†Present address: WiCell Research Institute, PO Box 7365, Madison, WI 53707, USA.
The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana
Article first published online: 8 AUG 2005
The Plant Journal
Volume 43, Issue 5, pages 716–723, September 2005
How to Cite
Nathan Hancock, C., Kent, L. and McClure, B. A. (2005), The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana. The Plant Journal, 43: 716–723. doi: 10.1111/j.1365-313X.2005.02490.x
- Issue published online: 8 AUG 2005
- Article first published online: 8 AUG 2005
- Received 18 March 2005; revised 29 May 2005; accepted 7 June 2005.
- 120 kDa glycoprotein;
S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia × SI Nicotiana alata (S105S105 or SC10SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors.
Plants possess a variety of physical and biochemical mechanisms to control pollination, of which self-incompatibility (SI) mechanisms are the best understood. In the Solanaceae, Roseacea and Scrophulariaceae the specificity of pollen rejection in SI is controlled by the S-locus (de Nettancourt, 1997). When the S-haplotype (e.g. SA2) of the haploid pollen matches either of the two S-haplotypes in the diploid pistil (e.g. SA2 + SC10), the pollen is inhibited (de Nettancourt, 1977). S-specific products are required on both the pollen and pistil sides. S-RNase determines specificity in the pistil and the S-locus F-box protein (SLF) determines specificity in the pollen (Lee et al., 1994; Murfett et al., 1994; Sijacic et al., 2004). Each S-haplotype encodes unique S-RNase and SLF genes. S-RNase is secreted into the extracellular matrix of the transmitting tract and it is thought to physically interact with SLF after uptake into pollen (Luu et al., 2000; Qiao et al., 2004). In an incompatible cross (i.e. S-RNase and SLF from the same S-haplotype) this interaction results in S-specific pollen rejection.
In addition to S-RNase and SLF, S-specific pollen rejection requires non-S-factors. For example, an SA2-RNase transgene expressed in self-compatible (SC) Nicotiana tabacum or SC Nicotiana plumbaginifolia does not cause rejection of Nicotiana alataSA2-pollen. Thus, these SC species lack one or more factors required for S-specific pollen rejection (Beecher and McClure, 2001). At present, HT-B is the only cloned non-S-factor known to be required for S-specific pollen rejection (McClure et al., 1999). Plants with suppressed levels of HT-B do not perform S-specific pollen rejection (McClure et al., 1999; O'Brien et al., 2002). Kondo et al. (2002a,b) also found that many SC Lycopersicon species do not express functional HT-B protein. They suggested that mutations in HT-B may have resulted in the transition from SI to SC (Kondo et al., 2002a,b).
S-RNase and non-S-factors are also implicated in interspecific pollen rejection. It is clear that N. plumbaginifolia pollen is rejected by an S-RNase-dependent mechanism. Untransformed [SC N. plumbaginifolia × SI N. alata (SA2SA2)] hybrids reject N. plumbaginifolia pollen. However, suppressing SA2-RNase expression in N. plumbaginifolia × SI N. alata (SA2SA2) prevents rejection of N. plumbaginifolia pollen (Murfett et al., 1996). Transgenic SC N. plumbaginifolia expressing SA2-RNase do not reject N. plumbaginifolia pollen (McClure et al., 2000). Thus, like S-specific pollen rejection, N. plumbaginifolia pollen rejection requires non-S factors. Nonetheless, the mechanisms are distinct, since, unlike the S-specific pollen rejection typical of SI, almost any S-RNase functions in the rejection of N. plumbaginifolia pollen. The role of HT-B in the rejection of N. plumbaginifolia pollen has not been previously reported.
The pistil also functions to promote the growth of desirable pollen. One group of pistil proteins that may contribute to this function are the arabinogalactan proteins (AGPs). AGPs are a diverse group of proteins characterized by their high carbohydrate content (often >90%) and binding to β-glucosyl Yariv reagent (Cassab, 1998; Yariv et al., 1962). They are implicated in cell growth and expansion, because they are often localized to the cell surface and some cell types do not grow when their AGPs are disrupted (Serpe and Nothnagel, 1999). Most AGPs are tethered to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (Schultz et al., 2004). A specialized class of AGPs that are abundant in the transmitting tract are not anchored by GPI but instead have a cysteine-rich C-terminal domain that is homologous to the major pollen allergen from Olea europaea, Ole e I (Bacic et al., 1988; Bosch, 2002; Schultz et al., 1997; Sommer-Knudsen et al., 1996; Villalba et al., 1993). One such AGP in N. tabacum and N. alata pistils, the transmitting-tract-specific (TTS) protein, has been shown to bind to pollen tubes, incorporate into the pollen tube wall and promote growth of the pollen tube in vitro (Cheung et al., 1995; Wu et al., 1995, 2000). Studies show that growth of the pollen tube is reduced when expression of TTS is suppressed in N. tabacum styles, suggesting that TTS supports the growth of the pollen tube in vivo (Cheung et al., 1995).
Two similar proteins, the pistil extensin-like protein III (PELP III) and the 120 kDa glycoprotein (120K), share sequence homology with the C-terminal Ole e I-like domain of TTS (Goldman et al., 1992; Schultz et al., 1997). These glycoproteins are classified as hybrid AGPs because they contain both extensin and AGP motifs (Bosch et al., 2001). Transgenic experiments in which PELP III was suppressed in N. tabacum showed no change in the behavior of the pollen tube even though it integrates into the callose of growing pollen tubes (Bosch et al., 2003; de Graaf et al., 2003). No transgenic experiments investigating the role of 120K in the function of the style have been reported, but immunolocalization studies indicate that 120K enters growing pollen tubes (Lind et al., 1996).
TTS, PELP III and 120K also comprise the most abundant class of S-RNase-binding proteins retained on S-RNase Affi-Gel (Cruz-Garcia et al., 2005). We hypothesized that these AGPs may be non-S-factors essential for S-specific pollen rejection. Lind et al. (1994) first reported that 120K exhibited size polymorphisms among Solanaceae species (Lind et al., 1994). We extended this study by examining variation of 120K in SI and SC Nicotiana species to determine if there was a correlation between 120K mobility and SI. The results suggest that 120K may indeed have a role in SI. Therefore, we used RNA interference (RNAi) to suppress 120K in SC N. plumbaginifolia × SI N. alata (SC10SC10 and S105S105) hybrids. We found that hybrids with little or no 120K fail to show S-specific pollen rejection but retain the ability to reject N. plumbaginifolia pollen. From this, we conclude that 120K has a role in S-specific pollen rejection.
We tested selected Nicotiana species for size polymorphisms among TTS-, PELP III- and 120K-like glycoproteins. Figure 1 shows that the NaTTS antibody detects a smear from about 55 to 120 kDa. The extent of the smear and of the staining intensity shows limited variability in different species and accessions. The NaTTS polypeptide has a predicted molecular weight of 27.5 kDa; differential glycosylation accounts for the wide variation in apparent molecular weight (Cheung et al., 1995; Sommer-Knudsen et al., 1996; Wu et al., 2000). The PELP III-like proteins are also variably glycosylated but show more size polymorphism among the tested species. A previous study using an antibody made to the C-terminal segment of PELP III showed no apparent size difference between N. alata and N. tabacum (de Graaf et al., 2004). The different results may be attributed to differences in antibody specificity, electrophoresis conditions or Nicotiana cultivars. The 120K-like proteins stain as relatively distinct bands that vary from about 80 to 120 kDa in different plant materials. Interestingly, extracts from some populations do not react with the Na120K antibody. For example, Nicotiana forgetiana accession 21A does not show a 120K-like band, but other N. forgetiana accessions do show appropriately sized immunoreactive bands (data not shown). We cloned a 120K-like cDNA from the former (see below). In general, the 120K from SI species displays lower mobility than the 120K from SC species. For example, 120K-like proteins from SC N. tabacum, SC N. plumbaginifolia and SC Nicotiana longiflora show a lower apparent molecular weight than the 120K from N. alata and Nicotiana bonariensis (Figure 1). In addition, the 120K-like protein from SC Nicotiana langsdorfii accession 28A migrates slightly faster than the 120K-like protein from SI N. langsdorfii accession 28B. The SC N. plumbaginifolia × SI N. alata (SC10SC10) hybrid expresses two 120K-like bands that correspond to the 120K from each parent. This suggests that the differences in apparent molecular weight result from differences in protein sequence rather than differences in the glycosylation machinery.
We cloned and sequenced 120K-like cDNAs to determine whether there are consistent differences between SI and SC species in Nicotiana. Figure 2 shows an alignment of the predicted amino acid sequences from eight species. N. alata and SI N. langsdorffii 28B express two 120K isoforms. Figure 2 shows the form most similar to the published sequence (Lind et al., 1996), but they also express a form missing residues 152–174 that is similar to the other species shown. The mature 120K-like proteins include variable proline-rich regions extending to approximately position 280 in the alignment, and a more conserved cysteine-rich C-terminal domain of about 138 residues. The variable proline-rich region from SC species contains numerous additional indels and amino acid replacements when compared with the SI species. The cysteine-rich domains are more conserved except for a 10 amino acid deletion in the SC N. plumbaginifolia and SC N. longiflora sequences. The N. forgetiana 120K-like protein has an amino acid substitution in the area recognized by the 120K antibody that may prevent detection by immunostaining (Figure 1). None of the sequence differences between SI and SC accessions consistently correlate with pollination behavior. Moreover, the changes in amino acids alone (i.e. changes in peptide length) are not sufficient to explain the observed differences in apparent molecular weight.
S-specific pollen rejection
Since 120K-like proteins showed the most polymorphism between SI and SC species we used RNAi (Wesley et al., 2001) to test for a direct role in S-specific pollen rejection. We identified a 120 bp region of the gene for 120K with minimal homology to the genes for PELP III and TTS (Figure 3a). We transformed SC N. plumbaginifolia and crossed the resulting T0 plants with SI N. alata (S105S105 and SC10SC10). Figure 3(b) shows that suppression was largely restricted to 120K. Results are shown for progeny of four independent T0 plants (i.e. ZE, ZG, ZW and BA). While hybrids ZE × S105S105 and ZG × S105S105 show partial suppression of 120K, hybrids ZW × S105S105, BA × S105S105 and BA × SC10S105 are fully suppressed (i.e. show no detectable 120K). The latter hybrids also show a slight reduction in levels of PELP III and TTS.
Plants with suppressed 120K are deficient in S-specific pollen rejection. Compatibility was assessed semiquantitatively by counting the number of pollen tubes that reached the base of the style 60 h after pollination with SC10- and S105-pollen (Figure 4). The untransformed control hybrid allowed two or less S105-pollen tubes to reach the base of the style, but in a compatible cross many (i.e. 65 or more) SC10-pollen tubes were observed (Table 1). Transformed hybrids with partial suppression of 120K were similar to controls, but hybrids with no detectable 120K showed large numbers (about 50) of otherwise incompatible pollen tubes at the base of the style (Table 1, Figure 4). Although the number of otherwise incompatible pollen tubes was slightly lower than the number of fully compatible pollen tubes, it is clear that S-specific pollen rejection does not occur in the absence of 120K. Thus, like HT-B, 120K is a non-S-RNase factor required for S-specific pollen rejection.
N. plumbaginifolia pollen rejection
Since it has been shown that non-S factors are also required for interspecific pollen rejection, we tested HT-B and 120K for roles in N. plumbaginifolia pollen rejection. Figure 5 shows that the N. plumbaginifolia pollen tubes do not reach the base of the untransformed control style [SC N. plumbaginifolia × SI N. alata (SC10SC10)] 72 h after pollination. This rejection mechanism inhibits N. plumbaginifolia pollen tubes at 1 ± 0.2 cm. Hybrids with suppressed HT-B allowed the N. plumbaginifolia pollen tubes to reach the base of the style (about 3.8 cm). The SC N. plumbaginifolia × SI N. alata (SC10SC10) hybrids with fully suppressed 120K were similar to the control, only allowing the N. plumbaginifolia pollen tubes to grow 1.6 ± 0.2 cm (ZW) and 1.1 ± 0.3 cm (BA).
We regarded TTS, 120K and PELP III as good candidates for non-S-RNase factors that contribute to controlling pollination because they bind to S-RNase in vitro and interact with growing pollen tubes (Cheung et al., 1995; Cruz-Garcia et al., 2005; de Graaf et al., 2003; Lind et al., 1996). TTS and PELP III-like proteins did not display a great deal of polymorphism (Figure 1). 120K showed the most polymorphism in our experiments (Figure 1), and, moreover, it has been shown to enter pollen tubes (Lind et al., 1996). Thus, 120K emerged as the best candidate to have a specific role in pollen rejection.
The sequence differences (i.e. indels) of 120K-like proteins do not account for the large differences in mobility (Figure 2). This suggests that the size polymorphism results from differences in glycosylation. In addition to alteration in the glycosylated domain, SC N. plumbaginifolia and SC N. longiflora have a 10 amino acid deletion in the cysteine-rich domain. Other proteins containing the Ole e I domain, including Ole e I and LAT52, also contain this deletion (Twell et al., 1989; Villalba et al., 1993); thus, it would not be expected to inhibit protein folding or function.
Control pollinations show that suppression of 120K has no effect on compatible pollinations; thus, it is not absolutely required for growth of the pollen tube (Table 1, Figure 4). However, the results clearly show that suppression of 120K interferes with normal S-specific pollen rejection. We observed this effect in progeny from two independent transformants (ZW and BA, Table 1, Figure 4) that failed to reject two different S-haplotypes (i.e. SC10- and S105-pollen). Since the partially suppressed hybrids behaved similarly to controls, this suggests that high-level expression of 120K is not required for S-specific pollen rejection. This is somewhat surprising since 120K-like proteins are very abundant – and yet, fully suppressed hybrids clearly fail to show S-specific pollen rejection.
S-RNase-dependent rejection of N. plumbaginifolia pollen is similar to S-specific pollen rejection in many respects (Murfett et al., 1996). We showed that HT-B is required for both of these pollen rejection mechanisms and that 120K is not required for N. plumbaginifolia pollen rejection. While the role of HT-B in pollen rejection is unknown, these results suggest that it has a more general function in pollen rejection than 120K. It is also possible that PELP III or another similar protein can substitute for 120K in pollen rejection in N. plumbaginifolia.
Despite the homology between 120K, TTS and PELP III, these proteins do not have entirely redundant functions. Sequence analysis of this protein group does not immediately suggest the structural basis for the functional differences. In addition to sequence differences at the protein level, there are differences in glycosylation. SDS-PAGE analysis indicates that, unlike TTS and PELP III, 120K is relatively uniformly glycosylated and that it runs as a distinct band. At this point we cannot speculate on the specific roles of the glycan and protein moieties in 120K. Further experiments such as domain substitution or deletion may help resolve this question.
Our goal is to develop a comprehensive model of the SI mechanism by identifying all the factors required for S-RNase-dependent pollen rejection. It is now established that S-RNase and SLF determine the specificity of pollen rejection in SI (Sijacic et al., 2004). We and others have shown that HT-B is also required (McClure et al., 1999; O'Brien et al., 2002). Here, we showed that 120K is similarly required, but neither HT-B nor 120K contributes directly to the specificity of S-specific pollen rejection.
N. alata (SC10SC10 and S105S105 genotypes), N. tabacum cv. Praecox and N. plumbaginifolia (43B) were described previously (Beecher and McClure, 2001; Murfett et al., 1994). N. langsdorfii (inventory no. TW74, accession 28A), N. langsdorfii (inventory no. TW75, accession 28B), N. bonariensis (inventory no. TW28, accession 11), N. forgetiana (inventory no. TW50, accession 21A) and N. longiflora (inventory no. TW79, accession 30A) were obtained from the US Tobacco Germplasm Collection (Crops Research Laboratory, Oxford, NC, USA). Antisense HT-B hybrids were described previously (McClure et al., 1999).
Styles were weighed, ground in 4× SDS-PAGE loading buffer (Barker, 1998), boiled and centrifuged to remove debris. Extracts equivalent to 1.5 mg fresh weight were separated in 7.5% Tris-tricine gels (Schägger and von Jagow, 1987). Proteins were electroblotted and immunostained as described (Cruz-Garcia et al., 2005; Harlow and Lane, 1988).
Cloning and sequencing
Polyadenylated RNA was prepared from mature styles. cDNA was prepared using the SMART cDNA library construction kit (BD Biosciences, Palo Alto, CA, USA). The following oligonucleotides were used for PCR amplification of 120K homologs: 120F-CCTCTAATCATCGTCGGCCATGT, 120R-GGTCTTTCTAATAATGAAGAGCTCG, con C-term-GCACCAGATTTTCCACCATTGAAGTTTGTTGGGACAT, mid N-term-TCTCCCAAGAAAAGCCCCTCTAGCCCTACA, rev N-term-CTGAGCAGCCGGTGACGGAGA, repeat rev-GGAGGTGATGATCTAATAGGTGGTGGAGGTGGC, whole N-term-ACATGGCCGACGATGATTAGAGG, N1F-TGAAAGAATCGAAAGATCGTTAATAGACAAAGGTCA, N1R-TTGTTTACCGGTGTAGGGCTAGAGGGG, N2F-TCACCACCACCACAGGTTAAGTCGTCC, N2R-ATTGTTTTGGTGGTTGAACTGGTGGCG. PCR fragments were cloned using the pGEM vector (BD Biosciences). Sequence alignment was performed using the programs clustal w (Thompson et al., 1994) and align (Pearson, 1990) administered by the San Diego Supercomputer Center (http://workbench.sdsc.edu).
RNAi construct and plant transformation
A 120 bp sequence near the 5′ end of the 120K transcript was amplified with the N1F and N1R primers and cloned into pHANNIBAL (CSIRO Plant Industry, Canberra, Australia) (Wesley et al., 2001) as shown in Figure 3(a). N. plumbaginifolia was transformed as described (Beecher, 1999) except that the shooting and co-cultivation media contained 10 μg ml−1 6-(3-methyl-2-butenylamino) purine (2iP) and 0.3 μg ml−1 indole-3-acetic acid.
Mature flowers were pollinated with N. plumbaginifolia or N. alata (genotype S105S105 or SC10SC10) pollen. Pistils were stained with decolorized aniline blue as described (Kho and Baer, 1968) and photographed on an Olympus IX-170 microscope. Pollen tubes were counted using a Leica MZFLIII stereoscope. The length of the pollen tube was measured from the top of the stigma to where fewer than 10 pollen tubes could be seen. Pollinations were repeated five or more times.
Special thanks to Dr T. Holtsford for helpful advice, Dr Z. Zhang for assistance with plant transformation and Dr Katsuhiko Kondo for assistance with Figure 3. We thank Melody Kroll for assistance in preparing the manuscript. This work was supported by NSF grants 99-82686 and 03-15647, the NIH training grant GM08396-11 and the senior Monsanto graduate fellowship.
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