Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed F-box gene with haplotype-specific polymorphism.

Abstract

Gametophytic self-incompatibility in Rosaceae, Solanaceae, and Scrophulariaceae is controlled by the S locus, which consists of an S-RNase gene and an unidentified "pollen S" gene. An approximately 70-kb segment of the S locus of the rosaceous species almond, the S haplotype-specific region containing the S-RNase gene, was sequenced completely. This region was found to contain two pollen-expressed F-box genes that are likely candidates for pollen S genes. One of them, named SFB (S haplotype-specific F-box protein), was expressed specifically in pollen and showed a high level of S haplotype-specific sequence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the S specificity of pollen because it showed little allelic sequence polymorphism and was expressed also in pistil. Three other S haplotypes were cloned, and the pollen-expressed genes were physically mapped. In all four cases, SFBs were linked physically to the S-RNase genes and were located at the S haplotype-specific region, where recombination is believed to be suppressed, suggesting that the two genes are inherited as a unit. These features are consistent with the hypothesis that SFB is the pollen S gene. This hypothesis predicts the involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based gametophytic self-incompatibility system.

Full text

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The Plant Cell, Vol. 15, 771–781, March 2003, www.plantcell.org © 2003 American Society of Plant Biologists
Structural and Transcriptional Analysis of the Self-Incompatibility
Locus of Almond: Identification of a Pollen-Expressed F-Box
Gene with Haplotype-Specific Polymorphism
Koichiro Ushijima,
and Hisashi Hirano
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
b
Kihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, Yokohama
244-0813, Japan
c
Department of Pomology, University of California, Davis, California 95616
a,1
Hidenori Sassa,
b,1,2
Abhaya M. Dandekar,
c
Thomas M. Gradziel,
c
Ryutaro Tao,
a
b
a
Gametophytic self-incompatibility in Rosaceae, Solanaceae, and Scrophulariaceae is controlled by the
sists of an S-RNase gene and an unidentified “pollen
almond, the
S haplotype–specific region containing the S-RNase gene, was sequenced completely. This region was found
to contain two pollen-expressed F-box genes that are likely candidates for pollen
lotype–specific F-box protein), was expressed specifically in pollen and showed a high level of
quence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the
cause it showed little allelic sequence polymorphism and was expressed also in pistil. Three other
cloned, and the pollen-expressed genes were physically mapped. In all four cases,
genes and were located at the
S haplotype–specific region, where recombination is believed to be suppressed, suggesting
that the two genes are inherited as a unit. These features are consistent with the hypothesis that
This hypothesis predicts the involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based gameto-
phytic self-incompatibility system.
S locus, which con-
S ” gene. An
?
70-kb segment of the S locus of the rosaceous species
S genes. One of them, named SFB (S hap-
S
S
haplotype–specific se-
specificity of pollen be-
S haplotypes were
SFBs were linked physically to the S-RNase
SFB is the pollen S gene.
INTRODUCTION
Self-incompatibility (SI) in flowering plants is a genetic system
that prevents self-fertilization by enabling the pistil to reject pol-
len from genetically related individuals, thus promoting out-
crossing. Genetic studies have shown that most SI systems are
controlled by a single multiallele locus called the
an
S
allele of pollen matches that of the pistil, the pollen is rec-
ognized as “self” and rejected by the pistil (de Nettancourt,
2001). However, recent studies on pistil- or pollen-specific self-
compatible mutants (Thompson et al., 1991; Sassa et al., 1997;
Golz et al., 1999) and transformation experiments with solana-
ceous species (Lee et al., 1994; Murfett et al., 1994; Dodds et
al., 1999) demonstrate that SI phenotypes of pistil and pollen
are determined by different genes, called pistil
genes, respectively. Based on the findings that the
multigene complex, the term “haplotype” has been adopted to
denote variants of the locus, and the term “allele” is used to
denote variants of a given polymorphic gene at the
(McCubbin and Kao, 2000).
The families Rosaceae, Solanaceae, and Scrophulariaceae
S
locus. When
S
and pollen
S
locus is a
S
S
locus
display gametophytic self-incompatibility (GSI) and share the
same pistil
S
gene product, called the S-RNase (for review, see
McCubbin and Kao, 2000). S-RNase is a basic glycoprotein with
RNase activity (McClure et al., 1989). RNase activity of S-RNase
is required for the pistil to reject self-pollen (Huang et al., 1994;
Royo et al., 1994; McCubbin et al., 1997). The rRNA of incom-
patible pollen of
Nicotiana alata
sistent with a model postulating that the S-RNases act as intra-
cellular cytotoxins (McClure et al., 1990). On the other hand,
the pollen
S
gene of the RNase-based GSI remains to be iden-
tified. Circumstantial evidence suggests two conceivable mod-
els for the action of pollen
S
product: the gatekeeper model
and the inhibitor model (Thompson and Kirch, 1992; McCubbin
and Kao, 2000). In the gatekeeper model, the pollen
is assumed to be a gatekeeper located at the plasma mem-
brane of the pollen tube. The gatekeeper interacts specifically
with cognate S-RNase, assisting in its uptake into the pollen
cytoplasm, leading to the degradation of pollen RNA and the
arrest of self-pollen tube elongation. In the inhibitor model, all
S-RNases enter the pollen tube regardless of their
types; however, the pollen
S
product inhibits the activity of all
S-RNases except for the cognate S-RNase. As a consequence,
the cognate S-RNase remains active in the pollen tube, leading
to the degradation of self-pollen RNA. Golz et al. (1999, 2001)
produced pollen-part self-compatible mutants of
compatible pollination with irradiated pollen and found that a
deletion mutant for the pollen
S
is degraded in the style, con-
S
product
S
haplo-
N
.
alata
by in-
gene could not be recovered
1
These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail sassa@
yokohama-cu.ac.jp; fax 81-45-820-1901.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.009290.
2

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772The Plant Cell
by this screen. This finding suggests that the pollen
is essential for pollen tube elongation, which is consistent with
the inhibitor model but in conflict with the gatekeeper model. Im-
munocytological experiments showing the entry of the S-RNase
into the compatible pollen tube support the inhibitor model (Luu
et al., 2000).
The identification and characterization of the pollen
essential to understanding the molecular mechanisms of the
RNase-based GSI. The pollen
S
following features: first, tight linkage to the S-RNase gene; sec-
ond, a high level of
S
haplotype–specific sequence polymor-
phism; third, pollen-specific expression. The tight genetic link-
age between the pollen
S
gene and the S-RNase gene
suggested that chromosome walking would be one of the feasi-
ble approaches for the identification of the pollen
ever, conventional chromosome walking in Solanaceae is diffi-
cult because the solanaceous
S
centromere, a region abundant with repetitive sequences
(Coleman and Kao, 1992; Matton et al., 1995; Bernacchi and
Tanksley, 1997; Entani et al., 1999). McCubbin et al. (2000)
screened a BAC library of the
Petunia inflata
molecular markers linked to the S-RNase gene and walked an
S
locus of
Petunia
from multiple starting points. Although they
obtained 51 BAC clones spanning a
S
locus region, they did not construct a contig encompassing
the region that encodes
S
specificity, which is delimited by re-
combination breakpoints. Therefore, it is not clear whether the
genomic clones contain the pollen
2000).
We previously conducted chromosome walking on the
haplotype of almond, which belongs to the Rosaceae, and con-
structed the contigs covering an
S
-RNase gene (Ushijima et al., 2001). Genomic DNA gel blot
analyses revealed that the nucleotide sequence of the
region, which is defined by the two boundary markers NP79R
and NP182F and contains the S
verged and specific to each
S
haplotype. The regions outside
of the boundary markers were relatively conserved among dif-
ferent
S
haplotypes. The structural heteromorphism is the dis-
tinctive feature of loci consisting of coadapted gene complexes
in different genetic systems, including sporophytic SI of Brassi-
caceae (Ferris and Goodenough, 1994; Brown and Casselton,
2001; Kusaba et al., 2001). The heteromorphism of the region
containing the
S
specificity genes is believed to maintain the
tight association of the pistil
S
suggesting that the pollen
S
gene of almond is located in the
haplotype–specific region (Ushijima et al., 2001).
Here, we sequenced the
?
70-kb
gion using a shotgun strategy. Based on the prediction of open
reading frames (ORFs), two pollen-expressed genes were iso-
lated. One of the two genes, named
F-box protein), showed a high level of allelic polymorphism and
pollen-specific expression. Comparative analysis of the four
haplotypes showed that all
SFB
kb of the S-RNase genes. Distances between genes and mark-
ers are highly variable among the
heteromorphism of the region, which might ensure the tight as-
sociation of
SFB
and the S-RNase gene. These features of
S
product
S
gene is
gene is expected to have the
S
gene. How-
locus is located close to the
genome with 13
?
2-Mb region around the
S
gene (McCubbin et al.,
S
c
?
200-kb region around the
c
?
70-kb
c
-RNase gene, was highly di-
gene and the pollen
S
gene,
S
S
c
haplotype–specific re-
SFB
(
S
haplotype–specific
S
alleles are located within
?
30
S
haplotypes, showing the
SFB
are consistent with those of the expected pollen
encodes an F-box protein that is a component of a class of
ubiquitin ligase (SCF complex) (Deshaies, 1999), raising the
possibility that the ubiquitin/26S proteasome proteolytic sys-
tem plays a central role in the discrimination of self/nonself pol-
len in the RNase-based GSI.
S
gene.
SFB
RESULTS
Sequence Analysis of the
Haplotype–Specific Region
?
70-kb S
c
We previously predicted the boundaries of the almond
and suggested that the pollen
?
70-kb area of the
S
haplotype–specific region that also con-
tains the S
-RNase gene (Ushijima et al., 2001). To identify the
pollen
S
gene, the
S
haplotype–specific region was sequenced
completely using the shotgun strategy. Three genomic clones
covering the region, p79SCU (
?
pPdC182 (
?
36 kb), were used to construct the shotgun library
(Figure 1A). A total of
?
1000 sequences, which included PCR
products filling gaps between the shotgun contigs, were assem-
bled and gave a nucleotide sequence of 71,953 bp. The
sequence was annotated by RiceGAAS (Rice Genome Auto-
mated Annotation System [Sakata et al., 2002]; http://ricegaas.
dna.affrc.go.jp/index.html), which automatically analyzes large
sequences using several programs for the prediction and anal-
ysis of protein-coding sequences: BLAST (Basic Local Align-
ment Search Tool [Altschul et al., 1990]), AutoPredLTR (Sakata
et al., 2002), and GENSCAN (Burge and Karlin, 1997).
GENSCAN identified 12 ORFs (ORF1 to ORF12) in the se-
quenced region (Figure 1A). By this prediction, the three exons
of the S
-RNase gene were misidentified as different genes,
ORF1 and ORF2 (Table 1). AutoPredLTR search, which pre-
dicted a long terminal repeat (LTR) sequence, showed that the
?
70-kb region contained four pairs of LTRs (Figure 1A). Se-
quences between the LTRs were found to be similar to ret-
rotransposon-like sequences and were designated retro0 to
retro3 (Figure 1B, Table 1). All four retrotransposon-like se-
quences included several mutations (insertion or deletion),
causing their putative polyproteins to be truncated. Retro0 was
found to be disrupted by the insertion of retro1 to retro3 (Figure
1B), implying that retro0 was first inserted into the
type. Retro3 also was disrupted by the insertion of a 2.5-kb
DNA sequence. The inserted sequence showed high similarity
to the region at
?
15 to
?
18 kb (Figure 1A) but displayed no sig-
nificant homology with sequences in the public databases.
S
locus
S
c
gene might be present in an
c
c
c
7 kb), pPdC55 (
?
35 kb), and
?
70-kb
c
S
c
haplo-
cDNA Cloning of the Pollen-Expressed Genes Located at
the
S Haplotype
c
To clone the pollen-expressed genes at the sequenced
region, we designed 17 forward and 11 reverse primers based
on the prediction of ORFs. The primers were used for rapid am-
plification of cDNA ends (Frohman et al., 1988) with pollen
cDNA. Pollen from almond cv Nonpareil (
cause an
S
homozygous cultivar is not available. For most
ORFs, sequences of isolated cDNA clones were not identical
S
locus
S
c
S
d
) was used, be-
c

Page 3

Pollen-Expressed
S
Locus Gene of Almond 773
to, but were similar to, those of the corresponding ORFs, sug-
gesting that they constitute gene families and that those ORFs
are not expressed in pollen. However, sequences of two of
the cDNAs completely matched the corresponding ORF se-
quences (ORF1 and ORF3). The first exon of the predicted
ORF1 was isolated as a pollen-expressed gene and is called
ORF1 hereafter.
ORF1 was located 6.7 kb upstream of the S
(Figure 1A). The transcriptional orientations of ORF1 and the
S
-RNase gene were the same. The deduced amino acid se-
quence of ORF1 showed 29.3% homology with that of
c
-RNase gene
c
SLF
(
S
locus F-box) of
at the
Antirrhinum
F-box motif (Lai et al., 2002). We named ORF1
cus F-box of Prunus dulcis. PdSLF of the Sc haplotype, desig-
nated PdSLFc, contained the sequence of the cosmid end
probe NP79R, which defined the boundary of the S haplotype–
specific region (Ushijima et al., 2001). NP79R sequence has
been shown to be conserved in different S haplotypes of al-
mond (Ushijima et al., 2001). PdSLF of the Sd haplotype, desig-
nated PdSLFd, was amplified by PCR from the cosmid clone
of the Sd haplotype. The deduced amino acid sequences of
Antirrhinum

S
locus region and encodes a protein with an
(Figure 2A, Table 1).
SLF
is located
PdSLF
for
S
lo-
Figure 1. Sequence Analyses of the Sc Haplotype–Specific Region.
(A) Scheme of the Sc haplotype–specific region. The nucleotide sequence of the ?70-kb Sc haplotype–specific region was characterized by GEN-
SCAN, BLASTX, and AutoPredLTR search of RiceGAAS (Sakata et al., 2002). The A nucleotide of the putative initiation codon (ATG) of the Sc-RNase
gene (Ushijima et al., 1998) is positioned as ?1. Exons of the Sc-RNase gene and molecular markers NP79R and NP182F, which define the bound-
aries of the almond S locus (Ushijima et al., 2001), are represented by black boxes. Red, blue, and green boxes represent the results of GENSCAN,
BLAST, and AutoPredLTR searches, respectively. Each pair of LTRs is linked by lines.
(B) Scheme of the retrotransposon-like sequence-rich region (?29 to ?53 kb). Gray boxes represent LTR sequences. Hatched boxes represent the
region encoding the polyprotein of retro1, retro2, and retro3. White boxes represent the region for the retro0 polyprotein, into which retro1, retro2, and
retro3 were inserted.

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774The Plant Cell
PdSLFc and PdSLFd were highly similar to each other (95.1%
identity; Figure 2B), as were AhSLF-S2 and AhSLF-S2L of Anti-
rrhinum (97.9% identity; Lai et al., 2002), suggesting that they
are unlikely to control the S specificity of pollen.
The ORF3 cDNA also encoded a protein with an F-box motif
(Figure 3). ORF3 and the Sc-RNase gene were located in in-
verse orientation and separated by ?1.6 kb (Figure 1). ORF3
contained one intron at the 5? untranslated region (Figure 3).
Except for the N-terminal region containing the F-box motif se-
quence, ORF3 showed no significant homology with known
Table 1. Summary of Homology Search (BLASTX) Results for the ORFs Predicted by GENSCAN
Expression
ORF Homolog (Accession No.) PollenStyleDescription
ORF1 (1st exon)
ORF1 (7th exon)
ORF2
ORF3 (1st and 2nd exons)
ORF3 (3rd exon)
ORF4
ORF5
ORF6
ORF7
ORF8
ORF9
ORF10
ORF11
ORF12
Antirrhinum SLF-S2 (AJ297974)
Almond Sc-RNase (AB011470)
Almond Sc-RNase (AB011470)
Arabidopsis putative protein (AL138652)
Arabidopsis putative F-box protein (AC018907)
Unknown
Unknown
Unknown
Oryza sativa putative protein (AC084763)
Unknown
Zea mays retrotransposon (AF082133)
Nicotiana retrotransposon (CAA32025)
Arabidopsis retrotransposon (AC006248)
Arabidopsis Ser/Thr protein phosphatase (U80922)
Yes
No
No
No
Yes
No
No
No
No
No
Yes
Yes
Yes
PdSLF (Prunus dulcis S locus F-box protein)
N-terminal region of the Sc-RNase
C-terminal region of the Sc-RNase
NoSFB (S haplotype–specific F-box protein)
Designated retro1 in Figure 1
Designated retro2 in Figure 1
Designated retro3 in Figure 1
No
Figure 2. Amino Acid Sequence Comparisons between SLFs.
Deduced amino acid sequences were compared between PdSLFc and AhSLF-S2 (A) and between PdSLFc and PdSLFd (B). Sites that are conserved
or that have only conservative replacements (amino acid groups defined by Dayhoff et al. [1979]: C, STAPG, MILV, HRK, NDEQ, and FYW) are marked
with asterisks or dots, respectively.

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Pollen-Expressed S Locus Gene of Almond775
proteins in the public databases. ORF3 has no known localiza-
tion signal, suggesting that it is a cytoplasmic protein. Genomic
DNA gel blot analysis with the ORF3 probe gave an Sc haplo-
type–specific signal in all cultivars that contained the Sc-RNase
gene (Figure 4A). Based on this result, ORF3 was designated
SFB (S haplotype–specific F-box protein). Phylogenetic analy-
sis showed that SFB is a member of the A3 subfamily in the
plant F-box superfamily defined by Gagne et al. (2002) (data
not shown).
S Haplotype–Specific Sequence Polymorphism of SFB
To clone the alleles of SFB, PCR was conducted with the
primer pairs SC05MetRV/SCa05R or SC05MetRV/SFBTerHI
(Figure 3). Three clones were isolated from genomic DNA of cv
Mission (SaSb), Sauret No. 1 (SaSd), and Monterey (SbSd). Geno-
mic DNA gel blot analyses of the three clones showed the Sa,
Sb, and Sd haplotype–specific signals (Figure 4B), suggesting
that these SFB clones were derived from the respective S hap-
lotypes.
Because the three clones lacked both ends of the coding re-
gion, rapid amplification of cDNA ends was conducted using
pollen RNAs from different S haplotypes to obtain full-length
cDNA sequences of SFBa, SFBb, and SFBd. Deduced amino
acid sequences of the SFB alleles were aligned and compared
(Figure 5). The F-box motif was conserved at the N-terminal re-
gions of all SFBs. Sequence identities among SFBs were 68.4
to 76.4% (Table 2), which were comparable to those of almond
S-RNases (54.2 to 76.2%) (Ushijima et al., 1998; Tamura et al.,
2000). Although variable sites are dispersed in the primary
structure of the SFB, two regions at the C terminus are quite
variable, as they are in the hypervariable region of the S-RNase
(Ioerger et al., 1991; Ushijima et al., 1998).
Organ-Specific Expression of the S Locus Genes
To investigate the expression pattern of SFB and PdSLF, we
performed reverse transcriptase–mediated (RT) PCR analyses
using RNA from leaf and floral organs of Nonpareil as tem-
plates. RT-PCR products for the S-RNase gene and the actin
gene, which were used as controls, did not contain genomic
DNA–derived PCR products, which are expected to contain in-
trons and thus would be longer than the expected RT-PCR
products, suggesting that the templates were not contami-
nated with genomic DNA. In addition, the forward primer for
SFB was designed to include the junction of the two exons to
prevent amplification from a genomic DNA template (Figure 3).
The cDNA for S-RNase was amplified as expected from the
Figure 3. Nucleotide and Deduced Amino Acid Sequences of the cDNA for SFBc (ORF3).
The deduced amino acid sequence is shown under the nucleotide sequence of the cDNA. The arrowhead represents the position into which an 85-bp
intron sequence was inserted. The amino acid sequence of the F-box motif is underlined. Arrows represent the positions and directions of primers
that were used to amplify the alleles (see Figure 5) or to characterize organ-specific expression patterns (see Figure 6). The 5? region of primer
SC05MetRV (5?-TGATATCATTTTCTACAGGATGAC-3?) corresponds to the 3? end of the intron and is represented by a dotted line. Restriction sites
for EcoRV and BamHI were added to the 5? regions of primers SC05MetRV and SC05TerHI (5?-TGGATCCGTTTAATAATTATTGAG-3?), respectively.