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Molecular characterization of the S haplotypes lacking SLG in the genome of Brassica campestris (syn. rapa) L.

Masao Watanabe1, Go Suzuki2, Yoshinobu Takada1, Tomohiro Kakizaki1, Hiroshi Shiba3, Seiji Takayama3 and Akira Isogai3

1 Faculty of Agriculture, Iwate University, 020-8550, Morioka, Japan, http://www.iwate-u.ac.jp/ Email; MW nabe@iwate-u.ac.jp, YT u0202008@iwate-u.ac.jp, TK u0203004@iwate-u.ac.jp,
2 Division of Natural Science, Osaka Kyoiku University, 582-8582, Kashiwara, Japan, http://www.osaka-kyoiku.ac.jp/ Email gsuzuki@cc.osaka-kyoiku.ac.jp
3 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 630-0101, Ikoma, Japan http://www.naist.jp/ Email; HS h-siba@bs.aist-nara.ac.jp, ST takayama@bs.aist-nara.ac.jp, AI isogai@bs.aist-nara.ac.jp

Abstract

Self-incompatibility (SI) discriminating self vs. non-self pollen is regulated by S-locus genes in Brassica species. In most of the S haplotype, a highly polymorphic S-locus glycoprotein (SLG) gene is tightly linked to genes for the SI determinants, S-receptor kinase (SRK) and S-locus protein 11 (SP11), although the precise function of SLG in SI reaction has not been clarified. In the present study, we performed DNA gel blot analysis for S32, S33, and S36 haplotypes of B. campestris showing normal SI phenotypes, and concluded that there might be no SLG in their genome. RNA gel blot analysis of the SLG-less S haplotypes indicated the possible existence of eSRK transcripts in the stigma. Furthermore, we constructed a catalogue of cDNA clones, which were hybridized to SLG45 cDNA, in S36 haplotypes. These three S haplotypes are useful resources to discern the molecular mechanism of the SI reaction without SLG.

Media summary

The S haplotypes lacking SLG will provide useful information to understand the molecular mechanism of SI recognition reaction in Brassica species.

Key Words

Brassica campestris (syn. rapa), S locus glycoprotein (SLG), S receptor kinase (SRK), self-incompatibility (SI), S-locus region

Introduction

Self-incompatibility (SI) systems prevent self-pollination and promote outbreeding. In the SI response of Brassica, self-pollen is rejected at the surface of the papilla cells due to recognition of self and non-self pollen. This highly regulated system is sporophytically controlled by a single S locus with multiple alleles. During the last decade, the S locus region has been dissected to identify highly polymorphic SI genes, SLG (a gene for S-locus glycoprotein), SRK (S-receptor kinase), and SP11/SCR (a gene for S-locus protein 11 or S-locus cysteine-rich protein) (reviewed in Watanabe et al. 2001). SLG encodes a secreted glycoprotein (Takayama et al. 1987), SRK encodes a transmembrane receptor-like kinase, which consists of an extracellular SLG-like domain (S domain), a transmembrane domain, and a cytoplasmic kinase domain (Stein et al. 1991), and SP11/SCR encodes a small cysteine-rich protein (Schopfer et al. 1999; Suzuki et al. 1999). Based on the sequence diversity of the SI genes, S haplotypes are classified into class I and class II; the amino acid sequence similarity of SLG and SRK is almost 65% between classes and 80-90% within classes (Watanabe et al. 2001). Class-I S haplotypes are dominant over class-II ones on the pollen side (Hatakeyama et al. 1998c).

From gain-of-function experiments, it has been demonstrated that SRK and SP11/SCR are female and male determinants of the SI recognition, respectively (Takasaki et al. 2000; Takayama et al. 2000). Recent studies showed that SRK on the stigma surface could bind SP11/SCR as a ligand released from a self-pollen coat in an allele-specific manner (Takayama et al. 2001). For SLG's function, SLG has been reported to enhance the SI reaction in SRK9-introduced transgenic Brassica (Takasaki et al. 2000), and co-expression of SLG stabilizes SRK in transgenic plants (Dixit et al. 2000). However, further molecular analyses for SLG's function are necessary because several reports show that SLG was mutated or deleted in some S haplotypes of self-incompatible B. oleracea (T Suzuki et al. 2000).

To determine the regulation of SRK-mediated SI response without SLG, the SLG-less S haplotypes in B. campestris (syn. rapa) must be carefully analysed. In this study, we selected three class-I S haplotypes (S32, S33, and S36) of B. rapa to examine the occurrence of SLG-less genotypes from the following reasons: 1) the detection of a single EcoRI band was reported in DNA gel blot analysis with the SLG probe (Hatakeyama et al. 1998b), 2) no amplified fragments were detected by PCR with PS5 and PS15 primer sets which can amplify class-I SLGs (Nishio et al. 1996), and 3) SLG sequences were not described in Kusaba et al. (1997) which reported many SLG sequences cloned by PCR. Although Nou et al. (1993) reported the detection of SLG32, SLG33 and SLG36 proteins by the IEF immunoblot analysis, these IEF bands may have corresponded to SLR1 (S-locus related 1) proteins, which could cross-hybridize to the anti-SLG polyclonal antibody (Watanabe et al. 1992). We performed DNA and RNA gel blot analyses to confirm the lack of SLG in the S32, S33, and S36 haplotypes. Furthermore, we constructed a cDNA catalogue, which were homologous to SLG45, in the S36 haplotype.

Materials and Methods

Plant materials

B. campestris S32, S33, and S36 homozygotes (Nou et al. 1993) were grown in a glasshouse. For the RNA gel blot analysis, S8 and S9 homozygotes of B. campestris were also used (Watanabe et al. 1994). All these S haplotypes belong to class I.

DNA and RNA gel blot analysis

Total DNA was extracted from young leaf tissue of B. campestris. Two µg of the total DNA were digested with restriction enzymes, fractionated on 0.8% agarose gels, and transferred to nylon membranes (Roche Diagnostics, Mannheim, Germany). Hybridization and washing of the membrane, and detection of the hybridized probes were carried out. Isolation of poly (A)+ RNA, electrophoresis of denatured RNA samples and blotting to a nylon membrane (Roche Diagnostics) were performed. A DNA fragment encoding vacuolar H+-ATPase (V-ATPase) of B. campestris (Takasaki et al. 2000) was used as a control probe to check the amount of loaded RNA.

Results

We have previously showed that the SLG45 probe could hybridize with both SLG and SRK genes in class-I S haplotypes of B. campestris (Hatakeyama et al. 1998a; G Suzuki et al. 2000). By using the SLG45 probe, genomic DNA gel blot analyses with various combinations of nine restriction enzymes were conducted to determine whether or not the SLG gene exists in the genome of the S32, S33, and S36 haplotypes of B. campestris. In most of the combinations of the enzymes, a single band was detected in these S haplotypes (data not shown), although double bands, corresponding to SLG and SRK genes, were generally found in the other class-I S haplotypes in the similar blot analyses (Hatakeyama et al. 1998a; Suzuki et al. 2000). These single bands obtained from the S32, S33, and S36 haplotypes were most likely SRK genes since most of them were also hybridized with SRK45-KD probe which was derived from the kinase-domain region of SRK45 (data not shown). Minor bands might be due to existence of the restriction sites in the probe region. Based on these hybridized data, we estimated the physical map of S locus in S32 and S33haplotypes (Fig. 1).

Figure 1. Estimated physical maps of S-locus region in S32 (a) and S33 (b) haplotypes.

We performed RNA gel blot analysis to analyze expression of SRK in the stigma of the S32, S33, and S36 haplotypes (Fig. 2). The stigma mRNAs of the S8 and S9 haplotypes that possess both SLG and SRK genes were loaded on the same gel to compare the expression of SLG. In the case of the blot with the SLG45 probe, which can hybridize with both SLG and SRK, strong signals for SLG transcripts were observed in the S8 and S9 haplotypes and weak signals of SRK transcripts were detected in all five S haplotypes (Fig. 2a). The amount of SRK transcript was relatively low compared with SLG, and the SRK transcripts were not clear in the S8, S32, and S33 haplotypes; the difference of the signals of the SRK transcripts might have been due to difference of the loaded mRNAs (see the control signals of V-ATPase in Fig. 3) and the sequence diversity of the SRKs. The normal expression level of the SRK in the five S haplotypes could be undoubtedly confirmed in the case of the blot with the SRK45-KD probe (Fig. 2b). In the three SLG-less haplotypes (S32, S33, and S36), weak signals of 1.5-kb transcripts were detected by the SLG45 probe (Fig. 2a). The 1.5-kb transcripts were similar in size to SLG, and might have corresponded to eSRK, the alternative transcript of SRK that contains only the S-domain region. The amount of the eSRK transcript in the S32, S33, and S36 haplotypes was extensively lower than that of SLG in the S8 and S9 haplotype, and several times higher than that of SRK.

Figure 2. Detection of transcripts for SRK and eSRK in the SLG-lacking S haplotypes.

As next step, we constructed cDNA library of stigma in S36 haplotypes to construct a catalogue of SLG and SRK-like clones. Partial nucleotide sequences of about 100 clones, which were hybridized to SLG45 cDNA clone, were determined. Unfortunately, almost clones were homologous to SLR1 or SLR2, which were also highly expressed in stigma and were not related to SI response.

Conclusion

Although the S32, S33, and S36 haplotypes of B. rapa lack SLG, the SI phenotype of the three S haplotypes is normal (Nou et al. 1993). Recognition of self-pollen is determined by SRK and SP11 genes, and SLG is estimated to be not necessary for the self-recognition (Takasaki et al. 2000). However, the S haplotypes lacking SLG are a minority in Brassica; a large amount of SLG proteins are expressed in the stigma of most of the S haplotypes, suggesting that SLG has some function in the SI system. To date, SLG is believed to enhance the SI phenotype by stabilizing SRK (Takasaki et al. 2000). If SLG functions as a stabilizer of the SRK-SP11 complex, one of the possible explanations of the normal SI phenotype of S32, S33, and S36 is the existence of a substitute for the SLG protein, e.g., eSRK. The regulation of the stable SI phenotype in the SLG-less S haplotypes is one of the most interesting unsolved questions that should be clarified in the near future.

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