Rejection of S-Heteroallelic Pollen by a Dual-Specific S-RNase in Solanum chacoense Predicts a Multimeric SI Pollen Component

Corresponding author: Mario Cappadocia, Biology Department, University of Montreal, Montreal, Quebec H1X 2B2, Canada., mario.cappadocia{at}umontreal.ca (E-mail)

Communicating editor: D. CHARLESWORTH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

S-heteroallelic pollen (HAP) grains are usually diploid and contain two different S-alleles. Curiously, HAP produced by tetraploids derived from self-incompatible diploids are typically self-compatible. The two different hypotheses previously advanced to explain the compatibility of HAP are the lack of pollen-S expression and the "competition effect" between two pollen-S gene products expressed in a single pollen grain. To distinguish between these two possibilities, we used a previously described dual-specific S_11/13-RNase, termed HVapb-RNase, which can reject two phenotypically distinct pollen (P₁₁ and P₁₃). Since the HVapb-RNase does not distinguish between the two pollen types (it recognizes both), P₁₁P₁₃ HAP should be incompatible with the HVapb-RNase in spite of the competition effect. We show here that P₁₁P₁₃ HAP is accepted by S₁₁S₁₃ styles, but is rejected by the S_11/13-RNase, which demonstrates that the pollen-S genes must be expressed in HAP. A model involving tetrameric pollen-S is proposed to explain both the compatibility of P₁₁P₁₃ HAP on S₁₁S₁₃-containing styles and the incompatibility of P₁₁P₁₃ HAP on styles containing the HVapb-RNase.

SELF-INCOMPATIBILITY (SI) is a cell-cell recognition phenomenon used by higher plants to prevent inbreeding. In the most widespread type of SI [gametophytic SI (GSI)], the self-incompatibility phenotype is specified by a highly multiallelic S-locus, and the genotype of the haploid pollen determines its own incompatibility phenotype (DE NETTANCOURT 1977 , 2001). In the Solanaceae, the identity of the pollen component of the GSI is unknown, whereas the stylar product has been identified as an extracellular ribonuclease, S-RNase (MCCLURE et al. 1989 ) expressed in the transmitting tissue of the style (ANDERSON et al. 1986 ). Gain-of-function experiments have shown that expression of an S-RNase transgene is necessary and sufficient to alter the SI phenotype of the pistil but does not change the pollen phenotype (LEE et al. 1994 ; MURFETT et al. 1994 ; MATTON et al. 1997 ), and thus the identity of the pollen-S gene must be different from the S-RNase (KAO and MCCUBBIN 1997 ). S-RNases appear to contain two domains, an RNase activity domain essential for expression of the SI phenotype (HUANG et al. 1994 ) and a recognition domain involved in the specificity of the cell-cell recognition phenomenon. In closely related S-RNases, such as the S₁₁- and S₁₃-RNases (SABA-EL-LEIL et al. 1994 ), the recognition domain includes the amino acids found in the so-called hypervariable (HV) regions (IOEGER et al. 1991 ). The HV regions of these two S-RNases differ by only four amino acids, and transgenic plants where these four residues in the S₁₁-RNase were replaced with those of the S₁₃-RNase displayed an S₁₃ rather than an S₁₁ phenotype (MATTON et al. 1997 ). Curiously, replacement of only three of these four amino acids produced RNases, that are either nonfunctional (MATTON et al. 2000 ) or have the unusual property of dual specificity (i.e., able to reject both the phenotypically distinct P₁₁ and P₁₃ pollen; MATTON et al. 1999 ).

The availability of this unique dual-specific S-RNase (termed HVapb-RNase) allowed us to reevaluate the S-heteroallelic pollen (HAP) effect (also known as competitive interaction in diploid HAP). In many diploid species with monofactorial GSI, naturally or artificially produced tetraploids often display self-compatibility (LEWIS 1947 ; BREWBAKER 1954 ; DE NETTANCOURT 1977 , DE NETTANCOURT 2001 ). Differences in reciprocal crosses between SI diploids and their tetraploid counterparts indicate that the breakdown of SI is due to the pollen and not the stylar component (DE NETTANCOURT 1977 ). As first noted by LEWIS 1947 , only pollen that contains two different S-loci can bypass the SI barrier, an observation fully confirmed by recent molecular analyses in both Lycopersicon peruvianum (CHAWLA et al. 1997 ) and Nicotiana alata (GOLZ et al. 1999 ). The HAP effect requires only a second different S-locus whether carried by a centric fragment or not (VAN GASTEL 1976 ; DE NETTANCOURT 1977 ; GOLZ et al. 1999 , GOLZ et al. 2000 ).

In spite of more than 50 years of research since its first description, the HAP effect remains poorly understood. In particular, it is not known if it is caused by some peculiar features of a distinct pollen-S gene (still unknown), by some other component of the S-locus (MCCUBBIN and KAO 1999 ), or by gene inactivation (LEWIS 1961 ; VAN GASTEL 1976 ), resulting in nonexpression of the pollen-S components. Current models for the biochemical role of pollen-S, however, cannot readily explain HAP compatibility. Immunocytochemical analyses showing that S-RNases can enter pollen tubes of any genotype (LUU et al. 2000 ) have provided experimental evidence for models involving RNase inhibitors (THOMPSON and KIRCH 1992 ; KAO and MCCUBBIN 1996 ). These models postulate that all S-RNases can enter a pollen tube and that their RNase activity is inhibited, except for that corresponding to the S-haplotype of the pollen. Models where the pollen-S and the RNase inhibitor are on the same molecule (KAO and MCCUBBIN 1996 ) or on separate molecules (LUU et al. 2000 ) have both been proposed. All versions of the inhibitor model assume that pollen-S binding to the recognition domain of its cognate S-RNase is thermodynamically favored over binding to the RNase activity domain, so that it permanently precludes activity domain binding and permits RNase activity (KAO and MCCUBBIN 1997 ). In HAP, the two pollen-S should each preferentially bind to the recognition domains of their respective S-RNases, leaving the RNases active. The inhibitor models thus predict incompatibility for HAP, in contrast with experimental observations.

We report here that P₁₁P₁₃ HAP is accepted by styles containing the S₁₁- and S₁₃-RNases but rejected by styles expressing the dual S_11/13 HVapb-RNase. This demonstrates that pollen SI components are functional in HAP, thus ruling out gene inactivation. We propose that pollen-S acts as a tetramer and that heterotetramers, such as would be produced in HAP, are unable to block inhibitor binding and thus produce compatible pollen.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Solanum chacoense Bitt (2n = 2x = 24) plants of various S-constitutions were produced by crosses (VERONNEAU et al. 1992 ; BIRHMAN et al. 1994 ; VAN SINT JAN et al. 1996 ). Diploid genotypes V22 (S₁₁S₁₃), V28 (S₁₂S₁₃), and G4 (S₁₂S₁₄) were selected from crosses of two parental lines PI458314 (S₁₁S₁₂) and PI230582 (S₁₃S₁₄; Potato Introduction Station, Sturgeon Bay, WI), whereas L25 (S₁₁S₁₂) resulted from crosses between V22 and V28 (pollen parent). The dual-specific S-RNase that rejects both P₁₁ and P₁₃ pollen was produced by site-directed mutagenesis and is expressed as a transgene introduced into host plant G4 (S₁₂S₁₄; MATTON et al. 1999 ). HVapb plants thus reject four different pollen haplotypes, the P₁₂ and P₁₄ via the endogenous S-RNases from the untransformed host and the P₁₁ and P₁₃ using the dual-specific HVapb-RNase. All plants with a dual-specific phenotype contain wild-type levels of the HVapb-RNase in the styles (MATTON et al. 1999 ).

Tetraploids of genotypes L25 (S₁₁S₁₁S₁₂S₁₂), V28 (S₁₂S₁₂S₁₃S₁₃), and G4 (S₁₂S₁₂S₁₄S₁₄) were produced by leaf disc culture from the corresponding diploids as described (VERONNEAU et al. 1992 ), whereas tetraploids F20 (S₁₁S₁₁S₁₂S₁₃), F38 and F55 (S₁₁S₁₁S₁₃S₁₃), and F44 (S₁₂S₁₂S₁₂S₁₂) were selected from progeny of crosses between tetraploids L25 and V28 (pollen parent; QIN et al. 2001 ). Tetraploid plant 1022 (S₁₁S₁₁S₁₃S₁₃) was produced by leaf disc culture of a plant issued from a cross between V22 (S₁₁S₁₃) as pollen donor and SP10 (S₁₃S₁₃), an individual obtained by obligate selfing of parental line PI230582 (S₁₃S₁₄; RIVARD et al. 1994 ). The genotype of all plants used was verified by Western analyses of stylar extracts using antibodies against S₁₁-, S₁₂-, and S₁₃-RNases (MATTON et al. 1999 ; QIN et al. 2001 ), by Southern blot analyses, and by PCR analyses using S-allele-specific primers. All plants used were true tetraploids and not chimeric, as assessed by chloroplast number in stomatal guard cells (L1 layer), pollen size and chromosome number in pollen mother cells (L2 layer), and chromosome number in root meristems (L3 layer). All cytological analyses were performed as described (CAPPADOCIA et al. 1984 ). Compared with their diploid relatives, most autotetraploids showed some reduction in pollen fertility, as generally reported (SINGH 1993 ).

Crosses were performed under greenhouse conditions and were classified as compatible when almost all pollinations resulted in fruit formation and incompatible when no fruits developed. Because the nature of the study required a precise assessment of pollen tube behavior after pollinations, tube growth inside the styles was routinely monitored by UV fluorescence microscopy as described (MATTON et al. 1997 ). Incompatibility defined by the pollinations corresponded in all cases to pollen tube growth arrest in the styles.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Tetraploids derived from self-incompatible diploids are known to produce compatible S-heteroallelic pollen (HAP; DE NETTANCOURT 1977 ), and this was also observed with our S. chacoense tetraploids. As an illustration, the breeding behavior of tetraploids containing two different S-loci (plants G4, L25, V28, F38, F55, and 1022) or three (F20) is shown in Table 1. These plants all produce diploid pollen, about two-thirds of which contain two different S-alleles and are thus fully self-compatible (see pollinations along the diagonal). This behavior is in sharp contrast to the breeding behavior of diploid pollen containing only one type of S-allele, such as that produced by the S-homozygous tetraploid F44 (Table 1), which is incompatible with any plant containing the S₁₂ allele. In agreement with all previous studies, the compatibility of these tetraploids is due to their pollen, as their styles continue to block haploid pollen containing corresponding S-alleles (Table 2). It is important to note that pollen produced by the plant V22 (S₁₁S₁₃) is rejected by all the tetraploids expressing both S₁₁- and S₁₃-RNases, by transgenic plants expressing the HVapb-RNase (an S_11/13 specificity), but is accepted by the untransformed host plant G4 (S₁₂S₁₄). Note also that the pollen produced by the HVapb plants behaves identically to the pollen produced by the untransformed host since transgene expression is restricted to the style.

The dual-specific HVapb-RNase provides a unique tool with which to distinguish between gene inactivation and competition models for the HAP effect. If the HAP effect were caused by gene inactivation (LEWIS 1961 ; VAN GASTEL 1976 ), the P₁₁P₁₃ HAP would be as compatible with HVapb plants as with V22 (S₁₁S₁₃). In contrast, if competition between P₁₁ and P₁₃ pollen-S components present together in diploid pollen takes place, the P₁₁P₁₃ HAP pollen should be rejected by HVapb plants (just like normal haploid pollen) since our dual-specific HVapb-RNase rejects both P₁₁ and P₁₃ pollen. As shown in Table 3, no fruits are formed when pollen from plants with an S₁₁S₁₁S₁₃S₁₃ genotype (F38, F55, or 1022) is tested on styles of HVapb plants, and microscopic examination of these pollinated styles confirms full rejection of P₁₁P₁₃ HAP at midstyle (not shown). Since plants F38, F55, and 1022 all have different genetic backgrounds, indicating that HAP rejection is not restricted to a particular genotype, we conclude that the pollen components of the SI system must be fully expressed in HAP.

The genetic analysis also demonstrates that the dual-specific HVapb-RNase alone is responsible for HAP rejection. First, there is nothing unusual about plants F38, F55, and 1022, as their HAP is self-compatible (Table 1), compatible on V22 styles (Table 3), and their styles reject pollen from V22 (Table 2). Second, there are no breeding differences between the five independent transgenic plants expressing the HVapb-RNase, as P₁₁P₁₃ HAP was fully rejected by their pistils (Table 3). All the HVapb plants used here express wild-type levels of their transgene S-RNase (MATTON et al. 1999 ) and accept all other HAP combinations such as P₁₁P₁₂, P₁₂P₁₄, or P₁₂P₁₃ (Table 1). In addition, HVapb transgenic plants that do not express the transgene behave like the untransformed host G4 and do not reject HAP (not shown). Last, the rejection of the P₁₁P₁₃ HAP is unrelated to expression of more than two different S-RNases in the style, as neither tetraploid F20 (Table 1) nor a transgenic plant of S₁₂S₁₄ genotype expressing an additional S₁₁-RNase (MATTON et al. 1997 ) reject P₁₁P₁₃ HAP (not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Model for pollen-S action:
Any model for GSI must now explain the normal compatibility of HAP, as well as its incompatibility with the cognate dual-specific S-RNase, as shown here. To develop a working model, however, two additional observations must be taken into account. First, screens for compatible pollen produced after mutagenesis have uncovered a variety of pollen part mutants, some of which contained what was referred to as an additional S-allele while others apparently lack any S-allele (PANDEY 1967 ; VAN GASTEL 1976 ; DE NETTANCOURT 1977 ; GOLZ et al. 1999 ). Clearly, while an additional S-allele (pollen-S) could be analogous to HAP, deletion of pollen-S must be different. Thus, any model for SI must predict a compatible pollen phenotype either when two different pollen-S are expressed or when none is expressed. Second, as discussed above, at least part of the function of the SI system inside pollen tubes is likely to involve RNase inhibitors (RI). Although not yet reported for plants, RI are well known in animal systems (HOFSTEENGE 1997 ).

We recently proposed a model for GSI with two pollen components, one a general RI that can inactivate any S-RNase and the other an S-allele-specific product that maintains the activity of a specific S-RNase inside the pollen tube by blocking RI binding (LUU et al. 2000 ). Separation of the pollen-S blocker from the general RI was proposed to explain the compatibility of pollen mutants possibly lacking pollen-S (PANDEY 1967 ; VAN GASTEL 1976 ; GOLZ et al. 1999 ). Interestingly, Dr. T. Sims recently identified in Petunia hybrida, by the two-hybrid system, a nonpolymorphic S-RNase-binding protein with a RING-HC domain that could represent a possible candidate for the general inhibitor (T. SIMS and M. ORDANIC, unpublished results).

The multimeric nature of pollen-S:
From the results shown here, we deduce that only a multimeric pollen-S blocker can explain all aspects of the HAP phenotype. First consider the incompatibility reaction of haploid P₁₁ pollen growing in an S₁₁S₁₃ style (Fig 1A). The RI components are drawn as shaded arcs to mimic the structure of the mammalian RNase inhibitor (HOFSTEENGE 1997 ), the P₁₁ blockers as small shaded circles, and the S-RNases as large white ovals. S-RNases enter the pollen tubes from the styles (LUU et al. 2000 ) and, in this illustration, we assume that eight S-RNases of any type present in the style will enter. We also assume that there are sufficient blockers in a pollen tube to bind to their cognate S-RNases, and therefore the P₁₁ pollen tube contains eight P₁₁ tetramers in addition to the S₁₁- and S₁₃-RNases. All the S₁₁-RNases bind the P₁₁ blocker (favored thermodynamically over RI binding; KAO and MCCUBBIN 1997 ). Since blocker binding precludes RI binding, S₁₁-RNase remains active and incompatibility results. The RI binds the S₁₃-RNase because no P₁₃ blocker is present in the P₁₁ pollen, but inhibition of the S₁₃-RNase activity has no effect on the incompatibility phenotype since the active S₁₁-RNase causes pollen rejection.

View larger version (51K): In this window In a new window Download PPT slide   Figure 1. A model for GSI derived from the HAP effect. (A) Haploid P11 pollen growing in an S11S13 style is incompatible because the ribonuclease inhibitors (RI; shaded arcs) are prevented from binding to the S11-RNases (open ovals at left), which have entered the pollen tubes from the styles, by the P11 blockers (small shaded circles) present in the pollen tubes. Since P13 blockers are absent, RI binds S13-RNases (open ovals at right). (B) S11- and S13- RNases from an S11S13 style enter diploid P11P13 HAP, but RI binding cannot be fully prevented because a binomial distribution of tetramer types results in a lower number of P11 and P13 homotetramers than would be found in either haploid pollen type. (C) The dual-specific HVapb-RNase does not discriminate between P11 and P13 blockers and thus binds all of the heterotetrameric blockers. This results in incompatibility because RI is prevented from binding.

The multimeric nature of the blocker is irrelevant for the incompatibility phenotype of normal (haploid) pollen but is essential to explain the compatibility of HAP (see next section for the choice of tetramers over dimers). When P₁₁P₁₃ HAP grows in S₁₁S₁₃ styles (Fig 1B), S₁₁- and S₁₃-RNases enter the pollen tube as before. Once again, blockers will compete with the RI for binding to their cognate S-RNases. However, even if HAP produces the same number of P₁₁ (small circles) or P₁₃ blockers (small squares) as would haploid, the random assembly of monomers into tetramers would produce homotetramers and heterotetramers in binomial proportions, similar to the 1:4:6:4:1 ratio observed for lactate dehydrogenase tetramers (MARKERT 1963 ). In this case, only 1 out of the 16 blocker tetramers in P₁₁P₁₃ HAP would be a P₁₁ homotetramer and thus only one of the S₁₁-RNases entering the pollen tube would remain active. If heterotetramers were inactive, the other S₁₁-RNases would be inhibited because the hybrid blockers would no longer outcompete RI binding. The same argument holds for assembly of a P₁₃ homotetramer blocker and its binding to the S₁₃-RNase. Therefore, only a fraction (one-quarter) of the amount of RNase active in haploid pollen (Fig 1A) would be active in HAP (Fig 1B). Is this reduction in active RNase sufficient to cause compatibility? It is generally accepted that a minimum threshold of S-RNase is required for pollen rejection. The threshold idea is derived from experiments in transgenic P. inflata (LEE et al. 1994 ) and data from natural SC Japanese pear mutants (HIRATSUKA et al. 1999 ), where S-RNase expression at one-third the normal level results in self-compatibility. Thus, a reduction in the amount of active S-RNases to one-quarter normal levels could indeed result in HAP compatibility.

How, then, might the dual-specific HVapb-RNase reject HAP? Since this S-RNase can bind either P₁₁ or P₁₃ (MATTON et al. 1999 ), it is unlikely to discriminate between any of the heterotetramers in P₁₁P₁₃ pollen (Fig 1C). Blocker binding then would be unaffected by the formation of heterotetramers and this RNase would remain fully active and reject the pollen. For clarity, we have drawn only the HVapb RNase in Fig 1C, although it must be kept in mind that these pollen tubes will also contain S₁₂- and S₁₄-RNases, which are present in the styles of the transformed plants at the same (wild-type) levels as the HVapb-RNase.

Theoretical support for a tetrameric pollen-S:
To buttress the intuitive argument provided above, we have also analyzed the predictions of a mathematical formulation for the amount of pollen-S, which takes into account the possibility of fractional activity of heteromers relative to homomers (b) and relative expression of pollen-S in diploid compared to pollen (a). In the expression defined below for k-mers, the amount of active pollen-S in diploid pollen (x_d) is a function of the amount of pollen-S normally expressed in haploid pollen (x_h):

(1)

This equation takes into account situations where pollen-S expression levels are less than in haploid plants (a < 1) as well as cases where heteromers are partially active (0 < b < 1). In the section above, we assumed that expression of pollen-S was the same in diploid and in haploid pollen (a = 1) and that heteromers are totally inactive (b = 0).

The quantity of active pollen-S in HAP must be <x_h for compatible crosses (with S₁₁S₁₃ plants) and x_h in incompatible crosses (with HVapb plants). To visualize the main conclusions of this model, we calculated the range of values of pollen-S expression (a) that satisfy these two requirements for various values of (k) and (b) (Table 4). Note that b = 1 for HVapb plants, since the dual-specific RNase cannot distinguish between P₁₁ and P₁₃. Two important conclusions can be unequivocally drawn from this analysis. First, the pollen-S cannot be a monomer (Table 4). Second, heteromers cannot be as active as homomers, as no value of a can produce compatibility with S₁₁S₁₃ and incompatibility with HVapb plants if b = 1.

The analysis also allows us to describe the conditions required for dimeric or tetrameric blocker activity. Were pollen-S dimeric, its expression in HAP would be restricted to ¹/₂ a < 1. Only two values of b are shown, but it is clear that as the activity of the heteromers (b) increases, the a value must decrease. If pollen-S were tetrameric, a wider range of pollen-S expression levels is permitted (¹/₂ a < 4). Thus, if pollen-S is a dimer, this model would require a reduced expression in diploid pollen. While pollen-S is as yet unknown and cannot be assayed, reduction of allele expression in polyploids has been reported for some genes (BIRCHLER and NEWTON 1981 ). However, we do not as yet have any evidence for a reduction in the levels of S-RNases (presumably tightly linked to the pollen-S gene at the S-locus) in tetraploids. Epigenetic silencing, resulting from the increase in ploidy level, could also account for a reduced level of pollen-S, although this phenomenon is usually restricted to the silencing of one of the original alleles (SCHEID et al. 1996 ). Furthermore, it is unclear why gene silencing would preferentially occur in HAP as opposed to homoallelic pollen. We therefore conclude that the blocker is probably a tetramer.

The model proposed here supports the proposal that the S-RNase-based GSI evolved from an RNase-based defense mechanism (KAO and MCCUBBIN 1996 ). Indeed, extracellular S-like RNases not involved in GSI have been described and their role in host defense has been suggested (LEE et al. 1992 ). If extracellular S-RNases can enter pollen tubes indiscriminately (LUU et al. 2000 ), then inhibitors for S-like RNases must have been necessary in pollen. GSI could indeed have derived stepwise from an ancestral self-compatible system as proposed (UYENOYAMA 1988 ). Initially, pollen tube RI would block the cytotoxicity of extracellular S-like RNases. These extracellular S-like RNases may already have evolved polymorphisms, which, if selected for a role in pathogen defense, would be neutral for pollen RI binding. Thus, we see self-incompatibility as having arisen by the development of an allele-specific recognition domain on a pollen protein that binds a particular stylar RNase and blocks RI binding.

Finally, our model suggests that the term "competition effect" may not accurately reflect the mechanism of HAP compatibility. Earlier interpretations of the phenomenon were that two different pollen components competed with each other for some limiting factor (DE NETTANCOURT 1977 ). In our view, the reduced activity of heteromers compared to homomers points to pollen-S itself as the limiting factor.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: CEA Cadarache/LEP/DEVM, 13108 Saint-Paul-lez Durance, France.
³ Present address: University of Nanjing, Department of Agriculture, 210095 Nanjing, China

	ACKNOWLEDGMENTS

We thank G. Teodorescu for plant care and are grateful to Professors T.-H. Kao, V. De Luca, and Dr. J Labovitz for helpful discussions on modeling SI and critical reviews of the manuscript. We also thank Dr. T. Sims for sharing unpublished data and an anonymous reviewer for suggesting the mathematical formula used to calculate pollen-S levels in diploid pollen. This work was supported by a fellowship from Program Québecois des Bourse d'Excellence, Québec (D.-T.L.) and by grants from Natural Sciences and Engineering Research Council of Canada (M.C.) and Fonds pour la Formation des Chercheurs et Aide à la Recherche (D.M. and M.C.).

Manuscript received March 2, 2001; Accepted for publication June 19, 2001.

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