Polyamines Regulate Strawberry Fruit Ripening byAbscisic Acid, Auxin, and Ethylene1[OPEN]

Jiaxuan Guo,a,2,3 Shufang Wang,a,2 Xiaoyang Yu,a Rui Dong,a Yuzhong Li,b Xurong Mei,b andYuanyue Shen3

aBeijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science andTechnology, Beijing University of Agriculture, Beijing 102206, ChinabWater Resources and Dryland Farming Laboratory, Institute of Agricultural Environment and SustainableDevelopment, Chinese Academy of Agricultural Science, Beijing 100081, P.R. China

ORCID ID: 0000-0002-1414-7324 (Y.S.).

Polyamines (PAs) participate in many plant growth and developmental processes, including fruit ripening. However, it is not clearwhether PAs play a role in the ripening of strawberry (Fragaria ananassa), a model nonclimacteric plant. Here, we found that thecontent of the PA spermine (Spm) increased more sharply after the onset of fruit coloration than did that of the PAs putrescine (Put)or spermidine (Spd). Spm dominance in ripe fruit resulted from abundant transcripts of a strawberry S-adenosyl-L-Metdecarboxylase gene (FaSAMDC), which encodes an enzyme that generates a residue needed for PA biosynthesis. ExogenousSpm and Spd promoted fruit coloration, while exogenous Put and a SAMDC inhibitor inhibited coloration. Based ontranscriptome data, up- and down-regulation of FaSAMDC expression promoted and inhibited ripening, respectively, whichcoincided with changes in several physiological parameters and their corresponding gene transcripts, including firmness,anthocyanin content, sugar content, polyamine content, auxin (indole-3-acetic acid [IAA]) content, abscisic acid (ABA) content,and ethylene emission. Using isothermal titration calorimetry, we found that FaSAMDC also had a high enzymatic activity with aKd of 1.73 1023

M. In conclusion, PAs, especially Spm, regulate strawberry fruit ripening in an ABA-dominated, IAA-participating,and ethylene-coordinated manner, and FaSAMDC plays an important role in ripening.

Fleshy fruit ripening involves complicated changesin sugar, texture, color, flavor, and aroma and iscontrolled by plant hormones. Based on studies in themodel plant tomato (Solanum lycopersicum), climac-teric fruit ripening is known to be controlled by eth-ylene (Alexander and Grierson, 2002). By contrast, theripening of strawberry (Fragaria ananassa), a modelnonclimacteric plant, is complex (Shen and Rose,2014) and controlled by abscisic acid (ABA; Li et al.,2011), auxin (indole-3-acetic acid [IAA]; Given et al.,1988), gibberellic acid (GA; Csukasi et al., 2011), eth-ylene (Merchante et al., 2013), jasmonate (JA; Concha

et al., 2013), and brassinosteroids (Chai et al., 2013).Polyamines (PAs) have been previously reported to beinvolved in strawberry fruit development (Tilak andRaymond, 1996), but whether PAs play a role in rip-ening is unknown.

PAs, which are ubiquitous aliphatic amines andbiogenic regulators, are involved in many physio-logical and developmental processes, includingplant growth, senescence, and stress (Tabor and Tabor,1984; Minocha, 1988; Mariani et al., 1989; Nambeesanet al., 2008). In plants, putrescine (Put) is convertedto spermidine (Spd) and then spermine (Spm) by Spdsynthase (SPDS)- and Spm synthase (SPMS)-catalyzedsequential additions of amino propyl residues. Decar-boxylated S-adenosyl-L-Met is required for the reactionsand is a product of SAM and the catalyst SAM decar-boxylase (SAMDC). Thus, SAMDC is a rate-limiting stepfor Spd and Spm synthesis (Mehta et al., 2002; Wei Huet al., 2006).

Notably, SAM acts as a common precursor for bothPA and ethylene biosynthesis (Apelbaum et al., 1981;Minocha, 1988; Larsson et al., 1997; Martin-Tanguy,2001). Although SAM is transformed preferentially intoPAs (Khan and Singh, 2010; de Dios et al., 2006), eth-ylene and PA biosynthesis do not compete (Kushadet al., 1988). SAM is homeostatically regulated, so as tomaintain higher rates in the simultaneous production ofethylene and PA (Van de Poel et al., 2013; Lasanajaket al., 2014). Overexpression of the gene encoding Orn

1 This work was supported by the China National Science Foun-dation (Projects 31040006, 31672125, and 41473004), the Beijing Nat-ural Science Foundation (6171001), and the Beijing Training Projectfor the Leading Talents in S&T (LJ201612).

2 These authors contributed equally to the article.3 Address correspondence to guojiaxuangjx@163.com or

sfmn@tom.com.The authors responsible for distribution of materials integral to

the findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org)are: Jiaxuan Guo (guojiaxuangjx@163.com) and Yuanyue Shen(sfmn@tom.com).

S.W., X.Y., and R.D. performed the experiments; Y.L. and X.M.analyzed the data; G.J. and Y.S. designed the research and wrotethe article.

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decarboxylase (ODC) promotes Put, Spd, and Spmsynthesis, inhibits ethylene emission, respiration andwater loss, and delays ripening but enhances tomatofruit quality (Gupta et al., 2013; Pandey et al., 2015).Overexpression of the SPDS gene in tomato fruitspromotes carotenoid and lycopene accumulation, aswell as ethylene production (Neily et al., 2011). No-tably, the increased expression of a yeast SAMDC(ySAMDC) gene in tomato fruits promotes conversionof Put to Spd and Spm, and influences multiple cellpathways and broad gene expression levels, therebyincreasing lycopene content, prolonging shelf life,and enhancing fruit juice quality (Mehta et al., 2002;Mattoo et al., 2006, 2007; Nambeesan et al., 2010;Kolotilin et al., 2011). High Spd and Spm contentsexhibit strong effects on various metabolic processes,including amino acid, sugar, and energy, and regulateripening rate and extent in tomato (Tassoni et al., 2006;Fatima et al., 2016). Changes in the contents of ripening-related physiological parameters, including sugar, eth-ylene, and lycopene, are positively related to Spd andSpm contents but negatively related to Put content,emphasizing that individual biogenic amines havedifferential roles in developing tomato fruits (Handaand Mattoo, 2010).

In addition to their roles in tomato fruit, PAs havebeen reported to function in other fruits. PAs play im-portant roles in grape (Vitis vinifera) berry ripening,including cell expansion and aroma development(Agudelo-Romero et al., 2013, 2014; Fortes et al., 2015).Exogenous PAs strongly inhibit peach (Prunus persica)fruit ripening, especially softening (Bregoli et al., 2002;Ziosi et al., 2006; Torrigiani et al., 2012), while higherPA concentrations coincide with fruit enlargementduring oil-palm (Elaeis guineensis) maturation (Tehet al., 2014). PAs also take part in raspberry (Rubusoccidentalis) and date (Phoenix dactylifera) fruit ripening(Diboun et al., 2015; Simpson et al., 2017). The pre- andpostharvest application of PAs delays ripening andextends shelf lives in mango (Mangifera indica), peach(Prunus persica), plum (Prunus domestica), and apple(Malus domestica) fruit, while controversial conclusionson the roles of PAs in ripening have also been reported(Law et al., 1991; Wang et al., 1993; Escribano andMerodio, 1994; Mattoo et al., 2002; Pérez-Amador et al.,2002; Torrigiani et al., 2004; Neily et al., 2011). Thus,more research is required to further understand theroles of PAs in fleshy fruit ripening.

Given that strawberry is regarded as an ideal modelplant for studying nonclimacteric fruit ripening (Liet al., 2011), we examined the roles of PAs duringstrawberry fruit ripening to better understand the mo-lecular mechanisms of nonclimacteric fruit develop-ment. We first measured PA contents in developingstrawberry fruits and investigated the effects of PAs onripening. Next, we cloned the strawberry SAMDC geneand identified its functions during ripening using virus-induced gene silencing (VIGS) and overexpression(OE). Finally, we analyzed the enzymatic activity ofSAMDC byHPLC and isothermal titration calorimetry.

Our results demonstrated that FaSAMDC positivelyregulates strawberry fruit ripening, and PAs, especiallySpm, play important roles in this process.

RESULTS

PA Contents in Developing Strawberry Fruits

According to a previous report (Jia et al., 2011), thedeveloping fruit of strawberry (F. ananassa cv ‘SweetCharlie’) can be divided into seven stages: small green(SG), large green (LG), degreening (DG), white (Wt),initial red (IR), partial red (PR; namely, turning), andfull red (FR; Fig. 1). HPLC analysis of Put, Spd, and Spmcontents is shown in Figure 1. Put contents graduallydecreased from SG to FR stages and reached the lowestlevels in FR fruits. Spd content was low from the SG toPR stages but increased slightly in FR fruits. Notably,Spm contents declined slightly in green fruits but in-creased sharply after the Wt stage and reached thehighest concentration in FR fruits. These results suggestthat PAs, especially Spm, play roles in strawberry fruitripening.

Effects of PAs and a SAMDC Inhibitor on StrawberryFruit Ripening

To further determine the role of PAs in strawberry fruitripening, we immersed 20 DG fruits still attached toplants in Put, Spd, and Spm, as well as methylgloxalbis(guanylhydrazine) (MGBG), a SAMDC inhibitor (Fig.2A). Sterile water was used as a control. Eight daysafter treatment, we observed the ratio of dark-red, red,and light-red fruits. Spm-, Spd-, Put-, MGBG-, andwater-treated fruits showed ratios of 17:2:1, 17:2:1, 1:3:16, 2:3:15,and 3:16:1, respectively (Fig. 2B). Thus, exogenous Spmand Spd promoted coloration, while MGBG and Putinhibited coloration compared to the control, which wassupported by anthocyanin contents (Fig. 2C). Analysis ofPA contents in these treated fruits showed that exoge-nous Spm and Spd significantly promoted endogenousSpm or Spd accumulation but not Put contents; in con-trast, exogenous Put and MGBG significantly inhibitedendogenous Spm or Spd accumulation, but significantlypromoted Put accumulation compared to the control(Fig. 2D). These results demonstrate that Spm and Spdpromote ripening, while Put inhibits ripening.

To understand the effects of PAs on coloration, con-tents of several ripening-related plant hormones, in-cluding ethylene, ABA, and IAA, were investigated.The results showed that in comparison to the control,exogenous Spm, Put, and MGBG promoted ethyleneemission, while Spd inhibited ethylene emission (Fig.2E); exogenous Spm promoted ABA accumulation,while Spd, Put, and MGBG inhibited ABA accumula-tion (Fig. 2F); exogenous Spm, Put, and MGBG pro-moted IAA accumulation, while Spd inhibited IAAaccumulation (Fig. 2G). These results suggest that theeffects of PAs on strawberry fruit coloration might berelated to ABA, ethylene, and IAA.

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RNA-Seq, Annotation, and Plant Hormone SignalTransduction Pathway Analyses

To maximize the transcriptional data obtainedfrom ripening strawberries, we used cDNA librariesfrom four stages (mixed and separate), i.e. LG, Wt,IR, and PR fruit, for RNA-seq. Approximately 5 to9 billion raw reads distributed among each li-brary and could be used for further analyses (Supple-mental Table S1; NCBI, SUB3724936). A total of 98,859unigenes were considered valid (Supplemental TableS2) and were annotated using 25 KOGs (eukaryoticorthologous groups; Supplemental Fig. S1). The topfive pathways with more abundant differentiallyexpressed genes (DEGs) were involved in ribosomeprocessing, spliceosome processing, protein processing,plant-pathogen interaction, and plant hormone signaltransduction (Supplemental Table S3). Based on GeneOntology analysis, a total of 31,792 unigenes were clas-sified into three groups: “biological process,” “cellularcomponent,” and “molecular function” (SupplementalFig. S2).To analyze the Kyoto Encyclopedia of Genes and

Genomes (KEGG) pathways of unigenes, we used theKEGG Automatic Annotation Server to obtain KEGGortholog numbers, which are mapped to correspondingKEGG pathways. Based on log2 expression level, a totalof 301 pathways with 14,025 unigenes were annotated.Based on DEG analysis between library pairs (LG-Wt,Wt-IR, and IR-PR), we found that the total number ofDEGs declined rapidly from LG to PR. More DEGswere downregulated between LG and Wt than wereupregulated, and more DEGs were upregulated from

Wt to IR than were downregulated. Notably, IR and PRhad similar quantities of up- and down-regulatedDEGs(Fig. 3A). These results suggest that metabolism tran-sitions occurred between green-white-red stages, sowhite fruit is a distinct stage.

Next, we focused on screening the DEGs involved inplant hormone signal transduction around the onset ofripening and found several important signaling DEGs,including genes related to IAA pathway (IAA2, ARF11,IAA16, IAA27, and LUX2), ABA pathway (PP2C37 andSnRK2), ethylene pathway (ACO1 and EBF1), JA (JAZ),and GA pathway (GID1; Fig. 3B). Notably, in conjunctionwith degreening and coloration, expression of IAA2,IAA16, IAA27, AUX2, and JAZ declined gradually, whileARF11 increased continually. Expression of PP2C andEBF1 increased during degreening but decreased duringcoloration. ACO1 expression increased during colorationwhereas GID1 expression increased during degreening,decreasedduring initial coloration, andfinally increased inthe ripening fruits (Fig. 3B). The fact that the most abun-dant hormone-related DEGs during ripening were relatedto IAA (five unigenes), ABA (two unigenes), and ethylene(twounigenes) suggests thatABA, IAA, and ethylenemayplay important roles in strawberry fruit ripening.

Roles of FaSAMDC in Strawberry Fruit Ripening

PA contents in developing fruit (Fig. 1) and phar-macological experiments (Fig. 2) suggest an importantrole of SAMDC, which is necessary for Spd and Spmbiosynthesis (Mehta et al., 2002; Wei Hu et al., 2006),in ripening. We investigated strawberry homologs of

Figure 1. Polyamine contents in seven developmental stages of strawberry fruit. Polyamine contents examined usingHPLC. Threefruits were used for detection in each stage. Error bars represent SE (n = 3).

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SAMDC in our RNA-seq data and found eight SAMDC-like transcripts, including their complementary se-quences. We divided these transcripts into four groups,of which comp72320_c0_seq1 and its complementarystrand, comp73015_c0_seq2, had substantially highermRNA expression levels than the others (Fig. 4A). Thistranscript encoded a protein with the SAM-decarbboxdomain (Supplemental Fig. S3) and thus was named

FaSAMDC (GenBank no. MG266892). Based on log2expression levels, the transcripts of FaSAMDC werehighly expressed in the LG fruits, declined duringdegreening, and increased rapidly during coloration(Fig. 4A). This trend of FaSAMDC expression levels wasfurther confirmed by quantitative PCR (qPCR) analysis(Fig. 4B). These results suggest a role of FaSAMDC inripening.

Figure 2. Effects of polyamines and an inhibitor on strawberry fruit coloration. Twenty degreening strawberry fruits still attachedto the plants were used for each independent treatment and immersed in 100 mM Put, Spd, Spm, or MGBG (n = 20, one repli-cation). Water was used as the control. Three fruits representative of the Put-, Spm-, Spd-, and MGBG-treated phenotypes wereused for detection of the physiological parameters (n = 3, three replications). A, Fruit phenotypes before (DG) and after treatment.B, The number of fruits per phenotype within each treatment. C, Anthocyanin contents. D, Polyamine contents. E, Ethyleneemission rates. F, ABA contents. G, IAA contents. Error bars represent SE (n = 3).The asterisk in same-colored columns indicates astatistically significant difference compared to the control (P, 0.05) after an ANOVA followed by Duncan’s multiple range tests.

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Figure 3. DEGs during the onset of strawberry fruit ripening as determined by RNA-seq. Fruit cDNA libraries from four devel-opmental stages, including LG,Wt, IR, and PR, were used for RNA-seq. A, The number of upregulated and downregulated DEGsbetween library pairs. B, Heat map and cluster analysis of the DEGs encoding proteins in plant hormone signaling pathways. Thedata for gene expression levels were normalized to a z-score with the formula log10 (FPKM+1) by color key and density plot.Green represents low expression and red represents high expression during ripening. IAA, auxin-responsive protein/transcriptionfactor; LUX, auxin transporter 2; JAZ, jasmonate ZIM (zinc-finger inflorescence meristem) domain; PP2C37/ABI37, 2C-typeprotein phosphatase 37/ ABA-insensitive 37; EBF1, EIN3 (ethylene insensitive 3) binding F-box 1; GID1, gibberellin receptor 1;ACO1, 1-aminocyclopropane-1-carboxylic acid oxidase 1; SnRK2, Ser/Thr-protein kinase 2.

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We then used RNA interference (RNAi) and OEtechniques (Jia et al., 2011, 2013) to determine the func-tion of FaSAMDC in ripening. Eight days after inocu-lation, RNAi-treated fruits had a chimeric phenotype(Fig. 5A),whilemost of theOE-treated fruits turneddarkred (18 dark red and 2 red; Figure 5C); in contrast,control fruits remained light red (Fig. 5B). qPCRanalysesshowed that the mRNA expression levels of FaSAMDCsignificantly decreased and increased in the RNAi andOE fruits compared to the control fruits (Fig. 5D), re-spectively. In addition, both vectors were detected ininfected sections (Fig. 5E). These results demonstratethat FaSAMDC plays a role in strawberry fruit ripening.

Manipulation of FaSAMDC Expression Affects SeveralPhysiological Parameters and CorrespondingGene Transcripts

Based on previous reports (Jia et al., 2013; Zhao et al.,2017; Zhang et al., 2017) and our RNA-seq data, the ex-pression levels of various genes and their correspondingphysiological parameters, including firmness (PG1 andPL1), anthocyanin content (CHS andDFR), sugar content(SUT1 and SS), polyamine content (ADC, ODC, SPMS,and SPDM), IAA levels and signaling (IAA2,ARF11, and

LUX2), ABA levels and signaling (ABI37, SnRK2, andNCED1), and ethylene emissions and signaling (ACO1and EBF1), were analyzed in the RNAi and OE fruits, inwhich FaSAMDC expression levels were down- andup-regulated over 80% comparedwith themixed controlfruits. The results showed that in comparison to thecontrol, both Spm and Spd contents were higher in OEfruits and lower in the RNAi fruits, but Put contentswere lower in the OE fruits and higher in the RNAi fruits(Fig. 6A). Ethylene emission rates decreased in bothRNAi and OE fruits (Fig. 6B). IAA (Fig. 6C) and antho-cyanin (Fig. 6E) contents declined in RNAi fruits butincreased inOE fruits. The contents of ABA (Fig. 6C) andsoluble sugars (Fig. 6F) increased in the OE fruits butdecreased in the RNAi fruits. Compared to the control,the expression of most genes, including PG1, PL1, CHS,DFR, SUT1, SS, ADC, ODC, SPDS, SPMS, ARF11,SnRK2, NCED1, and EBF1, was upregulated in the OEfruits and downregulated in the RNAi fruits, while theexpression of ABI37, IAA2, and AUX2 was down-regulated in the OE fruits and upregulated in the RNAifruits. Notably, ACO1 expression was downregulated inboth OE and RNAi fruits. Taken together, manipulationof FaSAMDC expression affected both synthesis-relatedPA gene transcript levels and PA contents. As a result,

Figure 4. Transcripts of FaSAMDC during straw-berry fruit ripening as determined by RNA-seq andqPCR expression analysis. A, Eight SAMDC gene-like contigs were found in the transcriptomic datafrom LG, Wt, IR, and PR fruits. B, The mRNA ex-pression levels of FaSAMDC in six fruit stages, in-cluding LG, DG, Wt, IR, PR, and FR. Actin mRNAwas used as an internal control. Columns with dif-ferent letters (a–f) indicate statistically significantdifferences (P , 0.05) after an ANOVA followed byDuncan’s multiple range tests. Error bars representSE (n = 3).

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altered FaSAMDC expression also affected ABA, ethyl-ene, and IAA levels and signaling, ripening-related geneexpression levels, and fruit ripening.

Prokaryotic Expression and Enzymatic Activity ofRecombinant FaSAMDC Protein

To investigate the activity of FaSAMDC, weexpressed the coding sequence of FaSAMDC in Esch-erichia coli cells. We purified the ;66-kD FaSAMDCprotein (Fig. 7A) and identified the protein with ananti-C-terminal GST antibody (Fig. 7B), resulting in aFaSAMDC protein with 97.1% purity and a concen-tration of 3.2 mg/mL. The combination of chemicalreactions and HPLC analysis revealed that the enzy-matic activity of FaSAMDC reached 1.88 units/mgbased on the standard curve analysis (SupplementalFig. S4). The SAM-buffer-FaSAMDC titration reac-tions in ITC200 indicated that one purified FaSAMDCmolecule could bind one molecule of SAM molecule,and the average Kd was 1.7 3 1023

M (Fig. 7C). Theseresults demonstrated that strawberry SAMDC proteinhas a high enzymatic activity.

DISCUSSION

PAs, in Particular Spm, Play a Role in StrawberryFruit Ripening

Polyamines do not simply act as protective molecules,but rather as signals in plant stress tolerance (Pál et al.,2015). Although much progress has been made in fleshfruit ripening (Alexander and Grierson 2002; Adams-Phillips et al., 2004; Prasanna et al., 2007; Jia et al., 2011;Li et al., 2011; Osorio et al., 2013; Seymour et al., 2013;Kumar et al., 2014; Shen and Rose, 2014; Liu et al.,2015; Gallusci et al., 2016), the role of PAs in the rip-ening of strawberry, a model nonclimacteric plant, isnot fully understood.

In this study, we found that exogenous Put inhibitedripening, while exogenous Spm and Spd both promotedripening (Fig. 2). Given that Spm content was highest inthe ripening fruits (Fig. 1), we speculated that Spm mayplay an important role in ripening. Since SAMDC isneeded for Spd and Spm biosynthesis (Mehta et al., 2002;Wei Hu et al., 2006), we manipulated its expression instrawberry fruits, finding that up- and down-regulationof FaSAMDC expression affected the abundance ofADC,ODC, SPDS, and SPDM transcripts, which led to lowerand higher (Spd + Spm)/Put ratio in the RNAi and OEfruits, inhibiting and promoting ripening, respectively(Fig. 5). In addition, strawberry FaSAMDCwas shown tohave a high enzymatic activity with a Kd of 1.7 3 1023

M

(Fig. 7). These results demonstrate that PAs, in particularSpm, play important roles in strawberry fruit ripening.

In tomato, enhanced expression of the gene encodingSAMDC in fruits increases the conversion of Put to Spdand Spm, promoting ripening. However, the ripeningfruits of such transgenic plants have more Spd thanSpm (Lasanajak et al., 2014). In contrast, the transgenicripening strawberry fruits contained higher levels ofSpm than Spd (Fig. 6). Given that tomato and straw-berry represent two different types of fruit ripening,climacteric and nonclimacteric, respectively, it is likelythat the roles of PA components vary with differenttypes of fruits during ripening.

Understanding Polyamine Mechanisms through ABA,Ethylene, and IAA during Strawberry Fruit Ripening

Ripening of strawberry fruits is not only controlledby ABA (Chai et al., 2011; Li et al., 2011; Jia et al., 2011,2013; Daminato et al., 2013; Li et al., 2013; Han et al.,2015), but also by several other hormones, includingIAA (Given et al., 1988), GA (Csukasi et al., 2011), eth-ylene (Merchante et al., 2013), JA (Concha et al., 2013),and brassinosteroids (Chai et al., 2013). Althoughstrawberry fruits do not show a peak in ethyleneemission during ripening (Given et al., 1988), this hor-mone plays a role in ripening (Sun et al., 2013). Agradual increase in ABA content is coupled with a de-crease in IAA in developing strawberry fruit, suggest-ing that the ABA/IAA ratio serves as a signal to triggerripening (Perkins-Veazie, 1995). IAA stimulates early

Figure 5. Silencing and overexpression of FaSAMDC in developingstrawberry fruit. Twenty DG fruit still attached to the plant were used forinoculations. A to C, Eight days after inoculation, FaSAMDC-VIGS(RNAi) fruit (A) showed chimeric phenotypes compared to the control(B), while the FaSAMDC-OE fruits (C) appeared dark red. D, FaSAMDCtranscripts in RNAi and OE fruit compared to the control. E, TRV vectordetection in VIGS fruit. Actin mRNA was used as an internal control.The asterisk in the columns indicates statistically significant differences(P , 0.05) compared to the control after an ANOVA followed byDuncan’s multiple range tests. Error bars represent SE (n = 3).

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Figure 6. Manipulation of FaSAMDC expression affects several physiological parameters and relevant gene transcripts. TheFaSAMDC transgenic fruits, in which the target gene was down (RNAi)- and up (OE)-regulated bymore than 80% compared withthe mixed control fruits, were used for analysis. A, Polyamine contents including Put, Spd, and Spm. B, Ethylene emissions. C,ABA and IAA contents. D, Fruit firmness. E, Anthocyanin contents. F, Soluble sugar contents. G, Ripening-related gene expressionlevels, including firmness (polygalacturonase [PG1] and pectate lyase [PL1]), anthocyanin (chalcone synthase [CHS] and dihy-droflavonol 4-reductase [DFR]), sugar (Suc transporter 1 [SUT1] and Suc synthase [SS]), polyamines (Arg decarboxylase [ADC],Orn decarboxylase [ODC], Spd synthase [SPDS], and Spm synthase [SPMS]), IAA (auxin-responsive protein 2 and factor 11 [IAA2and ARF11], and auxin transporter 2 [LUX2]), ABA (protein phosphatase 2C 37 [ABI37], Ser/Thr-protein kinase SRK2 [SnRK2],and 9-cis-epoxycarotenoid dioxygenase [NCED1]), and ethylene (1-aminocyclopropanecarboxylate synthase 1 [ACO1] andEIN3-binding F-box protein [EBF1]) genes. Actin mRNAwas used as an internal control. The asterisk in the same color or genecolumns indicates statistically significant differences (P, 0.05) compared to the control after an ANOVA followed by Duncan’smultiple range tests. Error bars represent SE (n = 3).

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receptacle expansion but inhibits later ripening (Givenet al., 1988), while ABA is a key inducer of ripening (Jiaet al., 2011). Our pharmacological tests revealed that PAregulation of strawberry fruit ripening was involved inABA, ethylene, and IAA (Fig. 2). Consistently, our RNA-seq data indicated that the hormone-related DEGs werealso involved in IAA,ABA, andethylenepathways (Fig. 3).In support of this, up- and down-regulation of FaSAMDCexpression promoted and inhibited ABA accumulationand inhibited and promoted IAA accumulation, respec-tively, while both inhibited ethylene emission in the RNAiand OE fruits (Figs. 5 and 6). These results demonstratethat PA regulation of strawberry fruit ripening is closelyrelated to ABA, ethylene, and IAA.Given that SAM serves as a common precursor in the

synthesis of PAs and ethylene (Apelbaum et al., 1981;Minocha, 1988; Larsson et al., 1997; Martin-Tanguy,2001), there may be an equilibrium between PA andethylene in strawberry fruit that is disrupted by themanipulation of FaSAMDC, thus inhibiting ethyleneemission (Fig. 6). We also note that the enhanced ex-pression of FaSAMDC in strawberry fruits resulted in anincrease in conversion of Put to Spd and then Spm and ahigh ratio of (Spm + Spd)/Put with the following con-sequences: (1) Expression levels were promoted in theNCED1 (key for ABA synthesis gene; Jia et al., 2011)and SnRK2 (a positive regulator gene of ABA signaling;Jia et al., 2013) genes but inhibited in the ABI37 (a neg-ative regulator gene of ABA signaling; Jia et al., 2013)gene. This synergistic regulation in ABA synthesis and

signaling contributes to a maximized role of ABA inripening. (2) Expression levels were inhibited in theLUX2 gene (key for IAA transport gene; Carrier et al.,2008), but separately inhibited and promoted in the IAA2(a negative regulator gene of IAA signaling; Liu et al.,2011) and ARF11 (a positive regulator gene of IAA sig-naling; Estrada-Johnson et al., 2017) genes, showing thatIAA levels and IAA signaling are not synergistic in rip-ening. (3) Expression levels were inhibited in the ACO1(key for ethylene synthesis gene; Liu et al., 1999) gene,but promoted in the EBF1 (a negative regulator gene ofethylene signaling; Yang et al., 2010) gene, showingthat ethylene synthesis and signaling were inhibited inripening. Based on these results, we propose a model forPA-mediated regulation of strawberry fruit ripeninginvolving ABA, ethylene, and IAA: In the developmen-tal strawberry fruit, the high Put contents inhibit ripen-ing and contribute to early fruit growth. With the onsetof fruit ripening, the high expression levels of SAMDCpromote conversion of Put to Spd and then Spm throughsynergistic expression of ODC, ADC, SPDS, and SPDM.As a result, the higher Spm contents promote ABA ac-cumulation and signaling and inhibit both IAA andethylene accumulation, while promoting IAA signalingbut inhibiting ethylene signaling. This leads to increasedexpression of softening-, anthocyanin-, and sugar-related genes, which promotes ripening (Fig. 8). In con-clusion, PAs, especially Spm, regulate strawberry fruitripening in an ABA-dominated, IAA-participating, andethylene-coordinated manner.

Figure 7. Purification, identification,and enzymatic activity of FaSAMDCprotein. A, Purification of the 66-kDrecombinant FaSAMDC protein. B,Immunoblot identification of the re-combinant FaSAMDC. C, Measure-ment of the affinity between SAMand the purified FaSAMDC proteinusing isothermal titration calorime-try. A typical and specific saturationcurve with stoichiometry (N) of 1:1was obtained, suggesting that oneSAM molecule could bind per puri-fied protein molecule with a disso-ciation constant of 1.7 3 1023

M.

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MATERIALS AND METHODS

Plant Material

Strawberry (Fragaria ananassa) plants (‘Sweet Charlie’) were grown in thegreenhouse during the spring season from 2015 to 2016. In total, 300 SG fruit on50 plants were tagged after flowering. Seven stages (SG, LG, DG, Wt, IR, PR,and FR) were collected at 7, 15, 20, 23, 25, 27, and 30 d after flowering, re-spectively. At every stage, 40 uniformly sized fruit were sampled and frozen inliquid nitrogen (280°C) until further use.

RNA-Seq, cDNA Synthesis, and Data Analysis

Three fruits (n = 3) of each stage were randomly selected for RNA isolationand cDNA synthesis. RNA was extracted from the receptacles of DG, Wt, IR,and PR fruits using the RNeasy plant mini kit (Qiagen). DNase digestion wasperformed by RNase-Free DNase (Qiagen), and cDNA synthesis was con-ducted using the RNA library prep kit (New England Biolabs). RNA-seq anddata analysis were carried out using Illumina HiSeq 2000 by the Yuanyi GeneCompany as previously described (Mortazavi et al., 2008; Ming et al., 2012;Wang et al., 2010; Benjamini and Yekutieli, 2001; Langmead and Salzberg,

2012) and using available genetic databases, including the NCBI nonredun-dant protein database, SWISS-PROT, TrEMBL, Cdd, pfam, KOG database,KEGG, and Gene Ontology based on an E-value of 1e25 and identity with30%. The experiment was repeated three times.

FaSAMDC Cloning

The obtained cDNA was used as a template for amplifying FaSAMDCwith primers (GenBank MG266892, not released; forward, 59-ATGGCTG-TACCGGTTTCTGC-39; reverse, 59-CTAGTTCTTCATGCTCAAAC-39). PCRwas performed with an annealing temperature of 58°C.The PCR fragment wasinserted into a T1 simple vector and then transformed into Escherichia coliDH5a(BioTeke). Positive colonies were sequenced by the Huada Company.

Construction of Recombinant Plasmids and Transfectionof Strawberry by Agroinfiltration

pTRV1 and pTRV2 were used for VIGS (Liu et al., 2002). A 650-bp cDNAfragment of FaSAMDC was amplified (primers: sense, 59-GGAATTCG-TAGCCCTGAC-39; antisense, 59-GGGGTACCGTTCTTCATGC-39) and inser-ted into pTRV2 using restriction enzymes EcoRI and KpnI. Agrobacteriumtumefaciens strain GV3101 containing pTRV1, pTRV2, or pTRV2-FaSAMDCwasinfiltrated into strawberry fruits. To generate the FaSAMDC overexpressionconstruct, the full-length cDNA of FaSAMDC was obtained by PCR (primers:forward, 59-GAATTCATGGCT GTACCGGTTTCTGC-39, EcoRI site underlined;reverse, 59-GGTACCTAGTTCTTCATGCTCAAACTC-39, KpnI site underlined).The cDNA was inserted into pCAMBIA1304 using EcoRI and KpnI. TheA. tumefaciens suspension was injected into 20 DG strawberry fruits attached tothe plants as previously described (Fu et al., 2005; Jia et al., 2011). The experi-ment was repeated one time.

Detection of TRV Vectors

Three infected fruits (n = 3) were used. The primers for TRV1 (sense, 59-TGCTCCTGAAAGTATGTTAGTGG-39; antisense, 59-CATCTCGGATGTCTCGACG-39)and TRV2 (sense, 59-CCGACTCATTGTCTTACCATAG-39; antisense, 59-TCTCCCGTTTCGTCCTTT-39) were used to detect TRV vectors in theinfiltrated strawberry fruits as previously described (Chai et al., 2011).The experiment was repeated three times.

qPCR

Three transgenic fruits (n = 3) were randomly selected from each stage forRNA isolation and cDNA synthesis as above. Gene expression levels wereanalyzed by real-time qPCR. The qPCR was performed using a Light Cycler96 real-time PCR system (Bio-Rad). The reactions (20 mL) contained 10 mL ofSYBR Premix ExTaq (TaKaRa), 0.4 mL of 10 mM forward-specific primer, 0.4 mLof 10 mM reverse-specific primer, and 2 mL of 1 mM cDNA templates. qPCR wasconducted with three biological replicates, and each sample was analyzed atleast in triplicate and normalized using Actin (Han et al., 2015) as an internalcontrol for relative expression levels confirmed by the form 2–DDCT (Livakand Schmittgen, 2001). The primers used for real-time PCR are shown inSupplemental Table S4. The experiment was repeated three times.

Effects of PAs and a SAMDC Inhibitor on Strawberry FruitRipening in Vivo

Twenty LG stage fruits still attached to plants were selected (n = 20) andimmersed independently into 100 mM Put, Spd, Spm, and MGBG for 20 s,respectively. Sterile water was used as a control. A total of four immersionswere performed for every treatment at 1-d intervals. Ten days after treatment,phenotypes were observed. The experiment was repeated one time.

Expression and Purification of FaSAMDCRecombinant Proteins

Expression and purification of FaSAMDCwere done in an E. coli expressionsystem. The coding sequence of FaSAMDC was amplified by PCR (forward,59-GAATTCATGGCTGTACCGGTTTCTG-39, EcoRI site underlined; reverse,59-GCGGCCGCCTAGTTCTTCATGCTCAAACTC-39, NotI site underlined) and

Figure 8. A model for PA regulation of strawberry fruit ripening throughABA, IAA, and ethylene. In developing strawberry fruit, the biosynthesis ofpolyamines (Put, Spd, and Spm) depends on ODC, ADC, SAMDS, SPDS,and SPDM, of which SAMDC is a rate-limiting step for Spd and Spm syn-thesis. The up-regulation of SAMDC expression positively regulates the ex-pression of ODC, ADC, SPDS, and SPDM, which promotes Spd and,especially, Spm accumulation while inhibiting Put accumulation. A highratio of (Spd + Spm)/Put accelerates ABA synthesis and signaling by pro-moting the expression of NCED1 (key to ABA synthesis) and SnRK2 (apositive regulator of ABA signaling) and inhibiting the expression of ABI37 (anegative regulator of ABA signaling).The high (Spd + Spm)/Put ratio alsodifferently regulates IAA transport and signaling by inhibiting the expressionof AUX2 (key to ABA transport) and IAA2 (a negative regulator of IAA sig-naling) and promoting the expression of ARF11 (a positive regulator of ABAsignaling). However, the high ratio inhibits ethylene synthesis and signalingby inhibiting the expression of ACO1 (key to ethylene synthesis) gene andpromoting the expression of EBF1 (a negative regulator of ethylene signaling)gene. Thus, PAs regulate strawberry fruit ripening in an ABA-dominated,IAA-participating, and ethylene-coordinated manner to promote ripening-related gene expression levels, including those for firmness (PG1 and PL1),anthocyanin content (CHS andDFR), and sugar content (SUT1 and SS). Thegraphic symbols (arrow, t-bar, and arrow with bar) represent promote, in-hibit, and no cooperation. Red arrows indicate promotion.

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inserted into expression vector PGEX-4T1 in frame with the N-terminalGST-tag fusion (BioTeke). The recombinant plastids were transformedinto E. coli for selection of transformants on LB plates with ampicillin(100 mg/mL). The FaSAMDC fusion protein was expressed and purifiedusing BeaverBeads GST, and immunoblotting was performed with anantibody of the C-terminal His tag using a one-step western kit HRP (anti-Mouse; Kangwei Company) following the manufacturer’s protocols.

Analysis of Enzyme Activity

The activity of FaSAMDCwas assayed byHPLCusing an enzyme activitystandard curve. SAMwas formulated at concentrations with 1.0, 0.8, 0.5, 0.4,and 0.1 mg/mL for construction of a SAM standard curve. To investigateenzymatic activity, the reaction of FaSAMDC protein (2 mg/mL) and SAM(25 mM) was carried out in a buffer (50 mM Tris-HCl, pH 8.0) at 30°C for10 min. After centrifugation, the supernatant (1 mL) was filtered with a 0.22-mmfilter, and the free SAM content was determined by HPLC. The C18-column(Zorbax Eclipse XDB-C18, 4.6 3 150 mm, 5 mm; Agilent) was used at 30°C for10 min at a flow rate of 0.6 mL per min, and the mobile phase was 0.01 mM

ammonium formate:methanol (v:v, 97:3). Effluent absorbancewasmonitored at260 nm. The injection volume was 10 mL. The experiment was repeated threetimes.

Determination of PA, Anthocyanin, and SolubleSugar Contents

Three fruits (n = 3) were used for determination of PA contents by HPLCafter the preparation and derivation of standard products. Based onRedmond and Tseng (1979), the improved method of acetylation of ben-zene was used. Samples (0.1 g) of individual PAs, including Put, Spd, andSpm, were dissolved with ultrapure water to 10 mL in volumetric flasks asstandards. The 20-mL solutions of Put, Spd, and Spm, individually, wereplaced into 10-mL centrifugal tubes, benzoyl chloride (10 mL) and 2 mol/Lsodium hydroxide (1 mL) were added, and the mixture was shaken for 20 susing a vortex mixer and then incubated at 37°C for 20 min. Saturatedsodium chloride solution was added, and the solution was extracted withdiethyl ether (2 mL). After centrifugation at 4°C at 1,500g for 5 min toseparate the layers, the upper organic phase was removed and evaporated.The residue was dissolved in methanol (1 mL) and filtered with a 0.45-mmfilter. Strawberry fruit (1 g, n = 3) was ground in liquid nitrogen to powderand then placed in a centrifuge tube (10 mL). Precooled perchlorate (vol-ume fraction 5%) was added to the centrifuge tubes, and the mixture wasshaken using a vortex mixer and then extracted in an ice bath for 1 h. Afterextraction, the mixtures were centrifuged at 4°C at 1,500g for 30 min. ThePA extraction from the supernatant (500 mL) was added to centrifuge tube(10 mL). The derivation of the supernatant was also as described above.The PA contents were examined by HPLC using the C18-column (ZorbaxEclipse XDB-C18, 4.6 3 250 mm, 5 mm; Agilent) at 30°C. The mobile phasewas methanol:water (v:v, 64:36) for 20 min at a flow rate of 0.7 mL per min.Effluent absorbance was monitored at 230 nm. The injection volume was10 mL. The experiment was repeated three times.

Three fruits (n = 3) were used for detection of anthocyanin and soluble sugarcontents by HPLC using a Zorbax Eclipse XDB-C18 column (4.6 3 150 mm,5 mm; Agilent) and Agilent Technologies 1200 Series, 6.5 3 300-mm Sugar-Pakcolumn (Waters), respectively, as previously described (Jia et al., 2011). Theexperiment was repeated three times.

Determination of ABA, IAA, and Ethylene Contents

Three strawberry fruits (n = 3) were used for ABA or IAA analysis. For ABAor IAA extractions, 1 g of receptacle was ground and homogenized in a solution(80% methanol, v/v) and then centrifuged at 10,000g for 20 min. The super-natants were eluted by the Sep-Pak C18 cartridge (Waters), after removal ofpolar compounds, then used for immune assays as described by Zhang et al.(2009).

Three uniform strawberry fruits (n = 3) were selected and placed in200-mL glass jars for 2 h at 25°C, then 1 mL of gas from the jar headspace waswithdrawn and injected into a gas chromatograph with a flame ionizationdetector and an activated alumina column (model 6890 N; Agilent) aspreviously described (Sun et al., 2013). The experiment was performedwith three replications.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data li-braries under the following accession numbers: Actin, AB116565; SAMDC,MG266892; PG1, AF380299; PL1, DQ076239; SUT1, JX013937; SS, AB275666; CHS,AY997297; DFR, AY695813; SPMS, XM_011471922; SPDS, XM_004297595; ADC,XM_004290251; ODC, XM_011464632; NCED1, HQ290318; ACO1, AJ851828;IAA2, XM_009340458; ARF11, XM_011459741; LUX2, XM_004302600; ABI37,XM_004307422; SnRK2, KJ748362; and EBF1, XM_004287259.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. KOG function classification of consensus se-quence.

Supplemental Figure S2. Gene Ontology classification.

Supplemental Figure S3. Putative conserved domains in the FaSAMDCprotein.

Supplemental Figure S4. The S-adenosyl-Met (SAM) standard curve.

Supplemental Table S1. Raw reads and clean reads from RNA-seq.

Supplemental Table S2. Summary of Illumina transcriptome assembly forstrawberry.

Supplemental Table S3. The top five pathways annotated with KEGGpathway analysis.

Supplemental Table S4. Primers used for qPCR.

ACKNOWLEDGMENTS

We thank Dr. Yu-Le Liu (Qinghua University) for the pTRV vectors and Dr.Qing Zhang (Beijing Key Laboratory for Agricultural Application and NewTechnique) for isothermal titration calorimetry analysis.

Received February 27, 2018; accepted March 5, 2018; published March 9, 2018.

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