and medium-chain a y acid–CoA ligase ac i i y (GO:0031956), which a e ela ed o lipid biosyn hesis. A T2, oli e ui s we e ac i ely g owing, which is e lec ed in he en ichmen o g ow h- ela ed GO e ms (GO:0046622, GO:0040009, GO:0035265, GO:0048437) and he close e ms GO:0020037 and GO:0044550. Genes wi h he e m GO:0020037 (heme-binding) belong o he cy och ome P450 amily, and o he s a e anno- a ed as pe oxides (Table S2). Speci ically, pe oxidases ha e been pa icula ly induced du ing he ea ly s age o oli e ui g ow h [38]. The genes en iching he e m GO:0048437 a e genes anno a ed as CYP78A5, a cy och ome P450 p o ein, which ha e been shown o s imula e cell p oli e a ion and p omo e seed g ow h [39]. A his ea ly s age o ui de elopmen , genes in ol ed in cellulose syn hesis and cell wall biogenesis (GO:0010330, GO:0016759, GO:0016760, GO:009832, GO:009833, and GO:009834) a e also p ominen , suppo ing he ui g ow h. In addi ion, en iched e ms ela ed o suc ose me abolism appea ed (GO:0005985 and GO:0016157), which is con- sis en wi h p e ious s udies ha also obse ed o e exp ession o genes encoding en- zymes ela ed o s a ch and suc ose syn hesis a 30 days AFB [2]. This makes sense, as oli e mesoca p cells equi e suga s o syn hesize oil [40]. In addi ion, he en ichmen anal- ysis e ealed genes ela ed o he hea -s ess esponse (GO:0070370 and GO:0005528), which could affec ui de elopmen , subsequen ui yield, and oil quali y [41,42]. Figu e 3. Clus e A. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p ocesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. In . J. Mol. Sci. 2024,25, 11150 5 o 21 The unc ional en ichmen analysis also e ealed genes ela ed o he ho mone e- sponse, such as auxin, gibbe ellin, jasmonic acid, e hylene, and salicylic acid, which a e e y impo an in plan g ow h and de elopmen . Two GO e ms ela ed o he syn hesis o indole-3-ace ic acid (GO:0103075, GO:0010279), he main auxin in plan s, and wo GO e ms ela ed o gibbe ellin (GO:0045544, GO:0009739) we e de ec ed. Bo h ho mones play a c ucial ole in he ui se and in cell di ision in he ea ly s ages o ui de elopmen [ 3 , 34 ]. Fo ins ance, in oma o and melon, an o e exp ession o auxin syn hesis- ela ed genes has been obse ed in he ea ly s ages o ui de elopmen , coinciding wi h an accumula ion o indole-3-ace ic acid, dec easing as he ipening p ocess p og esses [ 35 , 36 ]. Likewise, N,N-dime hylaniline monooxygenase (GO:0004499) is a la in monooxygenase, as well as indole-3-py u a e monooxygenase (GO:0103075). Bo h compounds in luence he na - u al syn hesis o gibbe ellin and auxins [ 36 ]. Among o he s, en iched e ms ela ed o mic onu ien s equi ed o ui de elopmen , such as i on and manganese, also appea ed in clus e A [37]. The second clus e (Figu e 4) was he esul o me ging clus e s 20 and 34 gene a ed by he DPGP algo i hm (Figu e S1), including a o al o 118 o e exp essed genes a T2 (1 mon h AFB). En ichmen analysis e ealed GO e ms ela ed o acyl–CoA biosyn hesis (GO:0046949) and medium-chain a y acid–CoA ligase ac i i y (GO:0031956), which a e ela ed o lipid biosyn hesis. A T2, oli e ui s we e ac i ely g owing, which is e lec ed in he en ichmen o g ow h- ela ed GO e ms (GO:0046622, GO:0040009, GO:0035265, GO:0048437) and he close e ms GO:0020037 and GO:0044550. Genes wi h he e m GO:0020037 (heme-binding) belong o he cy och ome P450 amily, and o he s a e anno a ed as pe oxides (Table S2). Speci ically, pe oxidases ha e been pa icula ly induced du ing he ea ly s age o oli e ui g ow h [ 38 ]. The genes en iching he e m GO:0048437 a e genes anno a ed as CYP78A5, a cy och ome P450 p o ein, which ha e been shown o s imula e cell p oli e a ion and p omo e seed g ow h [39]. In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 o 23 Figu e 4. Clus e B. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p o- cesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. Clus e C (Figu e 5) consis s o 407 genes o e exp essed du ing he ea ly s ages o ui de elopmen , including T1, T2, and T3. This pe iod encompasses he p ocess o cell di ision and expansion un il pi ha dening (a T3, he pi was ha dened). In his case, some biological p ocesses had al eady been highligh ed in clus e s A and B, as hey a e ela ed o cell di ision and expansion (GO:0009833, GO:0009834, GO:0009505, and GO:0016759). In addi ion o cellulose, his clus e includes he e m GO:0045492 o xylan biosyn hesis, which is also ela ed o cell wall biogenesis. Xylan is a hemicellulosic polysaccha ide abun- dan in he cell wall o oli e ui pulp [43]. The p esence o genes ela ed o cellulose and xylan compounds in clus e s A and B suppo s he hypo hesis ha hose compounds a e mo e abundan in un ipe ui and a e deg aded along he ipening p ocess [44]. Mo eo e , cell wall polysaccha ides a e he main ac o s esponsible o ui so ening du ing ipening [43,44], and emodeling o hese compounds along wi h he ligni ica ion could be in ol ed in ui abscission [45]. This ac may explain he appea ance o he lignin p ocess (GO:0046274 and GO:009809) and ui dehiscence (GO:0010047) oge he in ha g oup. The ea ly ui d op may be due o compe i ion o he ui s o nu ien s [46]. In he ‘Picual’ cul i a , he p ema u e ui d op has been p e iously obse ed a 217 days AFB and is cha ac e ized by an inc ease in he exp ession o genes om he MYB and bZIP (basic leucine zippe ) amilies, as well as a highe accumula ion o s e ols [10]. Despi e he occu ence o p ema u e ui d ops, ui d ops a e gene ically p og ammed o occu when he ui is ma u e. Figu e 4. Clus e B. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p ocesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. In . J. Mol. Sci. 2024,25, 11150 6 o 21 A his ea ly s age o ui de elopmen , genes in ol ed in cellulose syn hesis and cell wall biogenesis (GO:0010330, GO:0016759, GO:0016760, GO:009832, GO:009833, and GO:009834) a e also p ominen , suppo ing he ui g ow h. In addi ion, en iched e ms ela ed o suc ose me abolism appea ed (GO:0005985 and GO:0016157), which is consis en wi h p e ious s udies ha also obse ed o e exp ession o genes encoding enzymes ela ed o s a ch and suc ose syn hesis a 30 days AFB [ 2 ]. This makes sense, as oli e mesoca p cells equi e suga s o syn hesize oil [ 40 ]. In addi ion, he en ichmen analysis e ealed genes ela ed o he hea -s ess esponse (GO:0070370 and GO:0005528), which could a ec ui de elopmen , subsequen ui yield, and oil quali y [41,42]. Clus e C (Figu e 5) consis s o 407 genes o e exp essed du ing he ea ly s ages o ui de elopmen , including T1, T2, and T3. This pe iod encompasses he p ocess o cell di ision and expansion un il pi ha dening (a T3, he pi was ha dened). In his case, some biological p ocesses had al eady been highligh ed in clus e s A and B, as hey a e ela ed o cell di ision and expansion (GO:0009833, GO:0009834, GO:0009505, and GO:0016759). In addi ion o cellulose, his clus e includes he e m GO:0045492 o xylan biosyn hesis, which is also ela ed o cell wall biogenesis. Xylan is a hemicellulosic polysaccha ide abundan in he cell wall o oli e ui pulp [43]. In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 o 23 Figu e 5. Clus e C. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p o- cesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. The mos en iched biological p ocess in clus e C was ela ed o s e oid biosyn hesis (GO:0047787), which a e heal h-p omo ing compounds. In p e ious s udies on ui s o ‘Co a ina’ and ‘Tendellone’ oli e cul i a s, ansc ip s ela ed o s e oid syn hesis we e also de ec ed in he ea ly s ages o ui de elopmen [47]. Gene ally, his g oup includes a ious oxido educ ases (GO:0047787, GO:0035671, GO:0016722, GO:0016722, GO:0016491, and GO:0055114), which can ac a diffe en le els o he phenylp opanoid pa hway. Speci ically, wo o hem (GO:0016722 and GO:0052716) a e co ela ed wi h lig- nin biosyn hesis (GO:0009809 and GO:0046274). Lignin deposi ion and cellulose biosyn- hesis a e essen ial o pi ha dening [4], which occu ed be ween T2 and T3 in his s udy. In his ea ly s age o ui de elopmen , genes in ol ed in he syn hesis o isop enoids and e penoids (GO:0008299 and GO:0016114) a e also p ominen . The esul o g ouping 349 genes ha a e o e exp essed be ween T1 and T5 is he clus e D (Figu e 6), which also co esponds o he union o clus e s 9, 11, 16, 21, 24, 25, and 30 gene a ed by DPGP (Figu e S1). This g oup is mainly cha ac e ized by a peak o exp ession be ween T2 and T3 when pi ha dening akes place. These esul s a e in line wi h p e ious wo k con i ming ha p og ammed cell dea h (GO:0012502) and seconda y hickening o he cell wall a e cha ac e is ic o s one cell o ma ion in d upes, as well as he deposi ion o cellulose and lignin [4,48]. In his line, genes ela ed o he phenylp o- panoid pa hway and lignin syn hesis (GO:1903086 and GO:2000762) also s ood ou in clus- e D (Figu e 6). Al hough hey could also be ela ed o cell dea h p ocesses, his clus e includes genes in ol ed in au ophagy (GO:0010508) and esponse o high ligh in ensi y (GO:0009644). Howe e , i is mo e likely ha hese mechanisms a e associa ed wi h he esponse o abio ic s ess. Figu e 5. Clus e C. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p ocesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. The p esence o genes ela ed o cellulose and xylan compounds in clus e s A and B suppo s he hypo hesis ha hose compounds a e mo e abundan in un ipe ui and a e deg aded along he ipening p ocess [ 44 ]. Mo eo e , cell wall polysaccha ides a e he main ac o s esponsible o ui so ening du ing ipening [ 43 , 44 ], and emodeling o hese compounds along wi h he ligni ica ion could be in ol ed in ui abscission [ 45 ]. This ac may explain he appea ance o he lignin p ocess (GO:0046274 and GO:009809) and ui dehiscence (GO:0010047) oge he in ha g oup. The ea ly ui d op may be due o compe i ion o he ui s o nu ien s [ 46 ]. In he ‘Picual’ cul i a , he p ema u e ui d op has been p e iously obse ed a 217 days AFB and is cha ac e ized by an inc ease in he In . J. Mol. Sci. 2024,25, 11150 7 o 21 exp ession o genes om he MYB and bZIP (basic leucine zippe ) amilies, as well as a highe accumula ion o s e ols [ 10 ]. Despi e he occu ence o p ema u e ui d ops, ui d ops a e gene ically p og ammed o occu when he ui is ma u e. The mos en iched biological p ocess in clus e C was ela ed o s e oid biosyn hesis (GO:0047787), which a e heal h-p omo ing compounds. In p e ious s udies on ui s o ‘Co a ina’ and ‘Tendellone’ oli e cul i a s, ansc ip s ela ed o s e oid syn hesis we e also de ec ed in he ea ly s ages o ui de elopmen [ 47 ]. Gene ally, his g oup includes a ious oxido educ ases (GO:0047787, GO:0035671, GO:0016722, GO:0016722, GO:0016491, and GO:0055114), which can ac a di e en le els o he phenylp opanoid pa hway. Speci - ically, wo o hem (GO:0016722 and GO:0052716) a e co ela ed wi h lignin biosyn hesis (GO:0009809 and GO:0046274). Lignin deposi ion and cellulose biosyn hesis a e essen- ial o pi ha dening [ 4 ], which occu ed be ween T2 and T3 in his s udy. In his ea ly s age o ui de elopmen , genes in ol ed in he syn hesis o isop enoids and e penoids (GO:0008299 and GO:0016114) a e also p ominen . The esul o g ouping 349 genes ha a e o e exp essed be ween T1 and T5 is he clus e D (Figu e 6), which also co esponds o he union o clus e s 9, 11, 16, 21, 24, 25, and 30 gene a ed by DPGP (Figu e S1). This g oup is mainly cha ac e ized by a peak o exp ession be ween T2 and T3 when pi ha dening akes place. These esul s a e in line wi h p e ious wo k con i ming ha p og ammed cell dea h (GO:0012502) and seconda y hickening o he cell wall a e cha ac e is ic o s one cell o ma ion in d upes, as well as he deposi ion o cellulose and lignin [ 4 , 48 ]. In his line, genes ela ed o he phenylp opanoid pa hway and lignin syn hesis (GO:1903086 and GO:2000762) also s ood ou in clus e D (Figu e 6). Al hough hey could also be ela ed o cell dea h p ocesses, his clus e includes genes in ol ed in au ophagy (GO:0010508) and esponse o high ligh in ensi y (GO:0009644). Howe e , i is mo e likely ha hese mechanisms a e associa ed wi h he esponse o abio ic s ess. In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 o 23 Figu e 6. Clus e D. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p o- cesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. In hese in e media e s ages, ui de elopmen is also egula ed h ough genes in- ol ed in he conjuga ion o indole-3-ace ic acid (GO:0033473). This auxin, oge he wi h gibbe ellins (GO:0009740), is impo an o oli e ui de elopmen [13], suppo ing p e- ious indings in diffe en plan species in which bo h ho mones ha e an impo an ole in he egula ion o cell di ision and expansion in ui s [34]. In oli es, gibbe ellins ha e been sugges ed o egula e ui size and he p og ession o he ipening p ocess [13]. Be ween T1 and T5, oli e ui s emained g een, so hey had ac i e chlo oplas s pe - o ming pho osyn hesis. This ac is suppo ed by he en iched biological p ocess ela ed o he Ribulose–bisphospha e ca boxylase ac i i y (GO:0016984), which can be in ol ed in ui pho osyn hesis, p o iding addi ional suga s and o ganic compounds necessa y o be used o ui de elopmen and a y acid biosyn hesis in he oli e mesoca p [40,49]. Rela ed o his enzyma ic ac i i y, genes in ol ed in he biological p ocess GO:0019253 ( educ i e pen ose–phospha e cycle) ha e also been de ec ed. Clus e D is also en iched in CCAAT-binding ac o s (GO:0016602), which a e an- sc ip ion ac o s in ol ed in se e al p ocesses, highligh ing hei ole in de e mining he lowe ing ime, seed ma u a ion, and a y acid biosyn hesis [50,51]. These ansc ip ion ac o s in e ac wi h gibbe ellic acid and abscisic acid, also playing impo an oles in la- onoid biosyn hesis, pho omo phogenesis, pho osyn hesis, esponse o s ess, and ep o- duc i e de elopmen [50]. Figu e 6. Clus e D. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay In . J. Mol. Sci. 2024,25, 11150 8 o 21 lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p ocesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. In hese in e media e s ages, ui de elopmen is also egula ed h ough genes in- ol ed in he conjuga ion o indole-3-ace ic acid (GO:0033473). This auxin, oge he wi h gibbe ellins (GO:0009740), is impo an o oli e ui de elopmen [ 13 ], suppo ing p e i- ous indings in di e en plan species in which bo h ho mones ha e an impo an ole in he egula ion o cell di ision and expansion in ui s [ 34 ]. In oli es, gibbe ellins ha e been sugges ed o egula e ui size and he p og ession o he ipening p ocess [13]. Be ween T1 and T5, oli e ui s emained g een, so hey had ac i e chlo oplas s pe o ming pho osyn hesis. This ac is suppo ed by he en iched biological p ocess ela ed o he Ribulose–bisphospha e ca boxylase ac i i y (GO:0016984), which can be in ol ed in ui pho osyn hesis, p o iding addi ional suga s and o ganic compounds necessa y o be used o ui de elopmen and a y acid biosyn hesis in he oli e mesoca p [ 40 , 49 ]. Rela ed o his enzyma ic ac i i y, genes in ol ed in he biological p ocess GO:0019253 ( educ i e pen ose–phospha e cycle) ha e also been de ec ed. Clus e D is also en iched in CCAAT-binding ac o s (GO:0016602), which a e ansc ip- ion ac o s in ol ed in se e al p ocesses, highligh ing hei ole in de e mining he lowe ing ime, seed ma u a ion, and a y acid biosyn hesis [ 50 , 51 ]. These ansc ip ion ac o s in e ac wi h gibbe ellic acid and abscisic acid, also playing impo an oles in la onoid biosyn hesis, pho omo phogenesis, pho osyn hesis, esponse o s ess, and ep oduc i e de elopmen [ 50 ]. Clus e E includes 192 genes ha a e o e exp essed ela i e o he lowe ing s age, wi h an upwa d end as he de elopmen p ocess p og esses (Figu e 7). This clus e is he esul o joining clus e s 2, 6, 15, 18, and 22 gene a ed by DPGP (Figu e S1). In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 9 o 23 Clus e E includes 192 genes ha a e o e exp essed ela i e o he lowe ing s age, wi h an upwa d end as he de elopmen p ocess p og esses (Figu e 7). This clus e is he esul o joining clus e s 2, 6, 15, 18, and 22 gene a ed by DPGP (Figu e S1). Genes in ol ed in lipid oxida ion a e no ed in clus e E, bu hey a e genes anno a ed as lipoxygenase 2 (LOX2) (Table S2), which p oduce mainly 13-hyd ope oxides om lin- oleic and linolenic acids [52]. P e ious s udies abou lipoxygenase ac i i y in ‘Picual’ oli e ui s also desc ibed he up egula ion o LOX2 du ing ea ly ui de elopmen [3]. Lipox- ygenase enzymes a e esponsible o ola ile compounds which con ibu e o oli e oil a oma and can also ac as esis ance mechanisms [53]. Fo his eason, he biological p o- cess o esponse o he bi o es (GO:0080027) is also in his clus e . Figu e 7. Clus e E. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p o- cesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. This clus e has a b oad ep esen a ion o genes in ol ed in he i s s eps o he a y acid syn hesis pa hway, s a ing wi h he pho osyn he ic p ocess. The pho osys em II (GO:0009654) p o ides NADP which is educed o NADPH (GO:0070995) necessa y o a y acid biosyn hesis. Hence, he glycoly ic p ocess (GO:0006096) in ol es he b eak- down o ca bohyd a es in o py u a e wi h he concomi an educ ion in NADP o NADPH (GO:0006090) [54]. Py u a e is equi ed o ace yl–CoA biosyn hesis h ough he ac i i y o he py u a e dehyd ogenase complex (GO:0045254) [8,49]. The dihy- d olipoyllysine- esidue ace yl ans e ase (GO:0004742) is he E2 componen o he py- u a e dehyd ogenase complex (GO:0045254) and is essen ial o he oxida i e deca box- yla ion o py u a e o ace yl–CoA [55]. These wo me aboli es a e conside ed he main p ecu so s o a y acid biosyn hesis. Addi ionally, he enoyl-[acyl-ca ie -p o ein] educ- ase (GO:0016631) also uses NADPH as co- ac o o enoyl–ACP educ ion esul ing in Figu e 7. Clus e E. (A) Gene exp ession p o ile. Blue line ep esen s he mean alue o exp ession o he o al genes in he g oup. Blue shadow ep esen s he s anda d e o o gene exp ession. G ay lines ep esen he exp ession o indi idual genes. The ed ho izon al line ep esen s he h eshold sepa a ing posi i e om nega i e exp ession le els. (B) The 20 mos ep esen a i e biological p ocesses acco ding o he FDR and old en ichmen alues ob ained in ShinyGO 0.80. In . J. Mol. Sci. 2024,25, 11150 9 o 21 Genes in ol ed in lipid oxida ion a e no ed in clus e E, bu hey a e genes anno a ed as lipoxygenase 2 (LOX2) (Table S2), which p oduce mainly 13-hyd ope oxides om linoleic and linolenic acids [ 52 ]. P e ious s udies abou lipoxygenase ac i i y in ‘Picual’ oli e ui s also desc ibed he up egula ion o LOX2 du ing ea ly ui de elopmen [ 3 ]. Lipoxygenase enzymes a e esponsible o ola ile compounds which con ibu e o oli e oil a oma and can also ac as esis ance mechanisms [ 53 ]. Fo his eason, he biological p ocess o esponse o he bi o es (GO:0080027) is also in his clus e . This clus e has a b oad ep esen a ion o genes in ol ed in he i s s eps o he a y acid syn hesis pa hway, s a ing wi h he pho osyn he ic p ocess. The pho osys em II (GO:0009654) p o ides NADP which is educed o NADPH (GO:0070995) necessa y o a y acid biosyn hesis. Hence, he glycoly ic p ocess (GO:0006096) in ol es he b eakdown o ca bohyd a es in o py u a e wi h he concomi an educ ion in NADP o NADPH (GO:0006090) [ 54 ]. Py u a e is equi ed o ace yl–CoA biosyn hesis h ough he ac i i y o he py u a e dehyd ogenase complex (GO:0045254) [ 8 , 49 ]. The dihyd olipoyllysine- esidue ace yl ans e ase (GO:0004742) is he E2 componen o he py u a e dehyd ogenase complex (GO:0045254) and is essen ial o he oxida i e deca boxyla ion o py u a e o ace yl–CoA [ 55 ]. These wo me aboli es a e conside ed he main p ecu so s o a y acid biosyn hesis. Addi ionally, he enoyl-[acyl-ca ie -p o ein] educ ase (GO:0016631) also uses NADPH as co- ac o o enoyl–ACP educ ion esul ing in acyl–ACP [ 54 , 56 ]. This enzyme belongs o he enzyma ic complex a y acid syn hase (FAS), and i has been cha ac e ized in oli e ui in p e ious s udies, showing a simila exp ession pa e n [ 57 ]. P o eomic s udies desc ibed a peak o accumula ion a ound 110 days AFB [49]. Clus e F (Figu e 8) includes 54 genes mainly exp essed be ween T3 and T6, ha is, om pi ha dening o u ning pu ple. This clus e co esponds o clus e 17 gene a ed by DPGP (Figu e S1). Acco ding o he en ichmen analysis, he mos cha ac e is ic biological p ocesses a e hose ela ed o malona e me abolism. Speci ically, he malonyl–CoA syn- he ase is he enzyme equi ed o con e ing ee malona e in o malonyl–CoA (GO:0090409 and GO:009410). Go e ms ela ed o he inosi ol we e en iched in his clus e (GO:0019310 and GO:0050113). Inosi ol is a p ecu so o he phy ic acid p esen in he pulp o oli e ui , and i s con en dec eases acco ding o he ipeness [ 58 ]. Addi ionally, phy ic acid is necessa y o phospholipid o ma ion [1,54]. Genes ela ed o he phenylp opanoids pa hway a e also p esen in clus e F due o he ac i i y o he la onol 3-O-glucosyl ans e ase (GO:0047893). Likewise, e ms GO:0102425 and GO:0102360 a e obsole ed e ms synonymous wi h la onol 3-O-glucosyl ans e ase ac i i y (checked a h ps://www.ebi.ac.uk/QuickGO/, accessed on 24 July 2024). High ac i i y o he la onol 3-O-glucosyl ans e ase enzyme in he ui ’s ea ly s ages has been obse ed in p e ious s udies in which his ac i i y concu ed wi h a highe concen a ion o la onoids [ 2 , 59 ]. In his clus e , he di e en ial exp ession o genes in ol ed in la onoid biosyn hesis could be esponsible o he ui e aison [2,49]. Clus e F has he pa icula i y o showing se e al biological p ocesses ela ed o human heal h (GO:0003228, GO:0060297, GO:0007519, GO:0001947, GO:0048702, GO:0010830, GO:0072358). Genes included in hese biological p ocesses a e anno a ed as RNA-binding p o eins (Table S2) ha play a key pos - ansc ip ional ole in gene exp ession. In his case, genes speci ically coding o bm38 p o ein, con aining an RRM domain, ha e been s udied in animals, bu hei ole is unknown in plan s. None heless, se e al genes encoding o p o eins wi h he RRM domain o RNA ecogni ion ha e been desc ibed in plan s ela ed o he s ess esponse and he lowe ing ime egula ion and a ec ing he ui ipening p ocess [60]. Clus e G (Figu e 9) encompasses genes wi h a simila exp ession p o ile o clus e s E and F (Figu es 7and 8, espec i ely). The a e age exp ession o hese genes s a s upon pi ha dening (simila o clus e F) and ollows an inc easing end as ui ipening p og esses (simila o clus e E). In . J. Mol. Sci. 2024,25, 11150 16 o 21 In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 18 o 23 Figu e 13. Exp ession o coding genes o plas idial memb ane-bound FAD enzymes du ing oli e ui de elopmen . FAD6, as well as FAD2, could con ibu e o he linoleic acid con en in oli e ui , al hough o a lesse ex en [15,18,21,69]. The wo genes coding o FAD6 showed simila exp ession pa e ns dec easing du ing ui de elopmen (Figu e 13C), which is in line wi h p e ious s udies using qRT-PCR in he cul i a s ‘Picual’ and ‘A bequina’ [21], ‘Lec- cino’ and ‘Co a ina’ [18], and ‘Klon-14’ and ‘Abou Kanani’ [15]. Howe e , he exp ession pa e n o FAD6 genes seems o be cul i a -dependen because o he a iabili y obse ed du ing ui de elopmen in hese oli e cul i a s. Addi ionally, OeFAD7-1 and OeFAD7-2 ha e a c i ical ole in he linolenic acid con- en in oli e ui mesoca p [25]. The ole o OeFAD7-1 has been cha ac e ized in he ‘Ko o- neiki’ [26], ‘Picual’, and ‘A bequina’ cul i a s [25], whe eas OeFAD7-2 has been alida ed in he ‘Picual’ and ‘A bequina’ cul i a s [25]. In his wo k, single genes we e iden i ied coding o each FAD7 iso o m, and hei ansc ip s dec eased du ing ui de elopmen mainly in ea ly s ages (Figu e 13D,E). On he con a y, he exp ession pa e n in he ‘Pic- ual’ and ‘A bequina’ [25] and ‘Klon-14’ and ‘Abou Kanani’ cul i a s [15] showed a con- s an ansc ip le el o bo h genes du ing mesoca p de elopmen and ipening. Figu e 13. Exp ession o coding genes o plas idial memb ane-bound FAD enzymes du ing oli e ui de elopmen . FAD4 and FAD5 ha e no been s udied in oli e, possibly because hey a e in ol ed in he a y acid composi ion o plas idial lipids and, he e o e, no ela ed o he a y acid p o ile o he oli e oil. Bo h enzymes a e esponsible o he desa u a ion o palmi ic acid in he sn-2 posi ion o plas idial lipids, wi h FAD4 ac ing on phospha idylglyce ol o syn hesize ans-palmi oleic acid [ 27 ], while FAD5 ac s on monogalac osyldiacylglyce ol o yield del a-7 palmi oleic acid [ 28 ]. Th ee genes coding o FAD4 we e iden i ied in he ‘Picual’ oli e genome (Figu e 13A). These esul s a e in line wi h he p e ious desc ip ion o FAD genes in he ‘Fa ga’ cul i a , while he wild oli e has only wo genes coding o FAD4 [ 9 ]. The exp ession o genes coding o FAD4 was highe in he ea ly s ages o ui de elopmen . Howe e , he exp ession p o ile o Oleu 061Sc 1696g01055 di e ed om he o he wo genes coding o his iso o m (Figu e 13A). Simila esul s we e obse ed o genes coding o FAD5 (Figu e 13B), whe e he exp ession pa e n o Oleu 061Sc 3819g00007 was sligh ly di e en om he o he genes coding o he same iso o m in he ‘Picual’ cul i a . Ne e heless, he exp ession o Oleu 061Sc 2722g04011 and Oleu 061Sc 0657g07008 inc eased om lowe (T0) o yellowish ui (T5) and dec eased as he ui ipened. FAD6, as well as FAD2, could con ibu e o he linoleic acid con en in oli e ui , al hough o a lesse ex en [ 15 , 18 , 21 , 69 ]. The wo genes coding o FAD6 showed simila exp ession pa e ns dec easing du ing ui de elopmen (Figu e 13C), which is in line wi h In . J. Mol. Sci. 2024,25, 11150 17 o 21 p e ious s udies using qRT-PCR in he cul i a s ‘Picual’ and ‘A bequina’ [ 21 ], ‘Leccino’ and ‘Co a ina’ [ 18 ], and ‘Klon-14’ and ‘Abou Kanani’ [ 15 ]. Howe e , he exp ession pa e n o FAD6 genes seems o be cul i a -dependen because o he a iabili y obse ed du ing ui de elopmen in hese oli e cul i a s. Addi ionally, OeFAD7-1 and OeFAD7-2 ha e a c i ical ole in he linolenic acid con en in oli e ui mesoca p [ 25 ]. The ole o OeFAD7-1 has been cha ac e ized in he ‘Ko- oneiki’ [ 26 ], ‘Picual’, and ‘A bequina’ cul i a s [ 25 ], whe eas OeFAD7-2 has been alida ed in he ‘Picual’ and ‘A bequina’ cul i a s [ 25 ]. In his wo k, single genes we e iden i ied coding o each FAD7 iso o m, and hei ansc ip s dec eased du ing ui de elopmen mainly in ea ly s ages (Figu e 13D,E). On he con a y, he exp ession pa e n in he ‘Picual’ and ‘A bequina’ [ 25 ] and ‘Klon-14’ and ‘Abou Kanani’ cul i a s [ 15 ] showed a cons an ansc ip le el o bo h genes du ing mesoca p de elopmen and ipening. 3. Ma e ials and Me hods 3.1. RNAseq Da a and T ansc ip omic Analysis RNAseq da a om he biop ojec numbe PRJNA870905, a ailable in NCBI da abase, was used o his s udy. Speci ically, he da a co espond o lowe and ui samples o he oli e cul i a ‘Picual’ collec ed om h ee di e en ees on sou h- acing b anches. Sampling was pe o med a se en di e en imes, om ull blooming (T0: lowe ) o ull ma u i y, including ui a 15 days a e ull blooming (AFB) (T1: emb yo’s g ow h) and mon hly un il 6 mon hs AFB (T2: young d upe, T3: pi ha dened, T4: g een, T5: yellowish, T6: e aison, T7: comple ely pu ple). The samples we e s o ed a − 80 ◦ C un il p ocessing. RNA ex ac ion om ull samples (T0 o T3) o om mesoca p (T4 o T7) was pe - o med using he Spec um™ Plan ki (Me ck KGaA, Da ms ad , Ge many). A e pu i ica- ion, s and-speci ic sequencing was pe o med wi h he NGS Illumina pai -end echnology (150 bp ×2) by he company Sis emas Genómicos S.L. (Pa e na, Valencia, Spain). Fo ansc ip omic analysis, STAR 2.7 [ 70 ] was used o align eads o he e e ence genome o ‘Picual’ [ 65 ], and aligned agmen s we e coun ed wi h ea u eCoun s 2.0.6 [ 71 ]. The di e en ially exp essed genes we e iden i ied by applying EdgeR 4.2 [ 72 ], compa ing any s age o he ui de elopmen p ocess (T1–T7) o he lowe ing s age (T0). To conside a gene di e en ially exp essed, alues o p-adj < 0.01 and old change > 4 o < − 4 we e se . O e exp essed genes we e anno a ed using Sma3s 2 [ 73 ]. A e wa d, he exp ession p o ile o hose genes o e exp essing a any poin AFB (i.e., T1–T7) was analyzed using DPGP 0.1 [ 29 ] by clus e ing hose showing a simila exp ession pa e n. The no malized CPM alues ex ac ed om EdgeR 4.2 we e used as inpu o DPGP 0.1. The clus e s gene a ed by DPGP 0.1 showing simila exp ession p o iles we e eg ouped and plo ed using he ggplo 2 package 3.4.4 in Rs udio 4.2.1. These inal clus e s we e subjec ed o gene en ichmen analysis o biological p ocesses using ShinyGO 0.8 web ool (h p: //bioin o ma ics.sds a e.edu/go/ (accessed on 21 June 2024)) [ 74 ]. Fo his analysis, he o al o di e en ially exp essed genes, bo h up and down, as backg ound, we e conside ed as backg ound. 3.2. Fa y Acid Desa u ase Gene Iden i ica ion In his s udy, special emphasis has been gi en o hose genes in ol ed in a y acid desa u a ion. Fo his pu pose, sequences o genes coding o a y acid desa u ases we e sea ched on he Oli eT eeDB da abase (h ps://genomaoli a .dipujaen.es/db/index. php (accessed on 2 Ap il 2024)). Speci ically, homologous sequences o OepSAD1,Oep- SAD2 and OepSAD3 [ 17 ], OelSAD4 [ 18 ], OepFAD2-1,OepFAD2-2,OepFAD2-3,OepFAD2-4 and OepFAD2-5 [ 20 , 22 ], OepFAD3A and OepFAD3B, [ 25 ]A FAD4 [ 27 ], A FAD5 [ 28 ], Oep- FAD6 [ 23 ], OekFAD7-1 [ 26 ], and OepFAD7-2 [ 25 ] we e sea ched based on hei desc ip ion and alida ion in p e ious wo ks. In . J. Mol. Sci. 2024,25, 11150 18 o 21 4. Conclusions This wo k ep esen s an e o o cha ac e ize he ansc ip ional p o ile h oughou ui de elopmen , including in se e al s ages om lowe ing ( ull blooming) un il ui ipening (6 mon hs AFB). The objec i e is o cha ac e ize he impo ance o hose genes o e exp essed du ing oli e ui de elopmen . In gene al, a decay o gene exp ession has been obse ed h oughou ui de el- opmen . The e o e, he inclusion o samples close in ime o he ea ly s ages o ui de elopmen has p o ided no el in o ma ion abou he p ocesses occu ing a ha ime, which ha e usually been igno ed in mos o he wo k published so a . This wo k has highligh ed eigh clus e s o genes showing di e en exp ession p o iles. A he beginning o ui de elopmen (T1, clus e A), ho mones played an impo an ole, oge he wi h he syn hesis o cellulose as he p ima y componen o he cell wall and he accumula ion o suga compounds be o e pi ha dening (T2, clus e B). Genes in ol ed in he syn hesis o phenolic compounds we e de ec ed om he beginning o ui de elopmen (T2 and T3, clus e C), main aining hei ele ance h oughou he whole p ocess o ui de elopmen . None heless, he highe exp ession o genes in ol ed in he syn hesis o a y acids was obse ed a in e media e s ages (clus e s E and G), e en inc easing as he ui ipens. In he end, when he ui u ns comple ely pu ple (T6–T7, clus e H), cell wall deg ada ion p ocesses appea . Gene exp ession analysis has co obo a ed he complexi y o he a y acid syn hesis p ocess and he a iabili y o genes in ol ed in his p ocess h oughou oli e ui de elop- men . The e o e, a de ailed analysis o alida ed genes coding o he a y acid desa u ase enzymes esponsible o he a y acid composi ion has been ca ied ou . The iden i ica ion o genes coding o SAD,FAD2,FAD3,FAD4,FAD5,FAD6, and FAD7 in he ‘Picual’ genome has imp o ed he genome anno a ion. Mo eo e , he exp ession analysis o hese genes has co obo a ed hei ole in he syn hesis o a y acids in oli e ui s, excep o he genes coding o OeSAD4 and OeFAD2-3, which showed negligible exp ession h oughou he ui de elopmen . Supplemen a y Ma e ials: The suppo ing in o ma ion can be downloaded a : h ps://www.mdpi. com/a icle/10.3390/ijms252011150/s1. Au ho Con ibu ions: Concep ualiza ion, F.L. and J.M.M.-R.; me hodology, F.L. and A.S.; so wa e, A.S., J.G.-M. and M.M.; o mal analysis, A.S. and J.M.M.-R.; w i ing—o iginal d a p epa a ion, A.S. and F.L.; w i ing— e iew and edi ing, A.S., J.G.-M., J.M.M.-R., M.M. and F.L.; supe ision, F.L.; unding acquisi ion, F.L. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This wo k has been inanced by he Spanish Minis y o Science and Inno a ion (MCIN) PID2020-115853RR-C33. Da a A ailabili y S a emen : Da a is con ained wi hin he a icle and Supplemen a y Ma e ials. Acknowledgmen s: The echnical and human suppo p o ided by CICT o he Uni e sidad de Jaén (UJA, MINECO, Jun a de Andalucía, FEDER) is g a e ully acknowledged. Alicia Se ano is g a e ul o he suppo ecei ed in he call o Juan de la Cie a con ac s o he S a e Plan o Scien i ic and Technical Resea ch and Inno a ion 2021–2023, unded by he Eu opean Union-Nex Gene a ionEU. Con lic s o In e es : The au ho s decla e no con lic s o in e es . Re e ences 1. Conde, C.; Del o , S.; Ge ós, H. Physiological, Biochemical and Molecula Changes Occu ing du ing Oli e De elopmen and Ripening. J. Plan Physiol. 2008,165, 1545–1562. [C ossRe ] 2. 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