Miho Tatsuki, Corresponding Author tatsuki@affrc.go.jp Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanFor correspondence (e-mail tatsuki@affrc.go.jp).Search for more papers by this authorKazuo Soeno, Western Region Agricultural Research Center (WARC), NARO, Senyu, Zentsuji, Kagawa, 765-8508 JapanSearch for more papers by this authorYukihisa Shimada, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorYutaka Sawamura, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorYuko Suesada, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorHideaki Yaegaki, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorAkiko Sato, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorYusuke Kakei, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorAyako Nakamura, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorSongling Bai, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorTakaya Moriguchi, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorNaoko Nakajima, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this author Miho Tatsuki, Corresponding Author tatsuki@affrc.go.jp Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanFor correspondence (e-mail tatsuki@affrc.go.jp).Search for more papers by this authorKazuo Soeno, Western Region Agricultural Research Center (WARC), NARO, Senyu, Zentsuji, Kagawa, 765-8508 JapanSearch for more papers by this authorYukihisa Shimada, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorYutaka Sawamura, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorYuko Suesada, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorHideaki Yaegaki, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorAkiko Sato, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorYusuke Kakei, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorAyako Nakamura, Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813 JapanSearch for more papers by this authorSongling Bai, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorTakaya Moriguchi, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this authorNaoko Nakajima, Institute of Fruit Tree and Tea Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, 2-1, Tsukuba, Ibaraki, 305-8605 JapanSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text 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Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a traNSPoson-like sequence was found upstream of the PpYUC11 gene in the 5′-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of ‘Akatsuki’ and ‘Manami’ cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype. Introduction Peach (Prunus persica (L). Batsch) has a climacteric fruit, and generally increases ethylene production during ripening. Based on their fruit firmness and texture, peaches are classified as melting flesh, non-melting flesh, or stony hard types. In melting-flesh peaches, rapid softening occurs after harvest, resulting in fruit with a short shelf life. In non-melting-flesh peaches, softening is slow, and flesh firmness does not significantly reduce. The differences in softening between these two types of peaches have been attributed to the presence of endo-polygalacturonase (PG) activity during ripening (Pressey and Avants, 1978). In contrast with melting and non-melting peaches, stony hard peaches retain their flesh firmness even during the ripening stage on the tree or after harvest, although the change in fruit colour normally progresses concomitant with accumulating soluble solids (Haji etal., 2001). Genetic analysis has indicated that its peculiar characteristic involves a recessive stony hard (hd) locus (Yoshida, 1976), which is different from the melting (M)/non-melting (m) locus (Haji etal., 2005). We have previously reported that the fruit of melting-flesh (normal) peaches produce large amounts of ethylene at the late-ripening stage, i.e. system 2 ethylene production (Lelièvre etal., 1997), which is caused by high expression of PpACS1 (an isogene of 1-aminocyclopropane-1-carboxylic acid synthase, a key enzyme of ethylene biosynthesis) (Tatsuki etal., 2006). However, in stony hard peaches, the expression level of PpACS1 does not increase, and large amounts of ethylene are not produced. Our previous study has also shown that indole-3-acetic acid (IAA), a major auxin in plants, may be involved in system 2 ethylene production in peaches (Tatsuki etal., 2013). Peach fruit displays a double sigmoidal growth curve, with four stages (S1 to S4) (Zanchin etal., 1994). Cell growth in S1 and S3 is faster than that in S2 and S4. S2 is a pit-hardening stage and a period of slow growth. IAA concentration changes concomitant with these developmental stages in peach fruit. The IAA concentration is shown to be high in young fruit (S1) and then gradually decreases during fruit development, reaching its lowest levels just before the onset of the climacteric rise (S3) (Tatsuki etal., 2013). Thereafter, IAA concentration increases suddenly just before harvest time (S4) in normal-type peaches (Tatsuki etal., 2013). Treatment of S4 fruits of stony hard peaches with a synthetic auxin 1-naphthaleneacetic acid (NAA) stimulated PpACS1 gene expression, ethylene production and fruit softening (Tatsuki etal., 2013). These results suggest that the stony hard peach phenotype is caused by IAA deficiency at the late-ripening stage. Endogenous IAA can be supplied either by de novo synthesis or by release from conjugates (Ludwig-Müller, 2011; Korasick etal., 2013). Therefore, the deficiency of IAA in stony hard peaches can be explained by any of the following mechanisms: (i) defect in IAA biosynthesis, (ii) acceleration of IAA inactivation and degradation, (iii) imbalance in the metabolism of IAA storage forms (i.e. IAA conjugates or indole-3-butyric acid) to free IAA, and (iv) defect in IAA transport from biosynthetic tissues. Multiple pathways have been proposed for IAA biosynthesis. Possible l-tryptophan (Trp)-dependent IAA biosynthetic pathways are as follows: (i) indole-3-pyruvic acid (IPyA) pathway, (ii) indole-3-acetoamide (IAM) pathway, (iii) tryptamine (TAM) pathway and (iv) indole-3-acetaldoxime (IAOx) pathway (Woodward and Bartel, 2005; Pollmann etal., 2006; Chandler, 2009; Mano etal., 2010; Normanly, 2010; Zhao, 2010) (FigureS1). The best-characterized pathway in Arabidopsis thaliana L. is the IPyA pathway (YUCCA pathway), which consists of two successive reactions from Trp via IPyA (Stepanova etal., 2008, 2011; Tao etal., 2008; Mashiguchi etal., 2011; Won etal., 2011). The first step is catalysed by tryptophan aminotransferase of Arabidopsis 1 (TAA1), and its close homologues, TAA related 1 (TAR1) and TAA related 2 (TAR2) (Stepanova etal., 2008; Tao etal., 2008; Yamada etal., 2009), and the second step is catalysed by the flavin monooxygenase, YUCCA (Zhao etal., 2001; Mashiguchi etal., 2011; Stepanova etal., 2011; Won etal., 2011; Dai etal., 2013). It has been reported that one of the peach YUCCA isogenes, PpYUC11, is a candidate for the stony hard phenotype (Pan etal., 2015). However, the main pathway of IAA biosynthesis in the peach has not yet been established, and the intermediates or metabolites of IAA biosynthesis that exist in the fruit of normal and/or stony hard peaches are still unknown. In this study, we sought to elucidate the mechanism of IAA deficiency in the ripening fruits of stony hard peaches. We measured IAA, possible IAA intermediates, and IAA metabolites in normal and stony hard peaches. The level of IPyA was higher in stony hard than in normal peaches at the ripening stage. We searched for the causal gene that could be possibly related to auxin biosynthesis, and foundan insertion of a transposon in the 5′-flanking region of PpYUC11. The transposon is related to the suppressionof PpYUC11 induction at the late-ripening stage of stony peaches. The IAA level increases in the ripening stage of normal peaches, while its level does not increase during ripening fruit of stony hard peaches (Tatsuki etal., 2013). To understand the underlying molecular mechanisms, the possible intermediates or metabolites of the IAA biosynthetic pathway were analysed in both the normal peach, ‘Akatsuki’, and the stony hard peach, ‘Manami’, at three maturing stages (S2, S3 and S4) (Figure1, FigureS2). First, we examined which pathway is the major auxin biosynthetic pathway in peaches. The possible IAA intermediates, TAM and IAOx were not detected and trace amounts of IAM were detected (FigureS2), but IPyA and indole-3-acetaldehyde (IAAld) were detected in both ‘Akatsuki’ and ‘Manami’ fruit at all growth stages. Endogenous IAAld levels were abundant at S2 and then sharply decreased at S3 in both ‘Akatsuki’ and ‘Manami’, indicating no correlation with the different IAA levels between ‘Akatsuki’ and ‘Manami’ (FigureS2). These results suggest that the IPyA pathway (YUCCA pathway) functions as the major pathway of auxin biosynthesis in peach fruit of both ‘Akatsuki’ and ‘Manami’. Figure 1Open in figure viewerPowerPoint Endogenous levels of IAA biosynthesis intermediates and metabolites in peach mesocarp tissues. Flesh was collected from S2, S3 and S4 from ‘Akatsuki’ (normal) and ‘Manami’ (stony hard), and levels of IAA-related compounds were analysed by ultra-performance liquid chromatography (UPLC)-MS/MS. Trp, IAA, oxIAA, IAGlu, and IAAsp levels were quantified by using stable isotope-labelled internal standards. IPyA levels were analysed after derivatisation to the methoxime compounds. Trp: l-tryptophan, IPyA: indole-3-pyruvic acid, oxIAA: 2-oxo-indole-3-acetic acid, IAGlu: indole-3-acetyl-l-glutamic acid, IAAsp: indole-3-acetyl-l-aspartic acid, FW: fresh weight. Plotted values are the means±SE (n=3). Asterisks indicate significant differences within the same stage at p 0.05 using Tukey\'s test. The levels of Trp were not significantly different between ‘Akatsuki’ and ‘Manami’ at the three fruit growth stages. IPyA was more abundant in ‘Akatsuki’ than in ‘Manami’ at S2, but the differences between the varieties were smaller at S3. At the ripening stage (S4), the level of IPyA decreased in ‘Akatsuki’, but it was not altered between S3 and S4 in ‘Manami’. The IAA level at S4 was much higher in ‘Akatsuki’ than in ‘Manami’, and these results support those of our previous study (Tatsuki etal., 2013). Indole-3-acetyl-l-alanine (IAAla) and indole-3-acetyl-l-leucine (IALeu) are proposed to be reversible storage forms of IAA in Arabidopsis (Ludwig-Müller, 2011; Korasick etal., 2013). These conjugates were not detected in both ‘Akatsuki’ and ‘Manami’ fruit at all growth stages, indicating that these storage forms are not involved in auxin metabolism in the fruit of ‘Akatsuki’ and ‘Manami’. 2-Oxo-indole-3-acetic acid (oxIAA) is a major inactive form of IAA; indole-3-acetyl-l-glutamic acid (IAGlu) and indole-3-acetyl-l-aspartic acid (IAAsp) are also inactive forms of IAA. At S4, their levels were higher in ‘Akatsuki’ than in ‘Manami’. These differences may be caused by the level of IAA at S4. These data collectively suggest that the IAA biosynthetic pathway of stony hard peaches has a defect in the conversion of IPyA to IAA. Therefore, YUCCA activity should be reduced in ripening stony hard peaches. There are two possibilities that explain the defect in IAA biosynthesis in the stony hard fruit. One possibility is a defect in YUCCA gene expression, and the other is a defect in YUCCA enzyme activity. To identify which YUCCA gene is responsible for the phenotype, we analysed YUCCA gene expression in the developmental and ripening stages of the peach. It has been reported that the expression level of the YUCCA isogene, PpYUC11, is lower in stony hard peaches than in melting cultivars (Pan etal., 2015). There are nine YUCCA-like genes in the whole genome sequences of Peach v2.0 (Verde etal., 2017), and their expression patterns have been reported (Pan etal., 2015). Among these, the DNA sequence of Prupe.8G020600.1 is partial, and is very similar to the sequence of Prupe.1G468500.1; it is difficult to distinguish these two sequences. In the present study, we also could not isolate the cDNA for Prupe.1G054300.1 from either fruit (from immature to mature) or leaves. Therefore, we isolated the remaining six isogenes of YUCCA, and examined their expression levels at fruit development and maturing stages (Figure2a). The expressions of PpYUC1, -2, -6, and -8 were high during fruit growth and developmental stages (S1 and S2), but their expressions were low in ripening fruit (Figure2a). The expression of PpYUC9 was quite low in the fruit (FigureS3). PpYUC10 was expressed in both young fruit and ripening fruit of ‘Akatsuki’ and ‘Manami’. PpYUC11 was highly expressed in ripening ‘Akatsuki’ fruit, but it was not detectable in ‘Manami’. This result agrees with that of a previous study (Pan etal., 2015). These results indicate that low PpYUC11 expression may be responsible for the IAA deficiency in ripening stony hard peaches. We also conducted RNA sequence analysis of S3 and S4 peaches of ‘Akatsuki’ and ‘Manami’, and confirmed that PpYUC11 is the only differentially expressed gene among the known auxin biosynthetic genes (TableS1). Figure 2Open in figure viewerPowerPoint Relative transcript abundance of PpYUC isogenes and PpTAR2 in ‘Akatsuki’ (left) and ‘Manami’ (right) (a) and effects of NAA (left) and ethylene (right) on PpYUC11 expression in ‘Manami’ fruit (b). Gene expression levels were analysed by real-time RT-PCR. The steady-state levels were normalised to those of actin-7 (XM_007211382). Data are mean values ± SD of two individual experiments, each performed in triplicate. NAA: 1-naphthaleneacetic acid. TAA1 and TAR2 catalyse Trp to IPyA in Arabidopsis (Stepanova etal., 2008; Tao etal., 2008). PpTAA1 (Prupe.1G248300.1) and PpTAR2 (Prupe.5G168300.1) are homologous to Arabidopsis AtTAA1 and AtTAR2 respectively. The expression level of PpTAA1 was very low in the peaches (FigureS3). The changes in expression of PpTAR2 were smaller than those of the YUCCA isogenes during fruit development and ripening, except that PpTAR2 was slightly induced in ‘Manami’ fruit at 100days after full bloom (DAB) (Figure2a). We have previously reported that stony hard fruit can be softened by treatment with ethylene or auxin (Tatsuki etal., 2006, 2013). In the present study, we examined whether exogenous auxin or ethylene could induce expression of PpYUC11 in ‘Manami’ stony hard peaches. Although the expression of PpYUC11 was low at S4 in ‘Manami’ (Figure2a), the expression was increased in response to NAA or ethylene treatment (Figure2b). These results indicate that PpYUC11 gene expression is responsive to hormone treatments in the stony hard peach, but the gene expression is suppressed at the ripening stage. First, we compared the amino acid sequences of the PpYUC11 protein from ‘Akatsuki’ and ‘Manami’, and found them to be the same. Then, we examined whether PpYUC11 has YUCCA enzyme activity. Arabidopsis YUCCA protein converts IPyA to IAA invitro (Dai etal., 2013). PpYUC11 was expressed in Escherichia coli (FigureS4), and the crude extract was assayed for the ability to catalyse IPyA to IAA (Figure3). The lysates, including recombinant PpYUC11, produced large amounts of IAA after incubation with IPyA (Figure3d). In contrast, IAA was only slightly produced after incubation without lysate (Figure3b), with lysate from E.coli harbouring the control vector (Figure3c), or with heat-inactivated lysate from E.coli expressing recombinant PpYUC11 (Figure3e). Small amounts of IAA were produced in the absence of active PpYUC11 because IPyA is unstable and can be converted to IAA in the absence of the enzyme. These results indicate that PpYUC11 has YUCCA activity to convert IPyA to IAA. Figure 3Open in figure viewerPowerPoint Enzyme activity of recombinant PpYUC11. Enzyme reactions were performed in a reaction mixture containing E.coli lysates (50μg soluble protein), 3μmIPyA, 30μmFAD, and 1mmNADPH in Tris−HCl buffer (50 mM, pH 8.0) for 10min at 30°C, and then analysed by HPLC with a fluorescence detector (λex/λem = 280/355nm). Chromatogram of IAA authentic standard (a), enzyme reaction mixture without E.coli lysate (b), enzyme reaction mixture with E.coli lysate from control vector (c), enzyme reaction mixture with E.coli lysate expressing PpYUC11 (d), and enzyme reaction with heat-inactivated E.coli lysate expressing PpYUC11 (e). The arrow indicates the IAA peak. To examine the suppression mechanism of PpYUC11 in ripening stony hard fruit, the 5′-upstream region of PpYUC11 was amplified by polymerase chain reaction (PCR) and sequenced. The PpYUC11 gene from three stony hard peach varieties, ‘Manami’, ‘Odoroki’ and ‘Yumyeong’, has a 2569-bp transposon-like sequence insertion at −1721bp upstream of the transcriptional start site (TSS) (Figures4a and S5). In contrast, the alleles from the normal varieties, ‘Akatsuki’ and ‘Kawanakajimahakutou’, have heterozygous sequences; one allele has the transposon insertion, while the other does not (FigureS6). Then, we examined the correlation between the transposon insertion and the fruit firmness phenotype. In this study, the genotype with transposon insertion is denoted as hd-t, and that without the transposon is denoted as Hd. To distinguish the transposon insertion of 2569bp, two PCR primer sets were constructed (Figure4b). The F1 primer anneals upstream of the insertion, and the R1 primer anneals to the insertion sequence. If transposon sequences are inserted in the genome, the PCR amplification using F1 and R1 primers detects a product of 600bp (Figure4c). The R2 primer anneals downstream of the −1721 position, which is a common sequence for both hd-t and Hd alleles. PCR amplification using F1 and R2 primers should detect a product of 448bp and 3015bp, corresponding to Hd and hd-t alleles respectively. However, the latter primer set was used only to detect the Hd allele, because the 3015-bp PCR fragment (corresponding to the hd-t allele) was not detected under our PCR conditions (Figure4c). The stony hard peaches, ‘Manami’, ‘Odoroki’ and ‘Yumyeong’, produced the 600-bp product using the F1 and R1 primers, but did not produce a PCR product using the F1 and R2 primers. The normal flesh peaches, ‘Hakutou’, ‘Akatsuki’ and ‘Kawanakajimahakutou’, produced both the 600bp and 448bp products, using the F1-R1 and F1-R2 primer sets respectively. The normal flesh peaches, ‘Hakuhou’, ‘Saotome’, ‘Coral’, ‘Shanhai suimitsuto’ (‘Chinese Cling’), ‘NJN17’ and ‘Chimarrita’, did not produce the 600-bp product, but produced the 448-bp product using the F1 and R2 primers. The expression level of PpYUC11 at S4 was lower in the cultivars possessing the hd-t/hd-t genotype than in those possessing the hd-t/Hd and Hd/Hd genotypes (Figure4d). These data indicate that stony hard peaches with reduced PpYUC11 gene expression have a genotype homozygous for hd-t, and that normal types with high PpYUC11 gene expression have a genotype homozygous for Hd or heterozygous for hd-t/Hd (Table1). Figure 4Open in figure viewerPowerPoint Image of the PpYUC11 5′-flanking region of hd-t (upper) and Hd (lower) (a). White rectangle shows inserted sequences. +1 shows putative transcriptional start site. UTR: untranslated region. Detection of PpYUC11 hd-t and Hd alleles (b−d). (b) Primer positions for PCR to distinguish hd-t and Hd alleles. (c) PCR products separated by agarose gel electrophoresis. F1 and R1 primer set was used in (c: upper) and F1 and R2 primer set was used in (c) lower). 1, ‘Manami’; 2, ‘Odoroki’; 3, ‘Yumyeong’; 4, ‘Hakutou’; 5, ‘Akatsuki’; 6, ‘Kawanakajimahakutou’; 7, ‘Sakuhime’; 8, ‘Masahime’; 9, ‘Hikawahakuhou’; 10, ‘Okitsu’; 11, ‘Hakuhou’; 12, ‘Saotome’; 13, ‘Yoshihime’; 14, ‘Coral’; 15, ‘Shanhai Suimitsuto’; 16, ‘NJN17’; 17, ‘Chimarrita’. (d) Relative transcript abundance of PpYUC11 of S4 fruit. Ma, ‘Manami’; Odo, ‘Odoroki’; Aka, ‘Akatsuki’; Ka, ‘Kawanakajimahakutou’; Sao, ‘Saotome’; Haho, ‘Hakuhou’. Gene expression levels were analysed by real-time RT-PCR. The steady-state levels were normalized to the actin-7 (XM_007211382) signal. Data are mean values±SD of two individual experiments, each performed in triplicate. Identification of the segregation ratio of stony hard in F1 progenies and ht-d segregates with stony hard in offspring To confirm the relationship between ht-d genotype and stony hard phenotype, we conducted segregation tests and the segregation of stony hard in the offspring is shown in Table2. In a population of 28 offspring from ‘Kawanakajimahakutou’ (hd-t/Hd)בManami’ (hd-t/hd-t), 17 were classified as normal, with PpYUC11 genotypes of hd-t/Hd, and 11 were classified as stony hard, with genotypes of hd-t/hd-t. In a population of seven offspring from ‘Akatsuki’ (hd-t/Hd)בManami’ (hd-t/hd-t), three were classified as normal, with the genotype hd-t/Hd, and four were classified as stony hard, with the genotype hd-t/hd-t. The segregation ratios of these two populations were identified as at a ratio of 1:1, with statistically no discrepancy. All the offspring from ‘Odoroki’ (hd-t/hd-t)בManami’ (hd-t/hd-t) were classified as stony hard, and their genotypes were all hd-t/hd-t. In contrast, all the offspring from ‘Yoshihime’ (Hd/Hd)בMasahime’ (hd-t/Hd) were classified as normal, and their PpYUC11 genotypes were Hd/Hd and hd-t/Hd. Furthermore, the segregation ratio of both offspring of the cross ‘Kawanakajimahakutou’ (hd-t/Hd)× ‘Hikawahakuhou’ (hd-t/Hd) and ‘Kawanakajimahakutou’ (hd-t/Hd)× ‘Yoshihime’ (hd-t/Hd) was identified as 3 (normal):1 (stony hard), with statistically no discrepancy. Similar to the findings in the cultivars, the stony hard phenotype and genotype in the F1 progenies were completely consistent. These results indicated that the transposon insertion genotype correlated with the stony hard phenotype. Table 2. Segregation of stony hard phenotype and genotype of PpYUC11 in the F1 progeny naa Number of seedlings evaluated. Segregation of stony hard phenotype in the F1 progenybb Stony hard phenotype of the F1 progeny from cross combinations of ‘Odorokf’בManami’, ‘Akatsuki’×424-11 and ‘Kawanakajimahakutou’בManami’ were evaluated by sensory test and detection of ethylene using gas chromatography, and the other offspring were identified by sensory test. χ2 valuecc χ2 value indicates fit to the expected ratio. n.s. means not significant. Stony hard phenotype of the F1 progeny from cross combinations of ‘Odorokf’בManami’, ‘Akatsuki’×424-11 and ‘Kawanakajimahakutou’בManami’ were evaluated by sensory test and detection of ethylene using gas chromatography, and the other offspring were identified by sensory test. In a previous study, diverse (TC)n (n is the repeat number) microsatellites were found in the first intron of PpYUC11, and all stony hard peaches were homozygous for (TC)20 [shown here as simple sequence repeat (SSR)20/20] (Pan etal., 2015). We also investigated the SSR in the first intron of PpYUC11 of various cultivars. The (TC) repeat numbers that were observed were 20, 25, 28 and 33, and the stony hard cultivars were SSR20/20 (Table1). Most genotypes of SSR20 coincided with the transposon insertion (hd-t), and were consistent with the results of a previous report (Table1, Pan etal., 2015). However, the (TC) repeat number of the Hd genotype was not consistent with the result reported by Pan etal. (2015) (Table1). For example, we found that the (TC) repeat numbers of the Hd allele of the ‘Okitsu’ and ‘Hakutou’ varieties originating from Japan were 25 and 28 respectively, but Pan etal. (2015) reported these as 29 and 26 respectively. Furthermore, one cultivar, ‘Chimarrita’, which was introduced from Brazil, had a genotype that was not consistent with SSR and transposon insertion; ‘Chimarrita’ was homozygous for Hd/Hd, but its SSR was heterozygous (i.e. 20/25). Therefore, we examined the genotypes of the progeny of ‘Chimarrita’. In a population of 30 offspring from the cross ‘Kawanakajimahakutou’ (hd-t/Hd, SSR20/25)בChimarrita’ (Hd/Hd, SSR20/25), all their phenotypes were classified as normal (Table2), which was consistent with ‘Chimarrita’ being homozygous for Hd/Hd. One normal-type selection, from the ‘Kawanakajimahakutou’בChimarrita’, 403-19 cross (genotype was unknown, because the tree had already been felled), was crossed with ‘Sakuhime’ (hd-t/Hd, SSR20/25) (FigureS7). In 10 offspring from this crossing, the genotypes were either hd-t/Hd or Hd/Hd, and all of the phenotypes were normal (Table3). Therefore, we assumed that 403-19 was homozygous for Hd/Hd. Among the 10 offspring, some genotype discrepancies for transposon insertion and SSR in the first intron were observed. The phenotypes of 447-3, -4, -7 and -10 were normal (Table3, FigureS8), but the microsatellite genotypes were SSR20/20, which are regarded as stony hard genotypes (Pan etal., 2015). Furthermore, the 447-5 genotype was Hd/Hd, SSR20/25, which was the same as that of ‘Chimarrita’. These results indicated that the genotype of the transposon insertion is more suitable for distinguishing the phenotype of stony hard than is the genotype of the first intron microsatellite. Auxins play crucial roles in fruit growth and development (Pattison etal., 2014). In peaches, a possible relationship between auxins and fruit development has been reported (Miller etal., 1987; Agusti etal., 1999; Ohmiya, 2000). We have shown previously that an increased level of auxin is required for rapid fruit softening at the late-ripening stage of peaches (Tatsuki etal., 2013). In the present study, we showed that a transposon insertion in the 5′-flanking region of one of the isogenes of a key enzyme of auxin biosynthesis, YUCCA, clearly correlated with the stony hard peach phenotype. IAA is a major natural auxin in higher plants. Recently, the IAA biosynthetic pathway was elucidated in Arabidopsis (Stepanova etal., 2008, 2011; Tao etal., 2008; Yamada etal., 2009; Mashiguchi etal., 2011; Won etal., 2011). However, multiple auxin biosynthesis pathways have been proposed (FigureS1), and the main pathway in many other plant species is still obscure. In this study, we demonstrated that the hd-t/hd-t genotype is responsible for the stony hard fruit phenotype and auxin deficiency in fruit at the ripening stage. Therefore, the IPyA pathway (YUCCA pathway) is the main auxin biosynthesis pathway in the ripening stage of ‘Akatsuki’ and ‘Manami’ peaches. We also demonstrated that IAA de novo synthesis in mesocarp tissues is likely to be responsible for auxin production to support fruit ripening. Of additional importance, IAGlu and IAAsp, irreversibly inactive IAA amide conjugates, were detected in fruit (Figure1), while IAAla and IALeu, reversible IAA amide conjugates, were not detected at any stages of peach fruit development, suggesting that IAA is not supplied from storage forms during peach ripening. YUCCA catalyses the conversion of IPyA to IAA, which has been proposed to be the rate-limiting step in auxin biosynthesis in Arabidopsis (Zhao etal., 2001). YUCCA is a multi-gene family, and Arabidopsis has 11 YUCCA isogenes. Their functions overlap during development (Cheng etal., 2006, 2007), because the knockout of single YUCCA isogenes did not show obvious developmental defects in Arabidopsis. However, multiple inactivations of YUCCA isogenes produced severe defects in various developmental processes (Cheng etal., 2006, 2007). In peaches, several YUCCA homologous genes were found in the whole genome sequences of Peach v2.0 (Verde etal., 2017). During peach fruit development, six YUCCA isogenes were expressed, and their expression patterns were different (Figure2a). The concentration of IAA was high at the early stage of fruit development, and gradually decreased during fruit development, reaching its lowest levels just before the onset of the climacteric rise in both normal and stony hard cultivars (Figure1, Tatsuki etal., 2013). The expressions of PpYUC1, -2, -6 and -8 were high at the fruit development stage. In ripening fruit at S4, only PpYUC10 and -11 were expressed. It seems that there were no remarkably visible differences in fruit development and ripening processes between stony hard and normal peaches, except for fruit firmness at the ripening stage. Therefore, auxin biosynthesis in stony hard peaches seems to operate normally except during fruit ripening, but the reduced expression of PpYUC11 at the ripening stage characterized the stony hard trait. It remains unclear why IAA levels were low in stony hard because PpYUC10 is also expressed in ripening fruit. The level of IPyA was high at S4 in ‘Manami’ (Figure1), which indicates that YUCCA activity in the tissues was low. Therefore, one possible reason is that PpYUC10 enzymatic activity (e.g. specific activity) would be low. PpYUC10 is also expressed in young fruit, where the other isogenes (PpYUC1, 2, 6 and 8) were also expressed (Figure2). Therefore, low PpYUC10 enzymatic activity could be masked by other isozyme activities. PpYUC10 is probably a functional flavin monooxygenase, which contain the conserved motifs for binding the flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) cofactors (Hou etal., 2011). Therefore, detailed biochemical analysis of PpYUC10 is required. AtYUC10 and -11 (Arabidopsis), and OsYUC11 (rice), orthologue of PpYUC10 and -11, are expressed in endosperm tissue (Cheng etal., 2007; Abu-Zaitoon etal., 2012) and they are assumed to be related to embryogenesis and endosperm development (Zhang etal., 2018). Therefore, PpYUC11 could be associated with synthesis IAA during seed and fruit maturation. The transposon inserted in the 5′-flanking region of PpYUC11 was a non-autonomous DNA transposon belonging to the CACTA superfamily. The CACTA transposons have flanking terminal inverted repeats, which are normally 10–28 bp long and terminate in a conserved 5′-CACTA-3′ motif (Wicker etal., 2003) (Figures4a and S5). Almost the same sequence as the transposon inserted in PpYUC11 was found in the first intron of PpDAM6 (FigureS9), which is a MADS-BOX gene associated with lateral bud endodormancy (Yamane etal., 2011; Zhebentyayeva etal., 2014). In PpDAM6, a 2604-bp DNA fragment was found only in low-chill peach cultivars, not in high-chill ones. The expression level of PpDAM6 was lower in low-chill peach cultivars than in high-chill cultivars, which negatively correlated with bud burst percentages (Yamane etal., 2011). Transposable element (TE) insertion affects the near gene expressions (Weil and Wessler, 1990; Hayashi and Yoshida, 2009; Fernandez etal., 2010) and is subjected to epigenetic modification (Bennetzen and Wang, 2014). Recent comprehensive analyses of A. thaliana and rice indicated that expression of the gene increased or decreased by the insertion of methylated TE (Hollister and Gaut, 2009; Naito etal., 2009; Meng etal., 2016; Quadrana etal., 2016). In the present study, the inserted site was 1721bp upstream from the TSS. Expression of PpYUC11 was not detected in the ripening fruit of stony hard peaches, but was detected in the seeds (Pan etal., 2015), and NAA-treated or ethylene-treated fruits (Figure2b). Therefore, only ripening-dependent transcription was defective in stony hard peaches, presumably because the transposon insertion affected only ripening-specific cis-elements of PpYUC11. Considering numerous reports on TE methylation, the reduced expression of PpYUC11 may be caused by the changed epigenetic status of the cis-element of PpYUC11. In melon, the heavily methylated transposon (hAT) insertion at the 3′ flanking region changes the methylation status of linked gene and resulted in reduced gene expression (Martin etal., 2009). The altered methylation status for each plant organ and growth stage in the gene flanking region resulted in changed levels in gene expression (Manning etal., 2006; Martin etal., 2009). The changed methylation status of PpYUC11, which might be caused by hormone treatment, would result in increased expression. Almost all peaches of Japanese breeding cultivars that are grown today have originated from ‘Hakutou’. The PpYUC11 genotype of nearly all Japanese breeding cultivars was consistent with their phenotype (TableS2), and their hd-t allele could have originated from ‘Hakutou’. Several cultivars and accessions have been introduced from other countries, and these PpYUC11 genotypes were also consistent with the phenotype (TableS2). The origin of the hd-t allele of PpYUC11 is not clear, but it may have originated from other cultivars that were introduced from China and/or other countries. In conclusion, we found that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in peaches, and is disrupted in the stony hard cultivar to inhibit fruit softening at the ripening stage. Expression of PpYUC11 was suppressed in ripening stony hard peaches due to the insertion of a transposon in the 5′-upstream region of the PpYUC11 gene. Plants of P. persica (L.) Batsch were grown at the National Institute of Fruit Tree and Tea Science, Japan. For measurement of IAA biosynthesis intermediates and metabolites in peach mesocarp tissues, ‘Akatsuki’ and ‘Manami’ fruit were sampled at 66 and 63 DAB (S2), 96 and 102 DAB (S3), and 110 and 121 DAB (S4) respectively. Samples were cut into 3-mm cubes and portions were frozen in bulk with liquid nitrogen. The ‘Akatsuki’ and ‘Manami’ fruit, at various developmental stages, were the same samples as used in our previous report (Tatsuki etal., 2013). Each sample of 1day (‘Akatsuki’) and 0day (‘Manami’) after full bloom was the whole flower. These were used for extraction of total RNA and real-time RT-PCR. Leaves were sampled for isolation of genomic DNA. NAA and ethylene treatment of stony hard was performed according to previous reports (Tatsuki etal., 2013). In general, recognizing melting in peach fruit after the harvesting stage is straightforward. However, it is difficult to differentiate between immature melting and stony hard fruit. For more precise evaluations, the stony hard phenotype of F1 progenies from crossings of ‘Odoroki’בManami’, ‘Akatsuki’×424-11 and ‘Kawanakajimahakutou’בManami’ were evaluated by sensory test, measurement of ethylene production, and fruit firmness according to previous reports (Tatsuki etal., 2013); the other F1 progenies were identified by only the sensory test. Measurement of IAA biosynthesis intermediates and metabolites in peach mesocarp tissues Endogenous IAA, and its biosynthetic intermediates and metabolites were measured according to methods described previously (Kakei etal., 2015; Narukawa-Nara etal., 2016) with modifications. Method S1 describes the analysis of IAA-related compounds in detail. Total RNA was extracted from frozen samples by the hot borate method (Wan and Wilkins, 1994). First-strand cDNA was synthesised by reverse transcription of total RNA. To screen for peach YUCCA, TAA and TAR2 cDNAs, cDNA sequences of Arabidopsis were used as queries to find their homologous sequences in peach, using genome sequence data from P. persica Whole Genome v1.0 Assembly & Annotation. Primer sets were synthesised to isolate cDNA based on the identified peach YUCCA homology (TableS3). cDNAs of PpYUC1, 2, 6, 8, 9 and 10 and PpTAR2 were isolated from ‘Akatsuki’ young fruit, PpYUC11 was isolated from ‘Akatsuki’ ripening fruit, and PpTAA1 was isolated from ‘Akatsuki’ leaves. To confirm the TSS of PpYUC11, 5′ rapid amplification of cDNA ends (RACE) was performed by using the SMART™ RACE cDNA Amplification Kit (Clontech, CA, USA) as described in the manufacturer\'s protocol. Genomic DNA was isolated from peach leaves using the DNeasy Plant Mini Kit (QIAGEN). The fragments of the 5′-flanking region of PpYUC11 were amplified by PCR (LA Taq™, TaKaRa, Kyoto, Japan) with primer sets (TableS3) that were constructed based on sequence data from P. persica v1.0. Total RNA of ‘Akatsuki’ (88 DAB and 10 DAB) and ‘Manami’ (99 DAB and 112 DAB) fruit was used for RNA-seq. The extracted total RNA samples were treated with DNase I and then used for the construction of the RNA sequencing library. RNA sequencing libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer′s instructions. Paired-end and 90-base-long RNA sequencing were conducted using the HiSeq 2000 Sequencing System (Illumina). Approximately 22 million reads were obtained for each sample. The sequence reads were aligned to the P. persica reference genome, P. persica Whole Genome v1.0 Assembly & Annotation (Prunus_persica_v1.0_scaffolds.hardmasked.fa from www.rosaceae.org), with TopHAT2 software (Kim etal., 2013). Approximately 90% of clean reads were successfully mapped to the genome (TableS4). Gene expressions were estimated as read counts for each gene locus by cuffquant and cuffnorm v2.2.1 (Trapnell etal., 2012) using a gene annotation file (Prunus_persica_v1.0_genes.gff3). The sequence data were submitted to the NCBI Sequence Read Archive under accession number PRJNA437050. PpYUC11 cDNA, isolated from normal flesh ‘Akatsuki’ containing the complete coding region, was used as a template for PCR. The cDNA fragment was amplified by PCR using the primer set 5′-GCCGCATATGGCGGAGGAGGTGATTC-3′ and 5′-GCCGTCTAGATTACTTTTGTGCCATTAATGTAAGTCG-3′. The amplified fragment was cloned into the pCold I vector (TaKaRa). The cDNA fragments were then excised with NdeI and XhaI and subcloned into the pG-TF2 vector (TaKaRa). SoluBL21 E.coli (Genlantis, San Diego, CA) were transformed with the resulting plasmid. Expression of the recombinant protein was induced with 0.2mm isopropylthiogalactoside. The collected E.coli were suspended in BugBuster® (Merck) containing Benzonase® (Merck) and 0.5mg/ml lysozyme chloride. After centrifugation, the supernatant was diluted to 8mg/ml protein with 20mm Tris−HCl, pH 8.0, and used as a lysate for enzymatic assays. The PpYUC11 enzyme assay was conducted as described previously (Kakei etal., 2015) with minor modifications. The activity was measured in a reaction mixture containing E.coli lysates (50μg soluble protein), 3μm IPyA, 30μm FAD and 1mm NADPH in Tris−HCl buffer (50mm, pH 8.0). The mixture was incubated for 10min at 30°C and the reaction was stopped by adding 1/10 volume of 1N HCl. The reaction products (IAA) were analysed by high pressure liquid chromatography (HPLC) (Hitachi LaChrom Elite HPLC system) using fluorescence detection (λex/λem=280/355nm), equipped with a COSMOSIL 5C18-MS-II column (Nakalai Tesque Inc.). The preparations of first-strand cDNA and real-time RT-PCR were performed as described previously (Tatsuki etal., 2011) using a set of primers designed by DNASIS software (Ver. 3.0) (Hitachi Solutions, Tokyo, Japan) for each amplified gene (TableS5). Total RNA was extracted from the same samples of developmental and ripening fruit as used in Tatsuki etal. (2013). Real-time RT-PCR was performed with RNA samples isolated independently from two tissue sources, and each tissue source was assayed in triplicate. The primers were synthesized as follows: F1 (5′-TAAAGCCGCCCAAAAATAAA-3′), R1 (5′-TGGGAAGGAAGAAAATAGTCACA -3′), and R2 (5′-ATTTTCAACTTTCCCGAGCA -3′). PCR was performed using AmpliTaq Gold™ DNA polymerase (Applied Biosystems). Amplification was conducted as follows: 94°C for 12min, 35 cycles of (94°C for 0.5min, 55°C for 1min, and 72°C for 1min) and 72°C for 5min. The PCR products were analysed by agarose gel electrophoresis. SSR-PCR analysis was performed using the one-tube, single-reaction nested PCR method (Schuelke, 2000), with a 6-carboxyfluorescein (6-FAM)-labelled universal primer (Life Technologies) and the primer set 5′-GCTACGGACTGACCTCGGACCTATCTGGTATATAAGCTGAAACG-3′ and 5′-GTTTCTTACCTTTTTAGTTATTTCACCACAG-3′. Amplification was performed as follows: 94°C for 5min, 35 cycles of (94°C for 1min, 58°C for 1min and 72°C for 1min) and 72°C for 5min. A DNA standard (400HD ROX Size Standard, Life Technologies) was used to calculate the size of the PCR products. The PCR products were separated using an Applied Biosystems 3130xl Genetic Analyzer (Life Technologies). Size calculations were performed with GeneMapper® software version 5.0 (Life Technologies). This work was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and development for next-generation technology). We thank Drs. S. Terakami and M. Kunihisa (NIFTS) for advice on SSR analysis, Dr. T. Yamane (NIFTS) for providing ‘Akatsuki’ and ‘Kawanakajimahakutou’ fruit and K. Amano (NIFTS) and Y. Shikata (WARC) for technical support. tpj14070-sup-0001-FigS1.pdfPDF document, 101.8 KB FigureS1. Proposed IAA biosynthetic and metabolic pathway in plants. tpj14070-sup-0002-FigS2.pdfPDF document, 164.9 KB FigureS2. Measurement of IAAld and IAM in peach mesocarp tissues. tpj14070-sup-0003-FigS3.pdfPDF document, 163.4 KB FigureS3. Relative transcript abundance of PpYUC9 and PpTAA1 in ‘Akatsuki’ (left) and in ‘Manami’ (right). tpj14070-sup-0004-FigS4.pdfPDF document, 127.9 KB FigureS4. SDS-PAGE of E.coli lysate expressing recombinant PpYUC11. tpj14070-sup-0005-FigS5.pdfPDF document, 80.2 KB FigureS5. Genome sequence data of hd-t allele of the PpYUC11 gene from ‘Odoroki’. tpj14070-sup-0006-FigS6.pdfPDF document, 75.1 KB FigureS6. Genome sequence data of Hd allele of the PpYUC11 gene from ‘Kawanakajimahakutou’. tpj14070-sup-0008-FigS8.pdfPDF document, 128.5 KB FigureS8. Detection of PpYUC11 hd-t and Hd alleles in some cultivars and 447 selections. tpj14070-sup-0009-FigS9.pdfPDF document, 171.4 KB FigureS9. Sequence alignments of transposons inserted into PpYUC11 and PpDAM6. tpj14070-sup-0010-TableS1.pdfPDF document, 13.3 KB TableS1. RNA sequence analysis of YUCCA gene expression in S3 and S4 fruits of ‘Akatsuki’ and ‘Manami’. tpj14070-sup-0011-TableS2.pdfPDF document, 40.3 KB TableS2.PpYUC11 genotypes and fruit phenotypes. tpj14070-sup-0012-TableS3.pdfPDF document, 35.4 KB TableS3. Oligonucleotide primers used to isolate cDNAs and genomic DNA fragments. tpj14070-sup-0013-TableS4.pdfPDF document, 11.5 KB TableS4. Mapping results of RNA sequencing analysis. tpj14070-sup-0014-TableS5.pdfPDF document, 16.4 KB TableS5. Oligonucleotide primers used in real-time RT-PCR. 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