Journal of Experimental Botany, Vol. 54, No. 380, pp. 123-130,
January 1, 2003
© 2003 Oxford University Press
S-RNase complexes and pollen rejection
Received 22 May 2002; Accepted 24 September 2002
1 Department of Biochemistry, Facultad de Química, National Autonomous University of México, Conjunto ‘E’ Paseo de la Investigacio’n Cientifica, Ciudad Universitaria, 04510 México D.F., México
2 Department of Biochemistry, University of Missouri-Columbia, 117 Schweitzer Hall, Columbia, MO 65211, USA
3 To whom correspondence should be addressed. Fax: +1 573 882 5635. E-mail: mcclureb{at}missouri.edu
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Abstract |
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 Top Abstract IntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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Biochemical interactions between the pollen and the pistil allow plants fine control over fertilization. S-RNase-based pollen rejection is among the most widespread and best understood of these interactions. At least three plant families have S-RNase-based self-incompatibility (SI) systems, and S-RNases have also been implicated in interspecific pollen rejection. Although S-RNases determine the specificity of SI, other genes are required for the pollen rejection system to function. Progress is being made toward identifying these non-S-RNase factors. HT-protein, first identified as a non-S-RNase factor that was required for SI in Nicotiana alata, has now been implicated in other species as well. In addition, several pistil proteins bind to S-RNase in vitro. One hypothesis is that S-RNase forms a complex with these proteins in vivo that is the active form of S-RNase in pollen rejection.
Key words: Interspecific incompatibility, pollen–pistil interactions, self-incompatibility, S-locus, S-RNase.
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Introduction |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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Flowering plants display elegant control over fertilization. Controls take many forms, from the structural barrier imposed by separate organs for receiving pollen and fertilization (i.e. the stigma and ovule, respectively) to highly evolved plant-pollinator interactions. Nonetheless, plants are sessile and, consequently, pollen must be transported by agents (e.g. wind or animal vectors) over which the plant has little control. To offset this, biochemically-based pollen–pistil interactions have evolved to refine the plant’s control over fertilization. Broadly, these interactions support the growth of desirable pollen and inhibit fertilization by undesirable pollen. For instance, pollen from a different species should be prevented from fertilizing an ovule because the progeny are not likely to be successful; but con-specific pollen that is likely to give rise to fit progeny should be encouraged. Thus, discrimination between pollen types based on their genetic identity is central to controlling fertilization. Such discrimination is understood to emerge from interactions between specific factors expressed by the pollen and the pistil. Considerable progress has been made toward identifying these factors, particularly those that function on the pistil side.
The biggest advances toward understanding pollen rejection have been made by exploiting the highly specific self-incompatibility (SI) systems displayed by many plant species. SI systems are genetically controlled mechanisms that promote outcrossing within a species. Compatibility depends on interactions between factors transcribed in the pistil and in pollen or anthers. In many systems, the determinants of specificity are encoded at a single locus, the S-locus. Commonly, SI is determined on the male side by products expressed in the gametophyte; this is called gametophytic SI. Pollen is rejected when the single S-allele expressed in the pollen is the same as either of the two S-alleles expressed in the diploid pistil (de Nettancourt, 2001).
The identification of highly-expressed pistil-specific genes has yielded potential factors involved in supporting the growth of desirable pollen. For example, TTS (transmitting tract specific) glycoprotein, described in Nicotiana tabacum and N. alata, is highly expressed in the mature transmitting tract (Cheung et al., 1993, 1995; Wu et al., 1995, 2000). Growing pollen tubes remove the glycans from TTS and could use them as a growth substrate. Furthermore, antisense inhibition of TTS expression causes reduced pollen tube growth, strongly supporting a role in compatible pollination. Other pistil glycoproteins have been shown to interact directly with pollen tubes, but their role in pollination is still unknown (Lind et al., 1996; de Graaf, 1999; Bosch, 2002).
As understanding advances, the focus of research has shifted from identifying factors associated with pollination to discovering how interactions between them relate to compatibility. For instance, direct interactions between S-locus products expressed in the pollen and the pistil have been demonstrated in Brassica (Cabrillac et al., 2001; Takayama et al., 2001). Additional factors, not coded at the S-locus, are required for Brassica SI and also interact with S-locus products. In Nicotiana, proteins that determine the specificity of SI on the pistil side, S-RNases, bind to a family of glycoproteins that interact with pollen tubes (McClure et al., 2000); TTS glycoprotein is among the bound proteins. The hypothesis is that S-RNase exists as a complex with other pistil proteins in vivo and that this complex functions in SI. This molecular interaction implies a connection between two of the broad functions of the pistil described above: rejection of undesirable pollen in SI and supporting growth of desirable pollen.
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Self-incompatibility systems |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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Three SI systems have been studied extensively at the molecular level. The SI system in the Brassicaceae is the only one where specificity determinants have been described in both the pollen and the pistil. SI plants in this family express an S-receptor kinase (SRK) in the stigmatic papillae that interacts with a component in the pollen coat (S-locus cysteine rich protein, SCR) to control compatibility. The SRK and SCR genes are physically linked. Each different allele of the S-locus (often referred to as an S-haplotype in the Brassicaceae) encodes a distinct pair of polypeptides. An incompatible interaction occurs when pollen and pistil components from the same allele or haplotype bind on the papillar cell surface. In the Brassicaceae, the pollen component is expressed in sporophytic cells of the tapetum. Therefore, the interaction is always between sporophytically expressed products, hence the name sporophytic SI (Nasrallah, 2002).
Two gametophytic SI systems have been studied extensively at the molecular level. Here, the determinant of specificity on the pollen side is expressed by the gametophyte itself. Pollen is rejected when its single S-allele is the same as either of the two S-alleles in the diploid pistil (de Nettancourt, 2001). In Papaver, 15 kDa S-proteins are expressed in the stigma and located in the cell wall (Foote et al., 1994). The S-proteins are recognized by a putative receptor in the pollen and, thus, prevent growth of pollen tubes with an S-allele that matches one of the S-alleles in the stigma. P. rhoeas pollen tubes treated in vitro with purified S-proteins show rapid changes in both their tip-focused calcium gradient and a reorganization of their actin cytoskeleton (Snowman et al., 2000). Thus, in the Papaver system the S-proteins alone are sufficient to cause the pollen SI response.
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RNase-based self-incompatibility |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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The most phylogenetically widespread gametophytic SI system relies on ribonucleases called S-RNases (Igic and Kohn, 2001). S-RNases are basic glycoproteins of about 30 kDa that are secreted into the extracellular matrix of the stigma, transmitting tract, and the inner epidermis of the ovary (Anderson et al., 1986; Cornish et al., 1987; Anderson et al., 1989; McClure et al., 1993). S-allele-specific pollen rejection requires extremely high levels of S-RNase expression (Clark et al., 1990); the concentration of S-RNase in the extracellular matrix has been estimated at 10–50 mg ml–1 (Jahnen et al., 1989). High level expression of cloned S-RNases in an appropriate genetic background causes S-allele-specific pollen rejection (Lee et al., 1994; Murfett et al., 1994; Matton et al., 1997; Zurek et al., 1997). It has also been shown that the S-RNase ribonuclease activity, but not glycosylation, is required for pollen rejection (Huang et al., 1994; Karunanandaa et al., 1994). Thus, S-RNases are thought to function as highly specific cytotoxins that inhibit the growth of incompatible pollen. Each allele of the S-locus encodes a distinct S-RNase that acts as a specific cytotoxin inhibiting the growth of pollen bearing the same S-allele (reviewed in McCubbin and Kao, 2000). Consistent with this model, S-RNases are potent inhibitors of translation (Gray et al., 1991). Furthermore, radioactive tracer experiments show that pollen RNA is degraded after incompatible pollination (McClure et al., 1990).
Plant transformation experiments have shown that S-RNases determine the specificity of SI in the pistil (Lee et al., 1994; Matton et al., 1997; Murfett et al., 1994; Zurek et al., 1997). For example, expressing SA2-RNase from N. alata in N. langsdorffiixSCN. alata hybrids causes rejection of SA2-pollen, but not SC10-pollen (Murfett et al., 1994). Similar results were reported in Petunia and Solanum (Lee et al., 1994; Matton et al., 1997). Together these results demonstrate that S-RNases are the determinants of S-allelic-specificity on the pistil side.
Five conserved sequence elements have been identified in S-RNases, but allelic specificity is surely encoded in sequences that vary between different S-alleles (Ioerger et al., 1991). Figure 1 shows the relative locations of conserved and variable regions in a generalized S-RNase. By comparing solanaceous S-RNase sequences, Ioerger et al. (1991) identified two ‘hypervariable’ regions designated HVa and HVb. Ishimizu et al. (1998) identified four regions of rosaceous S-RNases that appear to be under positive selection, of which two overlap with HVa and HVb. A recent analysis of sequences from the Scrophulariaceae also found that the HVa and HVb regions were highly variable, but did not find evidence of diversifying selection (Vieira and Charlesworth, 2002).
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Experiments in Nicotiana and Petunia suggest that HVa and HVb are not sufficient for discrimination between typical S-RNases (Kao and McCubbin, 1996; Zurek et al., 1997), but are sufficient to discriminate between two very closely related S-RNases in Solanum chacoense (Matton et al., 1997). Interestingly, the recently reported crystal structure of SF11-RNase from N. alata (Ida et al., 2001) shows HVa and HVb are adjacent and solvent exposed. Since the allelic specificity determinants in S-RNase presumably interact with the specificity determinants on the pollen side (i.e. pollen-S), their location in the three-dimensional structure is important. For instance, if HVa and HVb form a discrete, localized structure that is sufficient for pollen recognition, then pollen-S may form a limited contact. Alternatively, if, as some experiments suggest (Zurek et al., 1997), non-contiguous regions are also important for recognition, then much more extensive contacts with pollen-S would probably be required.
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S-RNase and interspecific pollen rejection |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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Interspecific unilateral incompatibility (UI) occurs when pollinations between species are successful in one direction but not the other. SI species frequently show UI relationships with related self-compatible (SC) species (de Nettancourt, 2001). Typically, pollen from the SI species is compatible on the SC pistil, but the reciprocal pollination is rejected. Such relationships are said to follow the SIxSC rule (Lewis and Crowe, 1958; de Nettancourt, 2001).
While there are many UI systems that do not follow the SIxSC rule, it holds often enough and in enough different plant families to suggest a connection between at least some types of interspecific pollen recognition and SI. Hiscock and Dickinson (1993), who examined crossing relationships in the Brassicaceae, found a strong association between SI and rejection of pollen from SC species. Martin (1967) showed a relationship between SI and UI in the progeny of a cross between SC Lycopersicon esculentum and SI L. hirsutum. Mapping studies and QTL analyses in Lycopersicon also support a major role for the S-locus in UI (Chetelat and de Verna, 1991; Bernacchi and Tanksley, 1997). In N. bonariensis, S-alleles react differently in interspecific pollinations, directly implicating the S-locus in interspecific pollination (Pandey, 1973, 1981). It is striking that the SIxSC rule describes crossing relationships in both the Brassicaceae and the Solanaceae where the underlying SI mechanisms are totally different. The broad applicability of the rule suggests that this dual role for the S-locus (i.e. in determining compatibility within and between species) is a common, if not universal, feature. However, it is also clear that other mechanisms contribute to interspecific cross compatibility (Hogenboom, 1984; Mutschler and Leidl, 1994). As more pollen and pistil factors that control compatibility within species are identified and cloned, it will be possible to test for their roles in interspecific compatibility on a case-by-case basis.
Plant transformation has been used to show directly that S-RNase, the determinant of specificity in SI, can also cause UI between N. alata and the SC species N. plumbaginifolia and N. tabacum. N. plumbaginifolia follows the SIxSC rule in crosses with N. alata. SI accessions of N. alata reject pollen from N. plumbaginifolia, but SC accessions do not. N. tabacum is an example of a SC species that does not follow the SIxSC rule in crosses with N. alata; its pollen is rejected by both SI and SC accessions of N. alata.
Rejection of pollen from N. plumbaginifolia closely resembles SI. S-RNase is required for rejection of N. plumbaginifolia pollen and for SI, but is not sufficient for either mechanism. Additionally, non-S-RNase factors are also required, and are referred to as ‘factor-dependent’ mechanisms (McClure et al., 2000). Evidence for these factors is that S-RNase expression in purely SC backgrounds (i.e. N. plumbaginifolia and N. tabacum) does not cause either S-allele-specific rejection of N. alata pollen or rejection of N. plumbaginifolia pollen. However, when non-S-RNase factors are supplied in trans by crossing transgenic N. plumbaginifolia plants expressing S-RNase with a SC accession of N. alata, then these two pollen rejection mechanisms function normally (Murfett et al., 1996).
S-RNase causes rejection of pollen from N. tabacum through a different genetic mechanism. Expression of either SA2- or SC10-RNase in the purely SC genetic backgrounds is sufficient to cause N. tabacum pollen rejection (Murfett et al., 1996). Thus, the non-S-RNase factors from the N. alata background are not required, and this is referred to as ‘factor-independent’ pollen rejection. It should be emphasized that this name serves only to highlight the distinction between different rejection mechanisms. It is likely that even ‘factor-independent’ pollen rejection requires interactions between S-RNase and other pistil factors.
Thus, in Nicotiana, S-RNase is implicated in rejecting pollen from SC species that follow the SIxSC rule and species that do not. This demonstrates that S-RNase is implicated in multiple pollen rejection mechanisms. Still, there is a clear difference in specificity between SI and UI. In intraspecific crosses, S-RNase causes rejection of only a single pollen-S genotype. Interspecific pollen rejection is less specific; almost any S-RNase causes rejection of pollen from SC species.
To investigate the latter, four S-RNases and RNaseI from E. coli were expressed in transgenic plants. RNaseI was chosen because its size and charge are similar to S-RNases and it is active in the extracellular periplasmic space of E. coli (Meador and Kennell, 1990). SA2-RNase and RNaseI constructs were transformed into N. plumbaginifolia and crossed with SC N. alata to test their effects on ‘factor-dependent’ pollen rejection in N. plumbaginifoliaxSC N. alata hybrids. Both RNases were expressed at the same level. SA2-RNase functioned normally, but RNaseI did not cause rejection of N. plumbaginifolia pollen (Beecher et al., 1998). The specificity of ‘factor-independent’ pollen rejection was tested by expressing four RNases in N. tabacum. In these experiments, SC10-RNase was used as the ‘normal’ S-RNase control that functions in both SI and N. plumbaginifolia pollen rejection. S9811-RNase is functional in SI, but is unusual because it does not cause rejection of N. plumbaginifolia pollen. SCon5-RNase was used because it is a chimeric S-RNase that is an active ribonuclease, but is not functional in SI (Zurek et al., 1997). Finally, E. coli RNaseI was used as a representative non-S-RNase. All three S-RNases were effective in ‘factor-independent’ N. tabacum pollen rejection, but RNaseI was not (Beecher and McClure, 2001). Together, these experiments show that RNase activity alone is not sufficient for either type of RNase dependent UI pollen rejection. Therefore, even though UI is not as specific as SI, S-RNases still appear to have some special adaptation that allows them to function in SI.
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Factors not linked to the S-locus |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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While the S-locus encodes the determinants of specificity in SI, in most systems additional factors are also required. The Papaver system is an exception. Purified P. rhoeas S-proteins expressed in E. coli elicit a rapid, S-allele-specific SI response in vitro (Foote et al., 1994). Thus, in this system, it is clear that no other factors are required. However, in Brassica and the Solanaceae, factors that are not linked to the S-locus affect SI. These factors are sometimes referred to as modifiers because they influence SI (i.e. modify the response). Genetic evidence for such modifier factors comes from studies showing that the activity of the S-locus depends on genetic background (Martin, 1968; Ai et al., 1991; Bernatzky et al., 1995). A growing number of modifiers have been identified and, in some cases, there is evidence for how they interact biochemically with specificity determinants.
Modifiers can be placed in three groups based on how they interact with specificity determinants (McClure et al., 2000). Group 1 factors directly affect the expression of S-locus genes. These might include specific transcription factors or factors affecting a critical post-transcriptional modification of a specificity determinant. Group 2 factors are required for pollen rejection, but do not affect accumulation or structure of the specificity determinants. They interact genetically or biochemically with S-locus products, but have no general role in pollination. Group 3 factors are required for pollen rejection but have a wider role in pollination. The distinctions between these different groups of factors are important for understanding their effects on compatibility and for designing strategies to identify them. For example, mutations in Groups 1 or 2 factors could cause a change from SI to SC behaviour, but would have no effect in a SC species. However, a null mutation in a Group 3 factor might result in sterility. Thus, a mutational strategy that relies on screening for loss of SI will only detect Group 1 and Group 2 factors.
The greatest progress in identifying and characterizing modifier genes has been made in the Brassica system. Goring’s group used the SRK-kinase domain as bait in the yeast two-hybrid system to identify interacting proteins (Bower et al., 1996; Gu et al., 1998). ARM repeat containing protein 1 (ARC1) is a stigma-specific protein that interacts with phosphorylated SRK and can be phosphorylated by SRK in vitro. Antisense inhibition of ARC1 caused breakdown of SI, thereby, confirming its role in pollen rejection (Stone et al., 1999). Since antisense inhibition of ARC1 resulted in compatibility, it is not required for pollen tube germination or tube growth per se. Thus, it is a Group 2 factor. Two thioredoxin genes (THL1 and THL2) that interact specifically with SRK were also identified using the two-hybrid system (Bower et al., 1996). While these genes are not stigma-specific, Cabrillac’s elegant set of biochemical experiments suggest that they may be directly involved in SI (Cabrillac et al., 2001). They showed that incompatible pollination caused phosphorylation of SRK in vivo and in vitro. Phosphorylation was inhibited by stigma extracts, and the inhibitory activity could be mimicked by Spirulina thioredoxin or by one of the Brassica thioredoxins (THL1, Bower et al., 1996). In Cabrillac’s model, thioredoxin inhibits SRK autophosphorylation. Stimulation by incompatible pollen is thought to release thioredoxin from SRK allowing phosphorylation and, ultimately, pollen rejection (Cabrillac et al., 2001). Since the thioredoxin genes identified by Bower et al. (1996) are not stigma-specific, it is likely that they perform similar functions in other genetic pathways.
There is genetic evidence that modifier genes are also required in RNase-based pollen rejection systems. Tsukamoto et al. (1999) described a Group 1 factor segregating in a Uruguayan population of P. axillaris, which has an allele-specific effect on S-RNase expression. A classic study from Kao’s group showed that Strawberry Daddy, a cultivar of P. hybrida, expressed a functional S-allele. Thus, Strawberry Daddy is defective for a Group 2 factor since S-RNase expression is not affected and the cultivar is SC (i.e. the factor is not required for pollen tube growth). Bernatzky et al. (1995) showed that S-RNases from L. hirsutum are expressed after backcrossing into an L. esculentum background, but do not function in pollen rejection. Thus, L. esculentum is missing one or more Group 2 factors needed for pollen rejection.
Group 2 factors are clearly required in Nicotiana as well. The factors required for ‘factor-dependent’ inter- and intra-specific pollen rejection are Group 2 factors. For example, transgenic N. plumbaginifolia plants express high levels of SA2-RNase, but do not reject pollen from untransformed N. plumbaginifolia or SA2-pollen from N. alata (Murfett et al., 1996). Since the same transgene causes pollen rejection when expressed with factors from N. alata, it appears that N. plumbaginifolia is defective for one or more non-S-RNase Group 2 factors. A mutant that appears to be a defect in a Group 2 factor has also been observed. The recessive defect causes SI plants to be SC, but does not affect S-RNase expression (McClure et al., 2000).
One non-S-RNase Group 2 factor that is missing or defective in N. plumbaginifolia was recently cloned. An N. alata cDNA library was screened for sequences expressed in N. alata but not in N. plumbaginifolia. One clone, designated HT, was selected for characterization based on its expression pattern. Accumulation of HT-transcript lags slightly behind SC10-RNase in SI N. alata SC10SC10. Just before the pistil becomes competent to reject SC10-pollen, SC10-RNase transcript accumulates to 60% of its level at maturity, but HT-transcript is present at only 5% of its final level. Slightly later, coinciding with the onset of SI, HT-transcript accumulates rapidly. The cDNA sequence revealed that the HT-protein is 101 amino acids long and has an unusual stretch of asparagine and aspartate residues (ND-domain) near the C-terminus (McClure et al., 1999).
An antisense experiment showed that HT-protein is required for S-allele-specific pollen rejection. An antisense HT-construct was transformed into N. plumbaginifolia, and individual transformed lines were crossed with SI N. alata SC10SC10 to form N. plumbaginifoliaxSI N. alata SC10SC10 hybrids. Untransformed hybrids showed normal S-allele-specific pollen rejection. Progeny from five independent transformants showed no detectable HT-protein expression and accepted SC10-pollen. The antisense plants expressed normal levels of SC10-RNase. Thus, HT-protein is a Group 2 factor.
Kondo et al. (2002) recently described HT sequences from Lycopersicon and Solanum. They expressed S6-RNase from SI L. peruvianum in SC L. esculentum. Similar to the situation in N. plumbaginifolia, they found normal S-RNase expression, but the transformed plants failed to reject S6-pollen from L. peruvianum. Both these Lycopersicon species and Solanum chacoense were shown to possess two HT-genes, HTA and HTB. Both genes are defective in L. esculentum; one contains a frameshift and the other contains a nonsense mutation. This suggests that defective HT-genes may contribute to the failure of pollen rejection in L. esculentum.
The Lycopersicon and Solanum HT sequences are easily recognizable as homologues of the N. alata sequence. Figure 2 shows an alignment of the HT-A and HT-B sequences from L. peruvianum with the N. alata sequence. All three sequences have highly homologous N-terminal sequences and an ND-domain near the C-terminus. N-terminal sequencing of the N. alata HT-protein gave the sequence RDMVDPSISL (McClure et al., 1999). The 24 amino acids upstream of this sequence, the putative secretion signal, are about 75% identical between the N. alata sequence and the Lycopersicon and Solanum sequences. The rest of the proteins (i.e. the mature proteins) are only about 32% identical. All the proteins also contain a sequence similar to TLQKIGG. This motif contains the N-terminus of some small HT-related polypeptides identified in N. alata. This sequence conservation suggests that processing of HT-protein may be important. The ND-domains are flanked on one side by a CXXCXC domain and by a sequence CXXXCC at the extreme C-terminus. The significance of these cysteine residues is not known. Aside from the obvious preponderance of asparagine and aspartate residues, the ND-domains do not show strict sequence conservation. This domain most likely serves a structural or physical role, perhaps based on flexibility or charge. While these sequence comparisons place constraints on speculations about the regions of HT-protein that may be important for its function, they do not provide direct insight. Although HT-proteins are implicated in S-allele-specific pollen rejection, their exact function remains unknown.
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S-RNase complexes |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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S-RNase binding studies provide a different approach to identifying potential non-S-RNase factors required for pollen rejection. The yeast two-hybrid system has been used to clone an S-RNase binding protein from petunia pollen (Sims and Ordanic, 2001). S-RNase immobilized on Affigel on an affinity matrix has been used to identify S-RNase binding proteins in style extracts from N. alata (McClure et al., 2000). Crude extracts prepared in low salt buffers were passed over the affinity matrix followed by extensive washing with detergent. Bound proteins were eluted with a low pH buffer and neutralized for further analysis. When style extracts from SI N. alata SC10SC10 were passed over SC10-RNase Affigel and analysed by SDS-PAGE, the major binding proteins migrated near 11 kDa, 35 kDa, and a broad high molecular weight band near 100 kDa. Similar proteins were retained when SC N. alata extracts were analysed, except the 35 kDa band was not present. A qualitatively similar pattern of binding proteins was observed between pH 5.2 and 8.8. Matrices prepared with S-RNase retained far more protein than control matrices prepared with BSA or E. coli RNaseI (McClure et al., 2000). RNaseI was an especially good control; it has a similar charge and mass to SC10-RNase, but was not active in pollen rejection. Thus, binding to S-RNase Affigel is specific.
Immunoblot analysis and cDNA cloning have identified the major S-RNase binding proteins: the 11 kDa, 35 kDa, and high molecular weight species. The 35 kDa band retained from SI N. alata SC10SC10 binds to an SC10-RNase monoclonal antibody, and, thus, corresponds to SC10-RNase itself. Purified SC10-RNase also binds to the matrix suggesting a direct interaction. Preliminary evidence from native PAGE also suggests that S-RNases interact to form multimers (CN Hancock, B McClure, unpublished data).
The 11 kDa protein, p11, was purified for N-terminal sequencing and cloned. Interestingly, p11 copurified with proteins in the high molecular weight binding proteins. Sequence analysis revealed similarity to a class of copper binding proteins known as phytocyanins (Nersissian et al., 1998).
The high molecular weight band proved to contain at least three glycoproteins. The mobility of this band in low percentage SDS-PAGE was similar to the TTS glycoprotein described in N. tabacum (Cheung et al., 1993, 1995; Wu et al., 1995). The high molecular weight fraction bound anti-TTS antibody (gift of Alice Cheung, University of Massachusetts). A high salt-extractable protein similar to TTS has been described in N. alata (Sommer-Knudsen et al., 1996). However, this protein is not extracted under the low-salt conditions used here. Based on immunostaining, solubility characteristics, and its mobility in SDS-PAGE, the S-RNase binding protein was identified as NaTTS, the N. alata homologue of TTS (Wu et al., 2000). TTS protein shares a similar cysteine-rich C-terminal domain with at least two other pistil proteins, the 120 kDa glycoprotein from N. alata (Lind et al., 1994) and PELPIII from N. tabacum (de Graaf, 1999). Antipeptide antibodies were prepared and used to show that both the 120 kDa glycoprotein and NaMG-15, the N. alata homologue of PELPIII, were present in the high molecular weight S-RNase Affigel bound fraction. Thus, all three of these glycoproteins (NaTTS, the 120 kDa glycoprotein, and NaMG-15) are S-RNase binding proteins. This brings to five the total number of identified proteins (i.e. including S-RNase and p11). The non-S-RNase binding proteins may be regarded as putative Group 3 factors. Since TTS protein has been shown to be involved in supporting growth of compatible pollen tubes in N. tabacum (Cheung et al., 1995; Wu et al., 1995), the S-RNase binding proteins may be important in pollen rejection as well as other pistil functions.
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Implications of S-RNase complexes |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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One hypothesis is that S-RNase binding proteins form a complex with S-RNase in the style extracellular matrix, and that this complex is the functional form of S-RNase in pollen rejection. The three glycoproteins in the bound fraction have all been shown to interact with pollen tubes. TTS glycoprotein stimulates pollen tube growth in vitro. In vivo, TTS associates with the surface of pollen tubes and is deglycosylated by growing pollen tubes (Wu et al., 1995). PELPIII associates with the pollen tube wall and callose (de Graaf, 1999). The 120 kDa glycoprotein appears to be taken up into the pollen tube cytoplasm (Lind et al., 1996). Thus, by forming complexes with these glycoproteins, S-RNase may associate with pollen tubes indirectly.
If this is correct, it would imply that no ‘receptor’ for S-RNase is needed. This is consistent with the common observation that extremely high levels of S-RNase expression are required for pollen rejection. One estimate is that S-RNase accumulates to 50 mg ml–1 in the transmitting tract matrix, a concentration of about 1.5 mM (Jahnen et al., 1989)! Thus, if an S-RNase receptor does exist, it would have a very low affinity. Alternatively, the high concentration of S-RNase could be required because it interacts with other very abundant components of the extracellular matrix. If this model is correct, then perhaps NaTTS, the 120 kDa glycoprotein, and NaMG-15 are these abundant components. Preliminary estimates suggest that, together with S-RNase, these glycoproteins constitute about 80% of the soluble protein in the style (CH Hancock, B McClure, unpublished data).
This model helps explain how S-RNase can be involved in rejecting pollen from SC species. There would be no pressure on such species to maintain a mechanism for interacting with S-RNase. However, if S-RNase is complexed with proteins involved in supporting pollen tube growth (i.e. glycoproteins such as NaTTS), then their pollen could be forced into association with it.
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Conclusion |
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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Specific pollen rejection systems have been very useful for gaining a molecular level appreciation for pollen–pistil interactions. S-RNase-based systems are widespread and function at both the interspecific and intraspecific levels. To understand them fully it is necessary to identify the non-S-RNase factors required to form a functional pollen rejection system.
Both genetic and biochemical approaches have been successful. Each approach has its advantages and will identify different types of factors. Genetic approaches are well suited to the identification of Group 1 or 2 factors, those that function only in pollen rejection, because the absence of such factors leads to failure of pollen rejection (i.e. compatibility). A biochemical approach has identified S-RNase binding proteins. These are putative Group 3 factors that may be involved in pollen rejection and in supporting compatible pollen tube growth.
The formation of complexes between S-RNase and compatibility factors has implications beyond pollen rejection. It suggests that pollen rejection systems and compatibility systems are networked. Perhaps they should simply be regarded as different aspects of a single system, one that provides fine control over plant fertilization.
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Work in the authors’ laboratories has been supported by the US National Science Foundation Grants 9604645 and 9982686, the University of Missouri Molecular Biology Program, and CONACYT grant J31752-N.
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TopAbstractIntroductionSelf-incompatibility systemsRNase-based self-incompatibilityS-RNase and interspecific pollen...Factors not linked to...S-RNase complexesImplications of S-RNase...ConclusionReferences |
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