ASCORBATE PEROXIDASE6 Protects ArabidopsisDesiccating and Germinating Seeds from Stress andMediates Cross Talk between Reactive Oxygen Species,Abscisic Acid, and Auxin1[C][W][OPEN]

Changming Chen, Ilya Letnik, Yael Hacham, Petre Dobrev, Bat-Hen Ben-Daniel, Radomíra Vanková,Rachel Amir, and Gad Miller*

Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel (C.C.,I.L., B.-H.B.-D., G.M.); Laboratory of Plant Science, Migal Galilee Research Institute, Kiryat Shmona 12100,Israel (Y.H., R.A.); Tel Hai College, Upper Galilee 12210, Israel (Y.H., R.A.); and Institute of ExperimentalBotany Academy of Sciences of the Czech Republic, 16502 Prague 6, Czech Republic (R.V., P.D.)

A seed’s ability to properly germinate largely depends on its oxidative poise. The level of reactive oxygen species (ROS) inArabidopsis (Arabidopsis thaliana) is controlled by a large gene network, which includes the gene coding for the hydrogenperoxide-scavenging enzyme, cytosolic ASCORBATE PEROXIDASE6 (APX6), yet its specific function has remained unknown.In this study, we show that seeds lacking APX6 accumulate higher levels of ROS, exhibit increased oxidative damage, and displayreduced germination on soil under control conditions and that these effects are further exacerbated under osmotic, salt, or heatstress. In addition, ripening APX6-deficient seeds exposed to heat stress displayed reduced germination vigor. This, together withthe increased abundance of APX6 during late stages of maturation, indicates that APX6 activity is critical for the maturation-dryingphase. Metabolic profiling revealed an altered activity of the tricarboxylic acid cycle, changes in amino acid levels, and elevatedmetabolism of abscisic acid (ABA) and auxin in drying apx6 mutant seeds. Further germination assays showed an impairedresponse of the apx6 mutants to ABA and to indole-3-acetic acid. Relative suppression of abscisic acid insensitive3 (ABI3) andABI5 expression, two of the major ABA signaling downstream components controlling dormancy, suggested that an alternativesignaling route inhibiting germination was activated. Thus, our study uncovered a new role for APX6, in protecting maturedesiccating and germinating seeds from excessive oxidative damage, and suggested that APX6 modulate the ROS signal crosstalk with hormone signals to properly execute the germination program in Arabidopsis.

Seed development and seed germination are twocritical phases in the plant life cycle. Dehydration andrehydration during seed development or during ger-mination are associated with high levels of oxidativestress (Dandoy et al., 1987; Rajjou et al., 2012). Over-accumulation of reactive oxygen species (ROS) can causeoxidative damage to a wide range of cellular componentsand cause DNA damage, reducing the seed’s ability togerminate (Bailly et al., 2008; Chen et al., 2011; Parkheyet al., 2012). An optimal range of ROS levels is required

for successful germination (Bailly et al., 2008). Below this,germination is suppressed (e.g. in dormant seeds), andabove it, cellular oxidative damage accumulates, delay-ing or inhibiting germination. This concept, termed theoxidative window of germination, demonstrates the du-ality of ROS in seed physiology (Bailly et al., 2008). Ex-periments in rice (Oryza sativa), grasses, and Arabidopsis(Arabidopsis thaliana), in which suppression of ROSproduction inhibited germination, demonstrate the re-quirement of ROS for germination (Sarath et al., 2007;Leymarie et al., 2012; Liu et al., 2012). It was furthersuggested that ROS accumulation during a period ofdry storage following harvest, so called after-ripening,acts as a key signal in changing proteome oxidation toprepare the embryo for germination (Job et al., 2005;Oracz et al., 2009). Arabidopsis nondormant seeds, inwhich dormancy was alleviated by after-ripening or lighttreatment, produced more ROS than dormant seedsduring imbibition (Leymarie et al., 2012).

The commitment of seeds to germination is deter-mined during the seeds’maturation on the mother plant,with desiccation, accumulation of storage proteins, andtranscription of genes that are translated during imbibi-tion (Rajjou et al., 2004, 2012; Finch-Savage and Leubner-Metzger, 2006; Finkelstein et al., 2008; Holdsworth et al.,

1 This work was supported by the Marie Curie Actions-International Career Integration Grant (grant no. 293999), the IsraelScience Foundation (grant no. 938/11), and the Czech Science Foun-dation (project no. 206/09/2062).

* Address correspondence to gad.miller@biu.ac.il.The 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:Gad Miller (gad.miller@biu.ac.il).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OPEN] Articles can be viewed online without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.114.245324

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2008). Therefore, the potential of seeds for rapid uniformemergence and development under a wide range of fieldconditions (i.e. seed vigor) greatly depends on the properexecution of seed maturation- and desiccation-relatedprocesses (Finch-Savage and Leubner-Metzger, 2006).ROS also play a key regulatory role in the germination

program under favorable conditions (Sarath et al., 2007;Liu et al., 2010; Bahin et al., 2011; Ye and Zhang, 2012).Germination begins with the release of dormancy, whichis controlled by abscisic acid (ABA) and the activationof GA, which control germination-promoting signals(Finch-Savage and Leubner-Metzger, 2006; Finkelsteinet al., 2008).Recent studies in Arabidopsis and barley (Hordeum

vulgare) have shown that hydrogen peroxide (H2O2) me-diates the regulation of ABA catabolism, antagonizes ABAsignaling, and promotes GA synthesis and its germinationprogram (Liu et al., 2010; Bahin et al., 2011; Ishibashiet al., 2012; Krishnamurthy and Rathinasabapathi, 2013).Furthermore, dormancy release, in both dry and imbibedstates, has been associated with ROS production and thespecific oxidation of embryonic proteins, of fatty acids,and of mRNA molecules (Job et al., 2005; Oracz et al.,2007, 2009; Bazin et al., 2011). Protein carbonylation, themost prevalent type of protein oxidation caused by ROS,has been shown to target a specific set of embryo pro-teins and was suggested to be part of the dormancyalleviation mechanism in sunflower (Helianthus annuus)and Arabidopsis seeds (Job et al., 2005; Oracz et al., 2007,2009). Recent studies also support interactions betweenROS, ethylene, cytokinin, and auxin in controlling seedgermination and early seedling development (Oracz et al.,2009; Liu et al., 2010; Subbiah and Reddy, 2010; He et al.,2012; Krishnamurthy and Rathinasabapathi, 2013; Linet al., 2013). All of these accumulated findings indicatethat ROS signals play key roles in seed developmentand germination and demonstrate the diversity andcomplexity of ROS function.Because ROS metabolism and signaling are central

in dormancy and germination control, a tight regulationis required to properly execute these programs whileavoiding oxidative stress. In Arabidopsis, there are over150 enzymes dedicated to reducing ROS types, such asH2O2, superoxide ions, and others, to their lesser reac-tive forms (Mittler et al., 2004; Miller et al., 2008, 2010).Ascorbate peroxidases (APXs) comprise a small familyof nine enzymes in Arabidopsis that use ascorbic acid(AA) as a substrate to reduce H2O2 to water (Mittleret al., 2004; The Arabidopsis Information Resource 10).Of the three cytosolic ascorbate peroxidases (cAPXs),the functions of APX1 and APX2 are relatively wellestablished (Panchuk et al., 2002; Davletova et al., 2005;Suzuki et al., 2013a). APX1 is the most abundant APX,which functions in protecting cellular components,including chloroplasts, from oxidative damage as wellas modifying cellular and intracellular ROS signals(Davletova et al., 2005; Vanderauwera et al., 2011;Suzuki et al., 2013b). In contrast, the APX2 expressionlevel is constitutively very low under normal conditionsbut is highly induced in response to high temperatures

and increased light intensity (Panchuk et al., 2002;Suzuki et al., 2013a). However, very little is knownabout the expression of the third cAPX, APX6, and itsfunction is practically unknown. In this work, we iden-tified the function of APX6 in seeds using two inde-pendent Arabidopsis knockout lines. Here, we show thatAPX6 is important for protecting mature desiccatingseeds as well as germinating seeds from excessive oxi-dative stress and also functions in maintaining seedvigor under stress conditions. In addition, we discovereda novel interplay between ROS and ABA and betweenROS and auxin that could be interdependent.

RESULTS

Germination Phenotype of APX6-Deficient Mutants underFavorable and Stress Conditions

To study the role of APX6 in Arabidopsis, three in-dependent transfer DNA (T-DNA) insertion lines wereobtained from the Arabidopsis Biological Resource Cen-ter (Fig. 1A). Phenotypic evaluation of the identified ho-mozygous lines indicated relatively slower and poorergermination rates on soil as compared with the wildtype. The germination phenotype of apx6-1 and apx6-3was consistent, and these lines were later shown in re-verse transcription-PCR to be true knockout lines (Fig. 1B).In contrast, the 39 untranslated region insertion lineapx6-2 showed an inconsistent germination phenotypeand showed APX6 expression similar to the wild type(Fig. 1B). Therefore, only the apx6-1 and apx6-3 lines werefurther characterized.

Following germination, under favorable conditions, theseedlings and mature knockout plants were comparable

Figure 1. Gene structure and expression of APX6 in seeds. A, Genemap of the APX6 gene model. Exons are represented by boxes (un-translated regions in gray and coding sequence in black) and introns bythe black line. The T-DNA insertions into the gene are shown as tri-angles. Arrowheads below represent primers (P1–P4) used for absenceverification of the APX6 transcript expression in B. B, SemiquantitativePCR in dry seeds (DS) and in germinating seeds at 2 DAI. Col-0,Columbia-0.

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to wild-type plants, exhibiting similar growth rates andmature plant sizes (Supplemental Fig. S1).

Germination of freshly harvested nonstratified apx6-1and apx6-3 seeds on soil pellets resulted in a dramaticdelay and reduced level of germination, measured asthe appearance of cotyledons (Fig. 2A). Four days afterimbibition (DAI), only approximately 5% of the mu-tants were germinated, compared with 50% of the wildtype. The germination rate of apx6 mutants continuedsteadily and slowly, reaching less than 45% germinationat 8 DAI (Fig. 2A). Stratification treatment (48 h at 4°C)dramatically improved the germination rate, although itdid not completely reverse it (Fig. 2B). At 5 and 6 DAI,germination was 63% and 85% relative to the wild type,respectively (P, 53 1026 and P, 0.01, respectively). Incontrast, germination on plant growth medium (0.53Murashige and Skoog [MS] medium and 0.8% agar) didnot result in apparent differences in the rate of germi-nation (radicle emergence) between the wild type andapx6 mutants (Fig. 2C). This delayed-germination phe-notype on soil and the lack of it on MS medium wasconsistent.

Soil pellets are relatively hyperosmotic comparedwith MS media and the high-humidity environment in-side the plates. This and the results in Figure 2 pointed usto the possibility that APX6’s function might be in pro-tecting germinating seeds under low water potentials. Totest this hypothesis, we germinated the wild type andapx6 mutants on MS medium supplemented with sor-bitol (100 and 200 mM) and NaCl (50 and 100 mM; Fig. 3).Both apx6 lines showed severe inhibition of germinationin both treatments compared with the wild type. Theseresults clearly suggest that APX6 protects germinatingseeds from oxidative stress impediments that accom-pany osmotic stress.

We then evaluated the antioxidative impact of APX6in seeds by comparing ROS levels and oxidative damageaccumulated in the dry seeds of apx6 and the wild type

(Fig. 4). The levels of H2O2 and superoxide radicalsmeasured in dry seed extracts were 28% to 35% and 20%to 25% higher, respectively, in both apx6 lines comparedwith the wild type (Fig. 4, A and B). In addition, thehigher levels of ROS in the mutants were correlated withoxidative damage, as demonstrated by increased accu-mulation of carbonylated proteins (Fig. 4C). A generalperoxidase activity assay showed reduced activity indry seeds of apx6-1 (Supplemental Fig. S2), which is inagreement with the elevated level of H2O2 and increasedoxidative damage. We further measured AA and totalglutathione in dry seeds of the wild type and apx6-1(Supplemental Fig. S3). The level of total glutathioneaccumulated in wild-type seeds was approximately 25%higher on average compared with apx6-1. The level ofAA in apx6-1 was 5% lower and the level of dehydro-ascorbate (oxidized AA; DHA) was 25% higher on aver-age compared with the wild type, although thesedifferences were not significant. However, the ratio of re-duced to oxidized AA was significantly higher in thewild type (P , 0.05, Student’s t test). Seeds of apx6mutants were further tested for germination under oxi-dative stress on MS agar containing elevated concentra-tions of the superoxide-generating agent Paraquat. Theapx6 lines showed increased sensitivity, as demonstratedby the dose-response-delayed germination of the mutantrelative to the wild type (Fig. 4D), indicating thatthe apx6 lines are more sensitive to oxidative stress.Taken together, the results presented in Figure 4 andSupplemental Figure S3 suggested that there is a higheroxidative level in dry apx6 than in the wild type.

Expression Profile of cAPXs in Maturing andGerminating Seeds

A large degree of redundancy is assumed for theROS-gene network; however, some degree of speciali-zation is expected (Mittler et al., 2004). This redundancy

Figure 2. Germination phenotype of apx6 mutants under favorable conditions. A and B, Germination rates of freshly harvestedseeds on soil pellets (A) and following stratification treatment at 4˚C for 48 h (B). The images above were taken 6 DAI and 4 dafter stratification (DAS) as indicated. The germination rate was scored as cotyledon emergence. C, Germination rate on 0.53MS agar. Germination was scored as radicle emergence. The images above were taken at 48 HAI. SD values represent averagesof eight replicates of 50 seeds. Col-0, Columbia-0. [See online article for color version of this figure.]

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explains why the inhibition of germination in boththe wild type and apx6 mutants was not acute. AGenevestigator developmental expression graph of allnine Arabidopsis APXs revealed that, in seeds, only APX6shows an increase while the levels of all others decreaseor remain unchanged (https://www.genevestigator.com/gv/; Zimmermann et al., 2004; Supplemental Fig. S4). Thiscould lend support for a specific role of APX6 in seeds. AnElectronic Fluorescent Pictograph browser developmentalheat map in developing seeds further shows that APX6 isuniformly abundant in all the seed’s tissues (SupplementalFig. S5; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi;Winter et al., 2007). The Electronic Fluorescent Picto-graph browser profile of germinating seeds has revealedthat the level of APX6 decreased sharply already within3 h after imbibition (HAI; Winter et al., 2007). To learnmore about the expression of APX6 and the division oflabor among the three cAPXs, we determined their wild-type steady-state transcriptional levels in late maturingsiliques (stages 16–20; i.e. in mature desiccating seeds) aswell as in germinating seeds (0–72 HAI; Fig. 5). Real-timePCR analysis uncovered reciprocal trends in the expres-sion of APX1 and APX6; while the abundance of APX1declined as seeds matured and desiccated, the APX6mRNA level slowly accumulated (Fig. 5A), and the op-posite occurred during germination (Fig. 5B). The maxi-mal abundance of APX6 was detected in mature dryseeds, which was about 30-fold higher than the level of

APX1 (Fig. 5B). APX6 transcript levels decreased sharply,almost 20-fold, within 12 HAI, while the APX1 level slowlyincreased, reaching a comparable but relatively low level(Fig. 5B). The low levels of the three cAPXs persisted untilgermination was completed with the emergence of theradicle at 48HAI. Following germination, the level ofAPX1increased sharply, becoming once again the dominantcAPX. The APX2 level also increased during seed matu-ration; however, it still remained much lower comparedwith APX1 and APX6 and was undetectable throughoutgermination (Fig. 5). Taken together, the gradual replace-ment of APX1 with APX6 in desiccating seeds and con-trariwise in germinating seeds indicates specialization andtight control over the expression of cAPXs.

ABA Response and Metabolic Profiling in Seeds

The reversal of germination of apx6 seeds by strati-fication treatment (Fig. 2B) strongly pointed to the in-volvement of ABA. Cross talk between ABA and ROSwas previously shown in several independent studies(Sarath et al., 2007; Liu et al., 2010; Bahin et al., 2011;Ishibashi et al., 2012; Ye et al., 2012; Lariguet et al.,2013). To further investigate the possible relationshipbetween APX6 activity in seeds and ABA, freshly har-vested seeds of the wild type, apx6, and abi4 as a positivecontrol were germinated on MS medium containing

Figure 3. Germination assays of freshlyharvested seeds under hyperosmoticconditions: 0.53 MS medium supple-mented with NaCl or sorbitol. SD valuesrepresent averages of eight replicates of50 seeds. Col-0, Columbia-0. [Seeonline article for color version of thisfigure.]

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0.5 mM ABA (Fig. 6, A and B). ABA strongly inhibitedthe germination of apx6-1 and apx6-3 compared with thegermination rate of wild-type and abi4 seeds. Five DAI,only 54% of apx6 seeds showed emerged radicles (Fig. 6A),with only minor progression within the following4 d (Fig. 6B). Stratification treatment partially alleviated

the inhibition of ABA on the germination of apx6, re-sulting in a uniform and complete germination within7 d after stratification. Yet, apx6 germination and thedevelopment of the seedlings were still retarded com-pared with the wild type (Supplemental Fig. S6). Theseresults suggest that APX6 is involved in the release of

Figure 5. Developmental expression pattern ofcAPXs in the wild type. Relative expression ofAPX1, APX2, and APX6 was determined by real-time PCR analyses during the final stages of si-lique development (seed maturation; A) and seedgermination (B). The fold change values, pre-sented above and below the bars, are normalizedto the APX6 level in stage 16 siliques (in A) or tothe APX6 level in mature dry seeds (0 HAI; in B).SD values represent averages of three replicatesfor each stage. ND, Not determined.

Figure 4. ROS levels in dry seeds and germination response to oxidative stress. A, Relative levels of H2O2, normalized to thewild-type level, were measured using the Amplex Red fluorescence assay. B, Relative levels of superoxide radicals (O2∙

2) weremeasured as formazan accumulation in nitroblue tetrazolium staining. C, Protein oxidation assay, showing carbonylatedproteins (right) on a western blot of 10 mg of total seeds and Coomassie Blue staining (left) as a loading control. D, Germinationassay was conducted on 0.53 MS medium containing Paraquat (PQ). SD values for all samples represent averages of eightreplicates of 50 seeds. Asterisks indicate Student’s t test significance at *P , 0.05 and **P , 0.01. Col-0, Columbia-0. [Seeonline article for color version of this figure.]

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the ABA inhibitory effect during germination and per-haps also during early stages of seedling development.Next, we examined whether differences in the ex-

pression of ABA-related genes in dry and imbibed seedscould account for the hypersensitivity to ABA (Fig. 6,C and D). We examined the expression of the ABAsignal transduction genes ABI3, ABI4, and ABI5 and theexpression of the ABA response marker genes EARLYMETHIONINE-LABELLED6 (EM6) and RESPONSIVETO DESSICATION 29B (Rd29b) and dehydrin Late Em-bryogenesis Protein (LEA) (At2g21490). Apart from aslightly reduced EM6 level in apx6-1 imbibed seeds, nosignificant changes were detected in ABA responsemarker genes between apx6-1 and the wild type (Fig. 6,C and D). In contrast, the transcript levels of ABI3 andABI5 showed mild suppression in dry seeds (a decreaseof 22% and 35% relative to the wild type, respectively)that was further increased during imbibition (2.7- and2.4-fold decrease relative to the wild type, respectively).Conversely, ABI4 levels increased 45% and 75% relative

to the wild type in dry and imbibed seeds, respectively(Fig. 6, C and D).

Next, we performed liquid chromatography-massspectrometry analyses to measure the levels of ABA, itsmetabolic intermediates, as well as other phytohormonesthat may be involved in regulation of the germinationphenotype of apx6 (Table I). The ABA level in apx6-1was40% higher than in the wild type. Furthermore, the levelsof inactivated forms of ABA were higher in the mutant,particularly dihydrophaseic acid, which was more than5-fold higher (P, 0.0001, Student’s t test). These findingssuggest that apx6 seeds have a higher level of ABA me-tabolism during maturation compared with the wild type.

Furthermore, the level of the active GA4 was 79%higher (P, 0.005) in the mutant, while the level of GA19,the precursor of GA1, was similar in both seed types.Some effects on cytokinin metabolism were detected, asdifferences were found in active and storage forms ofcytokinins and several inactive forms. However, no cleartrend was observed. Interestingly, the most striking

Figure 6. Response of apx6mutants to ABA. A and B, Germination assay on 0.53MS medium containing 0.5 mM ABA (A) and arepresentative image showing germination at 9 DAI (B). SD values represent averages of eight replicates. C and D, Relativetranscript levels of ABA-responsive and signaling genes in dry seeds (C) and at 12 HAI (D). SD values represent averages of threereplicates. Asterisks indicate Student’s t test significance at *P, 0.05 and **P, 0.01. Col-0, Columbia-0. [See online article forcolor version of this figure.]

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finding was the changes observed in auxin homeo-stasis. Indole-3-acetic acid (IAA) level in apx6 seedswas almost twice that found in the wild type (P ,0.05). Furthermore, the level of IAA-Glu, the auxindegradation by-product, was 11 times higher in themutant (P , 0.005). Taken together, these metabolicchanges suggest that APX6 may play an importantrole in regulating major hormone metabolic pathwaysin desiccating seeds.

To gain a better understanding of the effect of APX6 onmetabolic processes in seeds, we performed a compara-tive nontargeted gas chromatography-mass spectrometry(GC-MS) analysis for several primarymetabolites (Table II),including analysis for amino acids (Table III). Principalcomponent analysis comparison of all metabolites in-cluded in Tables II and III showed divergent distributionfor apx6-1 and wild-type samples, demonstrating thedissimilarities between them (Supplemental Fig. S7).The most notable changes in the primary metabolitesappeared in the tricarboxylic acid cycle intermediates,succinate, citrate, fumarate, and malate, which were allsignificantly elevated in apx6-1 (Table II). These results

suggest that desiccating apx6 seeds had an alteredtricarboxylic acid cycle activity during development andthat an increased respiration rate might exist. In contrast,very little or no significant changes in the levels of sugarsand polyols were observed.

The levels of most amino acids were significantlyelevated in the mutant. Among the highly accumulatedamino acids (more than 2-fold) were Asn, Asp, and Pro(Table III). Asp is produced from Glu and oxaloacetate,which is also the tricarboxylic acid cycle intermediatelinking malate and citrate. This finding further corre-sponds with the accumulation in tricarboxylic acid cyclemetabolites (Table II) and suggests that the oxaloacetateproduction rate is elevated in apx6 seeds. Pro functionsas a compatible solute that is well known to accumulateduring drought and salt stress to counteract the re-duction in osmotic potential (Lehmann et al., 2010). Thelevels of Leu, Ile, and Val, collectively known as coor-dinately regulated branched-chain amino acids (BCAAs),similarly increased by 60% to 70% in apx6 seeds. LikePro, the three BCAAs are known to be involved in pro-tection during abiotic stresses, particularly osmotic stress

Table I. Levels of phytohormones in dry seeds

Values are means 6 SD (pmol g21 fresh weight) and ratios as indicated. SD values represents five replicates. Asterisks indicate Student’s t testsignificance at *P , 0.05 and **P , 0.01.

Group Compound Feature Wild Type apx6-1 Ratio, apx6-1:Wild Type

ABAs ABA Active 335.62 6 83.82 483.59 6 136.17 1.44PA Deactivated 2.38 6 1.03 3.65 6 1.16 1.53Dihydrophaseic acid Deactivated 4.50 6 0.87 23.10 6 4.98** 5.149OH-ABA Deactivated 0.37 6 0.29 0.86 6 0.64 2.32

Auxins IAA Active 330.80 6 78.02 636.68 6 174.61* 1.92IPA Precursor 55.28 6 11.07 67.67 6 19.78 1.22IAM Precursor 1.6 6 1.40 8.22 6 6.20 5.64IAN Precursor 1,919.9 6 1,122.84 1,028.79 6 184.31 0.54IAA-Asp Degradation 557.29 6 248.39 1,233.18 6 692.24 2.21IAA-Glu Degradation 2.96 6 1.69 33.58 6 23.79* 11.34OxIAA Degradation 8,193 6 2,090 12,100.48 6 2,777.42 1.48OxIAA-GE Degradation 257 6 94 436.12 6 179.87 1.70GA4 Active 55.50 6 17 99.14 6 27.73* 1.79

GAs GA19 Precursor 29.36 6 20 27.79 6 22.34 0.95Cytokinins tZ Active 1.53 6 0.62 2.23 6 0.36 1.46

tZR Active 4.13 6 1.06 4.54 6 1.10 1.10DZR Active 0.56 6 0.20 0.40 6 0.08 0.71iP Active 0.22 6 0.16 0.08 6 0.01 0.37iPR Active 0.97 6 0.32 0.90 6 0.25 0.92cZ Active 0.75 6 0.30 1.73 6 0.81 2.31cZR Active 5.07 6 1.76 6.80 6 1.41 1.34tZ7G Deactivated 2.26 6 0.43 2.52 6 0.75 1.11tZ9G Deactivated 0.43 6 0.14 1.00 6 0.32* 2.32DZ9G Deactivated 0.06 6 0.02 0.05 6 0.03 0.87iP7G Deactivated 49.36 6 12.25 64.96 6 9.94 1.32iP9G Deactivated 0.46 6 0.24 1.06 6 0.38* 2.29cZ7G Deactivated 6.46 6 0.98 11.17 6 3.11* 1.74tZRMP Precursor 1.43 6 0.39 1.24 6 0.29 0.87iPRMP Precursor 0.99 6 0.27 1.28 6 0.11 1.29tZOG Storage 0.51 6 0.10 0.36 6 0.10 0.72tZROG Storage 2.19 6 0.87 2.08 6 0.86 0.95DZROG Storage 0.25 6 0.11 0.30 6 0.14 1.20cZOG Storage 0.56 6 0.34 0.66 6 0.31 1.19cZROG Storage 0.42 6 0.22 1.57 6 1.11 3.79

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(Joshi et al., 2010). In contrast, Trp is the only amino aciddepleted in apx6 seeds, showing a 2.7-fold decreasecompared with the wild type (Table I). This reduction isinversely correlated with the accumulation of auxins,which is logical, since Trp is the precursor for auxinbiosynthesis.Auxin was most recently shown to promote seed

dormancy through the stimulation of ABA signal-ing (Liu et al., 2013). Our results in the apx6 mutant

point to a possible link between IAA and ABA throughH2O2. To test the possibility that IAA is involved in thereduced-germination phenotype of apx6 mutants, seedswere germinated on plant medium containing IAA.Both mutant lines showed clear inhibition at 50 mM andan almost complete suppression at 100 mM (P , 0.001;Fig. 7). This result suggests that there is interplay be-tween IAA and H2O2 in the ABA-mediated dormancythat is regulated by APX6 in seeds.

Table III. Levels of amino acids in dry seeds

Values are means 6 SD (nmol g21 fresh weight) and ratios as indicated. SD values represent five replicates. Asterisks indicateStudent’s t test significance at *P , 0.05, **P , 0.01, ***P , 5 3 1023, and ****P , 5 3 1024.

Amino Acid Wild Type apx6-1 Ratio, apx6-1:Wild Type

Ala 158.26 6 18.59 217.08 6 2.59**** 1.37Asn 134.57 6 58.47 290.98 6 86.70** 2.16Asp 127.64 6 26.59 403.31 6 126.01**** 3.16Cys 3.39 6 1.34 3.84 6 0.69 1.13Glu 776.44 6 136.10 1,149.97 6 211.60*** 1.48Gln 35.31 6 8.01 68.33 6 18.73*** 1.94Gly 233.84 6 129.56 255.62 6 174.45 1.09His 2.55 6 0.93 4.36 6 1.13* 1.71Homoserine 12.90 6 3.60 15.90 6 2.87 1.23Ile 110.16 6 17.65 174.54 6 24.64**** 1.58Leu 86.06 6 14.30 139.16 6 19.71**** 1.62Lys 5.80 6 1.00 10.47 6 3.41** 1.80Met 46.33 6 14.27 52.08 6 8.75 1.12Phe 158.19 6 45.92 240.10 6 36.42** 1.52Pro 83.76 6 21.09 237.77 6 17.94**** 2.84Ser 175.02 6 15.83 280.70 6 66.30*** 1.60Thr 96.65 6 9.51 146.62 6 37.41** 1.52Trp 480.54 6 77.42 177.25 6 31.61**** 0.37Tyr 54.50 6 7.30 65.75 6 14.40 1.21Val 245.46 6 38.87 411.542 6 65.52**** 1.68

Table II. Levels of primary metabolites in dry seeds

Values are means 6 SD (peak area per 1,000) and ratios as indicated. SD values represent five replicates. Asterisks indicate Student’s t test sig-nificance at *P , 0.05, **P , 0.01, and ****P , 5 3 1024.

Group Compound Wild Type apx6-1 Ratio, apx6-1:Wild Type

Sugars Fru monophosphate 2,725 6 871 3,633 6 1,679 1.3Gluconic acid 872 6 168 1,362 6 489* 1.6Glc biphosphate 3,629 5,050 6 1,155 1.4Glc monophosphate 25,475 6 9,644 34,960 6 6,984 1.4Suc 111,163 6 13,640 144,169 6 33,243 1.3

Polyols Erythritol 494 6 129 634 6 82* 1.3Glycerol 4,114 6 402 4,847 6 622 1.2Myoinositol 11,692 6 691 11,003 6 2,911 0.9Sorbitol 1,789 6 748 2,259 6 1,386 1.3Xylitol 161 6 16 150 6 39 0.9

Organic acids Citrate 4,727 6 839 10,510 6 2,214**** 2.2Fumarate 1,568 6 322 8,963 6 2,432**** 5.7Malate 2,294 6 321 6,071 6 1,625**** 2.6Succinate 337 6 54 471 6 83** 1.4Benzoate 3,245 6 346 4,801 6 489**** 1.5Nicotinic acid 1,687 6 148 3,177 6 327**** 1.9Palmitate 2,765 6 1,005 3,895 6 2,220 1.4Pyro-Glu 31,141 6 8,245 55,645 6 16,678** 1.8

Miscellaneous organic acid Ethanolamine 10,923 6 4,439 9,078 6 2,216 0.8Inorganic acids Phosphoric acid 1,066 6 203 2203 6 830** 2.1

Hydroxylamine 5,345 6 528 7,277 6 1,739* 1.4

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Response and Tolerance of Seeds to HeatStress Conditions

Desiccation (i.e. drought stress) and heat stress (HS)are two conflicting stresses, which are more harmfulwhen combined (Rizhsky et al., 2004; Suzuki et al.,2014). To test whether desiccating apx6 seeds are moresensitive to HS, we exposed 45-d-old flowering plantsbearing siliques (stages 17 and 18) to HS. Plants weresubjected to a daily regime of climbing temperatures,starting at 24°C and peaking at 39°C or 42°C at mid-day. The dry seeds were collected after 5 and 6 d.

The impact of the 42°C HS on seed vigor was testedby germination assays of freshly harvested stressedseeds on either soil pellets or MS agar under controlledconditions (Fig. 8, A and B). In both assays, apx6 seedsshowed dramatic retardation in germination comparedwith wild-type seeds (Fig. 8, A and B). Western-blotanalysis of dry seeds collected from 39°C- or 42°C-stressedplants revealed higher accumulation of the HS-responsiveproteins multiprotein bridging factor 1C (MBF1c) andheat shock proteins in apx6 seeds (Fig. 8C). In addition,APX1 and APX2 transcripts increased 2- and 2.5-fold,respectively, in apx6 seeds collected at 42°C (Fig. 8D).The higher accumulation of both HS-responsive APX1and APX2 indicates the activation of a compensatorymechanism to protect apx6 seeds from HS-associatedoxidative stress, which most likely prevents greater oxi-dative damage.

We next tested whether mature apx6 seeds are alsomore sensitive to HS during germination. For this pur-pose, seeds of the wild type and apx6-1 collected undercontrol conditions (i.e. 24°C) were exposed to 42°C for 6,12, 24, or 42 h. Following the HS period, the seeds weretransferred to 24°C for recovery and germination com-pletion. The results revealed that the longer the stressperiod persisted, the greater was the impairment in thegermination of apx6 compared with the wild type(Supplemental Fig. S8). Since APX6 expression de-clines sharply within a few hours after imbibition(Fig. 5B; Supplemental Fig. S5B), these findings suggestthat APX6 activity is required for seed thermotoleranceduring the initial stage of germination and furthersuggest that this stage is most vulnerable to stress. Takentogether, these results further support the function ofAPX6 as an important antioxidative mechanism thatprotects seeds from abiotic pressures during maturationand germination.

DISCUSSION

In this study, new varied roles were identified forcytosolic APX6 in Arabidopsis seeds during matura-tion and germination. We have shown that APX6 is amajor component of the antioxidative mechanism thatis important for seed vigor under favorable conditionsand even more so during stress.

The expression pattern of APX6 (Fig. 5; SupplementalFig. S5), together with the germination phenotypes ofthe mutants, highlight the specialized function of APX6

during seed desiccation and during the early stage ofgermination. The function of APX6 seems to be criticalduring the maturation-drying phase, in which the me-tabolism of the seed shifts from a general decrease inunbound metabolites to the accumulation of a set ofspecific metabolites (Angelovici et al., 2010). In addition,the stored reservoir of APX6 in the dry seed would servein protecting the embryo from excessive oxidative pres-sure that accompanies the increased respiratory metab-olism during imbibition. Furthermore, APX6 is requiredto protect seeds against osmotic stress (Fig. 3), and in itsabsence increased accumulation of Pro and BCAAs,which function in osmotolerance (Joshi et al., 2010;Lehmann et al., 2010), may provide partial compensation.

Multiple types of stress cause fluctuations in energythat ultimately converge and generate energy deficiencysignals, resulting in energy sensor activation (Baena-González and Sheen, 2008). This could be the case forapx6 seeds undergoing maturation drying under favor-able conditions and even more so during HS, whichfurther increases respiration rate, as indicated by the

Figure 7. Response of apx6 mutants to auxin. Freshly harvested seedswere germinated on 0.53 MS medium with IAA. Sample images ofseed germination (A) and histograms of germination at 5 DAI (B) areshown. SD values represent averages of eight replicates of 50 seeds.****Student’s t test significance at P , 5 3 1024. Col-0, Columbia-0.[See online article for color version of this figure.]

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elevated expression of the ALTERNATIVE OXIDASE 1aAOX1a) gene (Fig. 8D). Indeed, increased activation ofthe tricarboxylic acid cycle, as indicated by the accu-mulation of its intermediates (Table II), could increaseenergy generation (Angelovici et al., 2011; Galili, 2011)to compensate for energetic depletion in the absenceof APX6.

The relative elevation of most amino acids couldpotentially be attributed, at least in part, to an increasein the degradation of oxidized proteins (Fig. 4D), sincecarbonylated proteins are destined for degradation(El-Maarouf-Bouteau et al., 2013). Positive feedback fortricarboxylic acid cycle activity was recently shown dueto the contribution of catabolism of the Asp familypathway amino acids, Lys, Met, Thr, Ile, and Gly (Galili,2011). Activation of such positive feedback in apx6 seedsmight explain the fact that the level of Asp familyamino acids showed little or no increase compared withthe wild type, while the Asp level was 3-fold higher(Table III). Additionally, an increased flow through thetricarboxylic acid cycle could be achieved by the acti-vation of the g-aminobutyrate (GABA) shunt, generat-ing succinate from Glu via GABA. Activation of theGABA shunt is associated with stress conditions andseed desiccation (Angelovici et al., 2011; Fait et al., 2011)and has been shown to prevent ROS accumulation(Bouché et al., 2003). Our results showing that increasein cytosolic H2O2 in desiccating seeds triggers metabolicreprogramming indicate that APX6 is a major modu-lator of ROS signals in desiccating seeds. The new ROSsignal, seen in apx6 seeds and resulting in reducedgermination, revealed a strong cross talk with ABAsignaling. This was initially suggested by stratificationtreatment, which almost completely alleviated the ger-mination phenotype on soil (Fig. 2), and was furtherconfirmed by increased sensitivity to ABA (Fig. 6;Supplemental Fig. S6). Correspondingly, germination ofapx6 mutants was also hypersensitive to NaCl andsorbitol (Fig. 3). In contrast, ABA-insensitive mutants,such as abi4 and abi5, and ABA-deficient mutants, suchas aba2, precariously germinate in the presence of NaClor mannitol compared with wild-type seeds, which areprotected by dormancy governed by ABA (Quesadaet al., 2000; González-Guzmán et al., 2002; Tezukaet al., 2013).

Despite a 40% increase in the ABA content of dryapx6 seeds, the higher and more significant accumu-lation was observed for ABA breakdown products(Table I). This finding is in agreement with a previousreport showing that H2O2 mediates ABA catabolism(Liu et al., 2010). In addition, H2O2 was shown topromote GA biosynthesis (Liu et al., 2010; Bahin et al.,2011), and indeed, the level of GA4 was 80% higher inapx6 compared with the wild type. However, ABA andGA are antagonistic; therefore, increased ABA ho-meostasis cannot solely account for the germinationphenotype of apx6.

In contrast with ABA metabolic changes, only minordifferences were observed between the wild type andapx6 in the expression of the ABA-responsive marker

Figure 8. Effect of heat stress onmature drying seeds. A andB,Germinationassay on soil pellets (A) and on 0.53MSmedium (B) of seeds harvested fromplants grown under favorable conditions at 24˚C and seeds harvested fromplants exposed to HS (42˚C) for the last 5 to 6 d of their maturation (see“Materials andMethods”). SD values represent averages of eight replicates of50 seeds. C, Western blots showing HS-responsive proteins, MBF1c andHsp17.6, in dry seeds that experienced HS (39˚C and 42˚C) prior to harvesting(duringmaturation on themother plant). D, Relative transcript abundance ofthe three cAPXs and other stress-responsive genes in the 42˚C-stressed dryseeds. SD values represent averages of three replicates. Col-0, Columbia-0.

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genes EM6 and RD29b and the dehydrin LEA protein(Fig. 6, C and D). Interestingly, the transcript levels ofABI3 and ABI5were relatively lower in dry and imbibedapx6 seeds, yet these seeds display reduced germinationand enhanced ABA sensitivity.

ABI3 has long been recognized as a major regulatorof seed dormancy and the ABA inhibition of seed ger-mination (Bentsink and Koornneef, 2008). ABI5 functionsdownstream of ABI3, and in concert they promotedormancy and regulate the ABA-inducible expressionof LEA proteins such as EM1 and EM6 (Gampala et al.,2002; Lopez-Molina et al., 2002). Taken together, ourresults suggest that the germination phenotype in apx6mutants is not mediated by ABI3 but rather throughanother ABA-responsive signaling route.

In contrast, the level of ABI4was elevated in dry andimbibed apx6 seeds. ABI4 was recently shown to pos-itively regulate dormancy by promoting ABA synthe-sis (Shu et al., 2013). However, it is still debatablewhether ABI4 actually affects seed dormancy (Liu et al.,2013). Interestingly, recent evidence has shown thatABI4 is involved in redox regulation and oxidativechallenges in Arabidopsis leaves (Giraud et al., 2009;Foyer et al., 2012).

ABA synthesis and changes in ROS or redox arelinked through AA (Arrigoni and De Tullio, 2000;Foyer et al., 2012; Ye and Zhang, 2012; Ye et al., 2012).The activity of the 9-cis-epoxycarotenoid dioxygenaseenzyme responsible for the oxidative cleavage of neo-xanthin to xanthoxin in the ABA synthesis pathwaydepends on ascorbate (Arrigoni and De Tullio, 2000;Foyer et al., 2012). Nonetheless, in leaves of the AA-deficient mutant vitamin C defective1, the level of ABAwas 60% higher than in the wild type, due to anincrease in ABA synthesis transcripts, including thatof 9-cis-epoxycarotenoid dioxygenase (Pastori et al.,2003). Seeds of apx6 mutants contained slightly lessAA and more DHA than in the wild type, yet theirtotal is comparable in both seed types (SupplementalFig. S3). Potentially, the changes in the ratio betweenAA and DHA, acting as a redox couple signal, to-gether with elevation in ABI4 expression, may in-crease the synthesis rate of ABA and its accumulation.

In addition to the changes in ABA metabolism andsignaling, changes in auxin metabolism and signal per-ception (Table I; Fig. 7) were likely to affect the germi-nation phenotype of apx6. Since Trp is the principalprecursor for IAA biosynthesis routes (Korasick et al.,2013), its depletion in dry seeds of apx6 implies that theaccumulation in the mutant was due to de novo syn-thesis. The possibility that oxidative conditions in apx6seeds enhance auxin biosynthesis has yet to be examinedand will be tested in the near future.

It was recently shown that high levels of auxin andthe activation of IAA signaling enhance ABA-mediateddormancy by supporting the persistence of ABI3 ex-pression during imbibition (Liu et al., 2013). Our obser-vations indicate that both auxin and ABA are involvedin the germination phenotype of the apx6 mutants.However, since ABI3 and ABI5 expression were relatively

suppressed in apx6 seeds, it is likely that the cross talkbetween IAA and ABA does not involve activation ofthe ABI3 signaling route.

Interestingly, ABI4 was shown to regulate the de-velopment of Arabidopsis lateral roots by reducingpolar auxin transport (Shkolnik-Inbar and Bar-Zvi,2010). In addition, two recent studies revealed crosstalk between auxin homeostasis and ROS in seedgermination and primary root growth (He et al.,2012; Jiao et al., 2013). Whether ABI4 participates inthe regulation of auxin homeostasis in seeds remainsto be determined.

Altered levels of auxin may also influence the vigorof apx6 seeds under stress conditions and further feedback on ROS generation. The involvement of IAAcross talk with H2O2 in plant stress tolerance was recentlyreviewed; however, the mechanistic details are notwell understood (Krishnamurthy and Rathinasabapathi,2013). Accumulation of mitochondrial ROS in an Arabi-dopsis ABA overly sensitive mutant, abo6, deficientin a splicing regulator of mitochondrial complex I ren-dered it sensitive to germination on ABA. Furthermore,abo6 showed a decrease in auxin availability, and theaddition of auxin released the inhibition of germination(He et al., 2012). In contrast, the level of auxin homeo-stasis in APX6-deficient seeds increased, and the additionof IAA inhibited germination (Table I; Fig. 7). Therefore,we suggest that our findings point to a different node ofthis cross talk that is activated by an increase in cytosolicH2O2 and that is involved in dormancy, germinationcontrol, and the stress responsiveness of seeds. Thisfurther suggests that, under favorable conditions,the activation of this cross talk is suppressed by APX6activity.

Our study adds to recent reports portraying acomplex relationship between ROS, ABA, and otherhormones in seed physiology. The cross talk un-raveled in this study involving ROS, ABA, and IAAdoes not activate the ABI3-mediated dormancy pro-gram and, therefore, may constitute a novel interplaythat is associated with oxidative stress in desiccatingseeds. Consideration should be given to the involve-ment of other signals in the oxidative stress responsein seeds, since the levels of GA and several cytokininswere also altered in the mutant (Table I). APX6 emergesas the dominant ascorbate peroxidase in mature dry-ing seeds, protecting against stress and serving as amodulator of cellular signals. These newly discoveredspecialized roles in seeds for APX6, together with thereplacement of APX1 with APX6 in mature desiccatingwild-type seeds (Fig. 5), raise questions regarding thedifferences between these two enzymes. It might bethat APX6 is better suited to withstand severe desic-cation, a matter that will require further examina-tion. Understanding the degree of redundancy versusspecialization in family members such as APXs, per-oxiredoxins, and NADPH oxidases will immenselyincrease our understanding of the plant ROS net-work activity in developmental and physiologicalchallenges.

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MATERIALS AND METHODS

Plant Materials, Growth Conditions, Germination Assays,and Stress Treatments

Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used in this study.APX6 T-DNA insertion lines (apx6-1, WISCDSLOX321C09; apx6-2, WiscD-sLox466F10; and apx6-3, WiscDsLox337H07) were obtained from the Arabi-dopsis Biological Resource Center (https://abrc.osu.edu/). Homozygous lineswere PCR identified according to SIGnAL Laboratory recommendations(http://signal.salk.edu/tdnaprimers.2.html).

The abi4-1 mutant was used as a germination control in the presence ofABA.

In all germination assays, the seeds were kept at 23°C under continuous lowlight (50 mmol m–2 s–1) with 70% relative humidity. Freshly harvested seedswere surface sterilized (4 min in 50% [v/v] ethanol and 3.4% [v/v] bleachmixture, rinsed with 100% [v/v] ethanol, and dried on filter paper) and sownon 0.53 MS agar (0.8% [w/v]) or directly placed on soil pellets (Jiffy 7000). Allthe germination experiments were conducted within 1 week from harvestingto preserve dormancy. Approximately 50 seeds in six to eight replicates wereplaced on 0.53 MS plates per line per treatment. Plates were supplementedwith methyl viologen (Paraquat) for oxidative stress; with NaCl or sorbitol forosmotic stress; and with ABA or IAA (Sigma-Aldrich) for hormone sensitivitydetermination. The germination rate on MS agar was scored as radicleemergence observed with a dissecting microscope (Leica M80) and on soil ascotyledon greening. Germination on soil pellets took place without cover at24°C at 70% humidity. HS on imbibed seeds was applied by incubating at 42°Cfor 6 to 48 h, following by transfer to 24°C for germination to progress.

Mature seeds were collected from plants grown in mixed soil (3:2:1, peatmoss:vermiculite:perlite, v/v/v) and grown under controlled conditions ingrowth chambers (Percival E-30, AR-66; Percival Scientific): 24°C, 16/8-h light/dark cycle, 80 mmol m–2 s–1, and 70% relative humidity. The mutants and thewild type were always grown side by side. HS during seed maturation wasdone on 45-d-old plants bearing green mature stage 17 and 18 siliques (yellowsiliques were removed prior to treatment) grown in chambers as describedabove. Gradual HS was applied according to the following program: light, 6 to8 AM, 28°C; 8 to 10 AM, 32°C; 10 AM to 12 noon, 36°C; 12 noon to 4 PM, 39°C or42°C; 4 to 6 PM, 36°C; 6 to 8 PM, 32°C; 8 to 10 PM, 28°C; dark, 10 PM to 6 AM, 23°C.Seeds were collected after 5 and 6 d.

Molecular and Biochemical Analyses

RNA extraction from dry seeds was carried out according to the TRIzol-based method described previously (Meng and Feldman, 2010). All otherRNA extractions, PCR, complementary DNA synthesis, and real-time PCRwere done as described previously (Miller et al., 2009). First-strand comple-mentary DNA was synthesized from 1 mg of total RNA (treated with RNase-freeDNase; New England Biolabs) at 42°C with Promega MV-Reverse Transcriptase.Real-time PCR was performed on the Bio-Rad CFX96 Touch Real-Time PCRDetection System with 40 cycles and an annealing temperature of 60°C. Cyclethreshold values were determined by CFX Manager Software assuming 100%primer efficiency.

Primer sequences are listed in Supplemental Table S1. Extraction of totalprotein, western blotting, and protein oxidation analyses were done as de-scribed previously (Miller et al., 2007).

Determination of relative H2O2 concentrations and relative total peroxidaseactivity was performed as described previously (Xiong et al., 2007) with minormodifications. The dry seeds (2–3 mg) were crushed with 0.1 mL of 0.2 M

HClO4, incubated on ice for 5 min, and centrifuged for 10 min at 14,000g and4°C. The supernatant was neutralized with 0.2 M KOH and was centrifugedagain at 12,000g for 1 min. Quantification of H2O2 in extracts was performedusing a reaction with the Amplex Red reagent (Molecular Probes, Invitrogen)with three technical repeats and six biological replicates. Samples were mea-sured with the Synergy 4 fluorescence plate reader (Bio-Tek) using 530/590-nmexcitation/emission.

Relative peroxidase activity in total protein extracts from dry seeds wasdetermined according to Liu et al. (2010) with modifications. H2O2 was addedto a final concentration of 2 mM to extracts containing 200 mM Amplex Red onmicroplates, and fluorescence generation was measured as described above.The relative enzymatic activity of peroxidases was normalized to the totalamount of protein.

Relative superoxide concentration was determined according to Bournonvilleand Díaz-Ricci (2011) with minor modifications. About 5 mg of dry seeds was

crushed with 0.5 mg mL21 nitroblue tetrazolium prepared in 10 mM potassiumphosphate buffer, pH 7.8, for 1 h in the dark at room temperature. Fomazan wasextracted using 1 mL of 2 M potassium hydroxide:chloroform (1:1, v/v).Chloroformic extracts were light protected and completely dried in vacuum atroom temperature. The solid residue was dissolved in 350 mL of dimethylsulfoxide and 300 mL of 2 M potassium hydroxide at room temperature andimmediately analyzed with a Synergy 4 spectrophotometer. Formazan quanti-fication was performed at 630 nm.

AA levels were determined calorimetrically as described previously (Gillespieand Ainsworth, 2007) with the following minor adaptation for seeds: 40 mg of dryseeds was used for the extraction and was extracted to a final volume of 1 mL.

GC-MS and Data Analysis of Primary Metabolites,Including Amino Acids

Seeds from apx6-1 and wild-type lines were pooled from at least 100 pods.Free amino acids were extracted from 20 mg of dry seeds. The amino acidswere detected using the single-ion method of GC-MS, as described previously(Amira et al., 2005). For reduced glutathione determination, dry seeds (25 mg)were ground with a mortar and pestle and then extracted and analyzed byHPLC, as described previously (Matityahu et al., 2013). For primary metab-olite analysis, samples were prepared as described for the free amino acidsand 7 mL of a retention time standard mixture (0.2 mg mL21 n-dodecane,n-pentadecane, n-nonadecane, n-docosane, and n-octacosane) in pyridine. Inaddition, 4.6 mL of a retention time standard mixture of nor-Leu and ribitol(2 mg mL21) was added prior to trimethylsilylation. Samples were run on aGC-MS system (Agilent 7890A series gas chromatography system coupledwith Agilent 5975c Mass Selective Detector), and a Gerstel multipurposesampler (MPS2) was installed on this system as described previously (Matityahuet al., 2013). The data collected were obtained using the Agilent GC/MSDProductivity ChemStation software. All peaks above the baseline thresholdwere quantified and grouped according to retention time, with areas nor-malized to nor-Leu and ribitol. Substances were identified by comparison withstandards and were also compared with the commercially available electronmass spectrum libraries from the National Institute of Standards and Tech-nology and the Wiley Registry of Mass Spectral Data.

Plant Hormone Analysis

Seed samples were purified and analyzed essentially as described previ-ously (Dobrev and Kamínek, 2002; Dobrev and Vankova, 2012). Dry seeds (30mg) were homogenized with a ball mill (MM301; Retsch) and extracted in cold(220°C) extraction buffer consisting of methanol:water:formic acid (15:4:1,v/v/v). To account for sample losses and for quantification by isotope dilution,the following stable isotope-labeled internal standards (10 pmol per sample)were added: [13C6]IAA (Cambridge Isotope Laboratories), [2H6]ABA, [

2H2]GA4,[2H2]GA8, [

2H2]GA19, [2H5]trans-zeatin, [

2H5]trans-zeatin riboside, [2H5]trans-zeatin-7-glucoside, [2H5]trans-zeatin-9-glucoside, [2H5]trans-zeatin-O-glucoside,[2H5]trans-zeatin riboside-O-glucoside, [2H5]trans-zeatin riboside monophosphate,[2H3]dihydrozeatin, [2H3]dihydrozeatin riboside, [2H3]dihydrozeatin-9-glucoside, [2H6]isopentenyl adenine, [

2H6]isopentenyl adenosine, [2H6]isopentenyl

adenine-7-glucoside, [2H6]isopentenyl adenine-9-glucoside, and [2H6]isopentenyladenosine monophosphate (Olchemim). Extract was evaporated in a vacuumconcentrator (Alpha RVC; Christ). Sample residue was dissolved into 1 mL of0.1 M formic acid and applied to a mixed-mode reverse-phase cation-exchangesolid-phase extraction column (Oasis-MCX; Waters). Two hormone fractionswere eluted sequentially: (1) fraction A, eluted with methanol, containinghormones of acidic and neutral character (auxins, ABA, and GA); and (2)fraction B, eluted with 0.35 M NH4OH in 60% (v/v) methanol, containinghormones of basic character (cytokinins). Fractions were evaporated todryness in the vacuum concentrator and dissolved into 30 mL of 10% (v/v)methanol. An aliquot (10 mL) from each fraction was separately analyzed byHPLC (Ultimate 3000; Dionex) coupled to a hybrid triple quadrupole/linearion-trap mass spectrometer (3200 Q TRAP; Applied Biosystems) set in selectedreaction monitoring mode. Quantification of hormones was done using theisotope dilution method with multilevel calibration curves (r2 . 0.99). Dataprocessing was carried out with Analyst 1.5 software (Applied Biosystems).

Statistical Analysis

Principal component analysis of GC-MS data was done by MetaboAnalyst2.0 (http://metaboanalyst.ca; Xia et al., 2009, 2012) on the log-transformed

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(base 10) data sets and pareto scaling (mean centered and divided by thesquare root of SD of each variable) manipulation.

Arabidopsis Genome Initiative locus identifiers for the genes mentioned inthis article are as follows: APX1 (At1g07890), APX2 (At3g09640), APX6(At4g32320), UBIQUITIN5 (At3g62250), ACTIN2 (AT3G18780), MBF1c(At3g24500), ABI3 (At3g24650), ABI4 (At2g40220), ABI5 (At2g36270), DREBINDING FACTOR 1B (DREB1B) (At4g25490) DREB2B (At3g11020), EM6(At2g40170), LEA (At2g21490), Rd29b (At5g52300), and AOX1a (At3g22370).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Mature wild-type and apx6 plants grown undercontrol conditions.

Supplemental Figure S2. Total peroxidase activity in dry seeds of the wildtype and apx6.

Supplemental Figure S3. AA and glutathione levels in dry seeds.

Supplemental Figure S4. Genevestigator developmental expression plot ofAtAPXs.

Supplemental Figure S5. Electronic Fluorescent Pictograph browser devel-opmental expression pattern of cAPXs in the wild type.

Supplemental Figure S6. Germination of stratified seeds on 0.5 mM ABA.

Supplemental Figure S7. Principal component analysis of metabolites.

Supplemental Figure S8. Effect of HS during seed imbibition on germination.

Supplemental Table S1. Primer list and sequences.

Received June 16, 2014; accepted July 20, 2014; published July 21, 2014.

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