New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

Jou nal o Expe imen al Bo any, Vol. 67, No. 11 pp. 3523–3536, 2016 doi:10.1093/jxb/e w186 Ad ance Access publica ion 18 May 2016 This pape is a ailable online ee o all access cha ges (see h p://jxb.ox o djou nals.o g/open_access.h ml o u he de ails) RESEARCH PAPER New insigh s in o he pos - ansla ional modi ica ion o mul iple phosphoenolpy u a e ca boxylase isoenzymes by phospho yla ion and monoubiqui ina ion du ing so ghum seed de elopmen and ge mina ion IsabelRuiz-Balles a1, Guille moBaena1, Jacin oGandullo 1, Liqun Wang 2,3, Yi-MinShe2, William Cha lesPlax on4 and C is inaEche a ía1,* 1 Depa amen o de Biología Vege al, Facul ad de Biología, Uni e sidad de Se illa, A da Reina Me cedes nº 6, 41012 Se illa, Spain 2 Shanghai Cen e o Plan S ess Biology, Chinese Academy o Sciences, 3888 Chenhua Road, Shanghai 201602, China 3 S a e Key Labo a o y o C op Gene ics and Ge mplasm Enhancemen , Soybean Resea ch Ins i u e, Nanjing Ag icul u al Uni e si y, Nanjing, Jiangsu 210095, China 4 Depa men o Biology, Queen’s Uni e si y, Kings on, On a io K7L 3N6, Canada * Co espondence: [email p o ec ed] Recei ed 23 Decembe 2015; Accep ed 11 Ap il 2016 Edi o : Ge ha d Leubne , Royal Holloway, Uni e si y o London Abs ac Phosphoenolpy u a e ca boxylase (PEPC; E.C. 4.1.1.31) was cha ac e ized in de eloping and ge mina ing so - ghum seeds, ocusing on he ansc ip and polypep ide abundance o mul iple plan - ype phosphoenolpy u- a e ca boxylase (PTPC) genes, and he pos - ansla ional modi ica ion o each isoenzyme by phospho yla ion e sus monoubiqui ina ion du ing ge mina ion. We obse ed high le els o SbPPC4 (Sb07g014960) ansc ip s du ing ea ly de elopmen (s age I), and ex ensi e ansc ip abundance o SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) h oughou he en i e li e cycle o he seed. Al hough andem mass spec ome y (MS) analysis o immunopu i ied PTPC indica ed ha ou di e en PTPC isoenzymes we e exp essed in he de eloping and ge mina ing seeds, SbPPC3 was he mos abundan isozyme o he de eloping seed, and o he emb yo and he aleu one laye o ge mina ing seeds. In i o phospho yla ion o he di e en PTPC isoenzymes a hei conse ed N- e minal se yl phospho yla ion si e du ing ge mina ion was also es ablished by MS/MS analysis. Fu he mo e, h ee o he ou isoenzymes we e pa ially monoubiqui ina ed, wi h MS/MS pinpoin ing SbPPC2 and SbPPC3 monoubiqui ina ion a he conse ed Lys-630 and Lys-624 esidues, espec i ely. Ou esul s demons a e ha monoubiqui ina ion and phospho yla ion simul aneously occu in i o wi h di e en PTPC isozymes du ing seed ge mina ion. In addi ion, we show ha PTPC monoubiqui ina ion in ge mina ing so ghum seeds always inc eases a s age II (eme gence o he adicle), is main ained du ing he ae obic pe iod o apid cell di ision and ese e mobiliza ion, and emains ela i ely cons an un il s age IV–V when coleop iles ini ia e he o ma ion o he pho- osyn he ic issues. Key wo ds: Ge mina ion, monoubiqui ina ion, phospho yla ion, phosphoenolpy u a e ca boxylase, pos - ansla ional modi ica ions, seed de elopmen , seeds, So ghum bicolo , issue-speci ic gene exp ession. © The Au ho 2016. Published by Ox o d Uni e si y P ess on behal o he Socie y o Expe imen al Biology. Abb e ia ions: BTPC, bac e ial- ype phosphoenolpy u a e ca boxylase; COS, cas o oil seed; DTT, di hio h ei ol; Glc-6-P, glucose-6-phospha e; MALDI TOF MS, ma ix-assis ed lase deso p ion ioniza ion ime-o - ligh mass spec ome y; p110 and p107, 110 and 107-kDa polypep ides, espec i ely; PEP, phosphoenolpy u- a e; PEPC, PEP ca boxylase; PPCK, PEPC-kinase; PTPC, plan - ype phosphoenolpy u a e ca boxylase; PTM, pos - ansla ional modi ica ion. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/3.0/), which pe mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 3524 | Ruiz-Balles a e al. In oduc ion Phosphoenolpy u a e (PEP) ca boxylase (PEPC; EC 4.1.1.31) is an impo an , ighly egula ed, cy osolic me abolic enzyme ha plays a pi o al pho osyn he ic ole in p ima y CO2 ixa ion by C4 and C assulacean acid me abolism plan s, bu also has a a ie y o addi ional unc ions, including oles in seed de elopmen and ge mina ion (O’Lea y e al., 2011a). I ca alyses he i e e sible β-ca boxyla ion o PEP o o m oxaloace a e (OAA) and o hophospha e (Pi) (Cholle e  al., 2006). The plan - ype PEPC (PTPC) genes encode closely ela ed 100–110 kDa polypep ides ha ypically exis as Class-1 PEPC e ame s ha a e pos - ansla ionally egula ed by a combina ion o se e al allos e ic e ec o s, including Glc-6-P (ac i a ion) and L-mala e (inhibi ion), e e sible phospho yla ion (Nimmo, 2003; O’Lea y e  al., 2011a), and monoubiqui ina ion (Uh ig e al., 2008, Shane e al., 2013; Ruiz-Balles a e al., 2014). The me aboli e con ol is in eg a ed wi h he enzyme’s e e sible phospho yla ion a a highly conse ed se ine esidue loca ed nea he N- e minus o i s PTPC subuni s (Cholle e  al., 1996; Eche a ía and Vidal, 2003; O’Lea y e  al., 2011a). Phospho yla ion ac i- a es he enzyme by making i less sensi i e o eedback inhibi ion by mala e. Phospho yla ion is ca alysed by a PTPC-kinase (mo e commonly e e ed o as PEPC-kinase; PPCK), whe eas dephospho yla ion is ca alysed by a p o- ein phospha ase ype-2A (Nimmo, 2000; Izui e  al., 2004; O’Lea y e  al., 2011a). Bo h con ols a e in e ac i e and hus allow a ine modula ion o he enzyme’s kine ic p op- e ies as equi ed by he physiological con ex (Eche a ía and Vidal, 2003). Regula o y PTPC monoubiqui ina ion has been demons a ed in he endospe m o ge mina ing cas o oil seeds (COS) (Uh ig e al., 2008), in de eloping p o eoid oo s o P-de icien ha sh hakea (Shane e al., 2013), and in lily pollen (Igawa e al., 2010). A ecen s udy by ou g oup has shown monoubiqui ina ion o SbPPC3 in so ghum, a ce eal wi h s a ch-s o ing seeds (Ruiz-Balles a e al., 2014). Monoubiqui ina ion is inhibi o y as i esul s in inc eased Km (PEP) alues and enhanced sensi i i y o allos e ic inhibi o s (Uh ig e al., 2008; Shane e al., 2013; Ruiz-Balles a e al., 2014). In COS, PTPC monoubiqui ina ion and phospho yla- ion appea o be mu ually exclusi e, while PTPC polypep- ides a e al e na i ely phospho yla ed o monoubiqui ina ed in de eloping p o eoid oo s o ha sh hakea (Shane e  al., 2013). In addi ion, he elimina ion o pho osyn ha e sup- ply caused by depodding in COS igge s in i o dephos- pho yla ion and subsequen pa ial monoubiqui ina ion o PTPC polypep ides (Uh ig e  al., 2008; O’Lea y e  al., 2011b). P e ious s udies ha e indica ed ha PTPC poly- pep ides become in i o phospho yla ed in de-emb yona ed so ghum seed du ing imbibi ion (i.e. on he basis o a sig- ni ican inc ease in he enzyme’s IC50 o L-mala e and in i o 32P inco po a ion) (Nhi i e al., 2000). In o he s a ch-s o ing ce eal seeds such as ba ley and whea , he phospho yla ion o bo h PTPC polypep ides du ing ge mina ion has also been con i med by immunop ecipi a ion, ollowed by SDS- PAGE and au o adiog aphy o he in i o 32P-labeled PEPC (Osuna e al., 1999; Fe ia e al., 2008). As a as we know, no p e ious s udy has ocused on how hese pos - ansla ional modi ica ions (PTMs) a e combined in he di e en subuni s and isoenzymes o a PEPC’s p o ein amily. Deciphe ing how monoubiqui ina ion and phospho yla ion in luence each PTPC isoenzyme du ing he li e cycle o so ghum seeds was a majo objec i e o he cu en s udy. Bac e ial- ype PEPC (BTPC) genes encode la ge 116– 118 kDa polypep ides exhibi ing low (<40%) sequence iden- i y wi h PTPCs. BTPC lacks he conse ed N- e minal se yl phospho yla ion mo i cha ac e is ic o PTPCs, and in as- cula plan s i appea s o only exis as ca aly ic and egula- o y subuni s o no el Class-2 PEPC he e ome ic complexes composed o igh ly associa ed PTPC and BTPC subuni s (Igawa e al., 2010; O’Lea y e al., 2011a; Pa k e al., 2012). Using wes e n blo analysis and speci ic an i-BTPC-IgGs in c ude ex ac o ba ley seeds, Class-2 PEPC complexes ha e no been iden i ied (Fe ia e al., 2008). This is an in e es ing inding as he BTPC and Class-2 PEPC appea o play an impo an ole du ing COS de elopmen (Uh ig e al., 2008). Assessing, by MS/MS and qRT-PCR, he possible occu ence o BTPC (SbPPC6) and/o Class-2 PEPC du ing he cou se o he li e cycle o so ghum seeds was also an impo an objec i e o hiswo k. So ghum (So ghum bicolo L.Moench) is he wo ld’s i h mos impo an ce eal c op (www. ao.o g) and p o ides ood, eed, ibe , uel, and chemical/bio uel eeds ocks ac oss a ange o en i onmen s and p oduc ion sys ems (K eso ich e  al., 2005). So ghum encodes i e PTPC genes (Pa e son e al., 2009): Sb02g021090 and Sb04g008720 encode house- keeping and inducible SbPPC2 and SbPPC3, espec i ely (Lepiniec e al., 1993), whils Sb07g014960 and Sb03g035090 encode PTPC isoenzymes SbPPC4 and SbPPC5 bu hese ha e unknown unc ions. All o hese a e C3- ype PTPCs. Sb10g021330 encodes C4 pho osyn he ic SbPPC1 subuni s ha a e highly exp essed in so ghum lea es. In addi ion, Sb03g008410 (SbPPC6) encodes a single BTPC (Table1). In his s udy, he biochemical and immunological p op- e ies o PTPCs du ing de elopmen and ge mina ion o s a ch-s o ing so ghum seeds we e u he cha ac e ized. We also elucida ed he ansc ip abundance o PTPC genes, and de e mined subuni composi ion, ela i e abundance, and PTMs o each PTPC isoenzyme du ing ge mina ion. As a as we know, his ep esen s he mos de ailed s udy o da e ha examines bo h phospho yla ion and monoubiqui ina ion o a PTPC p o ein amily in any plan species. Fu he mo e, we show ha enhanced PTPC monoubiqui ina ion always accompanies adical eme gence du ing seed ge mina ion, and ha bo h p ocesses a e accele a ed in pa allel unde ae obic condi ions o ele a ed empe a u e. Ma e ials and me hods Plan ma e ial Ma u e so ghum [So ghum bicolo (L.) Moench, a . PR87G57 and a . PR88Y20, Pionee Hi-B ed, Spain] seeds we e s e ilized and ge mina ed o up o 96 h a 25 o 35ºC as p e iously epo ed (Nhi i PTMs o so ghum seed PEPC isozymes | 3525 e al., 2000). Fo immunological s udies o o analyze PEPC ac i i y, he aleu one laye s, s a chy endospe m (aleu one-endospe m), and emb yos we e ca e ully dissec ed a he indica ed imes pos -imbibi- ion. S e ilized so ghum seeds we e also subme ged in dis illed wa e (hypoxic condi ion) o placed on mois ened il e pape (no moxic condi ion) a 25 oC o 48 and 72 h. Ha es ed issues we e ozen in liquid ni ogen and s o ed a –80ºC un il used. De eloping seeds we e ob ained om so ghum plan s cul i a ed in a g eenhouse unde a 14 h day (25ºC)/10-h nigh (18ºC) cycle. Seeds we e ha es ed a a ious s ages o de elopmen , g ound o a powde unde liquid ni ogen, lyophilized, and s o ed a 4ºC o la e analyses. An ibodies Polyclonal an ibodies agains na i e C4-pho osyn he ic SbPPC1 om so ghum lea es (an i-C4 PTPC) we e p epa ed as desc ibed in Pacqui e al. (1995). An i-C19 and an i-N24 we e aised, espec i ely, agains syn- he ic pep ides co esponding o he C- e minal [(Y) EDTLILTMK GIAAGMQNTG] and he dephospho yla ed N- e minal [ERHHSIDAQLRALAPGKVSEE24(YG)] ends o SbPPC1 (i.e., so ghum C4-pho osyn he ic PTPC) as p e iously desc ibed (Ruiz- Balles a e  al., 2014). An i-COS PTPC was aised agains na i e Class-1 PEPC pu i ied om he endospe m o de eloping cas o oil seeds as desc ibed in T ipodi e al. (2005). Immunohis ochemis y Seeds ge mina ed a 24 h pos -imbibi ion we e ixed in 4% (w/ ) pa a o maldehyde and in 0.25% ( / ) glu a aldehyde dehyd a ed in a g aded se ies o aqueous e hanol solu ions, and embedded in Pa aplas Plus (Pan eac) as desc ibed by González e  al. (1998). Sec ions (15 µm hick) we e p epa ed using a mic o ome (model RM2165, Leica) and placed on poly-L-Lys-coa ed mic oscope slides. A e depa a inizing in xylol and ehyd a ing in dec easing concen- a ions o e hanol, sec ions we e blocked o 30 min in TBS bu e con aining 3% (w/ ) BSA. Then, 500µl o he same solu ion con ain- ing a ini y-pu i ied an i-C4 PTPC (3µg o p o ein) (Pacqui e al., 1995) o p e-immune se um was placed on he samples and incuba ed o e nigh a 4°C. Unbound an ibodies we e emo ed by h ee 10-min washes in TBS. Tissue sec ions we e hen incuba ed wi h alkaline phospha ase-conjuga ed goa an i-( abbi IgG)-IgG (Bio-Rad) o 2 h a 37°C. The eac ion o alkaline phospha ase was de eloped colo ime ically wi h a BCIP/NBT Liquid Subs a e Sys em (Sigma). P o ein ex ac ion F ozen powde (0.4 g) om whole de eloping o ge mina ed so - ghum seeds was g ound in a chilled mo a wi h sand in 1 ml o an ice-cold bu e ha con ained 100 mM T is-HCl (pH 7.5), 10 mM MgCl2, 1 mM EDTA, 20% ( / ) glyce ol, 14 mM β-me cap oe hanol, 1 mM phenylme hylsul onyl luo ide, 10µg ml−1 chymos a in, 10µM leupep in, 50 mM KF, 1 mM Na2MoO4, 1 mM Na3VO4−, and 50 nM mic ocys in-LR. Homogena es we e cen i uged a 17 000 g o 7 min and he supe na an s (0.5 ml) desal ed h ough 3-ml Sephadex G-25 spin columns equilib a ed in ex ac ion bu e (lacking PVPP and PVP) p io o enzyma ic analysis. Elec opho esis and immunoblo ing SDS-PAGE, non-dena u ing PAGE and in-gel PEPC ac i i y s ain- ing, immunoblo ing, and isualiza ion o an igenic polypep ides using an alkaline-phospha ase-conjuga ed seconda y an ibody wi h ch omogenic de ec ion we e pe o med as p e iously desc ibed (Ri oal e al., 2002; T ipodi e al., 2005). Al e na i ely, immuno e- ac i e polypep ides we e de ec ed using a chemiluminescence sys em (Supe Signal Wes Pico Rabbi IgGs; The mo Scien i ic) acco ding o he manu ac u e ’s ins uc ions in a Fuji ilm LAS3000 mini-sys- em. The immuno eac i e polypep ides we e quan i ied ia analysis o he scanned blo s using Mul i-Gauge V3.0 so wa e (Fuji ilm). De e mina ion o PEPC ac i i y and p o ein concen a ion PEPC ac i i y was assayed a 340 nm using a Molecula De ices Spec amax Kine ics Mic opla e eade . Op imized assay condi ions we e: 50 mM HEPES-KOH (pH 8.0), 2.5 mM PEP, 5 mM NaHCO3, 5 mM MgCl2, 0.15 mM NADH, 10% ( / ) glyce ol, 1 mM DTT, and 5 uni s ml−1 po cine hea mala e dehyd ogenase (0.2 ml inal olume). One uni o ac i i y is de ined as he amoun o PEPC ca alysing he p oduc ion o 1µmol o oxaloace a e min−1 a 25ºC. P o ein concen- a ions we e de e mined by he Coomassie Blue G-250 dye-binding me hod using bo ine γ-globulin as he s anda d (T ipodi e al., 2005). Immunop ecipi a ion and immunoa ini y ch oma og aphy Immunop ecipi a ion o PTPC polypep ides om cla i ied seed ex ac s was pe o med using an i-C4 PTPC as p e iously desc ibed (Osuna e al., 1999). Fo immunoa ini y ch oma og aphy, ozen whole 48-h ge mi- na ed so ghum seeds (5 g) we e homogenized (1:2.5; w/ ) using a Poly on PT-3100 homogenize in ice-cold bu e A ha con ained 100 mM HEPES-KOH (pH 7.5), 1 mM EDTA, 1 mM EGTA, 15% ( / ) glyce ol, 5 mM MgCl2,100 mM KCl, 10 mM NaCl, 25 mM NaF, 1mM Na3VO4−, 0.1% (w/ ) poly inylpolypy olidone, 0.1% ( / ) T i on X-100, 2 mM 2,2´-dipy idyl disul ide, and 10 µl ml−1 P o eCEASE-100 (G-BioSciences). Homogena es we e cen i- uged a 4ºC o 10 min a 48 500 g, and he supe na an luid e- cen i uged o 5 min and il e ed h ough a laye o Mi aclo h (Calbiochem). Cla i ied ex ac s we e p e-clea ed a 25ºC by elu ing a 0.5 ml min−1 h ough an AminoLink column (1 × 4 cm) ha had been p e-sa u a ed wi h 1 M T is-HCl (pH 7.4). Unbound p o eins we e immedia ely abso bed a 1 ml min−1 on o an an i-COS PTPC Table1. The so ghum PEPC gene amily. Names used in his wo k o he di e en PEPC genes, he equi alence wi h o he names, and assigned unc ions desc ibed by Lepiniec e al. (1993, 1994), and o hologs es ablished by Pa e son e al. (2009). Sb, So ghum bicolo ; S , So ghum ulga e; Zm, Zea mays; Os O yza sa i a. Name o gene Accession numbe a Named by Lepiniec e al. (1993) and Ruiz-Balles a e al. (2014) Known unc ion O hologs (Pa e son e al., 2009) SbPPC1 Sb10g021330 CP46 C4 PTPC, pho osyn he ic C4 gene X63756 (S PEPC1); NM001111948 (ZmPEPC1) SbPPC2 Sb02g021090 CP28 C3 PTPC, housekeeping gene X59925 (S PEPC4); NM001111968 (ZmPEPC2) SbPPC3 Sb04g008720 CP21 C3 PTPC, oo -inducible gene X65137 (S PEPC3); NM001112033 (ZmPEPC3 SbPPC4 Sb07g014960 - C3 PTPC, unknown so a Os08g0366000 SbPPC5 Sb03g035090 - C3 PTPC, unknown so a Os01g0758300 SbPPC6 Sb03g008410 - BTPC, as Class-2 PEPC Os01g0110700 a NCBI da abase 3526 | Ruiz-Balles a e al. immunoa ini y column (1 × 2 cm; p epa ed as desc ibed by Uh ig e al., 2008) ha had been p e-equilib a ed wi h PBS. Flow- h ough ac ions we e collec ed by elu ing he column a 1 ml min−1 wi h PBS un il A280 dec eased o baseline, and bound p o eins we e elu ed a 0.5 ml min−1 wi h 100 mM Gly-HCl (pH 2.8), and immedia ely neu alized (1-ml ac ions collec ed in o 0.1 ml o unbu e ed 1 M T is). A280 abso bing ac ions we e concen a ed o ~1 mg ml−1 and analyzed o hei polypep ide composi ion by SDS-PAGE and immunoblo ing. Mass spec ome y In he ini ial MALDI-TOF MS analysis, PTPCs a he di e - en s ages o seed de elopmen and ge mina ion we e ex ac ed and pu i ied by immunop ecipi a ion om he cla i ied ex ac s, ollowed by SDS-PAGE o he solubilized immunop ecipi a es (Osuna e al., 1999). P o ein-s aining bands o in e es we e manu- ally excised om mic o-p epa a i e gels using biopsy punches, hen educed, alkyla ed, and diges ed wi h ypsin acco ding o Sechi and Chai (1998). B ie ly, gel bands we e washed wice wi h wa e , sh unk o 15 min wi h 100% ace oni ile, and d ied in a Sa an SpeedVac o 30 min. Then he samples we e educed wi h 10 mM di hioe y h i ol in 25 mM ammonium bica bona e (NH4HCO3) o 30 min a 56ºC and subsequen ly alkyla ed wi h 55 mM iodoace a- mide in 25 mM NH4HCO3 o 15 min in he da k. Finally, samples we e diges ed wi h 12.5 ng µl−1 sequencing g ade ypsin (Roche Molecula Biochemicals) in 25 mM NH4HCO3 (pH 7.6) o e nigh a 37ºC. A e diges ion, 1µl o each sample was spo ed on o a MALDI pla e and allowed o ai -d y a oom empe a u e. Then, 0.4µl o 3 mg ml−1 α-cyano-4-hyd oxy-cinnamic acid as he ma ix (Sigma) in 50% ace oni ile was added on o he spo s and hey we e again allowed o ai -d y a oom empe a u e. Sample analyses we e pe o med on a 4800 Plus P o eomics Analyze MALDI-TOF/TOF mass spec ome e (AB Sciex, To on o, Canada). The ins umen was ope a ed in he posi i e e lec o mode wi h an accele a ing ol age o 20 000 V. Mass spec- a we e calib a ed in e nally using he pep ides o ypsin au olysis. Mass spec ome ic measu emen s yielded a lis o monoiso opic masses o he obse ed pep ides a a signal- o-noise a io g ea e han 10, which we e used o pep ide mapping. Pep ides o in e es we e u he analyzed by MS/MS in o de o assess hei sequences. Sui able p ecu so ions we e selec ed om he MS spec a and b oken down using collision induced dissocia- ion (CID) wi h a mosphe ic ai as he collision gas, a a collision ene gy o 1 kV and in he posi i e ion e lec o mode. The p ecu - so ions we e selec ed wi hin he mass windows o ±4 Da. The non- edundan NCBI da abase (downloaded on 24/2/2011; 13 135 398 sequences) and in-house da abases wi h he so ghum en ies om NCBIn (71 862 sequences) and he so ghum PEPC sequence only we e used o p o ein iden i ica ion, and he mass spec ome ic da a we e sea ched using he MASCOT 2.3 se e h ough he Global P o ein Se e 3.6 (AB Sciex). The sea ch pa ame e s we e se up o no limi a ion in axonomy, ypsin as he clea age enzyme, ca - bamidome hyla ion on cys eine as he ixed modi ica ion, and he oxida ion o me hionine and ubiqui ina ion (Gly-Gly adduc s) o lysine, phospho yla ion o se ine o h eonine as a iable modi i- ca ions. Mass ole ances o single MS and MS/MS we e se up o 80 ppm and 0.3 Da, espec i ely. Fo ypsin diges ion, one missed clea age was allowed. In such analyses, he p obabili y sco es g ea e han he sco e ixed by masco we e conside ed o be signi ican a a P- alue o <0.05. De no o sequencing o he MS/MS spec a o he pep ides was pe o med using he DeNo o ool so wa e (AB Sciex), he e ie ed pep ide sequences we e manually checked and alida ed. Asubsequen homology sea ch o hese sequences was ob ained by BLAST (www.ncbi.nlm.nih.go /BLAST). PTPC PTMs we e analyzed by a high-accu acy and sensi i - i y LC MS/MS. Coomassie blue-s ained polypep ides we e excised om SDS-PAGE gels, in-gel educed, alkyla ed, and diges ed wi h ypsin using s anda d p o ocols (Uh ig e al., 2008). T yp ic pep ides we e ex ac ed using ace oni ile/0.1% i luo oace ic acid (TFA) ( / , 60:40), and d ied using a Cen iVap e ige a ed cen- i ugal concen a o (Labconco Co p.). Pep ides we e econs i- u ed in 4µl o 0.1% ( / ) o mic acid (FA) and iden i ied using wo ins umen a ion me hods: nanoAcqui y ul a pe o mance LC sys ems (Wa e s, Mil o d, MA) coupled wi h a T ipleTOF 5600+ quad upole ime-o - ligh mass spec ome e (AB Sciex, Conco d, ON, Canada) a low-ene gy CID; and an O bi ap Fusion T ib id MS sys em (The mo Fishe Scien i ic Inc.Wa ham, MA) in highe ene gy collision induced dissocia ion (HCD) mode. The sample was apped by a 2G-V/MT T ap symme y C18 column (5μm pa icles, 180μm id × 20 mm leng h) a a low a e o 5μl min–1, and sepa- a ed on a BEH130 C18 analy ical column (1.7μm pa icles, 100µm id ×100 mm leng h) a 300 nl min−1 o 60 min. The mobile phase was se up o a linea g adien om 5–30% sol en B (0.1% FA in ace oni ile), ollowed by 85% sol en B o e 10 min o pep ide elu- ion. T ipleTOF MS and MS/MS scans we e acqui ed wi h a high esolu ion o 30.000, and he m/z egions o he MS su ey scan and MS/MS we e selec ed om m/z 350–1600 and m/z 100–1250, espec i ely, and by da a-dependen scanning o he op wen y ions a mul iply cha ged s a es o 2+, 3+ and 4+. Dynamic exclusion was se o a pe iod o ime o 30 s. Fo compa ison pu poses, simila analyses we e pe o med on he gel-sepa a ed p o eins using he O bi ap Fusion T ib id LC-MS sys em unde high-accu acy MS su ey scan a a esolu ion o 60.000 ollowed by sequen ial HCD scans a he wen y op ions. MS/MS da a om he wo ins u- men s we e sea ched agains he i idiplan ae (g een plan s) p o ein sequences in he NCBI da abase using he Masco Se e ( e sion 2.4.0, Ma ix Science, London, UK). The sea ch pa ame e s we e es ic ed o yp ic pep ides a a maximum o wo missed clea ages. Cys eine ca bamidome hyla ion was designa ed as a ixed modi ica- ion, and deamida ion o aspa agine and glu amine, oxida ion o me hionine, phospho yla ion o se ine/ h eonine/ y osine, ubiqui- ina ion o lysine we e conside ed as a iable modi ica ions. Mass ole ances we e se up o 30 ppm o T ipleTOF 5600 MS ions and 0.05 Da o MS/MS agmen ions, 20 ppm o O bi ap MS and 0.8 Da o he MS/MS agmen s. Pep ide assignmen s we e il e ed by an ion sco e cu o a 15, and he iden i ied MS/MS spec a we e also e i ied manually. RNA Ex ac ion and cDNA syn hesis To al RNA was isola ed om 100 mg o ozen, powde ed hal - emb yona ed ge mina ing seeds o whole de eloping seeds using he IQeasyTM Plan RNA Ex ac ion (In on Bio echnology). Ex ac ed nucleic acids we e DNase ea ed o elimina e genomic DNA. RNA was quan i ied using a NanoD op 2000 spec opho- ome e (The mo Scien i ic). Re e se- ansc ip ion eac ions we e pe o med using 1µg o pu i ied o al RNA, 1µl ImP om-IITM Re e se T ansc ip ase (P omega) and a eac ion bu e con aining 0.5 mM dNTP, 6 mM MgCl2, 20 uni s ecombinan RNasin® ibo- nuclease inhibi o , and 0.5µg oligo(d )15. qRT-PCR Quan i a i e polyme ase chain eac ion (qRT-PCR) was pe - o med in a inal olume o 20µl consis ing o 1µl o he cDNA, 10 µl o SensiFAST SYBR No-ROX ki (Roche) and 15 µM o he gene-speci ic p ime s pai s as ollows: SbPPC3 ( o wa d 5´– TGTTGAACAGTTTCTGGAACCTCTT-3´, e e se 5´-GCTTCA CAAGGGCAAGCCCAAAG-3´), SbPPC2 ( o wa d 5´-CCGCCT CGCAACACCTGAAACA-3´, e e se 5´-ACCGGGAGGTGGAA CCGTGT-3´), SbPPC4 ( o wa d 5´-TGAGCTTCGGGCACAAGC AGATG-3´, e e se 5´-GCTCCAAAGGCTCTAAGAACTGCT C-3´), and 18 S RNA ( o wa d 5´-GGGGAAACTTACCA GGTCCA-3´, e e se 5´-GGATGGCTCCGCATAGCTA-3´). PCR was conduc ed on he MiniOp iconTM Real-Time PCR De ec ion Sys em (Bio ad), and he h eshold cycles (C ) we e de e mined using Op icon Moni o TM analysis so wa e o all ea men s. To PTMs o so ghum seed PEPC isozymes | 3527 no malize he ob ained alues, 18S ibosomal RNA was used as in e nal con ol in each sample. This gene displays a s eady RNA le el ac oss he expe imen al condi ions. S a is ical analysis S a is ical analysis was pe o med using SigmaS a (Sys a So wa e Inc., San José, CA, USA). Da a we e analyzed using he S uden ’s - es o wi h he Mann-Whi ney U es . Means we e conside ed o be signi ican ly di e en a P<0.05. Resul s and discussion PEPC ac i i y, and PTPC in eg i y and cellula localiza ion PEPC was cha ac e ized h oughou he de elopmen , ma u- a ion, and ge mina ion o so ghum seeds, ocusing on he enzyme’s ac i i y, in eg i y, subuni composi ion, and immu- nolocaliza ion. S ages o so ghum seed de elopmen and ge mina ion a e desc ibed in Supplemen a y Fig. S1 (a JXB online); among hese, s age IV o de elopmen ma ks he beginning o seed desicca ion, whils s age II o ge mina ion ma ks he p o usion o he adicle and he comple ion o ge mina ion (Bewley e al., 2013). Du ing seed de elopmen , PEPC ac i i y o desal ed ex ac s inc eased by abou 3- old o each a maximum le el a s age II–III and hen dec eased (Fig.1). Du ing ge mina ion, PEPC ac i i y in he emb yo inc eased o a maximum a 72 o 96 h pos -imbibi ion, while in he aleu one/endospe m i did no change signi ican ly and was 10–15- old lowe han ha o emb yos (Fig.1). In ge mina ing COS, he p110 and p107 PTPC polypep- ides appea o be in i o unca ed by 19 amino acids such ha hei N- e minal phospho yla ion domain is absen (Uh ig e  al., 2008). In con as , non-monoubiqui ina ed p107 and monoubiqui ina ed p110 PTPCs om ge mina - ing so ghum seeds a 48 h pos -imbibi ion we e shown o be in ac and o he e o e con ain he phospho yla ion mo i (Ruiz-Balles a e al., 2014). In his s udy, we demons a e he in eg i y o PTPC polypep ides h oughou he li e cycle o so ghum seeds since he an i-C19 and an i-N24 bo h c oss- eac ed wi h he p110 and p107 (see Supplemen a y Fig. S2). PTPC was also immunolocalized in ge mina ing so ghum seed issues cha ac e ized by high me abolic ac i i ies (epi- co yl, adicle, aleu one laye , scu ellum, scu ellum’s epi he- lium) (Fig.2). Immunohis ochemis y s udies ha e localized PTPC in he p o ein bodies o whea g ains, whe e i was sug- ges ed o con ibu e o amino acid and p o ein biosyn hesis du ing g ain de elopmen (A aus e al., 1993). PTPC has also been localized in issues o de eloping and ge mina ing whea g ains cha ac e ized by high me abolic ac i i y (González e al., 1998). I is no ewo hy ha PEPC was de ec ed as an abundan p o ein in he aleu one laye using immuno-locali- za ion (Fig.2E). Consis en wi h his esul , he ole o PEPC in mala e p oduc ion by he ba ley aleu one laye has been desc ibed by Macnicol and Raymond, (1998). Collec i ely, hese esul s indica ed he s abili y and con- sis en unc ional ele ance o PTPC h oughou he li e cycle o so ghum seeds. Exp ession o PEPC genes in de eloping and ge mina ingseeds So ghum PEPC is encoded by a small mul igene amily con- sis ing o six genes: SbPPC2, SbPPC3, SbPPC4, SbPPC5 (all C3 PTPCs), and SbPPC1 (C4 pho osyn he ic PTPC) encode closely ela ed PTPCs, whils SbPPC6 encodes he dis an ly ela ed BTPC (Table 1) (Pa e son e al., 2009). In his p esen s udy, we used qRT-PCR o analyze he pa e n o hei an- sc ip abundance. SbPPC2, SbPPC3, and SbPPC4 ansc ip s we e p esen in de eloping and ge mina ing so ghum seeds (Fig. 3). T ansc ip s o SbPPC2, he housekeeping PTPC (Lepiniec e al., 1994), we e de ec ed du ing seed de elopmen and i s abundance inc eased sligh ly du ing he ma u a ion phase (Fig.3A). In he ge mina ing seeds, SbPPC2 showed maximal ansc ip abundance a 24–48 h pos -imbibi ion and hen dec eased (Fig.3B). By con as , SbPPC3 ansc ip le els we e maximum a he beginning o seed de elopmen , du ing he pe iod o cellula iza ion (Bewley e  al., 2013); his was ollowed by a dec ease o SbPPC3 ansc ip s un il s age III and hen an inc ease du ing he desicca ion pe iod wi h a peak a s age V (Fig.3C). SbPPC3 ansc ip s we e also de ec ed du ing ge mina ion wi h wo impo an peaks a 24 and 72 h pos -imbibi ion (Fig.3D). Finally, SbPPC4 ansc ip s we e qui e abundan a he beginning o de elop- men , sugges ing a possible speci ic ole o SbPPC4 du ing he phase o cellula iza ion (Fig.3E). Cellula iza ion is he massi e p oduc ion o new cells. Mala e is a ubiqui ous mol- ecule ha p o ides ca bon skele ons and educing powe and is needed o he syn hesis o s o age end-p oduc s by de el- oping seeds. SbPPC4 ansc ip s we e also e y abundan a 24–72 h pos -imbibi ion in ge mina ing seeds (Fig.3F). By con as , ansc ip s o SbPPC5 and SbPPC6 (BTPC) we e e y low du ing so ghum seed de elopmen and ge mina- ion and, as expec ed, no SbPPC1 ansc ip s we e de ec ed ( esul s no shown). These esul s show a no el pa e n o he s eady-s a e o ansc ip abundance o PTPC isogenes du - ing he li e cycle o so ghum seeds, wi h an impo an p es- ence o SbPPC3 and also an impo an con ibu ion o he SbPPC4 isogene o he cellula iza ion s age o de elopmen (s age I) and du ing ge mina ion. Di e en PTPC isoenzymes co-exis in de eloping and ge mina ingseeds Pep ide mass inge p in ing by MALDI-TOF MS o he p110 and p107 polypep ides immunop ecipi a ed using an i- C4 PTPC e ealed ha di e en PTPC isoenzymes co-exis in de eloping and ge mina ing so ghum seeds (Table2; as e - isks deno e con i ma ion o he iden i ica ion ia LC MS/MS sequencing o yp ic pep ides). SbPPC3 was he mos abun- dan du ing he cou se o he li e cycle o he seeds in bo h p107 and p110. SbPPC3 was highly abundan in he emb yo (Table2) and less abundan in he aleu one/endospe m o he ge mina ing seed; howe e , SbPPC3 was he only isoenzyme de ec ed in his issue (Table2). The housekeeping SbPPC2 (Lepiniec e al., 1993, 1994) was p esen in ea ly de elopmen only as a p107 (i.e. p obably non-monoubiqui ina ed) subuni 3528 | Ruiz-Balles a e al. (Table2, s ages Iand III) and was iden i ied as a p110 and p107 subuni in s age VI (Table2). SbPPC2 was also p e- sen as a monoubiqui ina ed p110 subuni in he emb yo o d y and 48-h pos -imbibi ion seeds, bu was no iden i ied in he aleu one (Table2). Finally, he SbPPC4 isogene was shown o be ansla ed in o he co esponding p107 PTPC subuni s in ea ly de elopmen (Table2, s ages Iand III), in ag eemen wi h he ansc ip p o iling esul s o Fig. 3E. In addi ion, SbPPC4 was also de ec ed in p110, indica ing he p obable monoubiqui ina ion o his PTPC isoenzyme. Al hough he p ecise physiological unc ion o SbPPC4 emains unclea , ou esul s suppo a ole o his isoenzyme du ing he ea ly s ages o seed de elopmen . Du ing he phase o cellula iza ion SbPPC4 is hypo hesized o gene - a e he C-skele ons needed as p ecu so s o syn hesis o new componen s such as amino acids/p o eins, and memb ane o s o age lipids. Finally, SbPPC5 was de ec ed only as p110 polypep ides in s age I(Table2). SbPPC6 polypep ides we e ne e de ec ed by MS/MS analysis, in ag eemen wi h he absence o SbPPC6 exp ession du ing so ghum seed de el- opmen and ge mina ion. No was a high molecula mass Class-2 PEPC he e o-oc ame e iden when cla i ied ex ac s om di e en s ages o de elopmen and ge mina ion we e subjec ed o non-dena u ing PAGE ollowed by in-gel PEPC ac i i y s aining (see Supplemen a y Fig. S3). Howe e , e a- me ic and dime ic Class-1 PEPC oligome s we e obse ed, Fig.1. Time cou se o PEPC ac i i y in de eloping and ge mina ing so ghum seeds. PEPC ac i i y was assayed a op imal pH (8.0), 2.5 mM PEP, and 30ºC in cla i ied ex ac s om: (A) whole seeds du ing de elopmen ; and (B) emb yos o (C) aleu one-endospe m o ge mina ing seeds. (A) PEPC ac i i y is exp essed on a pe seed basis (mU seeds−1) o on a p o ein basis (U mg−1 p o ein). Resul s a e means ±SE o h ee independen expe imen s. Mean alues ha a e no signi ican ly di e en (P<0.05, S uden ’s - es ) a e indica ed wi h he same le e s. Da a o aleu one-endospe m a e no signi ican ly di e en . PTMs o so ghum seed PEPC isozymes | 3529 wi h he dime ic o m being he mos abundan in i o (Supplemen a y Fig. S3A). This dime ic o m was no al e ed by de-ubiqui ina ion o he enzyme a e incuba ion wi h 2µM o he ca aly ic subuni o ubiqui in speci ic p o ease-2 (USP-2c) (Supplemen a y Fig. S3B). A numbe o impo an poin s eme ge om hese esul s, as ollows. (i) Mul iple C3 PTPC isoenzymes a e p esen du - ing he de elopmen and he ge mina ion o so ghum seeds. SbPPC3 appea s o ha e an essen ial ole as i is he iso- enzyme mos consis en ly p esen du ing he cou se o he li e cycle o he seeds , and was he only one de ec ed in he aleu one/endospe m. (ii) The SbPPC4 isozyme appea s o play a majo ole du ing he e y ea ly s age o seed de elop- men , known as he phase o cellula iza ion (Bewley e al., 2013).(iii) BTPC is no signi ican ly exp essed h oughou he li e cycle o he seeds, which is an impo an di e ence ela i e o de eloping COS, a non-g een oil seed, in which p118 BTPC polypep ides, and hus Class-2 PEPC complexes a e highly exp essed (O’Lea y e al., 2011b). Pos - ansla ional modi ica ions o he di e en Class-1 PEPC isoenzymes du ing ge mina ion In he endospe m o ge mina ing COS, p110 PTPC subuni s a e monoubiqui ina ed bu no phospho yla ed (Uh ig e al., 2008; O’Lea y e al., 2011b). On he o he hand, simul ane- ous p110 monoubiqui ina ion and p107 phospho yla ion ha e been desc ibed as an al e na i e PTM pa e n o Class-1 PEPC o imma u e p o eoid (clus e ) oo s o ha sh hakea (Shane e  al., 2013). Howe e , he coinciden occu ence Fig.2. Immunological localiza ion o PTPC in ge mina ing so ghum seeds. G ains we e imbibed o 24 h. Sec ions (15µm) we e p obed wi h: (A, B), p eimmune se um, o (C–F) polyclonal an i-C4 PTPC (SbPPC1). Abb e ia ions: al, aleu one laye ; ep, epico yl; ep , epi helium; sc, scu ellum; se, s a chy endospe m; , adicle; p, pe ica p. 3530 | Ruiz-Balles a e al. o bo h PTMs in so ghum seed p110 and p107 PTPC (i.e. SbPPC3) subuni s has only been shown in i o (Ruiz-Balles a e al., 2014). In his s udy, we show bo h PTMs also occu in i o wi h h ee o he ou PTPC isoenzymes p esen in ge mina ing so ghum seeds. To his end, PTPC polypep ides o cla i ied ex ac s om 48-h pos -imbibi ion ge mina - ing seeds we e isola ed by immunoa ini y ch oma og aphy wi h an i-COS PEPC (see Supplemen a y Fig. S4A, B). Coomassie blue R-250-s ained p110 and p107 we e excised om SDS gels o he immunopu i ied PTPC and indi idu- ally subjec ed o in-gel yp ic diges ion and de ailed analysis by LC MS/MS. AMasco da abase sea ch o p110 and p107 da ase s de i ed om nano HPLC T ipleTOF MS o hei yp ic pep ides (Table3) e ealed he p esence o SbPPC2, SbPPC3, and SbPPC4 PTPCs in p110 and p107, as well as SbPPC5 in p107. Consis en wi h ou ea lie esul s (Ruiz- Balles a e  al., 2014), p107 N- e minal yp ic pep ides o SbPPC3 con aining non-phospho yla ed Se 7 we e iden i ied by O bi ap LC MS/MS. Howe e , he monoubiqui ina ed SbPPC3 p110 subuni s we e also de e mined o be phospho- yla ed a Se 7 (Table3). Monoubiqui ina ed SbPPC2 and SbPPC4 (i.e. p110 subuni s), and deubiqui ina ed SbPPC5 (i.e. p107 subuni s) we e also shown o be phospho yla ed a hei conse ed N- e minal se yl phospho yla ion si es; i.e. Se 13, Se 10, and Se 11 o SbPPC2, SbPPC4, and SbPPC5, espec i ely (Table3). To he bes o ou knowledge, his is he i s e idence ha in i o phospho yla ion o di e en PTPC isoenzymes can simul aneously occu in any plan issue. MS/ MS also con i med he monoubiqui ina ion o SbPPC3 p110 subuni s a Lys624 (Table3), as p e iously epo ed (Ruiz- Balles a e al., 2014). In addi ion, T ipleTOF MS/MS analy- sis o he quad uply cha ged SbPPC2 p110 pep ide ion o m/z 455.7462 (LC e en ion ime 16.61 min) and he iply cha ged ion o m/z 507.9268 (LC e en ion ime 18.01 min), co e- sponding o esidues 623–637 (VAKDFGVKLTMFHGR) and 626–637 (DFGVKLTMFHGR), showed ha he masses o he C- e minal agmen s up o y7 ma ched he p edic ed alues, bu he mass shi o he agmen y8 by 114 Da indi- ca ed a ubiqui ina ion si e localized a Lys-630, whe e a cha - ac e is ic mass inc emen o 114 Da indica es a Gly-Gly mo i a achmen o ubiqui in a he side chain (Supplemen a y Fig. S5A, B). LC MS/MS measu emen s o he doubly and Fig.3. qRT-PCR analysis o PTPC ansc ip le els in de eloping and ge mina ing so ghum seeds. To no malize he alues, 18S RNA was used as in e nal con ol in each sample. Da a we e no malized o s age VI o de eloping seeds (A, C, E), o 14 h pos -imbibi ion ge mina ing seeds (B, D, F). Resul s a e means ±SE o a leas h ee independen expe imen s. Mean alues ha a e no signi ican ly di e en (P<0.05, S uden ’s - es ) a e indica ed wi h iden ical le e s. PTMs o so ghum seed PEPC isozymes | 3531 iply cha ged ions o m/z 769.3827 and m/z 513.2607 (LC e en ion ime 23.22 min) using a high- esolu ion O bi ap Fusion T ib id ins umen iden i ied he ubiqui ina ion o he oxidized pep ide a esidues 626–637 (Supplemen a y Fig. S5C, D). Unde high- esolu ion HCD agmen a ion, he mass shi o 114 Da on bo h he N- e minal b5 ion and he C- e minal y8 ion unambiguously con i med Lys-630 as p110’s monoubiqui ina ion si e in SbPPC2. This esidue p ecisely aligns wi h he Lys-624 and Lys-628 (a highly con- se ed PTPC esidue ha is immedia ely p oximal o ca aly ic esidues in ol ed in PEP binding) monoubiqui ina ion si es, espec i ely de e mined o p110 subuni s o so ghum seed SbPPC3 PTPC (Table3 and Ruiz-Balles a e al., 2014) and PTPC (RcPPC3) om ge mina ed COS (Uh ig e al., 2008). Collec i ely, hese esul s es ablish o he i s ime se - e al impo an poin s. (i) Di e en PTPC isoenzymes a e co-exp essed du ing so ghum seed ge mina ion and all o hem excep he p107 subuni s o SbPPC3 a e phospho yl- a ed du ing ge mina ion. (ii) Monoubiqui ina ion o he co - esponding conse ed Lys was con i med by MS/MS analysis o SbPPC3, and de e mined in his s udy o SbPPC2, dem- ons a ing ha his PTM occu s wi h mul iple PTPC iso- enzymes a a conse ed lysine esidue wi hin a single plan species. (iii) Monoubiqui ina ion and phospho yla ion can simul aneously occu on he same PTPC polypep ide and a e he e o e no mu ually exclusi e PTMs, as appea s o be he case wi h PTPCs om de eloping p o eoid oo s o ha sh hakea (Shane e al., 2013), as well as a ious g een and non-g een issues o he cas o oil plan (Uh ig e al., 2008; O’Lea y e al., 2011b). Is PTPC monoubiqui ina ion linked o speci ic s ages and/o en i onmen al condi ions du ing so ghum seed ge mina ion? Because o i s ecen disco e y (Uh ig e al., 2008), he e a e ew s udies abou he in luence o en i onmen al ac o s o bio ic/abio ic s esses on PTPC monoubiqui ina ion du ing seed de elopmen and ge mina ion. Elimina ion o pho- osyn ha e ansloca ion (a ype o abio ic s ess) om he shoo o he seed by depodding ( ui excision) o de elop- ing COS igge ed apid in i o p107 dephospho yla ion, ol- lowed by monoubiqui ina ion o 50% o he p107 subuni s o o m p110 cha ac e is ic o ge mina ing COS (O’Lea y e al., 2011b). So ghum is a ce eal cul i a ed in wa m en i onmen s and seeds ge mina e well wi hin a wide ange o empe a u e om 25–35ºC, wi h an op imum o o e 30ºC (Kanemasu e al., 1975). This allowed us o co ela e he e ec o empe a u e wi h ge mina ion and he pa e n o PTPC monoubiqui ina ion (i.e. appea ance o p110 polypep ides on PTPC immunob- lo s). To his end, seeds om wo so ghum a ie ies PR88Y20 Table2. Iden i ica ion o p107 and p110 PTPC iso o ms om de eloping and ge mina ing so ghum seeds. P o eins we e iden i ied ia MALDI-TOF/TOF MS pep ide mass inge p in ing o yp ic pep ides de i ed om immunop ecipi a ed samples o ep esen a i e s ages. As e isks indica e iden i ica ion ia MS/MS sequencing o yp ic pep ides and BLAST analysis. DPA, days pos -an hesis. STAGE Subuni P o ein name Accession numbe aSco e Sequence co e age (%) No. o Ma ching Pep ides I (7–12 DPA) p110 PEPC SbPPC3* gi|241931686 248 35 31 PEPC SbPPC4* gi|241940554 118 24 22 PEPC SbPPC5 gi|241930496 98 22 21 p107 PEPC SbPPC3 gi|241931686 325 45 39 PEPC SbPPC4 gi|241940554 114 26 21 PEPC SbPPC2 gi|241925556 107 23 20 III (16–20 DPA) p110 PEPC SbPPC3 gi|241931686 103 24 21 p107 PEPC SbPPC3 gi|241931686 206 33 29 PEPC SbPPC4 gi|241940554 66 19 16 PEPC SbPPC2 gi|241925556 61 17 15 VI (31–40 DPA) p110 PEPC SbPPC3 gi|241931686 60 10 13 PEPC SbPPC2* gi|241925556 36 9 8 p107 PEPC SbPPC3 gi|241931686 121 15 18 PEPC SbPPC2* gi|241925556 76 8 10 D yseed Aleu one/endospe m p110 PEPC SbPPC3 gi|241931686 30 13 9 p107 PEPC SbPPC3 gi|241931686 72 20 15 D yseed Emb yo p110 PEPC SbPPC3* gi|241931686 310 45 39 PEPC SbPPC2 gi|241925556 91 21 18 p107 PEPCSb PPC3 gi|241931686 283 42 48 PEPC SbPPC2 gi|241925556 63 16 14 48 h pos -imbibi ion Aleu one/endospe m p110 PEPC SbPPC3* gi|241931686 95 24 16 p107 PEPC SbPPC3 gi|241931686 67 16 15 48 h pos -imbibi ion Emb yo p110 PEPC SbPPC3 gi|241931686 305 44 38 PEPC SbPPC2 gi|241925556 54 15 14 p107 PEPC SbPPC3* gi|241931686 164 29 38 PEPC SbPPC2 gi|241925556 55 15 13 aNCBI da abase