JXB Advance Access published online on February 10, 2008
Journal of Experimental Botany, doi:10.1093/jxb/erm339
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Glycosylation of S-RNases may influence pollen rejection thresholds in Solanum chacoense
IRBV, Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, 4101 Sherbrooke est, Montréal, Québec, Canada H1X 2B2
* To whom correspondence should be addressed. E-mail: mario.cappadocia{at}umontreal.ca
Received 13 August 2007; Revised 27 November 2007 Accepted 30 November 2007
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Abstract |
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A survey of Solanum chacoense plants expressing an authentic S11-RNase transgene identified a line with partial compatibility to S11 pollen. By comparing fruit set to the S-RNase levels determined immunologically in single styles, the minimum level of S11-RNase required for full rejection of S11 pollen was estimated to be 18 ng per style. The S11-RNase threshold levels are thus considerably lower than those previously reported for the S12-RNase. Interestingly, these two allelic S-RNases differ dramatically in the extent of glycosylation, with the number of glycosylation sites varying from one (S11-RNase) to four (S12-RNase). It is suggested that reduced glycosylation of the S11-RNase may be related to the lower threshold for pollen rejection.
Key words: Gametophytic self-incompatibility, glycosylation, pistil-by-pistil analysis, S-RNase, Solanum chacoense, threshold
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Introduction |
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Self-incompatibility (SI) is a mechanism widespread among flowering plant species that promotes outbreeding by allowing the pistil of a flower to discriminate between genetically related (self) and unrelated (non-self) pollen (de Nettancourt, 1977). In Solanaceae, Rosaceae, and Scrophulariaceae, SI is of the gametophytic type, i.e. the phenotype of the pollen is determined by its haploid genotype and is controlled by a single multigenic S-locus, which includes the highly polymorphic male and female determinants to SI. The S-locus is inherited as a single segregating unit, with variants of the locus termed S-haplotypes and variants of the polymorphic genes residing in the S-locus called alleles (McCubbin and Kao, 2000).
In the families mentioned above, the pistil-expressed S-gene product is a secreted glycoprotein with RNase activity termed S-RNase (McClure et al., 1989) produced by the cells of the transmitting tract of the style and released in the extracellular matrix, where the pollen tubes grow. S-RNases possess a defined pattern of conserved (C1 through C5) and hypervariable (HVa and HVb) regions, and all S-RNases are glycosylated at a variable number of potential N-glycosylation sites. S-RNases from N. alata have been found to contain from one to five sites (Oxley et al., 1998) while those from S. chacoense contain from one to four sites (Qin et al., 2001). The one site conserved in all cases is located in the conserved C2 region in the Solanaceae (Singh and Kao, 1992), whereas in the Rosaceae it is located in the conserved RC4 region (Ishimizu et al., 1998). In spite of the fact that S-RNases have been studied for almost 20 years, the biological function of the glycosylation, and in particular that of the conserved glycosylation sites is still unknown. What is known, however, is that the enzymatic removal of the glycan side chains has apparently no effect on the enzyme activity of the native S-RNases in vitro (Broothaerts et al., 1991). It is also clear that glycosylation is not required for pollen rejection in vivo, as when the C2-glycosylation site of the Petunia hybrida S3-RNase was eliminated by site-directed mutagenesis, the mutated S3-RNase was able to reject S3 pollen totally (Karunanandaa et al., 1994). S-RNases must exert their cytotoxic activity inside the incompatible pollen tubes for pollen rejection to occur, since mutation of essential histidine residues within the active site produces inactive proteins with no RNase activity that are unable to elicit pollen rejection (Huang et al., 1994). In S. chacoense, immunolocalization studies have revealed that S-RNases enter the cytoplasm of both self and non-self pollen tubes (Luu et al., 2000), so at least part of the SI mechanism must involve the ability of pollen tubes to block the RNase activity of any non-self S-RNase.
The S-locus product expressed in pollen has been recently identified as an F-box protein, termed either S-locus F-box gene (SLF) or S-haplotype-specific F-box gene (SFB) (for details see Kao and Tsukamoto, 2004; McClure and Franklin-Tong, 2006). The usual role assigned to the F-box protein is to mediate protein–protein interactions, which in the context of the E3–ubiquitin ligase complex SCF, helps determine the specificity of the proteins targeted for degradation by the 26S proteasome (Bai et al., 1996). Both male and female determinants to SI have now been identified, and the current challenge is to determine how these molecules interact, and to explain how pollen growth is inhibited in an S-haplotype-specific manner. The more traditional models suggest that SLF/SFB interacts with both self and non-self S-RNases inside the pollen tubes. The maintenance of self S-RNase enzyme activity is proposed to occur via S-haplotype-specific interactions between their hypervariable regions and those of their cognate SLF/SFB partners, while all non-self S-RNases are targeted for ubiquitination and subsequent degradation (for details see Kao and Tsukamoto, 2004). A more recent model, based on the observation that S-RNases remain in the vacuole in compatible crosses and are only released into the pollen tube cytoplasm during incompatible pollinations, proposes S-RNase sequestration as a key element in pollen rejection (Goldraij et al., 2006). A further model is based on the finding that S-RNases bind more strongly to non-self SLFs than to self SLFs (Hua and Kao, 2006). This suggests that S-RNases enter the pollen tubes cytoplasm and are targeted for degradation by strong binding to the E3 ligase complex during compatible crosses, but remain free to degrade the RNA during incompatible crosses.
A number of factors are known to influence the pollen rejection phenotype, including the presence of other stylar factors, such as the small non-polymorphic HT-B (for details see Cruz-Garcia et al., 2005). However, the observation that different amounts of S-RNase can influence the pollen rejection phenotype has been made repeatedly in the literature (Clark et al., 1990; Lee et al., 1994; Murfett et al., 1994; Matton et al., 1997, 1999; Qin et al., 2001, 2005, 2006; Hiratsuka and Zhang, 2002) and has led to the S-RNase threshold hypothesis. This hypothesis posits that a minimum level of S-RNase is required for pollen rejection, and has been validated for the S12-RNase of S. chacoense using genotypes displaying sporadic self-compatibility (Qin et al., 2001). The observation that two S. chacoense genotypes expressing an S12-RNase, namely 314 and L25, displayed sporadic self-compatibility (Qin et al., 2001), followed by a style-by-style analysis of their S12-RNase levels (Qin et al., 2006), demonstrated that a variable S12 pollen rejection phenotype could be exploited to estimate the threshold, or the minimum level, of the S12-RNase required for pollen rejection. These studies showed that at least 80 ng of the S12-RNase are required for full rejection of pollen from S12 homozygous plants. Furthermore, since only 25 ng of S12-RNase is required to reject pollen from an S11S12 background, it appears that the RNase threshold differs when pollen donors of different genetic backgrounds are used (Qin et al., 2006). Those analyses are extended here to report the determination of S11-RNase threshold by means of style-by-style analysis of a transgenic plant line expressing low levels of the S11-RNase (Matton et al., 1997).
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Materials and methods |
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Plant materials and genetic crosses
The plants used in this study include two genetic lines of Solanum chacoense called 314 (S11, S12) and L25 (S11, S12) previously noted for their sporadic self-compatibility (Qin et al., 2001) and two transgenic plants called T12 and T35, which are a G4 (S12, S14) host line expressing an authentic S11-RNase construct from the chitinase promoter (thus with a stylar haplotype S11, S12, S14) (Matton et al., 1997). S12 thresholds for 314 pollen in L25 styles and S11 thresholds in T12 styles were determined as described (Qin et al., 2006).
In order to ensure that only flowers at an identical stage of development were used for both crosses and S-RNase quantification in styles, all open flowers were removed the afternoon preceding crosses and the collection of styles. Genetic crosses were made with freshly collected pollen from plant material grown in the Montreal Botanical Garden greenhouses under natural light conditions, and temperatures were constantly recorded. Pollen viability was estimated by staining with aceto-carmine.
Bacterial expression of S11- and S12-RNases standards
The cDNA fragment encoding S11-RNase was cloned by RT-PCR according to the manufacturer's instruction (Fermentas Inc. MD) using primers 5'-ATGTTTAAATCACTGCTTAC-3' and 5'-TCAAGGACGAAAAAATATTTT-3'. This fragment, a wild type S11-RNase sequence without the N-terminal signal peptide (22 amino acids), was subcloned into the expression vector pQE30 (Qiagen, Valencia, CA). The sequence was confirmed by sequencing and named pQE30-S11-HisWT. For the S12-RNase, the previously described clone pQE30-S12-His
C2, which lacks an essential histidine residue in the active site, was used (Qin et al., 2006). Two plasmids were transformed separately into competent M15 cells and protein expression induced by adding IPTG to a final concentration of 1 mM. The cultures were grown at 37 °C or 28 °C for 4–5 h before the cells were harvested. The target proteins were purified with Ni-NTA resin (Qiagen, Maryland, USA), electrophoresed on SDS-PAGE, and then eluted from gel slices using an Electro-Elutor (Bio-Rad, California, USA) following the manufacturer's protocol. Both RNases were pure, based on Coomassie blue staining after SDS-PAGE and western blot analysis. The concentration of purified S11 and S12 proteins was determined by the Micro BCA Protein Assay Kit (Pierce Inc., Illinois, USA) following the manufacturer's instructions using BSA as standard. One batch of each purified protein was used as the standard for all gels to calibrate S-RNases in styles of S. chacoense.
Western blots and quantification analysis of S-RNases in individual styles
For western blot analysis, plant styles were collected and immediately frozen in liquid nitrogen. The western blots and quantification analysis on individual styles were performed essentially as described (Qin et al., 2006). Briefly, proteins were extracted from each individual style in 50 µl extraction buffer (0.05 M TRIS pH 8.0, 1 mM DTT, 1 mM EDTA, 0.05 M CaCl2, 1 mM PMSF). The proteins were electrophoresed on SDS–PAGE and transferred to nitrocellulose membrane and stained with 2% (w/v) Ponceau red as a control for a uniform protein loading and transfer; S-RNase measurements from samples with visibly different protein loads were excluded from our analyses. The membrane was blocked by an overnight incubation with 1% w/v BSA fraction V (Sigma) and 2% skimmed milk in TBS-T (TRIS-buffered saline containing 0.05% Tween 20 v/v), incubated for 2 h at room temperature with a 1:1000 dilution of either polyclonal anti-S11 (Matton et al., 1999) or anti-S12 (Qin et al., 2001), rinsed three times with TBS-T, and incubated with 4 µl of 0.5 µCi mmol–1 I125 of a goat anti-rabbit secondary antibody (Perkin-Elmer) in 10 ml BSA-TBS for 2 h. After washing three times with TBS-T, membranes were exposed to a Phosphor screen for 12–76 h at room temperature and the screen imaged using a PhosphorImager scanner (Amersham Biosciences). The data on the scanner images were quantified using software supplied by the manufacturer. Both antibodies used are specific for their respective substrates and no cross-reaction was detected.
Protein analysis and deglycosylation reaction
Proteins were extracted from each individual style in 50 µl deglycosylation buffer (20 ml TRIS–HCl pH 7.4, 20 mM 2-mercaptoethanol, 150 mM NaCl, 1 mM MnCl2, 1 mM CaCl2, 1% Triton X100, 1 mM PMSF, added just before use) (Matton et al., 1997). The N-linked glycan side-chains were enzymatically removed by digestion with Peptide:N-Glycosidase F (PNGase F; New England Biolabs) according to the manufacturer's instructions typically using 500 units PNGase F for every 50 µl style sample. NP-40 was added to a final concentration of 1%, the samples denatured at 100 °C for 10 min and then incubated with l µl (500 units) PNGase F at 37 °C for 3 h. For partial digestion of the glycan side chains, differing amounts (varying from 0.5 to 500 units) of PNGase F were added to the reaction. Reactions were stopped by heating at 100 °C for 5 min with 4x SDS loading buffer. To determine the migration rate and molecular weight of S-RNases, prestained markers (Bio-Rad, CA) were mixed directly with each protein sample. Protein sequences were analysed for N-linked glycosylation sites with the NetNGlyc 1.0 Server at http://www.cbs.dtu.dk/services/NetNGlyc/.
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The minimum level of S11-RNase required for pollen rejection is very low
In a previous study (Qin et al., 2006), sporadic self compatibility of an S12 allele in some genetic backgrounds was exploited to estimate the threshold levels of an S-RNase required for pollen rejection. However, this approach could not be used to estimate the S11-RNase threshold because all naturally occurring S11 plants fully reject S11 pollen. Indeed, even bud pollinations of these plants conducted up to three days before anthesis failed to produce any S11S11 progenies. The lowest amount of S11-RNase measured in these immature buds was 53 ng, suggesting that the threshold for the S11-RNase could be significantly lower than that of the S12-RNase. To address this issue using the same logic used for the S12-RNase, a series of transgenic lines expressing the S11-RNase that had been generated previously were re-examined (Matton et al., 1997). Our attention was drawn to a line (T12) noted for its low levels of mRNA of S11-RNase and partial compatibility (two fruits out of 15 pollinations) (Matton et al., 1997). In order to assess if T12 continued to display partial compatibility towards the S11 pollen, a preliminary test was conducted. When crossed with pollen from line 314 (S11, S12), two fruits were obtained out of 10 pollinated flowers. Since the S12 pollen from pollen donor line 314 is fully rejected by the endogenous S12-RNase of the host plant used for transgenosis (Matton et al., 1997), any observed compatibility is solely due to the passage of S11 pollen. Line T12 is thus partially compatible for the S11-RNase.
The minimum level of S11-RNase required for pollen rejection is pollen-genotype-dependent
Previous work with the S12-RNase indicated that different pollen genotypes respond differently to a given amount of S-RNase (Qin et al., 2006). Thus, T12 was also pollinated with pollen from line L25, which has the same S-constitution (S11, S12) as line 314 but a different genetic background. In an experiment involving 12 crosses with each pollen type, five fruits were obtained using pollen from line 314, whereas no fruits at all were set with L25 pollen. It was concluded from this that the ability to resist rejection by S11-RNase does indeed depend on the genetic background of the pollen, in agreement with previous observations with the S12-RNase (Qin et al., 2006). In addition, since the phenomenon has now been observed both in plants carrying the natural occurring S12-RNase and in transgenics with the S11-RNase, it is likely to be a general feature of SI. This observation also suggests that the threshold for pollen rejection is likely to be lower for L25 than for 314 pollen, since pollen from L25 was completely rejected while pollen from 314 was partially compatible.
In order to test the relationship between the partial compatibility of the T12 line toward S11 pollen from line 314 and the levels of S11-RNase in the styles, some styles were harvested for protein analysis at the same time as other flowers were pollinated. As a control plant, styles were also taken from the T35 line that is fully incompatible with S11 pollen (Matton et al., 1997). The levels of S11-RNase in line T35 were uniformly high (Fig. 1A) and more than 30-fold higher than those measured in the partially compatible line T12 (Fig. 1B). It is concluded from this that the partial compatibility of the T12 line is correlated with insufficient accumulation of S11-RNase in the styles.
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To estimate the minimum amount of stylar S-RNase required for pollen rejection, the range of S11-RNase values in Fig. 1B were redrawn in ascending order (Fig. 2A). In this representation, a vertical dotted line is placed at a position where the number of plants with low S-RNase levels (to the left of the line) roughly corresponds to the percentage of fruits formed using plants pollinated in parallel with the protein analysis (three fruits from 16 pollinations). The minimum amount of S11-RNase needed for pollen rejection is thus estimated as
18 ng from this analysis. To test the validity of this estimate, the entire experiment was repeated three additional times (Fig. 2B–D). In each case, the number of styles with less than 18 ng S11-RNase is in good agreement with the pollen rejection phenotype as determined by fruit set with 314 pollen (Table 1). The average S-RNase levels in the four experiments also shows a good correlation to the degree of incompatibility (Fig. 2E). Interestingly, as was found for the S12-RNase, pollen genotype affects the threshold estimates. For example, the lowest value of S11-RNase that was detected during pollinations with L25 pollen (where no fruits were set) was 11 ng per style, indicating that this value still lies above the threshold level for this pollen genotype (Table 2).
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The glycosylation state of S11 and S12-RNases differs
One intriguing difference between the S11 and S12-RNases lies in their apparent size on SDS–PAGE (Fig. 3A). Indeed, while the molecular weight of the mature S11 and S12-RNases predicted from their cDNA sequences are very similar (22.6 kDa and 22.7 kDa, respectively), the proteins migrate with quite different apparent molecular weights (27 and 36 kDa, respectively). This difference results principally from differential glycosylation, as while complete removal of the sugar side chains with PNGase increases the mobility of both proteins, the difference in size between treated and untreated protein is greater for the S12-RNase than for the S11-RNase. Indeed, assuming a roughly 2 kDa size difference per sugar side chain, the two S12-RNase bands could be interpreted as glycosylation at either three or all four different sites predicted from the sequence. To test this, partial digestion of the glycan side chains was performed using decreasing dilutions of PNGase. By this method, the presence of four different glycosylated forms can be observed depending on the PNGase concentration used (Fig. 3B). These forms agree perfectly with the four glycosylation sites predicted from the primary structure (Fig. 3C), and confirm that each sugar group contributes
2 kDa to the protein apparent MW (Fig. 3D).
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Discussion |
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Our results using the transgenic line T12 with its partial rejection of S11 pollen have allowed the threshold for S11-RNase to be determined for two pollen types. These results clearly show that different S-RNases can have different thresholds for pollen rejection as, for pollen from a given donor genotype, there is a marked difference in the thresholds for the S11- and the S12-RNase. For example, rejection of pollen from line 314 (S11, S12) requires
40 ng of S12-RNase but only 18 ng of S11-RNase (Table 2). It is also clear that, for a given S-RNase, there is a difference in the ability of the pollen from plants of different genetic backgrounds to resist the cytotoxic effects of the RNase (Table 2). For example, while 18 ng of S11-RNase is required to reject pollen from line 314, less than 11 ng of S11-RNase appear sufficient to reject pollen fully from line L25. It is interesting, however, that the differences in pollen type appear to result in similar threshold differences for both S11- and S12-RNases. One such factor could be a general S-RNase inhibitor capable of binding any S-RNase as proposed previously (Luu et al., 2001) or, according to a more recent model for S-RNase-based SI, an inhibitor blocking the action of other factors playing an essential role in SI (McClure and Franklin-Tong, 2006).
It is tempting to attribute the different thresholds of S11- and S12-RNases required for pollen rejection to differential glycosylation. Clearly, the S12-RNase is more extensively glycosylated than the S11-RNase, as PNGase treatment decreases the apparent molecular weight of the S11-RNase by 2 kDa, versus the 9 kDa lost during deglycosylation of the S12-RNase. This difference reflects the larger number of glycan side chains in the S12-RNase. It is possible that a larger, more heavily glycosylated S-RNase may experience increased difficulty in penetrating into the pollen tube cytoplasm. For S. chacoense, it has previously been shown by immunoelectron microscopy that the S11-RNase accumulates inside pollen tubes to levels roughly 5-fold higher than those found in the transmitting tract (Luu et al., 2000). This hypothesis predicts that the S12-RNase would accumulate at lower levels in pollen tubes, and it would be interesting to test our anti-S12-RNase antibody to see if signal could be detected on cell sections.
Interestingly, recent microscopic observations using Nicotiana alata indicate that S-RNases accumulate in the pollen tube vacuole and are released into the cytoplasm as an all-or-nothing response to the presence of a cognate pollen partner to the SI system (Goldraij et al., 2006). Thus, while this model still assumes that cytotoxic effects of the S-RNase cause pollen rejection, differential release of S-RNases from the vacuole into the cytoplasm is unlikely to be the cause of the different thresholds. Indeed, it is likely that all S-RNases found in the vacuole would be released concurrently from vacuoles where the HT-B modifier protein had been stabilized (by as yet undefined components) during an incompatible cross. To date, it is not known if the presence of additional oligosaccharide side chains may interact with modifier proteins such as the small asparagine-rich HT-B (McClure et al., 1999) or the large 120 kDa glycoprotein (Hancock et al., 2005), although the 120 kDa protein has been shown to bind S-RNase in vitro (Cruz-Garcia et al., 2005).
In this study, the choice was made to analyse S-RNase levels as they can be quantitatively measured style-by-style, and it is clear from our results that the threshold amount of S-RNase required for pollen rejection is lower for the S11- than for the S12-RNase. However, our proposal that the extent of glycosylation influences the S-RNase threshold does have a number of important caveats. First, the specific activity of the S11- and S12-RNases has not been directly measured and may differ between the two. Different specific activites have been shown for S-RNases in Petunia (Broothaerts et al., 1991; Karunanandaa et al., 1994) and Nicotiana (McClure et al., 1989), although nothing is yet known for S-RNases in S. chacoense. Interestingly, as some of these studies also show that deglycosylation does not alter the specific activity of several different S-RNases (Broothaerts et al., 1991), it seems reasonable to conclude that the extensive glycosylation of the S12-RNase would not appreciably reduce its enzyme activity. Second, it is noted that the transgenic plants expressing the S11-RNase represent a genetic background in which the S12-RNase is fully incompatible, and for which the threshold thus cannot be ascertained. It is therefore a formal possibility that in this genetic background, the activity of modifier proteins such as HT-B and the 120 kDa protein may be different. Indeed, the role played by HT-B or the 120 kDa protein has not yet been characterized in S. chacoense. SI is a complex system, and until additional molecular probes become available, many of the complexities may pass unnoticed unless revealed by exceptional genotypes. The two sporadically compatible plants which allowed determination not only of the S12-RNase threshold but also the previously unsuspected influence of the pollen genetic background on the threshold represent an excellent example of this (Qin et al., 2006).
Finally, one of the most surprising aspects of the low S11-RNase threshold shown here is that the 1000 ng S11-RNase in the styles can exceed the required threshold by over 50 times. This vast excess of S-RNase may act as a reinforcing mechanism of the pollen rejection phenotype, and could help explain why self-compatible genotypes do not seem to accumulate in natural populations (Richman and Kohn, 1996). It would also explain the observation that S-RNase-based SI characteristically appears as a qualitative rather than a quantitative genetic trait, as an excess of S-RNase would ensure that the switch-like behaviour of the SI response at threshold levels would never be observed. At present, the generalization of this type of S-RNase excess in natural populations is not known. Clearly, much more work is required to understand fully the intricacy of the SI system.
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Acknowledgements |
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We are grateful to G Teodorescu for plant care. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (MC).
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References |
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