Plant Biotechnology 30, 119–123 (2013)
DOI: 10.5511/plantbiotechnology.13.0109a

Original Paper

Identification of an S-RNase binding protein1 (SBP1)
homolog of apple (Malus×domestica)
Mai F. Minamikawa1,†, Daisuke Fujii 1,†, Hiroyuki Kakui 1, Nobuhiro Kotoda2,
Hidenori Sassa1,*
1

Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan; 2 Apple Breeding and Physiology
Research Team, National Institute of Fruit Tree Science, Morioka, Iwate 020-0123, Japan* E-mail: sassa@faculty.chiba-u.jp Tel: +81-47-308-8967 Fax: +81-47-308-8720
Received December 12, 2012; accepted January 9, 2013 (Edited by S. Nakano)

Abstract Many flowering plants exhibit self-incompatibility (SI) to prevent inbreeding and promote outcrossing. This
self/non-self discrimination mechanism is controlled by the S locus, which contains separate genes for pistil and pollen
specificities. In the gametophytic SI (GSI) of Rosaceae, Solanaceae and Plantaginaceae, the pistil S determinant, S-RNase,
encodes extracellular ribonuclease which is thought to act as a cytotoxin to the self pollen tube, while the pollen S
determinant is the F-box gene called SLF/SFB/SFBB. In Petunia (Solanaceae), SLF is reported to be a component of thenoncanonical E3 ubiquitin ligase complex with S-RNase binding protein1 (SBP1) and Cullin1 (CUL1), and interact with
non-self S-RNases to ubiquitinate them for degradation. Here, we isolated an apple (Malus×domestica) homolog of SBP1
(MdSBP1) from pollen RNA by RT-PCR. MdSBP1 included a RING-HC domain required for E3 ubiquitin ligase activity,and showed 64.0–68.2% amino acid identities with solanaceous SBP1 proteins. Expression analysis showed that MdSBP1
was expressed in all the organs analyzed. We detected an interaction between recombinant MdSBP1 protein and S-RNase ofapple using a pull-down assay.
Key words:

Self-incompatibility, S-RNase binding protein, pollen, apple, Rosaceae.

Self-incompatibility (SI) is a mechanism adopted
by many flowering plants to prevent inbreeding andpromote outcrossing. The S-RNase-based gametophytic
self-incompatibility (GSI) of Rosaceae, Solanaceae andPlantaginaceae is controlled by a single multiallelic
S locus which contains separate genes for pistil andpollen specificities. When the S haplotype of a pollen
matches one of the two S haplotypes of the diploid
pistil, the pollen is recognized as self and rejected (deNettancourt 2001). The specificities of pistil and pollen
are controlled by separate but tightly linked genes
located at the S locus, S-RNase and the F-box gene
called SLF/SFB/SFBB, respectively (Franklin-Tong 2008;
Kao and Tsukamoto 2004; Meng et al. 2011; Sassa et al.
2010; Tao and Iezzoni 2010). The pistil S determinant,
S-RNase, encodes extracellular ribonuclease (Anderson
et al. 1986; McClure et al. 1989; Sassa et al. 1996, 1997;
Tao et al. 1997; Xue et al. 1996) which is thought to betaken up by pollen tubes and act as a cytotoxin to selfpollen (Goldraij et al. 2006; Luu et al. 2000). In Petunia
of Solanaceae and species of Rosaceae tribe Pyreae,i.e., apples (Malus×domestica) and pears (Pyrus spp.),multiple F-box genes SLFs/SFBBs are implicated inpollen-part specificity (De Franceschi et al. 2011; Kubo et
al. 2010; Kakui et al. 2011; Minamikawa et al. 2010; Saito
et al. 2012; Sassa et al. 2007).
SLF has been predicted to act as a component of theE3 ubiquitin ligase complex and interact with non-self
S-RNases to ubiquitinate them for degradation (Huang
et al. 2006; Qiao et al. 2004a, 2004b; Sijacic et al. 2004).
Canonical E3 complex comprises Skp1, Cullin1, F-box
protein and Rbx1 (Cardozo and Pagano 2004). In
Petunia inflata, however, SLF-containing E3 ubiquitin
ligase is reported to be a noncanonical SCF-like complex
which includes S-RNase binding protein1 (SBP1) inplace of Skp1 and Rbx1 (Hua and Kao 2006; Hua et al.
2008). SBP1 contained a RING-HC domain found in
Rbx1 (Deshaies and Joazeiro 2009) and was considered
to play the roles of Skp1 and Rbx1, binding of F-box
protein and ubiquitin activating enzyme E2, respectively(Hua and Kao 2006). SBP1 was first identified as an
S-RNase binding protein in Petunia hybrida by yeast
two-hybrid screening of an anther cDNA library (Simsand Ordanic 2001). The petunia SBP1 included a RING-

Abbreviations: GSI, Gametophytic self-incompatibility; RNase, ribonuclease; SBP1, S-RNase binding protein1; MBP, maltose binding protein.
†
These authors contributed equally to this work.
This article can be found at http://www.jspcmb.jp/
Published online May 16, 2013

Copyright © 2013 The Japanese Society for Plant Cell and Molecular Biology

120

An S-RNase binding protein1 homolog of apple

HC domain and interacted with S-RNase, and showed
no S haplotype-specific sequence polymorphism. SBP1
homologs have been identified in Solanum chacoense(O’Brien et al. 2004) and Nicotiana alata (Lee et al.
2008); however, SBP1-like protein has not yet beencharacterized outside Solanaceae. Here, we isolated anapple homolog of SBP1 from pollen RNA and named it
MdSBP1. MdSBP1 included a RING-HC domain and was
expressed in all the organs analyzed. An in vitro binding
assay showed interaction between MdSBP1 and S-RNase.

Materials and methods

and sequence-verified. The NdeI-SalI fragment of MdS9-RNase
and a SalI–BamHI fragment of the coding sequence of StrepII
tag (WSHPQFEK) were cloned into NdeI and BamHI sites of
pET15b (Novagen, Madison, WI, USA). The construct was
introduced into Escherichia coli strain SHuffle T7 (New England
BioLabs, Beverly, MA, USA). The recombinant MdS9-RNase
protein was expressed in E. coli, solubilized from inclusion
bodies with a buffer containing 8 M urea, and purified by
HisTrap FF Crude column (GE Healthcare, Little Chalfont,
Buckinghamshire, UK). The purified MdS9-RNase protein was
dialyzed against a buffer containing 0.5 M Arg-HCl to remove
urea, and used to immunize a rabbit to obtain antiserum.

Plant materials

Pull-down assay

Leaves and floral organs of an apple cultivar ‘Fuji’ (S1S9) werecollected in spring and stored at −80°C until used.

The open reading frame of MdSBP1 was amplified by PCR
using primers XbMdSBP1 (5′-CCG ​CTC ​GAG ​GAT ​CCA ​TGG ​CTG ​T TC ​CCC ​AGCACC-3′) and RMdSBP1Xb (5′-GCT ​CTA ​GAT ​TAC ​AAA ​TAT ​ACC ​TCC ​ATG ​CTG ​ATA ​AAC ​-3′). The
MdSBP1 fragment was then cloned into pColdIIMBP (Heangand Sassa 2012) at BamHI and XbaI sites for expression of
maltose binding protein (MBP)-fused MdSBP1 protein (MBP:
MdSBP1). pColdIIMBP is a derivative of pCold II (Takara Bio,
Otsu, Japan) and contains the coding sequence of MBP. The
construct was introduced into BL21 (DE3) pLysS (Novagen).
pColdIIMBP was also transferred to BL21 (DE3) pLysS for
expression of MBP as a negative control of the pull-down assay.
Expression of MBP: MdSBP1 and MBP proteins were induced
by addition of 0.5 mM isopropyl-β-d-thio-galactopyranoside(IPTG) (Wako) and culture at 15°C for 24 h. Crude proteins
were extracted from bacteria suspended in a binding buffer(50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA and 1 mM
DTT) by sonication followed by centrifugation, and reactedwith amylose resin (New England BioLabs). Style proteins of
the apple cultivar ‘Fuji’ (S1S9) were extracted with an extraction
buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2,
0.3% β-mercaptoethanol), and incubated with the proteinbound amylose resin at 4°C for 2 h. The beads were thenwashed five times with a washing buffer (50 mM Tris-HCl, pH
7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.01% Triton
X-100). Bound proteins were eluted from the beads by heating
at 65°C for 5 min in 30 µl of 2×SDS loading buffer (100 mM
Tris-HCl pH 6.8, 4% SDS, 12% β-mercaptoethanol and 20%
glycerol), and separated by SDS-PAGE with a 13% gel anddetected by anti-apple S9-RNase antiserum.

RACE and sequence analysis
RNA was isolated from the leaves and the floral organs of‘Fuji’ (S1S9) as described by McClure et al. (1990). Total RNA
samples were treated with DNaseI (Nippongene, Tokyo, Japan),and used for RACE and RT-PCR as described by Ushijima etal. (2003). A partial EST sequence homologous to solanaceous
SBP1 was selected from apple EST libraries (Moriya et al. 2012).
A gene-specific primer MdSBP1-5RACEr1 (5′-CAG ​GAA ​ATC ​AAT ​GGA ​CGA ​TAT ​T-3′) was designed based on the EST
sequence, and used for 5′RACE with pollen cDNA of ‘Fuji’(S1S9) as a template. A primer MSB5URHS (5′-AGT ​TGT ​GCC ​TTT ​CAC ​ACA ​AGC ​-3′) was designed from the sequences of
the 5′RACE clones, and used to amplify full length cDNA forthe apple SBP1 homolog from pollen cDNA of ‘Fuji’ (S1S9) by
3′RACE.
The amino acid identities among SBP1 proteins wereanalyzed by GENETYX-MAC (version 16; Genetyx, Tokyo,
Japan). The amino acid sequences of SBP1 proteins werealigned using Clustal W (Thompson et al. 1994). A neighborjoining tree was constructed (Saitou and Nei 1987) based on thealignment using MEGA ver. 5.05. (Tamura et al. 2011).

RT-PCR
Expression of MdSBP1 was analyzed by RT-PCR with genespecific primers ctMdSBP1-a (5′-CTA ​TGG ​CTG ​T TC ​CCC ​AGC ​ACC ​-3′) and MdSBP1Fjr1 (5′-TTT ​ATA ​TGA ​TGT ​ATG ​GCT ​TTG ​AAT ​-3′). Actin 2 (GenBank accession number
GU830959) used as control was amplified using primers
PbActin2f1 (5′-ATG ​GCC ​GAT ​GCT ​GAG ​GAC ​ATT ​CAA ​CCC ​CTCG-3′) and PbActin2r1 (5′-ATT ​GGC ​ACA ​GTG ​TGA ​CTC ​ACA ​CCA ​TCA ​CCAG-3′).

Production of antiserum against apple S-RNase
A cDNA clone of MdS9-RNase (synonym of Sc-RNase; Sassa etal. 1996) was amplified by PCR using FMdSpRNNd (5′-CAT ​ATG ​TAC ​GAT ​TAT ​TTT ​CAA ​TTT ​ACG ​-3′) and RMdS9RNSal(5′-GTC ​GAC ​ATA ​CAG ​AAT ​ATT ​ATT ​GGT ​GGG ​-3′), cloned
to the EcoRV site of pZErO-2 (Invitrogen, Carlsbad, CA, USA),
Copyright © 2013 The Japanese Society for Plant Cell and Molecular Biology

Results and discussion
Identification of the apple SBP1 homolog

To isolate the apple SBP1 homolog, we searched theapple EST libraries (Moriya et al. 2012) and selected apartial cDNA sequence homologous to solanaceous
SBP1. Based on the sequence, primers were designed
for RT-PCR. We obtained an SBP1 homolog from apple
pollen RNA and named it MdSBP1 (Figures 1, 2). The
amino acid identities among SBP1 proteins were 64.0–

M. F. Minamikawa et al.

		

Figure 1. Amino acid sequence alignment of MdSBP1 and other SBP1 homologs of Solanaceae. Amino acid sequences were aligned using Clustal
W. Conserved sites and relatively conservative sites are marked with asterisks and dots, respectively. The RING-HC finger motif detected by Pfam
(http://pfam.sanger.ac.uk) is denoted by a box. The triangles indicate the cysteine-histidine signature of the motif. The underline indicates thepredicted coiled-coil region detected by Pfam. Md, Malus×domestica; Na, Nicotiana alata; Pi, Petunia inflata; Ph, Petunia hybrida; Sc, Solanum
chacoense. Accession numbers: NaSBP1 (EU591514), PiSBP1 (DQ250022), PhSBP1 (AF223395), ScSBP1 (AY545464)
Table 1. Amino acid sequence identities (%) among MdSBP1 andother SBP1 homologs of Solanaceae.

Figure 2. Neighbor-joining tree of SBP1 proteins. The tree wasconstructed based on the aligned deduced amino acid sequencesof SBP1 proteins (Figure 1). The numbers at the nodes are bootstrap
values for 100 bootstrap resamplings.

68.2% (Table 1). Sequence analysis showed that MdSBP1
included two probable protein–protein interaction
domains, coiled–coil and RING-HC domains (Figure 1),
the same as solanaceous SBP1 proteins. The coiled–coilregion of petunia SBP1 was reported to be required forbinding to SLF (Hua and Kao 2006), while the RINGHC domain is thought to be required for E3 ubiquitin
ligase activity (Deshaies and Joazeiro 2009). Thesefeatures are consistent with the idea that MdSBP1 is acomponent of noncanonical E3 ligase, and is involvedin ubiquitinylation of S-RNase in apple pollen, as
hypothesized in P. inflata (Hua and Kao 2006).
SBP1 of Nicotiana alata, NaSBP1, was reported
to interact with the C-terminal domain of pistil
arabinogalactan proteins (AGPs), transmitting tractspecific glycoprotein (TTS) and 120-kDa glycoprotein(120K), by yeast two-hybrid assay and in vitro binding
assay (Lee et al. 2008). An in vitro binding assay also
showed that the helical and RING domain of NaSBP1
were sufficient for binding to TTS and 120K. Lee et al.

NaSBP1PiSBP1PhSBP1ScSBP1

MdSBP1NaSBP1PiSBP1PhSBP1

67.6
68.2
68.0
64.0

—
92.5
91.8
89.0

—
99.4
91.4

—
90.6

(2008) suggested that binding between NaSBP1 and thepistil AGPs may contribute to signaling and trafficking
processes inside pollen tubes. Thus, MdSBP1 may also be
involved in the processes inside pollen tubes, although
TTS and 120K-like proteins have not yet been identifiedin styles of apple.
Expression pattern of MdSBP1

RT-PCR analysis revealed that MdSBP1 was expressedin all the organs analyzed (Figure 3), the same as
solanaceous SBP1 homologs (Hua and Kao 2006; Leeet al. 2008; O’Brien et al. 2004; Sim and Ordanic 2001).
The expression pattern suggests that MdSBP1 is involvedin general cellular function besides a possible role inpollination.
Interaction of MdSBP1 with S-RNase

We examined the interaction between MdSBP1and S-RNase using an in vitro binding assay. MBPfused MdSBP1 (MBP: MdSBP1) and MBP (negative
control) proteins were expressed in E. coli and reactedwith amylose resin. The recombinant protein-boundbeads were then incubated with the apple style extract.

Copyright © 2013 The Japanese Society for Plant Cell and Molecular Biology

121

122

An S-RNase binding protein1 homolog of apple(B, 24380004) from the Ministry of Education, Science, Sports
and Culture of Japan. M. F. M and H. K. were supported by JSPS
Research Fellowship for Young Scientists.

References
Figure 3. RT-PCR analysis of MdSBP1 expression. MdSBP1 wasamplified by RT-PCR. Amplification consisted of 35 or 25 cycles for
MdSBP1 and Actin, respectively. RT, Reverse Transcriptase; Lf, Leaf; Pd,
pedicel; Sp, sepal; Pt, petal; An, Anther; Ov, Ovary; St, style.

Figure 4. In vitro binding assay for MdSBP1 and S-RNase. MBP:
MdSBP1 or MBP (negative control) were expressed in E. coli andreacted with amylose resin. The recombinant proteins-bound beads
were incubated with the style extract. Bound proteins were separated
by SDS-PAGE and detected by anti-MdS9-RNase antiserum (top).
Approximately equal recombinant protein loading was checkedby Ponceau-S staining of the blot before immunological detection(bottom). The triangle and the asterisk indicate MBP: MdSBP1 and
MBP, respectively. The arrow and diamond indicate specific and nonspecific signals, respectively.

Bound proteins were separated by SDS-PAGE anddetected by anti-apple S9-RNase antiserum. The result
showed that MdSBP1 interacts with S-RNase (Figure4). Given that MdSBP1 is homologous to solanaceous
SBP1, includes RING-HC domain and interacts with
S-RNase, biochemical function of MdSBP1 may besimilar to that of solanaceous SBP1 proteins. Although
the biological significance of the interaction between
SBP1 and S-RNase is not clear even in Solanaceae, it
was suggested that SBP1 of Petunia hybrida, PhSBP1,
could be a candidate for the non-allele-specific inhibitorof all S-RNase since it was expressed in pollen andshowed no polymorphism in different S alleles (Huaet al. 2008; Sims and Ordanic 2001). The RING finger
protein was also reported to contribute to ubiquitination
specificity (Deshaies and Joazeiro 2009; Noureddine etal. 2002; O’Brien et al. 2004). Biological implication of
the interaction between MdSBP1 and S-RNase, andinteractors with MdSBP1 other than S-RNase should be
experimentally analyzed to clarify the role of MdSBP1.
Acknowledgements
This work was supported by a Grants-in-Aid for Scientific Research

Copyright © 2013 The Japanese Society for Plant Cell and Molecular Biology

Anderson MA, Cornish EC, Mau S-L, Williams EG, Hoggart
R, Atkinson A, Bönig I, Grego B, Simpson R, Roche PJ, et al.
(1986) Cloning of cDNA for a stylar glycoprotein associated withexpression of self-incompatibility in Nicotiana alata. Nature 321:
38–44
Cardozo T, Pagano M (2004) The SCF ubiquitin ligase: insights intoa molecular machine. Nat Rev Mol Cell Biol 5: 739–751
De Franceschi P, Pierantoni L, Dondini L, Grandi M, Sanzol J,
Sansavini S (2011) Cloning and mapping multiple S-locus
F-box genes in European pear (Pyrus communis L.). Tree Genet
Genomes 7: 231–240
de Nettancourt D(2001) Incompatibility and incongruity in wild
and cultivated plants. Springer, Berlin
Deshaies RJ, Joazeiro CAP (2009) RING domain E3 ubiquitin
ligases. Annu Rev Biochem 78: 399–434
Franklin-Tong VE(2008) Self-incompatibility in flowering plants—
evolution, diversity, and mechanisms. Springer, Berlin
Goldraij A, Kondo K, Lee CB, Hancock CN, Sivaguru M, VazquezSantana S, Kim S, Phillips TE, Cruz-Garcia F, McClure B (2006)
Compartmentalization of S-RNase and HT-B degradation in
self-incompatible Nicotiana. Nature 439: 805–810
Heang D, Sassa H (2012) Antagonistic actions of HLH/bHLH
proteins are involved in grain length and weight in rice. PLoS
ONE 7: e31325
Hua Z, Kao TH (2006) Identification and characterization ofcomponents of a putative petunia S-locus F-box-containing E3ligase complex involved in S-RNase-based self-incompatibility.
Plant Cell 18: 2531–2553
Hua Z-H, Fields A, Kao TH (2008) Biochemical models for
S-RNase-based self-incompatibility. Mol Plant 1: 575–585
Huang J, Zhao L, Yang Q, Xue Y (2006) AhSSK1, a novel SKP1-likeprotein that interacts with the S-locus F-box protein SLF. Plant J
46: 780–793
Kakui H, Kato M, Ushijima K, Kitaguchi M, Kato S, Sassa H (2011)
Sequence divergence and loss-of-function phenotypes of S locus
F-box brothers genes are consistent with non-self recognition bymultiple pollen determinants in self-incompatibility of Japanesepear (Pyrus pyrifolia). Plant J 68: 1028–1038
Kao TH, Tsukamoto T (2004) The molecular and genetic basesof S-RNase-based self-incompatibility. Plant Cell 16(Suppl):
S72–S83
Kubo K, Entani T, Takara A, Wang N, Fields AM, Hua Z, Toyoda
M, Kawashima S, Ando T, Isogai A, et al. (2010) Collaborative
non-self recognition system in S-RNase-based selfincompatibility. Science 330: 796–799
Lee CB, Swatek KN, McClure B (2008) Pollen proteins bind to the
C-terminal domain of Nicotiana alata pistil arabinogalactan
proteins. J Biol Chem 283: 26965–26973
Luu DT, Qin X, Morse D, Cappadocia M (2000) S-RNase uptake
by compatible pollen tubes in gametophytic self-incompatibility.
Nature 407: 649–651
McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ,
Sakiyama F, Clarke AE (1989) Style self-incompatibility geneproducts of Nicotiana alata are ribonucleases. Nature 342:
955–957
McClure BA, Gray JE, Anderson MA, Clarke AE (1990) Self-

M. F. Minamikawa et al.

		

incompatibility in Nicotiana alata involves degradation of pollen
rRNA. Nature 347: 757–760
Meng X, Sun P, Kao TH (2011) S-RNase-based self-incompatibility
in Petunia inflata. Ann Bot (Lond) 108: 637–646
Minamikawa M, Kakui H, Wang S, Kotoda N, Kikuchi S, Koba T,
Sassa H (2010) Apple S locus region represents a large cluster ofrelated, polymorphic and pollen-specific F-box genes. Plant Mol
Biol 74: 143–154
Moriya S, Iwanami H, Kotoda N, Haji T, Okada K, Terakami S,
Mimida N, Yamamoto T, Abe K (2012) Aligned genetic linkage
maps of apple rootstock cultivar ‘JM7’ and Malus sieboldii‘Sanashi 63’ constructed with novel EST-SSRs. Tree Genet
Genomes 8: 709–723
Noureddine MA, Donaldson TD, Thacker SA, Duronio RJ (2002)
Drosophila Roc1a encodes a RING-H2 protein with a uniquefunction in processing the Hh signal transducer Ci by the SCF
E3 ubiquitin ligase. Dev Cell 2: 757–770
O’Brien M, Major G, Chantha S, Matton DP (2004) Isolationof S-RNase binding proteins from Solanum chacoense:
Identification of an SBP1 (RING finger protein) orthologue. Sex
Plant Reprod 17: 81–88
Qiao H, Wang H, Zhao L, Zhou J, Huang J, Zhang Y, Xue Y(2004a) The F-box protein AhSLF-S2 physically interacts with
S-RNases that may be inhibited by the ubiquitin/26S proteasome
pathway of protein degradation during compatible pollination in
Antirrhinum. Plant Cell 16: 582–595
Qiao H, Wang F, Zhao L, Zhou J, Lai Z, Zhang Y, Robbins TP,
Xue Y (2004b) The F-box protein AhSLF-S2 controls the pollen
function of S-RNase-based self-incompatibility. Plant Cell 16:
2307–2322
Saitou N, Nei M (1987) The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4:
406–425
Saito T, Sato Y, Sawamura Y, Shoda M, Takasaki-Yasuda T,
Kotobuki K (2012) Dual recognition of S1 and S4 pistils by S4sm
pollen in self-incompatibility of Japanese pear (Pyrus pyrifolia
Nakai). Tree Genet Genomes 8: 689–694
Sassa H, Hirano H, Nishino T, Koba T (1997) Style-specific selfcompatible mutation caused by deletion of the S-RNase gene inJapanese pear (Pyrus serotina). Plant J 12: 223–227
Sassa H, Kakui H, Minamikawa M (2010) Pollen-expressed F-box

gene family and mechanism of S-RNase-based gametophytic
self-incompatibility (GSI) in Rosaceae. Sex Plant Reprod 23:
39–43
Sassa H, Kakui H, Miyamoto M, Suzuki Y, Hanada T, Ushijima
K, Kusaba M, Hirano H, Koba T (2007) S locus F-box brothers:
multiple and pollen-specific F-box genes with S haplotypespecific polymorphisms in apple and Japanese pear. Genetics 175:
1869–1881
Sassa H, Nishio T, Kowyama Y, Hirano H, Koba T, Ikehashi H(1996) Self-incompatibility (S) alleles of the Rosaceae encode
members of a distinct class of the T2/S ribonuclease superfamily.
Mol Gen Genet 250: 547–557
Sijacic P, Wang X, Skirpan AL, Wang Y, Dowd PE, McCubbin AG,
Huang S, Kao TH (2004) Identification of the pollen determinantof S-RNase-mediated self-incompatibility. Nature 429: 302–305
Sims TL, Ordanic M (2001) Identification of a S-ribonucleasebinding protein in Petunia hybrida. Plant Mol Biol 47: 771–783
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S(2011) MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum
parsimony methods. Mol Biol Evol 28: 2731–2739
Tao R, Iezzoni AF (2010) The S-RNase-based gametophytic selfincompatibility system in Prunus exhibits distinct genetic andmolecular features. Sci Hortic (Amsterdam) 124: 423–433
Tao R, Yamane H, Sassa H, Mori H, Gradziel TM, Dandekar AM,
Sugiura A (1997) Identification of stylar RNases associated withgametophytic self-incompatibility in almond (Prunus dulcis).
Plant Cell Physiol 38: 304–311
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W:
improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific
gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano
H (2003) Structural and transcriptional analysis of the selfincompatibility locus of almond: identification of a pollenexpressed F-box gene with haplotype-specific polymorphism.
Plant Cell 15: 771–781
Xue Y, Carpenter R, Dickinson HG, Coen ES (1996) Origin ofallelic diversity in antirrhinum S locus RNases. Plant Cell 8:
805–814

Copyright © 2013 The Japanese Society for Plant Cell and Molecular Biology

123