multi-omics analysis reveals sequential roles for …...multi-omics analysis reveals sequential...

Multi-omics Analysis Reveals Sequential Roles for ABAduring Seed Maturation1

Frédéric Chauffour,a Marlène Bailly,a François Perreau,a Gwendal Cueff,a Hiromi Suzuki,a Boris Collet,a

Anne Frey,a Gilles Clément,a Ludivine Soubigou-Taconnat,b,c Thierry Balliau,d Anja Krieger-Liszkay,e

Loïc Rajjou,a,2 and Annie Marion-Polla,2,3

aInstitut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique, AgroParisTech, Centre Nationalde la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, FrancebInstitute of Plant Sciences Paris-Saclay (IPS2), Centre National de la Recherche Scientifique, Institut Nationalde la Recherche Agronomique, Université Paris-Sud, Université d’Evry, Université Paris-Saclay, 91192Gif-sur-Yvette, FrancecInstitute of Plant Sciences Paris-Saclay (IPS2), Centre National de la Recherche Scientifique, Institut Nationalde la Recherche Agronomique, Université Paris-Diderot, Sorbonne Paris-Cité, 91192 Gif-sur-Yvette, FrancedUniversité Paris-Saclay, Unité Mixte de Recherche Génétique Quantitative & Evolution Le Moulon, InstitutNational de la Recherche Agronomique, Université Paris Sud, Centre National de la Recherche Scientifique,AgroParisTech, La Plateforme d’Analyse Protéomique de Paris Sud Ouest, 91190 Gif-sur-Yvette, FranceeInstitute for Integrative Biology of the Cell (I2BC), Commissariat à l’Énergie Atomique, Centre National de laRecherche Scientifique, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France

ORCID IDs: 0000-0001-7873-9866 (F.C.); 0000-0002-3362-9361 (H.S.); 0000-0003-0188-9932 (B.C.); 0000-0001-7141-4129 (A.K.-L.);0000-0001-9739-1041 (L.R.); 0000-0003-1733-1984 (A.M.-P.).

Abscisic acid (ABA) is an important hormone for seed development and germination whose physiological action is modulatedby its endogenous levels. Cleavage of carotenoid precursors by 9-cis epoxycarotenoid dioxygenase (NCED) and inactivation ofABA by ABA 89-hydroxylase (CYP707A) are key regulatory metabolic steps. In Arabidopsis (Arabidopsis thaliana), both enzymesare encoded by multigene families, having distinctive expression patterns. To evaluate the genome-wide impact of ABAdeficiency in developing seeds at the maturation stage when dormancy is induced, we used a nced2569 quadruple mutant inwhich ABA deficiency is mostly restricted to seeds, thus limiting the impact of maternal defects on seed physiology. ABAcontent was very low in nced2569 seeds, similar to the severe mutant aba2; unexpectedly, ABA Glc ester was detected in aba2seeds, suggesting the existence of an alternative metabolic route. Hormone content in nced2569 seeds compared with nced259 andwild type strongly suggested that specific expression of NCED6 in the endosperm is mainly responsible for ABA production. Inaccordance, transcriptome analyses revealed broad similarities in gene expression between nced2569 and either wild-type ornced259 developing seeds. Gene ontology enrichments revealed a large spectrum of ABA activation targets involved in reservestorage and desiccation tolerance, and repression of photosynthesis and cell cycle. Proteome and metabolome profiles in drynced2569 seeds, compared with wild-type and cyp707a1a2 seeds, also highlighted an inhibitory role of ABA on remobilization ofreserves, reactive oxygen species production, and protein oxidation. Down-regulation of these oxidative processes by ABA mayhave an essential role in dormancy control.

Biological processes taking place during seed devel-opment are controlled by endogenous and exogenoussignals and, after dispersal or harvest, determine seedlongevity, germination vigor, and successful establish-ment of seedlings in a variable range of environmentalconditions (Penfield and MacGregor, 2017). In manydicots, including Arabidopsis (Arabidopsis thaliana),embryogenesis starts after the double fertilization thatgives rise to the diploid embryo and the triploid en-dosperm. At the heart stage, when morphogenesis iscompleted, the embryo grows at the expense of theendosperm, and reserve storage takes place. At the endof this maturation phase, the Arabidopsis embryo issurrounded by a single layer of endosperm and a seedcoat (or testa) of maternal origin, which is composed ofdead dry tissues (North et al., 2010). Abscisic acid

(ABA) has been extensively reported to regulate manyaspects of seed development, mainly during matura-tion. Its roles at earlier stages are poorly documented;nevertheless, reduced synthesis in the abscisic acid defi-cient2 (aba2) mutant has been shown to delay embryo-genesis and endosperm cellularization (Cheng et al.,2014). After embryogenesis, ABA inhibits embryogrowth and positively regulates reserve accumulationand at later stages induces primary dormancy anddesiccation tolerance. Analysis of seed phenotypes ofABA metabolism or signaling mutants in many speciespointed out its crucial action in preventing vivipary andallowing seed dispersal in a dormant state (Finkelsteinet al., 2008; Nambara et al., 2010; Graeber et al., 2012).Dormancy is an adaptive trait limiting germinationunder environmental conditions that would promote

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http://orcid.org/0000-0001-7873-9866

http://orcid.org/0000-0001-7873-9866

http://orcid.org/0000-0002-3362-9361

http://orcid.org/0000-0002-3362-9361

http://orcid.org/0000-0003-0188-9932

http://orcid.org/0000-0003-0188-9932

http://orcid.org/0000-0001-7141-4129

http://orcid.org/0000-0001-7141-4129

http://orcid.org/0000-0001-9739-1041

http://orcid.org/0000-0001-9739-1041

http://orcid.org/0000-0003-1733-1984

http://orcid.org/0000-0003-1733-1984

http://orcid.org/0000-0001-7873-9866

http://orcid.org/0000-0002-3362-9361

http://orcid.org/0000-0003-0188-9932

http://orcid.org/0000-0001-7141-4129

http://orcid.org/0000-0001-9739-1041

http://orcid.org/0000-0003-1733-1984

http://crossmark.crossref.org/dialog/?doi=10.1104/pp.19.00338&domain=pdf&date_stamp=2019-05-21

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optimal germination in nondormant seeds. The dor-mancy depth determines the timing of germinationunder seasonally varying conditions at which seedlingsurvival and growth are the most favorable (Burghardtet al., 2016). In Arabidopsis, seed dormancy of the mostcommonly used accession, Columbia-0, is relativelylow and can be released by a few weeks of after-ripening (dry storage) or by stratification (cold imbibi-tion). Genetic studies demonstrated that dormancydepth correlates well with ABA levels; it is increased inmutants defective for ABA catabolism and reduced inbiosynthesis mutants (Finkelstein et al., 2008).The ABA metabolism pathway has been largely un-

covered (Nambara and Marion-Poll, 2005). The firststep specific to ABA biosynthesis is the cleavage of cis-isomers of the oxygenated carotenoids, violaxanthinand neoxanthin, by a 9-cis epoxycarotenoid dioxygen-ase (NCED) that is encoded in Arabidopsis by a genefamily of five members, namely, NCED2, NCED3,NCED5, NCED6, and NCED9. The 15-carbon product,xanthoxin, is then transported from plastids to cytosoland converted into abscisic aldehyde by a short-chainalcohol dehydrogenase, encoded by the ABA2 gene.The final biosynthesis step is catalyzed by an abscisicaldehyde oxidase, requiring activation of its molybde-num cofactor for activity. ABA inactivation occurs ei-ther by oxidation or conjugation. The major route is 89hydroxylation by the CYP707A subfamily of P450monooxygenases, which is followed by the spontane-ous isomerization of 89-hydroxy-ABA into phaseic acid(PA). PA has been recently shown to retain some bio-logical activity, and complete inactivation occurs afterits conversion into dihydrophaseic acid (DPA) by a PAreductase (Weng et al., 2016). ABA conjugation into ABAGlc ester (ABA-GE) is catalyzed by glucosyl-transferases,and subsequentABA-GE hydrolysis by twob-glucosidases,BG1 and BG2, contributes to ABA production (Lee et al.,2006; Xu et al., 2012). In developing and imbibed seeds of

Arabidopsis, spatiotemporal regulation of specificmembers of NCED and CYP707A gene families has amajor role in modulating ABA levels and regulatingdormancy depth and germination timing, as deducedfrom phenotypes of multiple nced or cyp707a mutants(Lefebvre et al., 2006; Millar et al., 2006; Okamoto et al.,2006; Frey et al., 2012).There is now strong evidence that members of a

multigene family encoding pyrabactin resistance (PYR)/PYR-like (PYL)/regulatory components of ABA receptor(RCAR) are ABA receptors, which sequester and in-hibit protein phosphatases 2C (PP2C) when ABA ispresent. PP2C inactivation allows phosphorylationof Sucrose non-fermenting1-related kinases2, whichin turn phosphorylate Basic Leucine Zipper Domaintranscription factors of the ABA-INSENSITIVE5 (ABI5)/ABA-responsive element-binding protein/ABA-responsive element-binding factor family, which bind toABA response elements in promoter sequences of ABA-inducible genes (Cutler et al., 2010). The germinationphenotypes of either PP2C or SnRK2 multiple mutantssuggest that the ABA signaling pathway involving thePYR receptor and PP2C/SnRK2 phosphorylationcascade operates in dormancy regulation of Arabi-dopsis seeds (Fujii and Zhu, 2009; Nakashima et al.,2009). Moreover, recent studies reported the inter-action of DELAY OF DORMANCY1 (DOG1), a majorregulator of seed dormancy whose molecular func-tion remains elusive, with a subset of clade A PP2Cphosphatases, resulting in their inhibition (Née et al.,2017; Nishimura et al., 2018).In this study we exploited the distinctive expression

patterns of NCED genes in developing seeds to assessthe tissue-specific origin of ABA production. Our re-sults highlight the importance of endospermic ABAand also suggest the existence of alternative pathwaysfor ABA-GE synthesis in seeds. Previous transcriptomestudies have focused on seed imbibition and dormancyrelease. Here, we evaluated the genome-wide impact ofABA deficiency during seed development, in relationwith dormancy induction. Comparative analysis ofthe dry seed proteome and metabolome of the defi-cient nced2569mutant with those of the wild type andoverproducing cyp707a1a2 correlated well with dif-ferential gene expression in developing seeds. Thedata reported here strongly support the positive ef-fect of ABA on reserve storage and desiccation tol-erance and interestingly also reveal its importance inthe repression of photosynthesis, cell cycle, reserveremobilization, and oxidative processes to preventpremature germination and promote dormancy.

RESULTS

ABA Levels Are Similarly Reduced in nced2569 andaba2 Seeds

The ABA-deficient aba2 has been often used to studythe role of ABA-regulated biological processes. ABA

1This work was supported by the Agence Nationale de la Recher-che (grant no. ANR-2010-BLAN-1233-01 ABSIG; and LabEx SaclayPlant Sciences-SPS grant no. ANR-10-LABX-0040-SPS to Institut Jean-Pierre Bourgin), the European Commission (grant no. EU FP7-KBBEEcoSeed-311840; and the EU Marie-Curie FP7 COFUND People Pro-gramme Agreenskills postdoctoral fellowship to H.S.), and theFrench Ministère de l’Enseignement Supérieur et de la Recherche(doctoral fellowship to F.C.).

2Senior author.3Author for contact: [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Annie Marion-Poll ([email protected]).

F.C., L.R., and A.M.-P. conceived the research; L.R. and A.M.-P.supervised the experiments; F.C., M.B., H.S., B.C., A.F., and A.K.-L.performed experiments; F.C., M.B., and F.P. performed proteomeand/or hormone analysis; L.S.-T., G.Cu., T.B., and G.Cl. performedtranscriptome, proteome, and metabolome analysis, respectively;F.C., M.B., F.P., H.S., A.K.-L., L.R., and A.M.-P. analyzed data; F.C.,L.R., and A.M.-P. wrote the article.

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Genome-Wide Impact of ABA Deficiency in Seeds


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levels are severely reduced in both vegetative and re-productive tissues, and aba2 exhibits typical pheno-types associated with ABA deficiency, i.e. increasedplant water loss and reduced seed dormancy (Léon-Kloosterziel et al., 1996; Nambara et al., 1998).However, the aba2 mutation also has major effects onseed development and plant growth, resulting in re-duced plant size and poor seed yield (Cheng et al.,2002, 2014). We previously described a triple mu-tant nced569 whose dormancy was strongly reduced,without visible impact on plant development andseed production (Frey et al., 2012). Because AtNCED3has been described to have the major contribution toincreased ABA accumulation in response to waterstress (Iuchi et al., 2001; Tan et al., 2003), we reasonedthat the quadruple mutant nced2569 might also ex-hibit a reduction in dormancy depth and seed ABAcontent, without deleterious effects on plant devel-opment and seed yield. Comparison of plant size andflowering time of nced2569, aba2-2, and wild-typeArabidopsis indicated that NCED3 activity was suf-ficient for normal development of nced2569 plants(Supplemental Fig. S1).

In nced2569 developing siliques harvested from 6 to18 d after pollination (DAP) and in dry seeds, ABAcontent was strongly decreased compared to that inthe wild type and, at all developmental stages,ABA levels in nced2569 siliques were as low as those inaba2-2 (Fig. 1A). This suggested a minor contributionof NCED3 activity to ABA accumulation in these tis-sues, either from on-site synthesis or transported fromvegetative tissues. In our previous work, NCED6 wasshown to be specifically expressed in the endospermduring seed development and nced6mutation reducedABA accumulation in dry seeds (Lefebvre et al., 2006).Therefore, the contribution of NCED6 expression, asa proxy of ABA production in the endosperm, wasassessed here by comparing ABA content in nced2569and nced259 siliques. Surprisingly, ABA levels innced259 siliques were similar to those in the wild type,suggesting that NCED6 activity in the endosperm wasmainly responsible for the observed ABA accumula-tion. In developing siliques, ABA was shown to bemostly accumulated in seeds (Kanno et al., 2010). Toascertain that ABA measured here in siliques wellreflected seed contents, we dissected wild-type de-veloping seeds from silique envelopes and confirmedthis previous observation (Supplemental Fig. S2A).

Alternative Catabolic Routes Are Used in Mutant Seeds

It is well established that rates of both synthesis anddegradation modulate ABA accumulation; therefore,the content of the most abundant catabolites, DPA andABA-GE, was assessed in nced mutants compared towild type and aba2-2. In accordance with their im-paired ABA synthesis and low seed ABA content,DPA levels were strongly reduced in nced2569 andaba2-2, compared to wild type and nced259 (Fig. 1B),but unexpected differences in ABA-GE accumulation

were observed between nced2569 and aba2-2 (Fig. 1C).In contrast to nced2569 siliques that did not accumu-late significant ABA-GE amounts, in aba2 siliques, thisconjugate was detected at similar levels to wild type at10 DAP and at higher levels at 14 and 18 DAP.Therefore, it suggested that this conjugate could beproduced in this mutant that is unable to convertxanthoxin into abscisic aldehyde.

A more expected observation was the respectivesimilarity of ABA, DPA, and ABA-GE profiles innced259 siliques compared to wild type, which indi-cated that ABA catabolism was unaffected in thismutant. Interestingly, in both genotypes, DPA andABA-GE levels were maximal at 10 DAP, in contrast toABA levels that peaked at 14 DAP. Thus, at early de-velopmental stages, active ABA hydroxylation andconjugation would likely limit ABA accumulation(Fig. 1). DPA accumulation was also observed in en-velopes of wild-type siliques, although at lower levelscompared to dissected seeds. The higher amounts ofcatabolites, compared to ABA, at 10 DAP in both seedsand siliques suggested either active ABA productionin these tissues at this stage or/and translocation fromvegetative tissues, before subsequent hydroxylation/conjugation (Supplemental Fig. S2). In contrast to seeds,low ABA-GE levels were detected in wild-type silique

Figure 1. ABA biosynthesis and catabolism in ncedmutants. A–C, ABA(A), DPA (B), and ABA-GE (C) levels in developing siliques at 6, 10, 14,and 18 DAPand dry seeds of aba2-2, nced2569, and nced259mutants,compared to wild type. Means of three biological replicates are shownwith SE (n = 3). Two independent experiments were performed withsimilar results. DW, dry weight; WT, wild type.

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envelope (Supplemental Fig. S2C), indicating that ABAhydroxylation was predominant.In cyp707a1a2 siliques that are defective for ABA 89-

hydroxylase activity, ABA levels were increased andDPA levels reduced compared to wild type (Fig. 2, Aand B). Interestingly, in contrast to wild type, highcontents of ABA-GE and 79-hydroxy ABA were mea-sured, with maximal levels at 10 DAP for ABA-GE,and from 10 to 18 DAP for 79-hydroxy ABA (Fig. 2, Cand D). Moreover, the detection of high amounts of 79-hydroxy ABA in cyp707a1a2 siliques suggested that itsproduction did not require CYP707A1 or CYP707A2activity. The severe reduction in 89-hydroxylation incyp707a1a2 siliques was therefore partly compensatedby the alternative production of other metabolites thatwere barely detected in wild-type siliques.

Seeds of nced2569 and aba2 Mutants Exhibit SimilarDormancy Levels

Dormancy of the quadruple mutant nced2569 wascompared to that of nced259, aba2-2, cyp707a1a2, and

wild type. In accordance with their decreased ABAaccumulation, nced2569 seeds exhibited reduced dor-mancy levels, because germination rates at harvestwere 15% to 30% and reached 90% after 2 weeks of drystorage, as also observed in aba2 seeds (Fig. 3). Op-positely and in good agreement with the very highABA contents observed in developing siliques and dryseeds, cyp707a1a2 seed dormancy was strongly en-hanced and was not released after two months of drystorage. Despite ABA, DPA, and ABA-GE levels innced259 seeds being very similar to those of the wildtype, dormancy depth was lower. At harvest, germi-nation rates were very similar to wild type (,10%);however, dormancy release was faster (Figs. 1 and 3),thus suggesting that minor ABA pools may fine-tuneseed dormancy. Furthermore, comparison of germi-nation rates of nced2569 seeds with those observedin the four combinations of triple mutants confirmedthe contribution of ABA production from NCED5,NCED6, and NCED9 activity to dormancy depth(Supplemental Fig. S3).

Transcriptome Variations in Developing Seeds Correlatewith ABA Contents

The regulation of seed transcriptome by endoge-nous ABA has been previously studied in dry andimbibed seeds of aba2 and cyp707a1a2a3 mutants inrelation with dormancy release (Okamoto et al.,2010). However, ABA has also a very prominent

Figure 2. ABA biosynthesis and catabolism in cyp707a1a2 mutants.A–D, ABA (A), DPA (B), ABA-GE (C), and 79-hydroxy ABA (D) levels indeveloping siliques at 6, 10, 14, and 18 DAP and dry seeds ofcyp707a1a2mutants, compared to wild type. Means of three biologicalreplicates are shownwith SE (n = 3). Two independent experiments wereperformed with similar results. DW, dry weight; WT, wild type.

Figure 3. Dormancy is reduced in nced2569 and aba2-2. Germinationof nced269, nced2569, aba2-2, cyp707a1a2, and wild-type seeds,sown after 2 weeks of dry storage (A). Germination 4 d after sowing ofdry seeds stored at room temperature during 0–8 weeks (B). Means ofthree biological replicates are shown with SE (n = 3). Two independentexperiments were performed with similar results. WT, wild type.







role in dormancy induction, which we were inter-ested to investigate here by analyzing the impact ofABA deficiency on the global transcriptome in de-veloping nced2569 seeds, isolated from dissected si-liques. Because NCED6 activity in the endospermseverely reduced ABA content, we decided to eval-uate the specific impact of nced6 mutation, by com-paring nced2569 not only to wild type, but also tonced259. We focused on two mid-developmentalstages, 10 and 14 DAP, when ABA increase was ob-served (Fig. 1) and dormancy establishment isreported to occur in Arabidopsis seeds (Karssen et al.,1983). In parallel, ABA levels were analyzed in seedsamples at the two chosen stages, with relative ABAcontents in isolated seeds corroborating those mea-sured in whole siliques (Supplemental Fig. S4; Fig. 1).

At 10 DAP, transcriptome analysis revealed that 288transcripts were significantly down-regulated (.1.5-fold, false discovery rate [FDR], P , 0.05) in nced2569compared to either wild type or nced259. Most ofthese were down-regulated to a similar extent inboth comparisons, and only 41 of them were down-regulated by ,1.3-fold in either of the comparisons(Supplemental Table S1). A smaller number of tran-scripts (98) were significantly up-regulated (1.5-fold orgreater) in nced2569 compared to either wild type ornced259. Again, gene regulation was very similarin both comparisons, and only 13 transcripts were,1.3-fold more abundant in one or other comparison(Supplemental Table S2). A large overlap betweendifferentially regulated transcripts was therefore ob-served in nced2569 compared to either wild type ornced259, in accordancewith similar relative differencesin ABA levels (Supplemental Tables S1 and S2; Fig. 1).

At 14 DAP, a larger number of transcripts were dif-ferentially regulated. In nced2569, 853 transcripts weresignificantly down-regulated (.1.5-fold, FDR, P ,0.05) compared to either wild type or nced259. A largefraction of these (542) were down-regulated to a similarextent in both comparisons, .1.3-fold (SupplementalTable S3). Like at 10 DAP, a smaller number of tran-scripts (441) were significantly up-regulated (.1.5-fold)in nced2569 compared to either wild type or nced259.Again, gene regulation was very similar in both com-parisons, with an overaccumulation (.1.3-fold) of 334transcripts (Supplemental Table S4), suggesting thattranscriptome variations mainly correlated with ABAlevels.

ABA Deficiency Negatively Impacts on Reserve Storageand Desiccation Transcript Accumulation

Gene ontology (GO) enrichment was analyzed usingThaleMine tools (https://apps.araport.org/thalemine/begin.do). Among the down-regulated transcriptsin nced2569 at 10 DAP, enrichment was found in GOterms related to lipid and protein storage (Table 1;Supplemental Table S5). Accumulation of transcriptsencoding seed storage albumins (SESA), SESA1, SESA3,

SESA4, and SESA5, was strongly reduced, from 2- to10-fold. Seven genes of the deoxythymidine diphos-phates-4-dehydrorhamnose 3,5-epimerase (RmlC)-like cupins superfamily were also down-regulated;this family notably encodes the cruciferins, CRU1/CRA1 and CRU2, which transcript accumulationwas strongly decreased (4- to 5-fold). Among lipidstorage proteins, transcripts encoding oil-body associ-ated proteins, five encoding oleosins, two caleosins(CLO), and two hydroxysteroid dehydrogenases,were down-regulated at variable levels, up to 7-foldfor hydroxysteroid dehydrogenase1 and CLO2/Peroxygenase2. Enrichment was also observed forgenes related to seed development, among whichthose encoding late embryogenesis abundant (LEA)genes were strongly down-regulated, i.e. LEA28(5-fold), LEA29 (5-fold), LEA48 (4-fold), and LEA42(2.5-fold). Accumulation of other transcripts associ-atedwith desiccation tolerancewas also reduced, such asAT1G48130/PER1 encoding 1-Cys peroxiredoxin1 andseveral transcripts encoding aquaporins (SupplementalTable S5).

Among the down-regulated transcripts at 14 DAP,enrichment was found in GO terms related to lipidsynthesis and accumulation (Table 1; SupplementalTable S5). Accumulation of transcripts related to lipidstorage, encoding the oil-body associated proteinOBAP1a, the four caleosin-related proteins CLO2,CLO3, CLO4, and AT1G70680, and the three oleosin-related proteins OLEO6, OLEO11, and GRP14, werereduced;2-fold in nced2569. In addition, a number ofgenes belonging to the GO terms cutin, suberine, andwax biosynthesis (KEGG pathway 00073), were con-comitantly down-regulated. These included the threefatty acid reductase genes (FAR), FAR1, FAR4, andFAR5, and five genes related to the cytochrome P450family 86, which expression was reduced up to 2-fold.Compared to 10 DAP, fewer genes encoding storageproteins were differentially expressed at 14 DAP innced2659 compared to the other genotypes; however,SESA1 and SESA5 transcripts were down-regulatedat both stages. In contrast, a higher number of LEAtranscripts were down-regulated at 14 DAP than at10 DAP, which included LEA35/EM1, LEA46,LEA30, LEA51/RAB18, LEA29, LEA33/XERO2/LTI30, LEA28, LEA50, LEA34/XERO1, LEA15,AT5G66780, AT4G01410, LEA9, LEA7, LEA42,AT1G17620, AT1G54540, AT5G45320, LEA48, andLEA14, listed from the most strongly down-regulated LEA35/EM1 (18-fold) to LEA14 (1.5-fold). Interestingly, the accumulation of transcriptsencoding two heat shock transcription factors,AT3G02990/HSFA1E and AT3G24520/HSFC1, wasalso reduced (Supplemental Table S3).

At this stage, an enrichment was also found in GOterms related to carbohydrate transport and, amongthe transcripts for which accumulation was reducedin nced2569, we found four members of the SWEET(a nodulin MtN3 family protein) Suc efflux trans-porter family (SWEET11, SWEET12, SWEET14, and


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https://apps.araport.org/thalemine/begin.do

https://apps.araport.org/thalemine/begin.do








Table 1. Differentially expressed transcripts encoding proteins involved in reserve storage, carbohydrate transport, and LEA

The proteins were mostly down-regulated in nced2569 compared to either wild type or nced259 (.1.5-fold; Supplemental Tables S1 and S3).Gene and protein names are indicated according to https://apps.araport.org/thalemine and LEA are numbered according to Candat et al. (2014).

AGI SymbolVariation

Protein Encoded10 DAP 14 DAP

AT4G27140 SESA1 DOWN DOWN SEED STORAGE ALBUMIN1AT4G27160 SESA3 DOWN — SEED STORAGE ALBUMIN3At4G27170 SESA4 DOWN — SEED STORAGE ALBUMIN4AT5G54740 SESA5 DOWN DOWN SEED STORAGE ALBUMIN5AT5G44120 CRA1 DOWN — CRUCIFERINA; RmlC-like cupin superfamily proteinAT1G03880 CRU2 DOWN — CRUCIFERIN2; RmlC-like cupin superfamily proteinAT1G03890 AT1G03890 DOWN DOWN RmlC-like cupin superfamily proteinAT2G28490 AT2G28490 DOWN — RmlC-like cupin superfamily proteinAT2G43120 AT2G43120 — DOWN RmlC-like cupin superfamily proteinAT3G04150 AT3G04150 — DOWN RmlC-like cupin superfamily proteinAT3G22640 PAP85 DOWN — RmlC-like cupin superfamily proteinAT4G36700 AT4G36700 DOWN — RmlC-like cupin superfamily proteinAT5G15120 AT5G15120 DOWN — RmlC-like cupin superfamily proteinAT5G39110 AT5G39110 — UP RmlC-like cupin superfamily proteinAT5G40420 OLEO2 DOWN — OLEOSIN2AT2G25890 OLEO6 DOWN DOWN OLEOSIN6AT3G01570 OLEO7 DOWN — OLEOSIN7AT5G61610 OLEO11 — DOWN OLEOSIN11AT5G07510 GRP14 DOWN DOWN GLY-RICH PROTEIN14AT5G07520 GRP18 DOWN — GLY-RICH PROTEIN18AT4G26740 ATS1 DOWN — SEED GENE1/PEROXYGENASE1/CALEOSIN1AT5G55240 PGX2 DOWN DOWN PEROXYGENASE2/CALEOSIN2AT2G33380 RD20 — DOWN RESPONSIVE TO DESICCATION20/CALEOSIN3AT1G70670 CLO4 — DOWN CALEOSIN4AT1G70680 AT1G70680 — DOWN Caleosin-related family proteinAT5G50600 HSD1 DOWN DOWN HYDROXYSTEROID DEHYDROGENASE1AT5G50770 HSD6 DOWN — HYDROXYSTEROID DEHYDROGENASE6AT1G05510 AT1G05510 — DOWN Naphthalene 1,2-dioxygenase subunit alpha (duf1264)AT2G18370 AT2G18370 — DOWN Bifunctional inhibitor/lipid-transferprotein/seed storage 2S albumin

superfamily proteinAT4G22610 AT4G22610 — DOWN Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin

superfamily proteinAT5G22500 FAR1 — DOWN FATTY ACID REDUCTASE1AT3G44540 FAR4 — DOWN FATTY ACID REDUCTASE4AT3G44550 FAR5 — DOWN FATTY ACID REDUCTASE5AT5G49900 AT5G49900 — DOWN Beta-glucosidase, GBA2 type family proteinAT5G57800 CER3 — DOWN Fatty acid hydroxylase superfamilyAT5G58860 CYP86A1 — DOWN Cytochrome P450, family 86, subfamily A, polypeptide 1AT4G00360 CYP86A2 — DOWN Cytochrome P450, family 86, subfamily A, polypeptide 2AT1G01600 CYP86A4 — DOWN Cytochrome P450, family 86, subfamily A, polypeptide 4AT5G23190 CYP86B1 — DOWN Cytochrome P450, family 86, subfamily B, polypeptide 1AT5G08250 AT5G08250 — DOWN Cytochrome P450 superfamily proteinAT1G08920 ESL1 — DOWN ERD (EARLY RESPONSE TO DEHYDRATION) SIX-LIKE 1AT1G22710 SUC2 DOWN — SUC-PROTON SYMPORTER2AT1G71890 SUC5 — UP Major facilitator superfamily proteinAT1G73220 OCT1 — DOWN ORGANIC CATION/CARNITINE TRANSPORTER1AT1G77210 STP14 — DOWN SUGAR TRANSPORTER14AT3G05160 AT3G05160 DOWN — Major facilitator superfamily proteinAT3G05165 AT3G05165 — DOWN Major facilitator superfamily proteinAT3G05400 AT3G05400 — DOWN Major facilitator superfamily proteinAT3G19930 STP4 — DOWN SUGAR TRANSPORTER4AT3G47420 G3Pp1 DOWN DOWN Putative glycerol-3-P transporter1AT5G26340 MSS1 — DOWN Major facilitator superfamily proteinAT3G48740 SWEET11 — DOWN Nodulin MtN3 family proteinAT4G25010 SWEET14 — DOWN Nodulin MtN3 family proteinAT5G13170 SAG29 DOWN DOWN SENESCENCE-ASSOCIATED GENE2AT5G23660 SWEET12 — DOWN Bidirectional sugar transporter SWEET12-like proteinAT4G23010 UTR2 — DOWN UDP-GAL TRANSPORTER2

(Table continues on following page.)





https://apps.araport.org/thalemine


SENESCENCE-ASSOCIATED GENE29), eight mem-bers of the major facilitator superfamily (EARLYRESPONSE TO DEHYDRATION SIX-LIKE 1, MSS1(a major facilitator superfamily protein), SUGARTRANSPORTER4, SUGAR TRANSPORTER14,AT3G05165, AT3G05400, AT3G47420, and OR-GANIC CATION/CARNITINE TRANSPORTER1),and six members of the aquaporin family (BETA-TONOPLAST INTRINSIC PROTEIN (TIP), PLASMAMEMBRANE INTRINSIC PROTEIN2;6, NOD26-'LIKE MAJOR INTRINSIC PROTEIN1;1, NOD26-LIKE MAJOR INTRINSIC PROTEIN1;2, TIP2;1,and TIP3;1). Several of these transcripts were alreadyreduced at 10 DAP (Table 1). In accordance witha possible reduction of sugar transport, a numberof transcripts encoding sugar metabolism enzymeswere less accumulated, notably Suc synthases, SUS2at 10 DAP, and SUS3, SUS5, and stachyose synthase,and trehalose-6-P synthase (TPS1, TPS8, and TPS10)at 14 DAP. Taken together, these observations were infavor of a positive role of ABA in reserve storage anddesiccation tolerance.

ABA Deficiency Positively Impacts on Photosynthesis andCell Cycle

In green seeds, such as in Arabidopsis, photosyn-thesis is active during seed maturation and has animportant contribution to germination vigor (Allorent

et al., 2015). Interestingly, among the up-regulatedtranscripts at 14 DAP, a strong enrichment in GOterms related to photosynthesis was observed in nced2569seeds compared to the other two genotypes, whereas asingle transcript related to these GOs was found differ-entially regulated at 10DAP (Table 2; Supplemental TableS5). Moreover, an important enrichment was also foundin GO terms related to plastid/chloroplast localization(113 transcripts in GO:0009507 and GO:0009536). In goodagreement with increased accumulation of transcriptsinvolved in light harvesting and photosynthesis, thetranscript encoding STAY GREEN2/NON-YELLOW-ING2, an enzyme of the chlorophyll degradation path-way, was down-regulated.

At the same developmental stage, an enrichmentwas also found in several GO terms related to celldivision (GO:0051301) and cell cycle (GO:0007049),the list of transcripts partially overlapped with tran-scripts related to the GO term tissue development(GO:0009888), for which an enrichment was also ob-served. Among these up-regulated transcripts, themost represented belonged to the cyclin family(Table 2; Supplemental Table S5).

ABA Deficiency Differentially Alters Signaling Pathways

At 10 DAP, among the small number of up-regulatedgenes, we found that three members of the PYR/PYL/RCAR family of ABA receptors, PYL2, PYL4, and PYL6,

Table 1. (Continued from previous page.)

AGI SymbolVariation


AT1G17810 BETA-TIP DOWN DOWN BETA-TONOPLAST INTRINSIC PROTEINAT4G19030 NIP1;1 — DOWN NOD26-LIKE MAJOR INTRINSIC PROTEIN1;1AT4G18910 NIP1;2 — DOWN NOD26-LIKE INTRINSIC PROTEIN1;2AT2G39010 PIP2;6 — DOWN PLASMA MEMBRANE INTRINSIC PROTEIN2;6AT4G35100 PIP3 — UP PLASMA MEMBRANE INTRINSIC PROTEIN3AT3G16240 TIP2;1 DOWN DOWN DELTA TONOPLAST INTEGRAL PROTEINAT1G73190 TIP3;1 DOWN — Aquaporin-like superfamily proteinAT1G52690 LEA7 — DOWN LATE EMBRYOGENESIS ABUNDANT7AT1G72100 LEA9 — DOWN Late embryogenesis abundant domain-containing proteinAT2G21490 LEA/LEA14 — DOWN DEHYDRIN LEAAT2G23110 LEA15 — DOWN Late embryogenesis abundant protein, group 6AT3G02480 LEA28 DOWN DOWN Late embryogenesis abundant protein (LEA) family proteinAT3G15670 LEA29 DOWN DOWN Late embryogenesis abundant protein (LEA) family proteinAT3G17520 LEA30 — DOWN Late embryogenesis abundant protein (LEA) family proteinAT3G50970 LTI30/LEA33 — DOWN LOW TEMPERATURE-INDUCED30AT3G50980 XERO1/ LEA34 — DOWN DEHYDRIN XERO1AT3G51810 EM1/LEA35 — DOWN LATE EMBRYOGENESIS ABUNDANT1AT4G21020 LEA42 DOWN DOWN Late embryogenesis abundant protein (LEA) family proteinAT5G06760 LEA4-5/LEA46 — DOWN LATE EMBRYOGENESIS ABUNDANT4-5AT5G44310 LEA48 DOWN DOWN Late embryogenesis abundant protein (LEA) family proteinAT5G53270 LEA50 — DOWN Seed maturation proteinAT5G66400 RAB18/LEA51 — DOWN RESPONSIVE TO ABA18AT1G17620 AT1G17620 — DOWN Late embryogenesis abundant (LEA) Hyp-rich glycoprotein familyAT4G01410 AT4G01410 — DOWN Late embryogenesis abundant (LEA) Hyp-rich glycoprotein familyAT5G45320 AT5G45320 — DOWN Late embryogenesis abundant proteinAT1G54540 AT1G54540 — DOWN Late embryogenesis abundant (LEA) Hyp-rich glycoprotein familyAT5G66780 AT5G66780 — DOWN Late embryogenesis abundant protein


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Table 2. Differentially expressed transcripts encoding proteins involved in photosynthesis and cell cycle

The proteins were mostly up-regulated at 14 DAP in nced2569 compared to either wild type or nced259 (.1.5-fold; Supplemental Tables S2 andS4). Gene and protein names are indicated according to https://apps.araport.org/thalemine.

AGI SymbolVariation


AT1G31330 PSAF — UP PHOTOSYSTEM I SUBUNIT FAT1G45474 LHCA5 — UP PHOTOSYSTEM I LIGHT HARVESTING COMPLEX PROTEIN5AT1G48510 AT1G48510 — UP Surfeit locus 1 cytochrome c oxidase biogenesis proteinAT1G50900 GDC1 — UP GRANA DEFICIENT CHLOROPLAST1AT1G52220 AT1G52220 — UP Curvature thylakoid proteinAT1G52230 PSAH2 — UP PHOTOSYSTEM I SUBUNIT H2AT1G54500 AT1G54500 — UP Rubredoxin-like superfamily proteinAT1G54780 TLP18.3 — UP THYLAKOID LUMEN PROTEIN18.3AT1G60600 ABC4 — UP ABERRANT CHLOROPLAST DEVELOPMENT4AT1G60950 FED A — UP 2Fe-2S ferredoxin-like superfamily proteinAT1G76100 PETE1 — UP PLASTOCYANIN1AT1G76570 LHCB7 — DOWN LIGHT-HARVESTING COMPLEX B7AT1G80480 PTAC17 — UP PLASTID TRANSCRIPTIONALLY ACTIVE17AT2G05100 LHCB2.1 — UP PHOTOSYSTEM II LIGHT HARVESTING COMPLEX

PROTEIN2.1AT2G26670 TED4 — DOWN REVERSAL OF THE DET PHENOTYPE4AT2G28605 AT2G28605 — UP Photosystem II reaction center PsbP family proteinAT2G40100 LHCB4.3 — UP LIGHT HARVESTING COMPLEX PHOTOSYSTEM IIAT2G42310 AT2G42310 — UP ESSS subunit of NADH:ubiquinone oxidoreductase (complex I)AT2G46820 PSI-P — UP PHOTOSYSTEM I P SUBUNITAT2G47450 CAO — UP CHAOSAT2G47940 DEG2 — UP DEGRADATION OF PERIPLASMIC PROTEINS2AT3G04550 AT3G04550 — UP Rubisco accumulation factor-like proteinAT3G22840 ELIP1 — DOWN EARLY LIGHT-INDUCIBLE PROTEINAT3G27690 LHCB2.3 — UP PHOTOSYSTEM II LIGHT HARVESTING COMPLEX

PROTEIN2.3AT3G47470 LHCA4 — UP LIGHT-HARVESTING CHLOROPHYLL-PROTEIN COMPLEX I

SUBUNIT A4AT3G50820 PSBO2 — UP PHOTOSYSTEM II SUBUNIT O-2AT3G56090 FER3 — UP FERRITIN3AT3G59400 GUN4 — UP GENOMES UNCOUPLED4AT4G01150 AT4G01150 — UP Curvature thylakoid 1A-like proteinAT4G04640 ATPC1 — UP ATPase, F1 complex, gamma subunit proteinAT4G05180 PSBQ-2 — UP PHOTOSYSTEM II SUBUNIT Q-2AT4G10340 LHCB5 — UP LIGHT HARVESTING COMPLEX OF PHOTOSYSTEM II 5AT4G11910 AT4G11910 — DOWN STAY-GREEN-like proteinAT4G21280 PSBQA — UP PHOTOSYSTEM II SUBUNIT QAAT4G27440 PORB — UP PROTOCHLOROPHYLLIDE OXIDOREDUCTASE BAT4G37925 NDHM UP — NADH DEHYDROGENASE-LIKE COMPLEX MAT5G01530 LHCB4.1 — UP LIGHT HARVESTING COMPLEX PHOTOSYSTEM IIAT5G28500 AT5G28500 — UP Rubisco accumulation factor-like proteinAT5G45040 CYTC6A — UP CYTOCHROME C6AAT5G51010 AT5G51010 — UP Rubredoxin-like superfamily proteinAT5G52970 AT5G52970 — UP Thylakoid lumen 15.0 kD proteinAT5G54270 LHCB3 — UP LIGHT-HARVESTING CHLOROPHYLL B-BINDING PROTEIN3AT5G55710 TIC20-V — UP TRANSLOCON AT THE INNER ENVELOPE MEMBRANE OF

CHLOROPLASTS 20-VAT5G61410 RPE — UP D-RIBULOSE-5-PHOSPHATE-3-EPIMERASEAT5G66570 PSBO1 — UP PS II OXYGEN-EVOLVING COMPLEX1AT1G15570 CYCA2;3 — UP CYCLIN A2;3AT1G20610 CYCB2;3 — UP CYCLIN B2;3AT1G20930 CDKB2;2 — UP CYCLIN-DEPENDENT KINASE B2;2AT1G26870 FEZ — UP NAC (No Apical Meristem) domain transcriptional regulator

superfamily proteinAT1G34460 CYCB1;5 — UP CYCLIN B1;5AT1G44110 CYCA1;1 — UP CYCLIN A1;1AT1G50490 UBC20 — UP UBIQUITIN-CONJUGATING ENZYME20AT1G65470 FAS1 — UP FASCIATA1

(Table continues on following page.)






https://apps.araport.org/thalemine


were up-regulated by ;2-, 2.4-, and 1.5-fold, respec-tively, in comparisons of nced2569 with either wildtype or nced259 (Supplemental Table S2). At 14 DAP,only PYL4 transcript was found significantly up-regulated (;2-fold; Supplemental Table S4). Thissuggested that reduction in ABA levels may becompensated to some extent by an increased sensi-tivity, thanks to overexpression of these three PYR/PYL/RCAR receptor genes. Moreover, the transcriptencoding BG1, the b-glucosidase (BGLU18) convert-ing ABA-GE into ABA, was also 2-fold up-regulated(Supplemental Table S4), suggesting that ABA defi-ciency may activate ABA release from stored conju-gated forms. Expression of these four genes at 14DAP was tested by quantitative PCR (qPCR), whichconfirmed that PYL4 was the most up-regulatedgene (Fig. 4). Despite Complete Arabidopsis Tran-scriptomeMicro Array (CATMA) analysis detecting asignificantly increased accumulation of PYL2 andPYL6 transcripts only at 10 DAP (FDR-adjusted Pvalue , 0.05), increased expression of these geneswas also observed at 14 DAP by qPCR. Interestingly,accumulation of transcripts encoding several PP2Cproteins, including ABI2, PP2CA/ABA HYPERSEN-SITIVE GERMINATION3, and HIGHLY ABA-INDUCED3, was reduced up to 2-fold in nced2569 at14 DAP (Supplemental Table S3). Concomitantly,expression of DOG1, which protein has been shownto interact with PP2C, was similarly reduced, to-gether with its related DOG-like genes (DOGL2,DOGL3, and DOGL4). Furthermore, a lower accu-mulation of ABI5 transcripts, which encode a majorABA-regulated transcription factor downstream ofSnRK2 kinase, was also observed, in good correlationwith that of its well-known targets, such LEA35/EM1(Table 1; Carles et al., 2002).

A number of other transcription factors were also dif-ferentially regulated at 10 and 14 DAP in nced2569 seedscompared to wild type (Supplemental Tables S1–S4). At

10 DAP, transcripts encoding six transcription factorswere up-regulated in nced2569 (MYB13/AT1G06180,RELATED TO ABI3/VIVIPAROUS1 2/AT1G68840,AGAMOUS-like22/SHORT VEGETATIVE PHASE/AT2G22540, CYTOKININ RESPONSE FACTOR5/AT2G46310, ABERRANT TESTA SHAPE/AT5G42630,and MADS AFFECTING FLOWERING 5/AT5G65080).In contrast, nine transcripts encoding proteins involved intranscriptional regulation were down-regulated (Ethylene-responsive transcription factor ERF023/AT1G01250,CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 5/AT1G26260, No apical meristemdomain containing protein NAC019/ANAC019/AT1G52890, WRKY1/AT2G04880, FERTILIZATIONINDEPENDENT SEED 2/AT2G35670, REPRESSOROF SILENCING 4/AT3G14980, NAC2/ANAC056/AT3G15510, OXIDATION-RELATED ZINC FINGER2/AT4G29190, and RELATED TO AP2 10/AT4G36900). Previous works showed that ANAC019acts as a positive regulator of ABA signaling (Jensenet al., 2010). The oilseed rape (Brassica napus) Bna-NAC56 transcription factor, the ortholog gene ofANAC056, has been shown to be induced by ABAand jasmonic acid (Chen et al., 2017). Another tran-scription factor, the CCCH zinc finger proteingene, OXIDATION-RELATED ZINC FINGER 2/AT4G29190, is also involved in ABA and jasmonic acidresponses (Lee et al., 2012). The ABA deficiency appears tonegatively affect defense pathways. Indeed, WRKY1/AT2G04880 involved in the salicylic acid signalingpathwaywas also down-regulated in nced2569 developing seeds.

Among 64 transcription factors up- or down-regulated at 14 DAP, at least 29 of them were de-scribed to be involved in hormonal responses(GO:0009725). In accordance with the tight interactionbetween the ABA and ethylene signaling pathways(Corbineau et al., 2014), transcription factors involvedin responses to these hormones were the most repre-sented. Nevertheless, ABA deficiency broadly affected

Table 2. (Continued from previous page.)

AGI SymbolVariation


AT1G76310 CYCB2;4 — UP CYCLIN B2;4AT1G78770 APC6 — UP ANAPHASE PROMOTING COMPLEX6AT2G26760 CYCB1;4 — UP CYCLIN B1;4AT2G45490 AUR3 — UP ATAURORA3AT3G19050 POK2 — UP PHRAGMOPLAST ORIENTING KINESIN2AT3G19590 BUB3.1 — UP BUB (BUDDING UNINHIBITED BY BENZYMIDAZOL)3.1AT3G57060 AT3G57060 — UP Binding proteinAT3G57860 UVI4-LIKE — UP UV-B-INSENSITIVE4-LIKE PROTEINAT4G05190 ATK5 — UP KINESIN5AT4G31805 AT4G31805 — UP WRKY family transcription factorAT4G33260 CDC20.2 — UP CELL DIVISION CYCLE20.2AT4G34160 CYCD3;1 — UP CYCLIN D3;1AT4G37490 CYCB1;1 — UP CYCLIN B1;1AT5G13840 FZR3 — UP FIZZY-RELATED3AT5G18700 AT5G18700 — UP Kinase family with ARM repeat domain-containing proteinAT5G48600 SMC3 — UP STRUCTURAL MAINTENANCE OF CHROMOSOME3AT5G50375 CPI1 — UP CYCLOPROPYL ISOMERASE


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the expression of other hormone signaling components(Table 3).

Protein Accumulation in Dry Seeds Correlates Well withTranscriptome Variations in Developing Seeds

To get a deeper insight into the biological functionsaffected by ABA content in developing seeds, proteo-mics and metabolomics were carried out on dry seeds.Because the transcriptome of nced569 appeared similarto that of thewild type, themore contrasting genotypes,nced2569, cyp707a1a2, and wild type, were chosen toassess the impact of altered ABA levels on the globalproteome and metabolome in dry seeds.Shotgun liquid chromatography-tandem mass spec-

trometry (LC-MS/MS) proteomics led to the identifi-cation of .1,500 proteins, among which 838 wereaccurately quantified based on extracted ion currents(Valot et al., 2011). Statistical analysis revealed the dif-ferential accumulation of 120 proteins, among which 68displayed a higher abundance in nced2569 seeds com-pared to either wild type or cyp707a1a2 (.1.3-fold, one-wayANOVA, P, 0.05; Supplemental Table S6). A largenumber of them (63) were more abundant in nced2569compared to cyp707a1a2, with 48 more abundant innced2569 versus wild type. Moreover, most of themwere also more abundant in wild type compared tocyp707a1a2. On the other hand, 46 proteins were moreabundant in cyp707a1a2 compared to nced2569 mutantand 15 were more abundant in cyp707a1a2 compared towild type (.1.3-fold, one-way ANOVA, P , 0.05;Supplemental Table S7). Interestingly, 25 of these pro-teins were significantly less abundant in nced2569 thanin wild type.Proteome modifications observed in mature dry

seeds were in good accordance with transcriptome

variations described above in developing seeds. In-deed, similar enrichments were found in GO terms,notably those associated with biological func-tions and metabolic pathways (Fig. 5; SupplementalTable S5). Among up-regulated proteins in nced2569,enrichments were found for GO terms related tometabolic processes. The strong enrichment in sev-eral GO terms associated with carbon fixation andrelated carbohydrate metabolism was in good corre-lation with that observed for Kyoto Encyclopedia ofGenes and Genomes (KEGG) pathways related toCalvin cycle and photosynthesis (KEGG:00710),pentose P pathway (KEGG:00030), glycolysis/gluconeogenesis (KEGG:00010), and Fru and Man me-tabolism (KEGG:00051). Moreover, among proteinssignificantly more abundant in nced2569, enrich-ment was observed in GO terms related to organicacid metabolic processes and FA metabolism. Inter-estingly, three enzymes involved in fatty acid(FA) b-oxidation showed a higher abundance innced2569, such as the peroxisomal acyl-CoA oxidase3(ACX3/AT1G06290), enoyl-CoA hydratase (ECHIA/AT4G16210), multifunctional protein2 (MFP2/AT3G06860), and NAD(P)-binding Rossmann-foldsuperfamily protein (AT1G24360), which could beassociated with the induction of key enzymes in-volved in reserve remobilization, namely isocitratelyase (ICL)/AT3G21720 and phosphoenolpyruvatecarboxykinase 1/AT4G37870, suggesting that the oilreserve remobilization previously reported duringlate maturation in oilseed rape and Arabidopsis seeds(Eastmond and Rawsthorne, 2000; Baud et al., 2002)was activated in nced2569. A parallel conclusion canbe drawn from the differential accumulation of pro-teins involved in glycolysis and/or gluconeogenesispathways, such as glyceraldehyde-3-P dehydrogen-ase B subunit (GAPB/AT1G42970), triosephosphate

Figure 4. ABA deficiency increases expressionof ABA receptors and BG1. Relative expressionlevels of PYL2, PYL4, PYL6, and BG1 in de-veloping seeds dissected from mutant andwild-type siliques at 14 DAP. The values aremeans of three biological replicates, presentedwith SE values. Expression levels were nor-malized with those of EF1a4 (At5g60390) andAt4g12590 reference genes. Significant differ-ences compared to wild type were analyzedby Student’s t test (*P , 0.05, **P , 0.01,***P , 0.001). WT, wild type.









isomerase (TIM/AT2G21170), Fru-bisphosphate al-dolase2 (FBA2/AT4G38970), Fru-1,6-bisphosphatase(FBP/AT1G43670), and high cyclic electron flow1(HCEF1/AT3G54050).

Among proteins up-regulated in cyp707a1a2 com-pared to the other genotypes, enrichment wasfound for GO terms related to seed development,reserve storage, and stress responses. These func-tions confirmed the central role of ABA in the con-trol of late maturation programs. In particular,abundance of four members of RmlC-like cupins su-perfamily (AT5G44120/CRU1/CRA1, AT1G03880/CRU2, AT2G18540, and AT2G28490) and albumin(AT5G54740/SESA5) was higher in cyp707a1a2 com-pared to nced2569 seeds. Moreover, the abundanceof cruciferin CRU1/CRA1 was also higher incyp707a1a2 seeds compared to the wild type. Fur-thermore, 10 LEA proteins were more abundantin cyp707a1a2 mutants (Supplemental Table S7).AT1G47980, annotated as a desiccation-like protein,

was similarly differentially accumulated (2.7-fold)in the two mutants.

Glucosinolate catabolism enzymes, nitrile specifierprotein2 (NSP2/AT2G33070) and thioglucoside gluco-hydrolase1 (TGG1/AT5G26000), were also found dif-ferentially accumulated. Strikingly, these two proteinswere respectively more abundant in nced2569 andcyp707a1a2 (Fig. 5; Supplemental Tables S6 and S7).TGG1 transcript was also overexpressed in nced2569seeds at 14 DAP (Supplemental Table S4). Glucosino-lates are sulfur- and nitrogen-containing secondarymetabolites. Their hydrolysis by myrosinases promotethe release of defense-related compounds, such as thi-ocyanate, cyanate, or nitrile. However, it has been alsosuggested that glucosinolates could act as sulfur- andnitrogen-sources, notably upon seed imbibition (Gallandet al., 2014). Thus, the accumulation of these proteinsmay indicate a premature resumption of metabolism innced2569 seeds, which would contribute to germinationactivation upon seed imbibition.

Table 3. Differentially expressed transcripts encoding proteins involved in hormone response pathways

The proteins were either up- or down-regulated in nced2569 compared to either wild type or nced259 (.1.5-fold at 14 DAP; Supplemental TablesS3 and S4).

AGI Symbol Variation GO Terms

AT1G04250 AXR3 UP Response to auxin; auxin-activated signaling pathwayAT1G26870 FEZ UP Response to auxinAT4G16780 HB-2 UP Response to auxin and to cytokininAT5G39860 PRE1 UP Gibberellic acid-mediated signaling pathway; response to brassinosteroid;

brassinosteroid-mediated signaling pathwayAT5G17490 RGL3 UP Gibberellic acid-mediated signaling pathway; response to gibberellin; negative

regulation of gibberellic acid-mediated signaling pathway; response toethylene; jasmonic acid-mediated signaling pathway; response to abscisicacid

AT5G25190 ESE3 UP Ethylene-activated signaling pathwayAT2G44840 ERF13 UP Ethylene-activated signaling pathwayAT5G65510 AIL7 UP Ethylene-activated signaling pathway; auxin-mediated signaling pathway

involved in phyllotactic patterningAT3G23050 IAA7 DOWN Response to auxin; auxin-activated signaling pathway; response to jasmonic acidAT5G17300 RVE1 DOWN Auxin-activated signaling pathwayAT1G79700 WRI4 DOWN Ethylene-activated signaling pathwayAT3G16280 AT3G16280 DOWN Ethylene-activated signaling pathwayAT3G50260 CEJ1 DOWN Ethylene-activated signaling pathwayAT3G54990 SMZ DOWN Ethylene-activated signaling pathwayAT4G06746 RAP2.9 DOWN Ethylene-activated signaling pathwayAT4G39780 AT4G39780 DOWN Ethylene-activated signaling pathwayAT3G61630 CRF6 DOWN Ethylene-activated signaling pathway; cytokinin-activated signaling pathwayAT1G13960 WRKY4 DOWN Response to ethylene; response to jasmonic acidAT3G15500 NAC3 DOWN Jasmonic acid mediated signaling pathwayAT1G61660 AT1G61660 DOWN Cellular response to abscisic acid stimulusAT1G01720 ATAF1 DOWN Negative regulation of abscisic acid-activated signaling pathwayAT1G17950 MYB52 DOWN Response to abscisic acidAT2G35940 BLH1 DOWN Response to abscisic acidAT2G46270 GBF3 DOWN Response to abscisic acidAT4G28110 MYB41 DOWN Response to abscisic acidAT5G65310 HB5 DOWN Response to abscisic acid; abscisic acid-activated signaling pathwayAT2G36270 ABI5 DOWN Response to abscisic acid; abscisic acid-activated signaling pathway; response to

gibberellinAT5G37260 RVE2 DOWN Response to abscisic acid; response to gibberellin; response to ethylene;

response to auxin; response to jasmonic acidAT2G36890 RAX2 DOWN Response to abscisic acid; response to gibberellin; response to jasmonic acid


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Figure 5. Differentially accumulated proteins and metabolites in dry seeds of ABA metabolism mutants. Proteins and metabo-lites involved in photosynthesis, glycolysis/gluconeogenese, energy, and amino acid and glucosinolate metabolism incyp707a1a2, wild type, and nced2569. ABA-regulated proteins are displayed within rectangles; metabolites are shown in blue.1,3-BPG, Glycerate-1,3-bisphosphate; 1,6 FBP, Fru-1,6-bisphosphate; 2-PGA, Glycerate-2-P; 3-PGA, glycerate-3-P; 4MTB,





Metabolite Analysis Reveals the Impact of ABA Levels onAmino Acid, Sugar, and Glucosinolate Contents

Themetabolomic analysis of nced2569, cyp707a1a2, andwild-type dry seeds was carried out using gaschromatography-MS analysis. In total, 274 metaboliteswere identified based on their retention times and theirmass spectra and a differential accumulation of 74 me-tabolites was detected among the three genotypes (one-way ANOVA, P, 0.05, Supplemental Tables S8 and S9).

Metabolomic analysis revealed a positive effect ofABA-deficiency on the accumulation of free primarymetabolites during late maturation (Fig. 5). Indeed,44 metabolites showed a higher level in nced2569compared to cyp707a1a2, among which 15 metabo-lites were also significantly more abundant com-pared to wild type (Supplemental Table S8). Aminoacids, organic acids, and sugars were predominantlyrepresented among these metabolites. Indeed, 11 ofthe 16 quantified amino acids, notably branched-chain amino acids (Val, Leu, and Ile) and shikimatepathway-related amino acids (Trp, Phe, and Tyr),displayed higher abundance in nced2569 and wildtype than in cyp707a1a2. Organic acids, as key inter-mediates of tricarboxylic acid (TCA)/glyoxylate cy-cles (namely citrate, aconitate, fumarate, and malate),were also more abundant in nced2569. In agreementwith the up-regulation of beta-oxidation and glyox-ylate cycle related enzymes, this observation sug-gested an increase of FA-derived carbon skeletonfluxes into energetic and remobilization pathways.

In good correlation with transcriptomics data, a num-ber of metabolites related to carbohydrate metabolismwere negatively impacted by ABA-deficiency, notablyGal metabolism-related sugars (e.g. Gal, galactinol,galactiosylglycerol, Fru, Suc, and myo-inositol). Sev-eral of these metabolites belong to the raffinose familyoligosaccharides (RFO; namely stachyose, raffinose,myo-inositol, galactinol, Suc, and Gal). RFO synthesisis closely linked to the acquisition of desiccation tol-erance during seed maturation (Bailly et al., 2001).Whereas galactinol, myo-inositol, and Gal displayedhigher abundance in nced2569, raffinose levels were

higher in cyp707a1a2 seeds, suggesting a differentialfine-tuning of RFO biosynthesis by ABA content.

In contrast, 21 metabolites showed a higher level incyp707a1a2 compared to both nced2569 and wild type(Supplemental Table S9). Interestingly, these includedMet-derived aliphatic glucosinolates and some of theirbreakdown products, e.g. 5-methylthio-pentanenitrile,6-methylthio-hexanenitrile, 4-methylthiobutylglucosinolate(4-MTB), and 5-methylthiopentylglucosinolate (5-MTP). Together with the differential accumulation ofproteins related to glucosinolate breakdown (Fig. 5;Supplemental Tables S5 and S6), these results furthersuggested a regulatory role of ABA on glucosinolatemetabolism.

Seed ABA Content Influences Oxidation Events

Reactive oxygen species (ROS) and nitric oxide(NO) have been reported to play pivotal roles in theregulation of seed dormancy and seed germination(Bailly et al., 2008; Arc et al., 2013). Some clues indi-cated that nced2569 mutant dry seeds had to copewith oxidative stress because antioxidant compoundsnamely, alpha- and gamma-tocopherols, were moreabundant (Supplemental Table S8). In the vitaminE group, alpha-tocopherol was reported as the mostefficient free radical trap. In addition, gamma-tocopherol was described to react with nitrogendioxide (NO2), resulting in NO production (Cooneyet al., 1993). Consistently, transcriptome data showedthat the gene expression of HAEMOGLOBIN 2/AT3G10520 encoding the nonsymbiotic hemoglobin2 was promoted in nced2569 developing seeds at 10and 14 DAP, indicating an amplified NO response.Thus, hydrogen peroxide (H2O2), superoxide anion(O2°2), and NO released by imbibed seeds wereassayed and our results supported the assumptionthat ABA-deficient seeds produced more radicalsthan overaccumulating seeds (Fig. 6). Indeed, nced2569seeds released more H2O2, O2°2, and NO thancyp707a1a2 seeds, whereas wild type was intermedi-ate. Dry seed protein oxidation profiles were also

Figure 5. (Continued.)4-methylthiobutyl glucosinolate; 5MTP, 5-methylthiopentyl glucosinolate; 5MTPN, 5-methylthiopentanenitrile glucosinolatebreakdown product; 6MTHN, 6-methylthiohexanenitrile glucosinolate breakdown product; a-KG, a-ketoglutarate (2-oxoglu-tarate); ACX3, Acyl-CoA oxidase3; ASP2, Asp aminotransferase2; AT1G24360, NAD(P)-binding Rossmann-fold superfamilyprotein; AT2G22230, Thioesterase superfamily protein; AT3G58610, Ketol-acid reductoisomerase; AT3G60750, Transketolase;CAC3, acetyl Coenzyme a carboxylase carboxyltransferase alpha subunit; CAT3, Catalase3; DHA, Dehydroascorbate; DHAP,dihydroxyacetone P; E-4P, Erythrose-4-P; ECHIA, Enoyl-CoA hydratase/isomerase A; FBA2, Fru-bisphosphate aldolase2; FBP, Fru-1,6-bisphosphatase; Fru-6P, Fru-6-P; GABA, g-aminobutyric acid; Glc-6P, Glc-6-P; Glycerol-3P, Glycerol-3-P; GSH, Glutathi-one; GSL, Glucosinolate; GSSG, Glutathione disulfide (oxidized glutathione); HCEF1, High cyclic electron flow1 (chloroplasticFru 1,6-bisphosphate phosphatase); MAML-4, Methylthioalkylmalate synthase-like4; MDAR1/6, Monodehydroascorbate re-ductase1/6; MDHA, Monodehydroascorbate; MPF2, Multifunctional protein2; NSP2, nitrile specifier protein2; OAA, Oxalo-acetate; OPHS, O-phospho-l-homo-Ser; PCK1, Phosphoenolpyruvate carboxykinase1; PEP, Phosphoenolpyruvate; PGAL,Glyceraldehyde 3-P; PSBO1, PS II oxygen-evolving complex1; R-5P, Ribose-5-P; RBCL, Ribulose-bisphosphate carboxylase largechain; RBCS1A, Ribulose bisphosphate carboxylase small chain1A; Ru-5P, Ribulose-5-P; Ru-BP, Ribulose-1,5-bisphosphate; S-7P, Sedoheptulose-7-P; SBP, Sedoheptulose-1, 7-bisphosphate; SBPASE, Sedoheptulose-bisphosphatase; TAG, Triacylglyceride;TGG1, Thioglucoside glucohydrolase1; TIM, Triosephosphate isomerase; Xu-5P, Xylulose 5-P AT1G24360, NAD(P)-bindingRossmann-fold superfamily protein.


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assessed by detection of carbonyl groups on alpha12S-cruciferin subunits using Anti-2,4-dinitrophenylhy-drazone (DNP) immunoassay (Fig. 6A). Alpha 12S-cruciferin subunits have been previously described asthe most sensitive proteins to carbonylation in Arabi-dopsis dry seeds (Job et al., 2005). Our results showed ahigher protein oxidation in nced2569 than in wild-typeseeds. In contrast, the rate of protein oxidation was verylow in cyp707a1a2 seeds, suggesting a negative correlationbetween ABA content and protein oxidation.Furthermore, it is worth noting that our proteomic

results also showed that several proteins involvedin antioxidant processes were more abundant innced2569 dry seeds (Fig. 5), highlighting the inductionof protective mechanisms and influence of ABA levelson ROS production and scavenging. ABA deficiencyhad a positive impact on the abundance of catalase3(CAT3/AT1G20620) and monodehydroascorbate re-ductases1 and 6 (MDAR1/AT3G52880 and MDAR6/AT1G63940). MDARs function as key enzymes ofmonodehydroascorbate recycling into ascorbic acid.Ascorbic acid plays an important role in the protectionagainst ROS and is involved in the reduction of toco-pheroxyl radical into alpha-tocopherol to prevent oxi-dative damage of cellular membranes.

DISCUSSION

NCED6 Expression in the Endosperm Has a MajorContribution to ABA Accumulation in Developing Seeds

ABA accumulation in Arabidopsis developing seedsand siliques has been previously shown to peak atmidmaturation, as observed here (Karssen et al., 1983;

Okamoto et al., 2006; Kanno et al., 2010). However, itsorigin has not been fully established. From crosses be-tween an ABA-deficient aba1 female and wild-typemale, genetic studies concluded that at early matura-tion stages ABA was synthesized in maternal tissuesand at later stages in zygotic tissues (Karssen et al.,1983). More recently, an in-depth analysis of ABAcontent in seeds dissected from siliques of F1 (aba2-2female and wild-type male) plants indicated that ABAwas produced by zygotic tissues. However, measure-ment of ABA contents in single F2 seeds from this crosssuggested dual maternal and zygotic origins (Okamotoet al., 2010). Maternal ABA can be provided by eithervegetative or reproductive (silique/testa) tissues. Asobserved here and previously reported (Okamoto et al.,2010), ABA accumulation in silique envelopes is low.However, the presence of DPA in these tissues suggestsactive ABA synthesis and catabolism, and ABA trans-location to seeds cannot be excluded.NCED3 has been described to contribute to ABA

synthesis in vegetative tissues, and be essential in waterstress responses (Tan et al., 2003; Urano et al., 2009).Upon drought induction, both transcript and proteinwere specifically detected in vascular tissues (Endoet al., 2008), suggesting a key role for NCED3 in ABAsupply to other tissues. However, the very low levels ofABA in isolated seeds at 10 and 14 DAP and both ABAand catabolites in nced2569 siliques suggest that ABAproduction from NCED3 activity and transport fromvegetative to reproductive tissues seeds is marginal.Furthermore, in agreement with its low expression,NCED3 does not significantly contribute to ABAsynthesis inside seeds. Among the five NCEDs,NCED6 transcripts are the most abundant in seeds

Figure 6. Seed ABA content influences oxidationevents. A, Detection of carbonylated proteins indry seeds: Coomassie blue protein staining of al-pha 12S-cruciferin subunits (top) and anti-DNPimmunoassay of alpha 12S-cruciferin subunits(bottom). Three biological replicates were ana-lyzed with similar results. B–D, O2°2 (B), H2O2

(C), and NO (D) released by imbibed seeds. Threebiological replicates were analyzed and mea-surements were performed using five seed sam-ples per biological replicate. Box plots weredrawn for groups of ROS and NO measurementsfor each genotype. The middle “grayed box”represents themiddle 50%of values for the group.The median marks the midpoint of the data and isindicated by the black line. Box plots display thedata distribution through their quartiles andwhiskers are used to indicate variability outsidethe upper and lower quartiles. DSW, dry seedweight before imbibition; WT, wild type.





and specifically detected in the endosperm from fer-tilization to maturation stages (Lefebvre et al., 2006)and NCED6 together with NCED5 and NCED9 wereshown to have a major role in dormancy regulationby ABA. Unexpectedly, comparison of ABA levels innced2569, nced259, and wild type strongly suggeststhat NCED6 activity in endosperm is responsible formost of the xanthoxin production that will give rise toABA in developing seeds. It also provides furtherevidence that zygotic tissues, and in particular en-dosperm, are likely the major source of ABA dur-ing seed maturation. Nevertheless, because nced6mutants exhibit only mild dormancy phenotypes(Lefebvre et al., 2006), ABA synthesis in restrictedembryo tissues is certainly essential for dormancyinduction.

ABA Catabolite Profiles Suggest New Routes in the ABAMetabolism Pathway

A previous study demonstrated that the CYP707Aenzyme is responsible for ABA hydroxylation at theC-89 and C-99 positions (Okamoto et al., 2011). In-deed, a decrease in both DPA and neoPA, the re-spective end-products of the 89- and 99-hydroxylationpathways, was observed in cyp707a1a3-developingsiliques and water-stressed plants compared to wildtype. Furthermore, ABA-GE and 79-OH-ABA levelswere higher in cyp707a1a3 seeds than in wild type, asobserved here in cyp707a1a2 seeds. ABA-GE has beendescribed as a storage form of ABA, the remobiliza-tion of which is under environmental control invegetative tissues (Lee et al., 2006; Xu et al., 2012;Ondzighi-Assoume et al., 2016). Because the expres-sion of the b-glucosidase gene BG1 was detected indeveloping seeds, this conjugate could be a reusableform of ABA also in seeds. In contrast to ABA-GE and99-OH-ABA, 79-OH-ABA synthesis remains obscure.Because its accumulation was observed here incyp707a1a2, as previously in cyp707a1a3 (Okamotoet al., 2011), it is unlikely to be a side product of 89-OH-hydroxylase activity. Nevertheless, it may have,together with ABA-GE, a role in removal of ABAexcess.

A very surprising observation was the accumulationof ABA-GE in aba2 mutant. Several studies suggestedthe existence of minor routes for ABA production fromxanthoxin, as reviewed by Endo et al. (2014). In tomato(Solanum lycopersicum) and Tex-Mex tobacco (Nicotianaplumbaginifolia) mutants defective for the conversion ofabscisic aldehyde into ABA, production of abscisic al-cohol has been observed, the conversion of which intoABA results in less severe phenotypes. In aba2mutants,residual ABA levels were found and the involvement ofnonspecific short-chain dehydrogenases/reductaseshas been hypothesized. Finally, the production of ABAfrom xanthoxin via xanthoxic acid has also been en-visaged as a possible minor route. The biosynthesissteps that would form ABA-GE from xanthoxin remain

to be investigated and the subsequent question iswhether this conjugate can be a source of ABA in thismutant. Because BG1 expression is detected at highlevels in developing seeds, starting from preglobularstage (http://www.bar.utoronto.ca), the existence ofthis alternative pathway may favor seed developmentand explain the residual dormancy of aba2 seeds.

ABA Differentially Regulates Specific Components of ItsOwn Signaling Pathway

ABA treatment has been described to down-regulateexpression of a majority of PYR/PYL/RCAR genes andoppositely up-regulate most PP2C (Santiago et al.,2009; Szostkiewicz et al., 2010; Gonzalez-Guzmanet al., 2012). In accordance, we found that PYL2,PYL4, and PYL6 expression was lower in wild typethan in nced2569 seeds at 10 and 14 DAP. Moreover,seed transcriptome available from globular embryoto mature green stages for PYR1 and PYL1 to PYL9(http://www.bar.utoronto.ca) and here for PYR1and PYL1 to PYL13, at 10 and 14 DAP, showed de-tectable expression levels for most of them. How-ever, only three were significantly up-regulated innced2569 seeds, suggesting specific transcriptionalregulation of the receptor gene family. Similar ob-servations were made for the PP2C family, amongwhich three members were down-regulated innced2569 seeds. Therefore, during seed development,combinatorial interactions between PP2C and PYR/PYL proteins may be modulated, in a tissue-specificmanner, by their transcriptional regulation by en-dogenous ABA. Two PP2Cs, AHG1 and AHG3, wererecently identified as DOG1-interacting proteins (Néeet al., 2017; Nishimura et al., 2018). Here we foundthat expression of DOG1 and DOG1-like genes wasdifferentially affected by ABA levels. This observa-tion suggests that interactions between DOG1 andABA signaling do not only exist at the protein level,but also at the transcriptional level through the up-regulation of DOG1 by ABA, which would hence re-inforce dormancy induction.

ABA Is a Repressor of ROS Production and ProteinOxidation in Seeds

Transcriptome analysis during seed development, ingood accordance with dry proteome data, provided clearevidence that ABA is a broad-spectrum regulator of seedmaturation, because ABA deficiency results in the down-regulation of many storage reserve and desiccation tol-erance genes. Furthermore, ABA has been previouslyshown to repress photosynthesis during late-maturation,notably by triggering chlorophyll degradation throughthe activation of ABA-related transcription factors, suchas ABI3 and ABI5 (Nambara et al., 1994; Delmas et al.,2013). Here the overaccumulation of a large number oftranscripts and proteins related to Calvin cycle and


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photosynthesis was observed in nced2569 seeds, thusfurther extending the inhibitory role of ABA on chloro-plast activity.Chloroplasts have been described as the major

generation site of ROS (Asada, 2006). Therefore, theactivation of chloroplast activity in nced2569 andconversely its reduction in cyp707a1a2, compared towild type, well correlate with the observed differencesin ROS production and protein oxidation. Thus, itsuggests that ABA reduces ROS levels in both devel-oping and imbibed seeds to prevent germination. Therepressive effect of ABA on oxidative processes inseeds contrast with observations in guard cells, inwhich ABA has been reported to induce ROS and NOproduction to promote stomatal closure, through theactivation of NADPH oxidases by SnRK2 (Waszczaket al., 2018). Recently, it has been shown that ABA alsopromotes the accumulation of ROS in Arabidopsisseedlings through an antagonist crosstalk with ethyl-ene (Yu et al., 2019). The production of radicals may berequired for the transition from heterotrophic (seed) toautotrophic (seedling) development and closely re-lated to acquisition of photosynthetic competence (Haet al., 2017). Because seeds, seedlings, and guard cellsshare upstream core ABA signaling components, dis-tinct ROS signaling pathways may operate in thesetissues.In seeds, ROS play a key role in the regulation of

germination performance through specific oxidationphenomena, notably targeting proteins (Arc et al.,2011). The role of protein oxidation has been investi-gated in the context of seed physiology, showingthat dormancy breaking is associated with an in-creased level in the carbonylation of specific pro-teins (Oracz et al., 2007; Arc et al., 2011). Furthermore,during germination, oxidized storage molecules wouldbe more easily accessible to degradation and used as fuelfor radicle protrusion and seedling establishment.Thus, the higher rate of protein oxidation in nced2569seeds, which may result from increased metabolic ac-tivity during seed development, would facilitate seedstorage protein mobilization and boost germinationupon imbibition. In contrast, the very low content ofoxidized proteins observed in cyp707a1a2 seeds wouldprevent the use of storage compounds and maintainmolecular locks imposing dormancy.

Metabolism Activation in ABA-Deficient Dry SeedsSupports the Premature Initiation ofGermination Programs

Seed dormancy reduction in ABA-deficient seedsmight be a consequence of the decreased repression byABA of cell-cycle, photosynthesis, and oxidative pro-cesses. In good accordance, the concomitant accumu-lation of free metabolites, such as sugars, amino acids,and organic acids and enzymes involved in these met-abolic pathways, suggested a premature metabolismresumption state in ABA-deficient dry seeds. Indeed,

high free amino acid content, as observed here forbranched-chain and shikimate pathway amino acids,may contribute to seed vigor by supporting proteinneosynthesis during early imbibition and fueling theTCA cycle for energy production (Fait et al., 2006).Furthermore, higher levels in intermediates of TCA andglyoxylate cycles (aconitate, citrate, fumarate, and ma-late) and the overaccumulation of the key enzyme ofglyoxylate cycle ICL likely implies an absence of re-pression byABAof energeticmetabolism that would berequired for dormancy induction. In accordance, ICLaccumulation has been shown to occur upon nondor-mant seed imbibition and also during dormancy release(Chibani et al., 2006; Arc et al., 2012). Globally, our re-sults showed an overaccumulation of free metabolitesand enzymes involved in reserve remobilization, innced2569 dry seeds compared to wild type andcyp707a1a2, before imbibition, thus likely facilitating aquick restart of primary metabolism and anabolic pro-cesses during seed germination.

ABA Influences Nitrogen and Sulfur Allocation throughGlucosinolate Metabolism

The role of ABA in glucosinolatemetabolism remainsobscure. Here we observed that a strong metabolicsignature of high ABA content in seeds was an increasein glucosinolate content. These active secondary me-tabolites, mainly found in Brassicaceae, derive fromeither Met or Trp. Their synthesis requires coordinationof several enzymes involved in amino acid, sulfur, andprimary metabolisms, which are subjected to a redoxregulation (He et al., 2009). Hydrolysis of the Glc moi-ety by the myrosinase leads to the production of de-fense compounds (thiocyanate, isothiocyanate, andnitrile). Glucosinolate synthesis is regulated bymethyljasmonate (Mikkelsen et al., 2003) and various abioticstresses such as salinity, drought, and temperature (DelCarmen Martínez-Ballesta et al., 2013). Furthermore,the myrosinases TGG1 and TGG2 activities have beenreported to participate in ABA and methyl jasmonatesignaling pathways in guard cells (Islam et al., 2009),suggesting possible auxiliary signaling roles in stressresponses. During germination of cabbage sprouts,exogenous ABA triggers glucosinolate breakdown byincreasing myrosinase activity and subsequent accu-mulation of hydrolysis products (Wang et al., 2015). Incontrast, our results indicated that, during seed devel-opment, ABA promoted both glucosinolate accumula-tion and catabolism. It can be hypothesized that, in thedeeply dormant cyp707a1a2 seeds, the high content ofglucosinolates may contribute to sulfur and nitrogenstorage for the production of defense compounds aslong as dormancy is maintained.In summary, the combination of hormone profiling

and omics approaches on ABA metabolism mutantsgave new insights on the role of ABA during seed de-velopment and highlighted the prominent role of en-dosperm to provide 15-carbon precursors of ABA. ABA





has been shown here to sequentially regulate multiplebiological processes that globally contribute to dor-mancy establishment. This study also emphasized theimportance of ABA in fine-tuning the protein oxidationprocesses and ROS production associated with dor-mancy release.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana [Columbia-0 accession]) wild-type andmutant seeds were surface-sterilized, sown in petri dishes containing Arabi-dopsis Gamborg B5 medium (Duchefa; http://www.duchefa.com) supple-mented with 30-mM Suc, and stratified at 4°C in the dark for 3 d. Petri disheswere then placed for 4 d in a growth chamber (16-h photoperiod, 50-mmol m22

s21 light intensity, 18°C, 60% relative humidity). Germinated seedlings weretransferred to soil (Tref Substrates; http://www.trefgroup.com) and, unlessotherwise stated, grown in a growth chamber (16-h light/21°C, 8-h dark/19°C,200mE.m22.s21; 65% relative humidity). In each experiment, all genotypes weregrown together.

As previously described, the nced6-1 mutant was identified among theSainsbury LaboratoryArabidopsis Transposant lines (Tissier et al., 1999), nced9-1 (SALK_033388) was obtained from the Salk database (Alonso et al., 2003;http://signal.salk.edu/cgi-bin/tdnaexpress), and nced5-2 (GK_328D05) wasobtained from the GABI-Kat mutant collection (http://www.gabi-kat.de; Liet al., 2007). These were used to generate the triple mutant nced569 (Freyet al., 2012). The mutant nced2-3 (SALK_090937) was obtained from the Salkdatabase and provided by the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info). The quadruple mutant nced2569 was identified in the F2progeny after crossing nced2-3with nced569. Themutants aba2-2 and cyp707a1a2were kindly provided by Dr E. Nambara. Phenotypic comparisons of allelicseries of nced2, nced5, nced6, nced9, cyp707a1, cyp707a2, and some combinationsof double and triple mutants have been previously reported (Lefebvre et al.,2006; Okamoto et al., 2006; Toh et al., 2008; Frey et al., 2012).

Germination Experiments

For dormancy assays, freshly harvested seedswere sown in triplicate in petridishes containing 0.5% (w/v) agarose and then placed in a growth chamber(continuous light, 25°C, 70% relative humidity). Germination was scored eachday based on radicle protrusion. After few weeks of dry seed storage at roomtemperature, dormancy release was analyzed on the same seed lots by scoringgermination 4 d after sowing. All genotypes were grown in a glasshouse with aminimum photoperiod of 13 h, assured by supplementary lighting, and threeindependent seed lots were harvested for each genotype; each seed lot wasobtained by pooling seeds from three to four plants.

Determination of ABA and Catabolite Contents

Siliques, isolated seeds, or envelopes and dry seeds were frozen in liquidnitrogen and then ground and freeze-dried. Samples were then extracted inacetone/water/acetic acid (80:19:1, v/v/v) with addition of 1 ng of d4-ABA,2 ng d3-PA, 8 ng d3-DPA, 10 ng d5-ABA-GE, and 5 ng d4-79OH-ABA isoto-pically labeled standards (National Research Council Canada Plant Biotech-nology Institute). Their ABA content was measured using LC-MS/MS (I-ClassUPLC system coupled with Xevo TQ-S, Waters; http://www.waters.com), asdescribed in Li-Marchetti et al. (2015). Multiple ReactionMonitoring transitionsfor ABA catabolites were analyzed as described by Chiwocha et al. (2003).

RNA Extraction and CATMA Analysis

Developing seeds were dissected from siliques, 10 and 14 d after tagging offlowers at fertilization stage, and immediately frozen in liquid nitrogen. TotalRNAs of three biological replicates were extracted using the RNeasy Plant MiniKit (Qiagen; http://www.qiagen.com). Transcriptome profiling was carriedout using Arabidopsis CATMA 6.2 arrays containing 30,834 probes corre-sponding to DNA coding sequences from The Arabidopsis Information Re-source (v8 annotation; including 476 probes of mitochondrial and chloroplast

genes) and 1,289 probes corresponding to EUGENE software predictions.Moreover, it included 5,352 probes corresponding to repeat elements, 658probes for miRNA, 342 probes for other RNAs (ribosomal RNA, transfer RNA,small nuclear RNA, and small nucleolar RNA), and finally 36 controls (IPS2POPS platform; http://ips2.u-psud.fr/en/platforms/spomics-interactomics-metabolomics-transcriptomics/pops-transcriptomic-platform.html). One tech-nical replicate with fluorochrome reversal was performed for each biologicalreplicate (i.e. four hybridizations per comparison). The labeling of comple-mentary RNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life ScienceProducts; http://www.perkinelmer.com) was performed as described byLurin et al. (2004). The hybridization andwashingwere performed according toNimbleGen Arrays User’s Guide (v5.1) instructions. Two-micron scanning wasperformed with an InnoScan 900 scanner (Arrayit; http://www.arrayit.com)and raw data were extracted using the software MapixR (InnopsysR; https://www.innopsys.com).

CATMA Array Data Analysis

For each array, the rawdata comprised the logarithm ofmedian feature pixelintensity at wavelengths 635 nm (red) and 532 nm (green). For each array, aglobal intensity-dependent normalization using the “loess” procedure (Yanget al., 2002) was performed to correct the dye bias. The dataframes were ana-lyzed with the software R (R Development Core Team, 2005). The “lm()” and“Anova()” functions from, respectively, “{stats}” and “{car}” packages wereused to calculate global P values. Adjusted P values were calculated by a FDRBenjamini-Hochberg correction, and a threshold of 0.05 was used to selectcandidates. Paired comparisons were done for each variable (probe) to deter-mine the sources of variation by the “TukeyHSD” function in the package “R{stats}.” Along with the fold change ratios, these paired comparisons resultswere used to determine gene expression profile variations among the threegenotypes.

Microarray data were submitted to the international repository Gene Ex-pression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo; Edgar et al.,2002) with accession numbers GSE68088 and GSE125573. All steps of the ex-periment, from growth conditions to bioinformatic analyses, are detailed in theCATdb database (Gagnot et al., 2008; http://tools.ips2.u-psud.fr.fr/CATdb/)with Project IDs RA12-02_ABA-seed and RA14-04_nced according to the“Minimum Information About a Microarray Experiment” standards.

RNA Analysis

Total RNA was prepared from frozen dissected developing seeds usingRNeasy Plant Mini Kit (Qiagen). Total RNA (1 mg) was used as a template tosynthesize cDNA. Reverse transcription and qPCR reactions were performed asdescribed in Plessis et al. (2011). Gene-specific primers are listed inSupplemental Table S10.

Protein Extraction and LC-MS/MS Analysis

Dry seeds (25mg)were ground in liquid nitrogen and total proteins extractedin TCA-acetone, as described by Méchin et al. (2007). Proteins were solubilizedin 400 mL of ZUT buffer (6 M of urea, 2 M of thiourea, 10 mM of dithiothreitol,30 mM of Tris-HCl at pH 8.8, and 0.1% (w/v) zwitterionic acid labile surfactant I[Protea Biosciences; https://proteabio.com]). Protein concentrations weremeasured using the 2DQuant Kit (GE Healthcare; https://www.gehealthcare.com) and adjusted to 4 mg.mL21 before digestion. Diluted proteins (10 mL) werereduced (10 mM of dithiothreitol present in ZUT buffer) for 30 min at 24°C andthen alkylated in 55 mM of iodoacetamide in 50 mM of ammonium bicarbonatefor 1 h at room temperature in the dark. Digestion was performed overnight at37°C with 600 ng of modified trypsin (Promega V5111; https://france.promega.com) dissolved in 50 mM of ammonium bicarbonate. Digestion wasstopped by adding 1% (v/v) trifluoroacetic acid. Protein samples were thenspeed-vacuum–dried and solubilized in 20 mL of loading buffer (0.1% [v/v]formic acid in water, and 2% [v/v] acetonitrile).

Tryptic peptides were separated on a NanoLC-Ultra system (Eksigent;https://sciex.com) coupled to a Q-Exactive Mass Spectrometer (ThermoElectron; https://www.thermofisher.com), as described by Balliau et al. (2018).Eluted peptides were analyzed on-line with a Q-Exactive Mass Spectrometer(Thermo Electron) using a nano-electrospray interface. Data were acquiredwith the software Xcalibur (v2.1; Thermo Fisher Scientific) with the followingdata-dependent parameters: a full MS scan covering 300–1,400 m/z range of


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http://www.perkinelmer.com

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mass-to-charge ratio (m/z) with a resolution of 70,000 and aMS/MS scan with aresolution of 17,500 and normalized collision energy = 30%. The MS/MS scanwas reiterated for the eight most abundant ions detected in the MS scan withdynamic exclusion set to 45 s.

Proteome Data Analysis

Data search was performed with the software X!Tandem (v. 2015.04.01.1;Craig and Beavis, 2004) against the Arabidopsis TAIR database (https://www.arabidopsis.org) and a homemade database containing contaminants (trypsin,keratins). Specific protein cleavage was fixed as tryptic cleavage with one au-thorized missed cleavage. Cys carbamidomethylation was set as a fixed mod-ification, and Met oxidation was set as a potential modification. Additionalpeptide identification was performed by submitting samples to the refinemodeof X!Tandem. In this second round, Ser, Thr, and Tyr phosphorylation, Gln andAsn deamidation, and Trp oxidation were set as possible modifications. Proteininference was performed by using X!TandemPipeline (v. 3.4.2; Langella et al.,2017). Selection of peptides and proteins was performed following the settings:peptide E value , 0.01, protein E value ,1025, one peptide identified perprotein. Peptide quantification was performed on extracted ion currents byusing the software MassChroQ (v. 2.2.1; Valot et al., 2011; http://pappso.inra.fr/bioinfo/masschroq/).

Data analysis was performed using the package MassChroQ for R. Peptidesshowing toomuchvariation of their retention time andassociatedwith too-largechromatographic peaks were removed. Normalization of peptide intensitieswas performed taking into account peptidemedian-retention time (Lyutvinskiyet al., 2013). Only protein-specific peptides present in at least two of the threebiological replicates were kept for further protein quantification. Proteinsquantified using fewer than two peptides were eliminated. Normalized peptideintensities were summed to obtain protein relative abundances. The finalnumber of quantified proteins obtained was 838. A differential analysis bycombining a one-way ANOVA and a multiple comparison procedure (Tukey’shonestly significant difference test) was performed on log10-transformed pro-tein abundances. Proteins with adjusted P values , 0.05 were considered asshowing significant differential abundance between genotypes.

Metabolite Profiling Using Gas Chromatography-MS

Dry seeds were ground in liquid nitrogen and lyophilized. Extraction, de-rivatization, analysis, anddataprocessingwereperformedasdescribedbyFiehn(2006) and Clément et al. (2018). Lyophilized samples (20 mg) were extractedin 1mL of cold water/acetonitrile/isopropanol (2:2:3, v/v/v) containing ribitol(4 mg.mL–1) in tubes placed in a Thermomixer (10 min, 4°C; Eppendorf) beforecentrifugation (20,000g, 5 min). Supernatants were collected and speed-vacuum–dried for 4 h. Then, 10 mL of 20 mg.mL–1 methoxyamine in pyridinewere added to the samples, which were incubated for 90 min at 28°C undercontinuous shaking. Silylation of metabolites was performed for 30 min at 37°Cwith N-methyl-N-trimethylsilyl-trifluoroacetamide (Sigma-Aldrich). Metabo-lites were analyzed on a model no. 7890A Gas Chromatograph (Agilent;https://www.agilent.com) coupled to a model no. 5975C Mass Spectrometer(Agilent) with the parameters described by Clément et al. (2018). Metaboliteswere annotated and their levels were normalized with respect to the ribitolinternal standard.

Metabolome Data Analysis

Raw Agilent datafiles were converted to NetCDF format and analyzed withAutomated Mass Deconvolution and Identification System (http://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:amdis). A home retention index/mass spectra library, built from the National Institute of Standards and Tech-nology, Golm, and Fiehn databases and standard compounds, was used formetabolite identification. Peak areas were then integrated using the softwareQuanLynx (Waters; http://www.waters.com) after conversion of the NetCDFfile to MassLynx format.

Statistical analysis was performed with a home-made R script using thepackages Car (v3.0-2) and Multicomp (v1.4-8; http://cran.r-project.org). Me-tabolites detected in at least 80% of the samples (i.e. 173) were selected for asubsequent quantitative analysis. One-way ANOVA and a multiple compari-son procedure (Tukey Honestly Significant Difference test) were carried outon log10-transformed metabolite abundances. Metabolites showing adjustedP values , 0.05 were considered.

Detection of NO Emission in Seeds

The cell membrane nonpermeable fluorescent probe 4-amino-5-methyl-amino-29,79-difluorescein (D1821; Sigma-Aldrich; https://www.sigmaaldrich.com) was used to measure NO production by imbibed seeds. This probe reactswith N2O3, which is the main NO autooxidation derivative and a robust indi-cator of NO production (Planchet and Kaiser, 2006; Liu et al., 2016; Nagel et al.,2019). The amount of N2O3 was determined in five dry seed samples perbiological replicate using sodium nitroprusside (S0501, Sigma-Aldrich;Supplemental Fig. S5) as standard, as described by Sechet et al. (2015), and threebiological replicates were analyzed for each genotype. Fluorescence intensitywas measured after incubation of seeds (5 mg) during 24 h at 25°C in the dark.

Hydrogen Peroxide and Superoxide Anion ProductionIn Seeds

Dry seeds (20 mg) were sown in petri dishes containing water-imbibed filterpaper (VWR) and placed at 25°C in the light during 24 h. No germination(radicle protrusion) was observed before seeds were collected and incubated in500 mL of 10 mM P buffer at pH 6.0, containing 5 mM of scopoletin (Sigma-Aldrich) and 10 U/mL of horseradish peroxidase (Sigma-Aldrich), and vigor-ously shaken for 1 h. Fluorescence measurements were performed using a CaryEclipse fluorometer (Agilent; excitation, 350 nm; emission, 420–500 nm). Alinear calibration curve was determined using increasing concentrations ofH2O2 (0 to 20 mM). Three biological replicates were analyzed, and measure-ments were performed using five seed samples per biological replicate.

For the determination of superoxide anion production measurements, seedsimbibed for 24 hwere incubated for 3 h at room temperature on a shaker in assaysolution containing 10 mM of P buffer at pH 6.0 and 500 mM of sodium 39-1-[phenylamino-carbonyl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (Sigma-Aldrich). Sodium 39-1-[phenylamino-carbonyl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reductionwas measured at 470 nm («470 = 24.2 mM21 cm21). The blanks were donewithout seeds and were used to correct for unspecific absorbance changes.Measurements were performed using three biological replicates and threetechnical replicates for each of them.

Protein Oxidation

Carbonylated protein profiles of dry mature seeds were determined by 1DPAGE of total protein extract followed by derivatization with 2,4-dinitro-phe-nylhydrazine and immunological detection of the DNP adducts with mono-clonal anti-DNP antibody (OxyBlot Oxidized Protein Detection Kit; Chemicon;www.merckmillipore.com) as described in Job et al. (2005).

Accession Numbers

Microarraydatawere submitted to the international repositoryGEO (http://www.ncbi.nlm.nih.gov/geo) with accession numbers GSE68088 andGSE125573.

Supplemental Data

The following materials are available.

Supplemental Figure S1. Stature of aba2, nced2569, and wild-type plants.

Supplemental Figure S2. ABA biosynthesis and catabolism in wild-type seeds.

Supplemental Figure S3. Germination of nced256, nced259, nced269,nced569, nced2569, aba2-2, and wild-type seeds.

Supplemental Figure S4. ABA levels in wild-type, nced2569, andnced259 seeds.

Supplemental Figure S5. Nitric oxide (NO) determination using sodiumnitroprusside (SNP) for calibration.

Supplemental Table S1. Transcriptome analysis at 10 DAP revealed285 transcripts that were significantly down-regulated (.1.5-fold,FDR, P , 0.05) in nced2569 compared to either wild type or nced259.




https://www.arabidopsis.org

https://www.arabidopsis.org

http://pappso.inra.fr/bioinfo/masschroq/

http://pappso.inra.fr/bioinfo/masschroq/

https://www.agilent.com

http://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:amdis

http://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:amdis

http://www.waters.com

http://cran.r-project.org

https://www.sigmaaldrich.com

https://www.sigmaaldrich.com


http://www.merckmillipore.com










Supplemental Table S2. Transcriptome analysis at 10 DAP revealed 98transcripts that were significantly up-regulated (.1.5-fold, FDR, P ,0.05) in nced2569 compared to either wild type or nced259.

Supplemental Table S3. Transcriptome analysis at 14 DAP revealed 853transcripts that were significantly down-regulated (.1.5-fold, FDR, P ,0.05) in nced2569 compared to either wild type or nced259.

Supplemental Table S4. Transcriptome analysis at 14 DAP revealed 441transcripts that were significantly up-regulated (.1.5-fold, FDR, P ,0.05) in nced2569 compared to either wild type or nced259.

Supplemental Table S5. GO enrichment (P , 0.05, Holm-Bonferroni)among differentially expressed genes (.1.5-fold) in nced2569 comparedto wild type and nced259, proteins (.1.3-fold) in nced2569 compared towild type and cyp707a1a2, and proteins (.1.3-fold) in cyp707a1a2 com-pared to wild type and nced2569.

Supplemental Table S6. Proteome analysis in dry mature seeds revealed68 proteins that were significantly up-regulated (one-way ANOVA, P ,0.05) in nced2569 compared to either wild type or cyp707a1a2. Ratios ,1.3 are indicated in gray.

Supplemental Table S7. Proteome analysis in dry mature seeds revealed52 proteins that were significantly up-regulated (one-way ANOVA, P ,0.05) in cyp707a1a2 compared to either wild type or nced2569. Ratios ,1.3 are indicated in gray.

Supplemental Table S8. Metabolome analysis in dry mature seedsrevealed the overaccumulation of 44 metabolites (one-way ANOVA,P , 0.05) in nced2569 (15 metabolites) or in both nced2569 and wildtype (29 metabolites), compared to cyp707a1a2. Ratios , 1.3 are indi-cated in gray.

Supplemental Table S9. Metabolome analysis in dry mature seedsrevealed the overaccumulation of 21 metabolites (one-way ANOVA,P , 0.05) in cyp707a1a2 compared to both wild type and nced2569. Ra-tios , 1.3 are indicated in gray.

Supplemental Table S10. Gene-specific primers used for expressionanalysis.

ACKNOWLEDGMENTS

Weare grateful toAmélieDegueuse, Hervé Ferry, and PhilippeMaréchal forplant culture, and to Corentin Moreau and Camille Roux for contribution toexperimental work.

Received March 19, 2019; accepted March 20, 2019; published April 4, 2019.

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