A Low Transcriptional Level of Se-RNase in the Se -haplotype Confers Self-compatibility in Japanese Plum

in Journal of the American Society for Horticultural Science

Most commercial cultivars of japanese plum (Prunus salicina Lindl.) exhibit S-RNase-based gametophytic self-incompatibility (GSI), although some self-compatible (SC) cultivars exist. In this study, we characterized S-RNase and SFB, the pistil and pollen S determinants of the specificity of the GSI reaction, respectively, from four S-haplotypes, including a SC (Se) and three SI (Sa, Sb, and Sc) S-haplotypes of japanese plum. The genomic organization and structure of the SC Se-haplotype appear intact, because the relative transcriptional orientation of its S-RNase and SFB and their intergenetic distance are similar to those of the other three SI S-haplotypes of japanese plum and other Prunus L. species. Furthermore, there is no apparent defect in the DNA sequences of Se-RNase and SFBe. However, a series of transcriptional analyses, including real-time reverse transcriptase–polymerase chain reaction, showed that the Se-RNase transcript levels in the pistil are significantly lower than those of the Sa-, Sb-, and Sc-RNases, although transcripts of SFBa, SFBb, SFBc, and SFBe are present at similar levels in pollen. Furthermore, no Se-RNase spot was detected in two-dimensional polyacrylamide gel electrophoresis profiles of stylar extracts of the cultivars with the Se-haplotype. We discuss the possible molecular basis of SC observed with the Se-haplotype with special reference to the insufficient Se-RNase accumulation incited by the very low transcriptional level of Se-RNase in pistils.

Abstract

Most commercial cultivars of japanese plum (Prunus salicina Lindl.) exhibit S-RNase-based gametophytic self-incompatibility (GSI), although some self-compatible (SC) cultivars exist. In this study, we characterized S-RNase and SFB, the pistil and pollen S determinants of the specificity of the GSI reaction, respectively, from four S-haplotypes, including a SC (Se) and three SI (Sa, Sb, and Sc) S-haplotypes of japanese plum. The genomic organization and structure of the SC Se-haplotype appear intact, because the relative transcriptional orientation of its S-RNase and SFB and their intergenetic distance are similar to those of the other three SI S-haplotypes of japanese plum and other Prunus L. species. Furthermore, there is no apparent defect in the DNA sequences of Se-RNase and SFBe. However, a series of transcriptional analyses, including real-time reverse transcriptase–polymerase chain reaction, showed that the Se-RNase transcript levels in the pistil are significantly lower than those of the Sa-, Sb-, and Sc-RNases, although transcripts of SFBa, SFBb, SFBc, and SFBe are present at similar levels in pollen. Furthermore, no Se-RNase spot was detected in two-dimensional polyacrylamide gel electrophoresis profiles of stylar extracts of the cultivars with the Se-haplotype. We discuss the possible molecular basis of SC observed with the Se-haplotype with special reference to the insufficient Se-RNase accumulation incited by the very low transcriptional level of Se-RNase in pistils.

Self-incompatibility (SI) is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing (de Nettancourt, 2001). In the Rosaceae, Scrophulariacae, and Solanaceae plant families, SI is gametophytically controlled by the S locus, which contains the genes that control the pollen and pistil specificities. The pistil determinant of the three families is S-RNase, which encodes a basic glycoprotein with ribonuclease activity that is present in the extracellular matrix of the style-transmitting tract. The F-box genes named SFB or SLF were found to be good candidates for the pollen S component in Prunus (Rosaceae) (Entani et al., 2003; Ushijima et al., 2003; Yamane et al., 2003b) and Antirrhinum L. (Scrophulariacae) (Lai et al., 2002). A subsequent transformation experiment demonstrated that SLF is the pollen S component in Petunia inflata R.E. Fries (Solanaceae) (Sijacic et al., 2004).

SI in Prunus interests pomologists and has been extensively studied because most Prunus fruit tree species are SI and unable to bear fruit parthenocarpically. The pistil S gene, S-RNase, has been characterized at the molecular level in most SI Prunus fruit tree species, including almond [P. dulcis (Mill.) D.A. Webb. (Ushijima et al., 1998)], sweet cherry [P. avium L. (Tao et al., 1999)], sour cherry [P. cerasus L. (Yamane et al., 2001)], japanese apricot [P. mume Siebold & Zucc. (Yaegaki et al., 2001)], and apricot [P. armeniaca L. (Romero et al., 2004)]. Recently, pollen S component SFB (S haplotype-specific F-box protein gene) has also been characterized in almond (Ushijima et al., 2003), sweet and sour cherries (Yamane et al., 2003b), japanese apricot (Entani et al., 2003; Yamane et al., 2003c), and apricot (Romero et al., 2004). The results from these studies have led to the development of not only molecular typing methods for S-haplotypes (Tao et al., 1999), but also molecular markers for self-compatibility (SC) in Prunus fruit tree species (Ikeda et al., 2004b; Tao et al., 2000, 2002a, 2002b).

There have been several reports on the characterization of japanese plum S-RNase and SFB. Yamane et al. (1999) first isolated a japanese plum S-RNase from the SI cultivar ‘Sordum’. Based on the S-RNase DNA sequence, Beppu et al. (2002, 2003) developed a polymerase chain reaction (PCR)-based S-haplotype typing system and identified 14 different japanese plum S-RNases corresponding to the Sa- to Sn-haplotypes. Later, Beppu et al. (2005) reported that one of the 14 S-haplotypes, the Se-haplotype, could be responsible for SC in some of the cultivars based on pollination and genetic studies. Tao et al. (2007) and Zhang et al. (2007) isolated SFB from several S-haplotypes. Because only partial sequences are available for S-RNase and SFB from all of the S-haplotypes of japanese plum, except for the Sa-haplotype (Tao et al., 2007), the nature of the SC conferred by the Se-haplotype is unclear.

In the present study, we thoroughly characterized the pistil and pollen S determinants of the specificity of the gametophytic self-incompatibility (GSI) reaction, S-RNase and SFB, respectively, from four S-haplotypes, including a SC (Se) and three SI (Sa, Sb, and Sc) S-haplotypes of japanese plum, to clarify the molecular basis of the SC observed in the Se-haplotype. Elucidation of the molecular basis of SC observed with Se-haplotype could lead to the development of not only the molecular markers for SC, but also the novel SC breeding strategies in japanese plum. A series of molecular analyses that were conducted in this study suggest that the SC in the Se-haplotype is incited by the accumulation of insufficient levels of Se-RNase incited by a very low Se-RNase transcription level in the pistil.

Materials and Methods

Plant material.

Twelve japanese plum cultivars were used in this study (Table 1). Young leaves, pollen grains, and styles with stigmas at the balloon stage of development were collected and stored at −80 °C until use.

Table 1.

S-haplotypes of 16 japanese plum cultivars.

Table 1.

Isolation of DNA.

Total DNA was isolated from young leaves as described previously (Yamane et al., 1999). In brief, leaves were ground to a powder using a mortar and pestle and homogenized in washing buffer [0.1 M HEPES (pH 8.0), 0.1% PVP, 2% mercaptoethanol]. The homogenate was centrifuged (6500 gn at 4 °C for 20 min) to collect the pellet, and the pellet was resuspended in washing buffer and centrifuged again. Next, the pellet was suspended in extraction buffer [0.2 m Tris-HCl (pH 8.0), 0.25 m NaCl, 25 mm ethylenediaminetetraacetic acid, 6% PVP], and one-half volume of 7.5 m ammonium acetate was added. The mixture was incubated on ice for 30 min, after which one volume of chloroform was added. After centrifugation (2500 gn at 4 °C for 15 min), the supernatant was transferred to a new tube and one volume of isopropanol was added. The mixture was centrifuged (8000 gn at 4 °C for 20 min) to collect the pellet. The resulting DNA pellet was dissolved in 10 mm Tris-HCl + 1 mm EDTA buffer (pH 8.0) (TE). After RNase treatment, the DNA solution was further purified by phenol/chloroform extraction and the DNA was precipitated with ethanol, rinsed with 75% ethanol, and dissolved in TE.

Construction and screening of a genomic library.

A fosmid library was constructed from genomic DNA of ‘Sordum’ (SaSb) and ‘Santa Rosa’ (ScSe) using the CopyControl Fosmid Library Production Kit (Epicentre, Madison, Wis.). The library was screened with a DIG-dUTP-labeled (Roche, Basel, Switzerland) probe corresponding to a partial fragment of the coding region of almond Sk-RNase or SFBk (Tao et al., 2007). Plasmid was extracted from positive colonies, and DNA sequences were determined with the BigDye Terminator version 3.1 Cycle Sequencing Kit and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif.). The deduced amino acid sequences of the SFBs and S-RNases were aligned using Clustal X (Thompson et al., 1997).

Analysis of the physical distances between S-RNase and SFB.

PCR reactions were performed to determine the physical distance between S-RNase and SFB in the Sb-, Sc-, and Se-haplotypes of japanese plum. For the Sb-haplotype, the primers PdS4-RNase-F4 and SFB-C2F (Table 2) were used along with ExTaq polymerase. The primers rtPS_ScF and rtPS_SFBcF (Table 2) were used with the Expand Long Template PCR System (Roche) for the Sc-haplotypes. Because it appeared that SFB and S-RNase are located in close proximity in the Sb- and Sc-haplotypes, the numbers of nucleotides flanked by the coding regions of the two genes were determined by sequencing. For the Se-haplotype, the distance between S-RNase and SFB was determined by estimating the positions of the two genes. The primers ccFos-F and ccFos-R, which are specific to the flanking regions of the cloning site of the CopyControl pCC1FOS cloning vector (Epicentre), were used in combination with the primers rt_PS_SeF and rt_PS_SFBeF (Table 2). The distance between the flanking region of the cloning site and the respective genes was determined by long PCR with the Expand Long Template PCR System. The distance between S-RNase and SFB was calculated from sizes of the PCR fragments and the inserted genomic DNA fragment. The transcriptional orientations of the genes were determined by PCR amplification with various combinations of primers designed to complement the flanking regions of the vector cloning site and gene-specific primers for SFB or S-RNase. All PCR reactions contained 1X PCR buffer, 300 μm of each dNTP, 3.75 mm MgCl2, 500 nm of each primer, 1 to 25 ng of template plasmid DNA, and 1 U Taq polymerase in a 15-μL reaction volume. Long PCR was performed using a program of an initial denaturation step at 94 °C for 2 min, 10 cycles of 94 °C for 10 s, 65 °C for 30 s, and 68 °C for 15 min, 20 cycles of 94 °C for 10 s, 65 °C for 30 s, and 68 °C for 15 min with an extension of an extra 20 s after each cycle and a final extension at 68 °C for 7 min. PCR products were separated on 1.5% or 0.6% agarose gels and visualized with ethidium bromide under ultraviolet light.

Table 2.

DNA sequences of oligonucleotide primers.

Table 2.

DNA gel blot analysis.

Six micrograms of genomic DNA were digested with HindIII, separated on a 0.6% agarose gel and transferred to a nylon membrane (Hybond-N; Amersham, Tokyo). The membrane was probed with a DIG-dUTP-labeled DNA fragment and washed under high-stringency conditions (twice at room temperature in 2X SSC, 0.1% SDS for 5 min, and twice at 68 °C in 0.1X SSC, 0.1% SDS) or low-stringency condition (twice at room temperature in 2X SSC, 0.1% SDS, and twice at 60 °C in 0.1X SSC, 0.1% SDS). Hybridization signals were visualized as described (Tao et al., 1999). The DIG-dUTP-labeled probe was synthesized by PCR using the Sa-RNase cDNA with the primers, Pru-C2 and Pru-C4R (Tao et al., 1999). For SFB probes, genomic clones of SFBa, SFBb, SFBc, and SFBe were used as templates with the primer sets, a forward primer SFB-C5F and reverse primers SFB-AR2, SFBBR3-PS, PSSFBcR, and PSSFBeR, respectively (Table 2).

Reverse transcriptase–polymerase chain reaction.

Total RNA was isolated from pollen grains, styles with stigmas at the balloon stage of development, and leaves of ‘Sordum’ (SaSb) and ‘Santa Rosa’ (ScSe) as described (Tao et al., 1999). One microgram of total RNA treated with DNase I (Invitrogen, Carlsbad, Calif.) was used for first-strand cDNA synthesis by SuperScript III RT (Invitrogen) with an adapter-dT primer. The primer combinations used to amplify the RT-PCR products from SFBa, SFBb, SFBc, and SFBe were PpSFB1F1 and PpSFB2R2, PsSFBb-F3 and PsSFBb-R3, rt_PS_SFBcF and rt_PS_SFBcR, and rt_PS_SFBeF and rt_PS_SFBeR, respectively (Table 2). The PCR mixture contained 1X ExTaq buffer, 200 μm of each dNTP, 400 nm of each primer, 20 ng of template cDNA, and 0.5 U of TaKaRa ExTaq polymerase (TaKaRa Bio, Ohotsu, Japan). PCR was performed using a program of 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s with an initial denaturation step at 94 °C for 2.5 min and a final extension step at 72 °C for 7 min. Expression of the actin gene was used as a control, using the ActF1 and ActR1 primers (Table 2). The control PCR conditions were identical to those of Yamane et al. (2003b).

Construction of a phylogenetic tree.

The coding DNA sequences for 23 S-RNases were used to construct a phylogenetic tree. The S-RNase sequences encompassed 17 Prunus S-RNases, including three japanese plum S-RNases that were identified in this study, three P. inflata S-RNases, and three Antirrhinum hispanicum Chav. S-RNases. Similarly, 17 Prunus SFBs, including three japanese plum SFBs that were identified in this study, seven P. inflata SLFs, and three A. hispanicum SLFs, were used to construct a phylogenetic tree. To construct an SFB/SLF tree, we also used eight Prunus SLFLs (S locus F-box genes with low allelic sequence polymorphism), the functions of which have yet to be clarified. The DNA sequences were aligned using CLUSTAL X (Thompson et al., 1997) and adjusted manually. The best-fit model of nucleotide sequence evolution was first determined in Modeltest (Posada and Crandall, 1998) and then used for a maximum likelihood tree search in PAUP* (Swofford, 2001).

Real-time reverse transcriptase–polymerase chain reaction for S-RNase in style and SFB in pollen.

Gene-specific primers for each S-RNase and SFB were designed using Primer Express (version 2.0; Applied Biosystems). The primer combinations used to amplify real-time RT-PCR products from SFBa, SFBb, SFBc, and SFBe were rt_PS_SFBaF and rt_PS_SFBaR, rt_PS_SFBbF and rt_PS_SFBbR, rt_PS_SFBcF and rt_PS_SFBcR, and rt_PS_SFBeF and rt_PS_SFBeR, respectively (Table 2). The primer combinations used to amplify real-time PCR products from Sa-RNase, Sb-RNase, Sc-RNase, and Se-RNase were rt_PS_SaF and rt_PS_SaRII, rt_PS_SbF and rt_PS_SbRII, rt_PS_ScF and rt_PS_ScR, and rt_PS_SeF and rt_PS_SeR, respectively (Table 2). As a control, ACTIN transcripts were quantified using two actin-specific primers designed for real-time RT-PCR analysis, qpActF and qpActR (Table 2). For real-time RT-PCR, 1 μL of cDNA solution, equivalent to the amount synthesized from 10 ng of total RNA, was used. Reactions were carried out in 96-well plates in a 25-μL reaction volume containing 12.5 μL of 2X SYBR ExTaq Master Mix (TaKaRa Bio), 0.2 μm each of forward and reverse primers, and 1 μL of cDNA solution. Three reactions were performed. The threshold cycle value (Ct) of each sample was automatically measured using SDS software (version 2.1; Applied Biosystems). After the PCR reaction, a dissociation curve analysis was performed to confirm that the observed fluorescence resulted only from the gene-specific amplification. Based on the Ct values, the original cDNA quantities of S-RNase and SFB were normalized using the ACTIN cDNA values and compared as relative amounts of their transcripts.

Two-dimensional polyacrylamide gel electrophoresis analysis of pistil proteins.

Acetone powders were prepared from styles with stigmas from ‘Sordum’ (SaSb), ‘Taiyo’ (SbSc), ‘Santa Rosa’ (ScSe), and ‘Rio’ (SaSe) as described previously (Tao et al., 1997). Crude extracts from the acetone powders were subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using nonequilibrium pH gradient electrophoresis in the first dimension and SDS-PAGE in the second dimension as described (Tao et al., 1997). After electrophoresis, the proteins in the gel were detected by silver staining using Sil-Best Stain for Protein/PAGE (Nacalai Tesque, Kyoto, Japan).

Results

Isolation and primary structures of SFB and S-RNase from four S-haplotypes.

The entire coding sequences for SFB and S-RNase of the Sb-haplotype of ‘Sordum’ (SaSb) and the Sc- and Se-haplotypes of ‘Santa Rosa’ (ScSe) were determined using fosmid clones obtained with probes designed for the almond Sk-RNase or SFBk. The deduced amino acid sequences of SFBb, SFBc, and SFBe were aligned with those of SFBa from japanese plum (Tao et al., 2007), SFB3 and SFB6 from sweet cherry, SFB1 and SFB2 from apricot, SFBa and SFBc from almond, and SFB1 and SFB7 from japanese apricot (Fig. 1). The amino acid identities among the 12 SFB alleles, including three japanese plum alleles identified in this study, range from 66.1% to 82.2%, which is comparable to the amino acid identities between japanese plum S-RNases and other Prunus S-RNases ranging from 49.1% to 78.9% (Table 3). All of the S-RNases from the four S-haplotypes, including the SC Se-haplotype, contain the two active sites of the T2/S-type RNases, including the two histidine residues essential for activity and five regions conserved in the Prunus S-RNases (Fig. 2). Similarly, all four SFBs contain the F-box motif within the N-terminal region as do other Prunus SFBs (Fig. 1). Two hypervariable hydrophilic regions that are under positive selection (Ikeda et al., 2004a) are also present in the C-terminal region (Fig. 1). No substantial difference was observed in the primary structures of the S-RNase and SFB from the SC Se-haplotype as compared with other S-RNases and SFBs from SI S-haplotypes.

Table 3.

Identities of the amino acid sequences of the Prunus S locus genes.z

Table 3.
Fig. 1.
Fig. 1.

Alignment of deduced SFB amino acid sequences. The deduced amino acid sequences were aligned using the Clustal X program (Thompson et al., 1997). The F-box motif, the variable regions V1 and V2, and the hypervariable regions HVa and HVb, are boxed (Ikeda et al., 2004a). Gaps are marked by dashes. Sharps (#) indicate the positions that have a single, fully conserved residue. EMBL/GenBank/DDBJ accession numbers are as follows: SFBa (Ps_SFBa; AB252409), SFBb (Ps_SFBb; AB252412), SFBc (Ps_SFBc; AB280792), and SFBe (Ps_SFBe; AB280794) of japanese plum (P. salicina); SFB3 (Pav_SFB3; AV096857) and SFB6 (Pav_SFB6; AB096858) of sweet cherry (P. avium); SFB1 (Par_SFB1; AY587561) and SFB2 (Par_SFB2; AY587562) of apricot (P. armeniaca); SFBa (Pd_SFBa; AB092966) and SFBc (Pd_SFBc; AB079776) of almond (P. dulcis); and SFB1 (Pm_SFB1; AB101440) and SFB7 (Pm_SFB7; AB101441) of japanese apricot (Prunus mume).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

Fig. 2.
Fig. 2.

Alignment of deduced S-RNase amino acid sequences. The deduced amino acid sequences were aligned using the Clustal X program (Thompson et al., 1997). The five conserved regions C1, C2, C3, RC4, and C5, and the hypervariable region RHV (Ushijima et al., 1998), which exist in rosaceous S-RNases, are boxed. Gaps are marked by dashes (—). Sharps (#) indicate positions that have a single, fully conserved residue. EMBL/GenBank/DDBJ accession numbers are as follows: Sa-RNase (Ps_Sa; AB252411), Sb-RNase (Ps_Sb; AB252413), Sc-RNase (Ps_Sc; AB280791), and Se-RNase (Ps_Se; AB280793) of japanese plum (P. salicina); S3-RNase (Pav_S3; AB010306) and S6-RNase (Pav_S6; AB010305) of sweet cherry (P. avium); S1-RNase (Par_S1; AY587561) and S2-RNase (Par_S2; AY587562) of apricot (P. armeniaca); Sa-RNase (Pd_Sa; AB026836) and Sc-RNase (Pd_Sc; AB011470) of almond (P. dulcis); and S1-RNase (Pm_S1; AB101438), and S7-RNase (Pm_S7; AB101439) of japanese apricot (Prunus mume).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

The relative transcriptional orientations of SFB and S-RNase and their physical distances in the Sb-, Sc-, and Sd-haplotypes were determined in this study (Fig. 3), and those of the Sa-haplotype had already been determined (Tao et al., 2007). In all four S-haplotypes, SFB is located downstream of S-RNase in a reverse transcriptional orientation, like in all of the other S-haplotypes of Prunus that have been investigated to date except for the S2-haplotype of P. armeniaca (Fig. 3; Ikeda et al., 2005; Romero et al., 2004; Tao et al., 2007; Ushijima et al., 2003). The estimated distances between SFB and S-RNase in the Sb-, Sc-, and Se-haplotypes are 539 base pairs (bp), 756 bp, and 18 kilobp (kbp), respectively, whereas that for the Sa-haplotype was described previously (Tao et al., 2007) as ≈41 kbp (Fig. 3). These results show that the distance between the two genes in the Sb-, Sc-, and Se-haplotypes is within the range of those in other SI Prunus S-haplotypes, and the SFBs are physically tightly linked to the S-RNases in japanese plum like in other Prunus species.

Fig. 3.
Fig. 3.

Schematic diagram illustrating the S-RNase and SFB gene organization in the S loci of four japanese plum S-haplotypes. The illustration of the locus of the Sa-haplotype is from Tao et al. (2007). The nucleotide “A’’ of the start codon of S-RNases and SFBs is at position +1. Black boxes represent exons in the S-RNases and SFBs. Arrows denote the direction of transcription.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

Genomic DNA blot and reverse transcriptase–polymerase chain reaction analysis of four SFBs.

Genomic DNA blot analysis showed that SFBa, SFBb, SFBc, and SFBe are cosegregated with the Sa-, Sb-, Sc-, and Se-haplotypes of japanese plum cultivars, respectively (Fig. 4). Under low-stringency washes at 60 °C with the Sa-RNase or SFBa probe, all cultivars yielded Sa-haplotype-specific bands corresponding to their S-haplotypes (Fig. 4A, B). With high-stringency washes at 68 °C with the SFBa, SFBb, SFBc, or SFBe probes, only cultivars with the respective S -haplotypes yielded hybridization signals (Fig. 4C–F).

Fig. 4.
Fig. 4.

Genomic DNA blot analyses for the Sa- and Sb-haplotypes of japanese plum. Genomic DNAs from 14 japanese plum cultivars were digested with HindIII, separated on a 0.6% agarose gel, and transferred to a nylon membrane. (A) Membrane hybridized with an Sa-RNase probe and washed under low-stringency conditions. (B) Membrane hybridized with an SFBa probe and washed under low-stringency conditions. (C) Membrane hybridized with an SFBa probe and washed under high-stringency conditions. (D) Membrane hybridized with an SFBb probe and washed under high-stringency conditions. (E) Membrane was hybridized with an SFBc probe and washed under high-stringency conditions. (F) Membrane was hybridized with an SFBe probe and washed under high-stringency conditions. Lanes: A, ‘Sordum’ (SaSb); B, ‘Burmosa’ (SaSb); C, ‘Rio’ (SaSe); D, ‘Terada’ (SaSf); E, ‘Botan’ (SaSm); F, ‘Oishinakate’ (SbSc); G, ‘Formasa’ (SbSd); H, ‘Frontier’ (SbSf); I, ‘Honey Rosa’ (SbSg); J, ‘Yonemomo’ (SbSh); K, ‘Bakemonosumomo’ (SbSi); L, ‘Harypickstone’ (SbSk); M, ‘Lantz’ (SbSl); N, ‘Santa Rosa’ (ScSe).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

RT-PCR using primers specific for SFBa, SFBb, SFBc, or SFBe revealed that the transcription of these SFBs is specific to pollen (Fig. 5), like found with other Prunus SFBs.

Fig. 5.
Fig. 5.

Expression analysis of the SFBs of four S-haplotypes of japanese plum. RT-PCR for SFBs and ACTIN was performed with total RNA from pollen, styles, and leaves of ‘Sordum’ (SaSb) and ‘Santa Rosa’ (ScSe).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

Phylogenetic analysis of SFBs and S-RNases.

To investigate the phylogenetic relationships of the S-RNases and SFBs of the japanese plum S-haplotypes with other reported S-RNases or SFB/SLFs, we constructed phylogenetic trees for the S-RNase and SLF/SFB from Prunus of the family Rosaceae, A. hispanicum, and P. inflata. All Prunus S-RNases fell into one clade, and the S-RNases from A. hispanicum and P. inflata were incorporated into the other two clades (Fig. 6). Similarly, all SFBs fell into one clade that was distinct from the clades of the SLFLs of Prunus and the SLFs of A. hispanicum and P. inflata (Fig. 7). As reported by Ushijima et al. (2004), the SLFLs of Prunus were phylogenetically less similar to the SFBs of Prunus than to the SLFs of Antirrhinum and Petunia Juss. Like with the S-RNases, no species-specific subgroup of Prunus SFBs was formed. Both S-RNase and SFB from the SC Se-haplotype fell into the clades of Prunus S-RNases and Prunus SFBs, respectively, confirming the finding that there are no substantial differences in the primary structures of Se-RNase and SFBe from other S-RNases and SFBs of SI S-haplotypes, respectively.

Fig. 6.
Fig. 6.

Phylogenetic tree of the S-RNases of Prunus, Antirrhinum, and Petunia. All phylogenetic reconstitution was conducted in PAUP* (Swofford, 2001) as described in the text. Numbers indicate the percentage of 100 bootstrap replicates in which a given group was found (values below 50% not shown). Sequence information for the S-RNases included in the tree is as follows: Sa-RNase (Ps Se; AB026981), Sb-RNase (Ps Sb; AB026982), Sc-RNase (Ps Sc; AB280791), and Se-RNase (Ps Se; AB280793) of japanese plum (P. salicina); S1-RNase (Pav S1; AB028153), S3-RNase (Pav S3; AB010306), S4-RNase (Pav S4; AB028154), S5-RNase (Pav S5; AJ298314), and S6-RNase (Pav S6; AB010305) of sweet cherry (P. avium); S1-RNase (Pm S1; AY587561), S2-RNase (Pm S2; AY587562), and S4-RNase (Pm S4; AY587564) of apricot (P. armeniaca); Sa-RNase (Pd Sa; AB026836), Sb-RNase (Pd Sb; AB011469), and Sc-RNase (Pd Sc; AB011470) of almond (P. dulcis); S1-RNase (Pm S1; AB101438) and S7-RNase (Pm S7; AB101439) of japanese apricot (P. mume); S2-RNase (Ah S2; X96465), S4-RNase (Ah S4; X96466), and S5-RNase (Ah S5; X96464) of Antirrhinum hispanicum; S1-RNase (Pi S1; M67990), S2-RNase (Pi S2; AF301533), and S3-RNase (Pi S3; M67991) of Petunia inflata.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

Fig. 7.
Fig. 7.

Phylogenetic tree of the SFB/SLFs of Prunus, Antirrhinum, and Petunia. All phylogenetic reconstitution was conducted in PAUP* (Swofford, 2001) as described in the text. Numbers indicate the percentage of 100 bootstrap replicates in which a given group was found (values below 50% not shown). Sequence information for the SFBs included in the tree is as follows: SFBa (Ps SFBa; AB252409), SFBb (Ps SFBb; AB252412), SFBc (Ps SFBc; AB280792), and SFBe (Ps SFBe; AB280794) of japanese plum (P. salicina); SFB1 (Pav SFB1; AB111518), SFB3 (Pav SFB3; AB096857), SFB4 (Pav SFB4; AB111521), SFB5 (Pav SFB5; AB111520), SFB6 (Pav SFB6; AB096858), SLFL1-S4 (Pav SLFL1S4; AB280953), SLFL2-S4 (Pav SLFL1S4S4; AB280954), and SLFL3-S4 (Pav SLFL3S4; AB280955) of sweet cherry (P. avium); SFB1 (Par SFB1; AY587563), SFB2 (Par SFB2; AY587562), and SFB4 (Par SFB4; AY587565) of apricot (P. armeniaca); SFBa (Pd SFBa; AB092966), SFBb (Pd SFBb; AB092967), and SFBc (Pd SFBc; AB079776) of almond (P. dulcis); SFB1 (Pm SFB1; AB101440), SFB7 (Pm SFB7; AB101441), SLFL1-S1 (Pm SLFL1S1; AB092623), SLFL1-S7 (Pm SLFL1S7; AB092624), SLFL2-S1 (Pm SLFL2S1; AB092625), SLFL2-S7 (Pm SLFL2S7; AB092626), and SLFL3-S7 (Pm SLFL3S7; AB092627) of japanese apricot (P. mume); SLF-S2 (Ah SLF2; AJ297974), SLF-S4 (Ah SLF4; AJ515534), and SLF-S5 (Ah SLF5; AJ515536) of Antirrhinum hispanicum; SLF1 (Pi SLF1; AY500390), SLF2 (Pi SLF2; AY500391), and SLF3 (Pi SLF3; AY500392) of Petunia inflata.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

Transcript accumulation of SFBe in pollen and Se-RNase in style.

Beppu et al. (2005) reported the involvement of the Se-haplotype in SC in japanese plum based on segregation analysis. Because insufficient expression of either SFB or S-RNase leads to SC in Prunus, we performed real-time PCR to determine the amounts of the corresponding transcripts. The relative transcript amounts of Se-RNase and those of the other three S-RNases, the Sa-, Sb- and Sc-RNases, in different lines, are shown in Figure 8A. The amount of Se-RNase transcript in styles of ‘Santa Rosa’ was significantly lower (less than 1/10,000) than in those of Sc-RNase. Similarly, significantly fewer Se-RNase transcripts accumulated than Sc- or Sa-RNase transcripts in the styles of ‘Beauty’ and ‘Rio’, respectively (Fig. 8A). Given that the amounts of Sa-RNase and Sb-RNase transcripts are comparable in ‘Sordum’ pistils, the significantly lower amount of Se-RNase transcripts in styles could be responsible for the SC observed in the Se-haplotype. The amounts of SFBe transcript were equivalent to those of SFBc in ‘Santa Rosa’ pollen, as was the case with SFBa and SFBb in ‘Sordum’ pollen (Fig. 8B).

Fig. 8.
Fig. 8.

Relative amounts of S-RNase and SFB transcripts. (A) Relative amounts of S-RNase transcripts in styles of the japanese plum cultivars ‘Sordum’ (SaSb), ‘Santa Rosa’ (ScSe), ‘Beauty’ (ScSe), and ‘Rio’ (SaSe) as analyzed by real-time RT-PCR. (B) Relative amounts of SFB transcripts in pollen grains of the japanese plum cultivars ‘Sordum’ (SaSb) and ‘Santa Rosa’ (ScSe) as revealed by real-time RT-PCR. See text for details. See text for details. Vertical bars indicate standard deviation.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

S-RNase protein assay.

The 2D-PAGE profiles of ‘Santa Rosa’ (ScSe), ‘Taiyo’ (SbSc), and ‘Rio’ (SaSe) styles contained major protein spots with molecular weights and isoelectric points similar to those of the S-RNases of the japanese plum ‘Sordum’ (Yamane et al., 1999) and other Prunus species, including almond (Tao et al., 1997), sweet cherry (Tao et al., 1999), and japanese apricot (Tao et al., 2002b) (Fig. 9). Although two S-RNase spots corresponding to the Sb and Sc alleles were observed in ‘Taiyo’, only a single S-RNase spot was visible in the 2D-PAGE profiles of ‘Santa Rosa’ and ‘Rio’ (Fig. 9). Because the S-RNase spots of ‘Santa Rosa’ and ‘Rio’ had different molecular masses and isoelectric point (pI) values, and these are thought to correspond to Sc-RNase and Sa-RNase, respectively, we concluded that the Se-RNase spot was missing in the 2D-PAGE profiles. This suggests that very little Se-RNase, if any, accumulates in the styles of cultivars with the Se-haplotype.

Fig. 9.
Fig. 9.

S-RNases of the japanese plum cultivars (A) ‘Sordum’ (SaSb), (B) ‘Taiyo’ (SbSc), (C) ‘Santa Rosa’ (ScSe) and (D) ‘Rio’ (SaSe). Stylar proteins were separated by 2D-PAGE and visualized by silver staining. S-RNases are indicated by arrows. The spatial distribution and relative intensities of the S-RNases in 2D-PAGE profiles are shown in E.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 132, 3; 10.21273/JASHS.132.3.396

Discussion

SI in Prunus is controlled by two genes, S-RNase and SFB, which are located in the S locus. The former controls pistil specificity and the latter controls the pollen specificity of the SI reaction. Although partial japanese plum S-RNase sequences have been deduced through PCR and characterized at the molecular level (Beppu et al., 2002, 2003; Sapir et al., 2004; Yamane et al., 1999), SFB remained to be cloned until very recently (Tao et al., 2007; Zhang et al., 2007). Tao et al. (2007) isolated full-length S-RNase and SFB of the japanese plum Sa-haplotype by screening a genomic library, and Zhang et al. (2007) isolated partial SFB sequences from eight different S-haplotypes by genomic PCR. The japanese plum Sa-haplotype and the peach S2-haplotype appear to have the same origin (Tao et al., 2007). The former appears to be an intact SI S-haplotype and the latter to be a mutated SC S-haplotype from the same ancestral S-haplotype. As indicated by our phylogenetic analysis and those of others (Ikeda et al., 2004b; Ushijima et al., 1998), the diversification of S-haplotypes apparently occurred before speciation in Prunus.

In this study, we isolated the S locus region of the Sb-, Sc-, and Se-haplotypes and characterized their full-length S-RNase and SFB. The molecular characteristics of the S-RNase and SFB of the three S-haplotypes support the idea that they encode factors that control the pistil and pollen specificity of the SI reaction in japanese plum, like in Sa-haplotype (Tao et al., 2007). The full-length coding sequences for S-RNase and SFB of the four S-haplotypes, including the SC Se-haplotype of japanese plum, were used to elucidate the molecular basis of SC observed with the Se-haplotype, because defects in either S-RNase or SFB often lead to SC in Prunus (Hauck et al., 2006a, 2006b; Tao et al., 2007; Tsukamoto et al., 2006; Ushijima et al., 2004; Yamane et al., 2003a). Although a series of molecular analyses, including a primary structural comparison, investigation of the gene organization in the S locus and a phylogenetic reconstruction indicated that both S-RNase and SFB are intact in the Se-haplotype, real-time RT-PCR analysis showed that the transcriptional level of Se-RNase is much lower than those of the S-RNases of the other three japanese plum SI S-haplotypes investigated. Although no difference was observed previously in the transcriptional levels of the Se-RNase and the other S-RNases attributable to the use of nonquantitative RT-PCR in our earlier study (Beppu et al., 2005), the use of real-time RT-PCR in this study made it possible to demonstrate the low Se-RNase transcription level. Furthermore, the 2D-PAGE analysis clearly showed that the low level of transcription leads to a very low level of Se-RNase in the style.

In the Rosaceae, the molecular bases of four stylar-part mutants have been reported. The S4-RNase of the S4sm-haplotype of japanese pear is completely deleted, whereas the respective S-RNases of the S6m-, S6m2-, and S13m-haplotypes of sour cherry have defects that affect their transcription or translation. The molecular defect in the S6m-haplotype was determined to be an insertion of a Mu-like element upstream of the S6-RNase that blocks S-RNase transcription (Yamane et al., 2003a). The S6m2- and S13m-haplotypes are disrupted by different defects: deletions of 1 bp and 23 bp in S6m2-RNase and S13m-RNase, respectively, leading to a premature stop codon, although transcripts of each are still present (Tsukamoto et al., 2006). Although the nature of the low level of Se-RNase transcription remains to be characterized, it results in a low level of Se-RNase accumulation in the style, possibly causing the SC observed in the Se-haplotype.

Qin et al. (2006) estimated the threshold amount of S12-RNase of Solanum chacoense Bitt. that is required for pollen rejection. They showed that 68 ng of S12-RNase per style did not confer SI, whereas 160 ng S12-RNase per style led to SI. This indicates that approximately half of the threshold amount of S-RNase required for a successful SI reaction failed to confer SI in S. chacoense. Because the amount of Se-RNase transcript was at least 10,000-fold lower than those of the SI S-RNases of japanese plum and no Se-RNase spot was found in the 2D-PAGE profile of the stylar extracts, the SC conferred by the Se-haplotype could be attributable to an insufficient level of Se-RNase transcript. Because SFBe is transcribed normally, the Se-haplotype may contain a functional pollen S allele and a nonfunctional stylar S allele, which would make the Se-haplotype a stylar-part mutant SC S-haplotype. However, because the original functional version of Se-haplotype has yet to be found, there is a possibility that SFBe is also nonfunctional.

In conclusion, we isolated and characterized complete coding sequences for S-RNase and SFB from three S-haplotypes of japanese plum, Sb, Sc, and Se, and deduced a possible molecular basis for the SC observed in the Se-haplotype. In addition to the previously characterized Sa-haplotype (Tao et al., 2007), full-length coding sequences for the SFBs and S-RNases of four S-haplotypes of japanese plum are now available. Furthermore, partial sequence information is available for the SFBs and S-RNases of several other S-haplotypes (Zhang et al., 2007). Because SC mechanisms in addition to that related to the Se-haplotype are thought to exist in japanese plum (Beppu et al., 2002, 2003), the gene sequence information could provide further insight into the molecular mechanisms of SC in this fruit species, which, in turn, would provide valuable information for SC breeding.

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Contributor Notes

Ms. Watari and Mr. Hanada contributed equally.

Corresponding author. E-mail: rtao@kais.kyoto-u.ac.jp.

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    Alignment of deduced SFB amino acid sequences. The deduced amino acid sequences were aligned using the Clustal X program (Thompson et al., 1997). The F-box motif, the variable regions V1 and V2, and the hypervariable regions HVa and HVb, are boxed (Ikeda et al., 2004a). Gaps are marked by dashes. Sharps (#) indicate the positions that have a single, fully conserved residue. EMBL/GenBank/DDBJ accession numbers are as follows: SFBa (Ps_SFBa; AB252409), SFBb (Ps_SFBb; AB252412), SFBc (Ps_SFBc; AB280792), and SFBe (Ps_SFBe; AB280794) of japanese plum (P. salicina); SFB3 (Pav_SFB3; AV096857) and SFB6 (Pav_SFB6; AB096858) of sweet cherry (P. avium); SFB1 (Par_SFB1; AY587561) and SFB2 (Par_SFB2; AY587562) of apricot (P. armeniaca); SFBa (Pd_SFBa; AB092966) and SFBc (Pd_SFBc; AB079776) of almond (P. dulcis); and SFB1 (Pm_SFB1; AB101440) and SFB7 (Pm_SFB7; AB101441) of japanese apricot (Prunus mume).

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    Alignment of deduced S-RNase amino acid sequences. The deduced amino acid sequences were aligned using the Clustal X program (Thompson et al., 1997). The five conserved regions C1, C2, C3, RC4, and C5, and the hypervariable region RHV (Ushijima et al., 1998), which exist in rosaceous S-RNases, are boxed. Gaps are marked by dashes (—). Sharps (#) indicate positions that have a single, fully conserved residue. EMBL/GenBank/DDBJ accession numbers are as follows: Sa-RNase (Ps_Sa; AB252411), Sb-RNase (Ps_Sb; AB252413), Sc-RNase (Ps_Sc; AB280791), and Se-RNase (Ps_Se; AB280793) of japanese plum (P. salicina); S3-RNase (Pav_S3; AB010306) and S6-RNase (Pav_S6; AB010305) of sweet cherry (P. avium); S1-RNase (Par_S1; AY587561) and S2-RNase (Par_S2; AY587562) of apricot (P. armeniaca); Sa-RNase (Pd_Sa; AB026836) and Sc-RNase (Pd_Sc; AB011470) of almond (P. dulcis); and S1-RNase (Pm_S1; AB101438), and S7-RNase (Pm_S7; AB101439) of japanese apricot (Prunus mume).

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    Schematic diagram illustrating the S-RNase and SFB gene organization in the S loci of four japanese plum S-haplotypes. The illustration of the locus of the Sa-haplotype is from Tao et al. (2007). The nucleotide “A’’ of the start codon of S-RNases and SFBs is at position +1. Black boxes represent exons in the S-RNases and SFBs. Arrows denote the direction of transcription.

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    Genomic DNA blot analyses for the Sa- and Sb-haplotypes of japanese plum. Genomic DNAs from 14 japanese plum cultivars were digested with HindIII, separated on a 0.6% agarose gel, and transferred to a nylon membrane. (A) Membrane hybridized with an Sa-RNase probe and washed under low-stringency conditions. (B) Membrane hybridized with an SFBa probe and washed under low-stringency conditions. (C) Membrane hybridized with an SFBa probe and washed under high-stringency conditions. (D) Membrane hybridized with an SFBb probe and washed under high-stringency conditions. (E) Membrane was hybridized with an SFBc probe and washed under high-stringency conditions. (F) Membrane was hybridized with an SFBe probe and washed under high-stringency conditions. Lanes: A, ‘Sordum’ (SaSb); B, ‘Burmosa’ (SaSb); C, ‘Rio’ (SaSe); D, ‘Terada’ (SaSf); E, ‘Botan’ (SaSm); F, ‘Oishinakate’ (SbSc); G, ‘Formasa’ (SbSd); H, ‘Frontier’ (SbSf); I, ‘Honey Rosa’ (SbSg); J, ‘Yonemomo’ (SbSh); K, ‘Bakemonosumomo’ (SbSi); L, ‘Harypickstone’ (SbSk); M, ‘Lantz’ (SbSl); N, ‘Santa Rosa’ (ScSe).

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    Expression analysis of the SFBs of four S-haplotypes of japanese plum. RT-PCR for SFBs and ACTIN was performed with total RNA from pollen, styles, and leaves of ‘Sordum’ (SaSb) and ‘Santa Rosa’ (ScSe).

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    Phylogenetic tree of the S-RNases of Prunus, Antirrhinum, and Petunia. All phylogenetic reconstitution was conducted in PAUP* (Swofford, 2001) as described in the text. Numbers indicate the percentage of 100 bootstrap replicates in which a given group was found (values below 50% not shown). Sequence information for the S-RNases included in the tree is as follows: Sa-RNase (Ps Se; AB026981), Sb-RNase (Ps Sb; AB026982), Sc-RNase (Ps Sc; AB280791), and Se-RNase (Ps Se; AB280793) of japanese plum (P. salicina); S1-RNase (Pav S1; AB028153), S3-RNase (Pav S3; AB010306), S4-RNase (Pav S4; AB028154), S5-RNase (Pav S5; AJ298314), and S6-RNase (Pav S6; AB010305) of sweet cherry (P. avium); S1-RNase (Pm S1; AY587561), S2-RNase (Pm S2; AY587562), and S4-RNase (Pm S4; AY587564) of apricot (P. armeniaca); Sa-RNase (Pd Sa; AB026836), Sb-RNase (Pd Sb; AB011469), and Sc-RNase (Pd Sc; AB011470) of almond (P. dulcis); S1-RNase (Pm S1; AB101438) and S7-RNase (Pm S7; AB101439) of japanese apricot (P. mume); S2-RNase (Ah S2; X96465), S4-RNase (Ah S4; X96466), and S5-RNase (Ah S5; X96464) of Antirrhinum hispanicum; S1-RNase (Pi S1; M67990), S2-RNase (Pi S2; AF301533), and S3-RNase (Pi S3; M67991) of Petunia inflata.

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    Phylogenetic tree of the SFB/SLFs of Prunus, Antirrhinum, and Petunia. All phylogenetic reconstitution was conducted in PAUP* (Swofford, 2001) as described in the text. Numbers indicate the percentage of 100 bootstrap replicates in which a given group was found (values below 50% not shown). Sequence information for the SFBs included in the tree is as follows: SFBa (Ps SFBa; AB252409), SFBb (Ps SFBb; AB252412), SFBc (Ps SFBc; AB280792), and SFBe (Ps SFBe; AB280794) of japanese plum (P. salicina); SFB1 (Pav SFB1; AB111518), SFB3 (Pav SFB3; AB096857), SFB4 (Pav SFB4; AB111521), SFB5 (Pav SFB5; AB111520), SFB6 (Pav SFB6; AB096858), SLFL1-S4 (Pav SLFL1S4; AB280953), SLFL2-S4 (Pav SLFL1S4S4; AB280954), and SLFL3-S4 (Pav SLFL3S4; AB280955) of sweet cherry (P. avium); SFB1 (Par SFB1; AY587563), SFB2 (Par SFB2; AY587562), and SFB4 (Par SFB4; AY587565) of apricot (P. armeniaca); SFBa (Pd SFBa; AB092966), SFBb (Pd SFBb; AB092967), and SFBc (Pd SFBc; AB079776) of almond (P. dulcis); SFB1 (Pm SFB1; AB101440), SFB7 (Pm SFB7; AB101441), SLFL1-S1 (Pm SLFL1S1; AB092623), SLFL1-S7 (Pm SLFL1S7; AB092624), SLFL2-S1 (Pm SLFL2S1; AB092625), SLFL2-S7 (Pm SLFL2S7; AB092626), and SLFL3-S7 (Pm SLFL3S7; AB092627) of japanese apricot (P. mume); SLF-S2 (Ah SLF2; AJ297974), SLF-S4 (Ah SLF4; AJ515534), and SLF-S5 (Ah SLF5; AJ515536) of Antirrhinum hispanicum; SLF1 (Pi SLF1; AY500390), SLF2 (Pi SLF2; AY500391), and SLF3 (Pi SLF3; AY500392) of Petunia inflata.

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    Relative amounts of S-RNase and SFB transcripts. (A) Relative amounts of S-RNase transcripts in styles of the japanese plum cultivars ‘Sordum’ (SaSb), ‘Santa Rosa’ (ScSe), ‘Beauty’ (ScSe), and ‘Rio’ (SaSe) as analyzed by real-time RT-PCR. (B) Relative amounts of SFB transcripts in pollen grains of the japanese plum cultivars ‘Sordum’ (SaSb) and ‘Santa Rosa’ (ScSe) as revealed by real-time RT-PCR. See text for details. See text for details. Vertical bars indicate standard deviation.

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    S-RNases of the japanese plum cultivars (A) ‘Sordum’ (SaSb), (B) ‘Taiyo’ (SbSc), (C) ‘Santa Rosa’ (ScSe) and (D) ‘Rio’ (SaSe). Stylar proteins were separated by 2D-PAGE and visualized by silver staining. S-RNases are indicated by arrows. The spatial distribution and relative intensities of the S-RNases in 2D-PAGE profiles are shown in E.

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