Stylar glycoproteins bind to S-RNase in vitro

S-RNases determine the specificity of S-specific pollen rejection in self-incompatible plants of the Solanaceae, Rosaceae, and Scrophulariaceae. They are also implicated in at least two distinct types of unilateral interspecific incompatibility in Nicotiana. However, S-RNase itself is not sufficient for most types of pollen rejection, and evidence for its direct interaction with pollen tubes is limited. Thus, non-S-RNase factors also are required for pollen rejection. As one approach to identifying such factors, we tested whether SC10-RNase from Nicotiana alata would bind to other stylar proteins in vitro. SC10-RNase was immobilized on Affi-gel, and binding proteins were analyzed by SDS-PAGE and immunoblotting. In addition to SC10-RNase and a small protein similar to lily chemocyanin, the most prominent binding proteins include NaTTS, 120K, and NaPELPIII, these latter three being arabinogalactan proteins previously shown to interact directly with pollen tubes. We also show that SC10-RNase and these glycoproteins migrate as a complex in a native PAGE system. Our hypothesis is that S-RNase forms a complex with these glycoproteins in the stylar ECM, that the glycoproteins interact directly with the pollen tubes and thus that the initial interaction between the pollen tube and S-RNase is indirect.     

RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen

Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives. For example, SI Nicotiana alata rejects pollen from SC N. plumbaginifolia, but the reciprocal pollination is compatible. This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection. However, the two systems differ in degree of specificity. In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype. Rejection of N. plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase. Here, we have tested whether a non-S-RNase can cause rejection of N. plumbaginifolia pollen. The Escherichia coli rna gene encoding RNaseI was engineered for expression in transgenic (N. plumbaginifolia × SC N. alata) hybrids. Expression levels and pollination behavior of hybrids expressing E. coli RNaseI were compared to controls expressing S_A2-RNase from N. alata. Immunoblot analysis and RNase activity assays showed that RNaseI and S_A2-RNase were expressed at comparable levels. However, expression of S_A2-RNase caused rejection of N. plumbaginifolia pollen, whereas expression of RNaseI did not. Thus, in this system, RNase activity alone is not sufficient for rejection of N. plumbaginifolia pollen. The results suggest that S-RNases may be specially adapted to function in pollen rejection.     

The S-locus and unilateral incompatibility

Plants have many ways to regulate the type of pollen that arrives on the stigma surface. Once there, further control mechanisms regulate compatibility. The latter controls are largely based on biochemical interactions that support compatible pollination and prevent incompatible matings. S-RNase-based self-incompatibility (SI) systems are the most phylogenetically widespread mechanisms for controlling pollination. Studies of Nicotiana establish a firm link between SI and unilateral interspecific incompatibility. Although implicated in both inter- and intraspecific compatibility, S-RNase operates through at least three distinct genetic mechanisms that differ in their dependence on non-S-RNase factors. Identification and characterization of these non-S-RNase factors is currently an area of active research. Searching for genetic and biochemical interactions with S-RNase can identify candidate non-S-RNase factors. HT-protein is one factor that is required for S-allele-specific pollen rejection in the Solanaceae. Major style arabinogalactan proteins such as TTS interact biochemically with S-RNase. These glycoproteins are known to interact with compatible pollen tubes and have long been suggested as possible recognition molecules. Their binding to S-RNase implies a link between stylar systems for compatibility and incompatibility. Thus, genetic and biochemical studies suggest a highly networked picture of pollen-pistil interactions.     

S RNase and Interspecific Pollen Rejection in the Genus Nicotiana: Multiple Pollen-Rejection Pathways Contribute to Unilateral Incompatibility between Self-Incompatible and Self-Compatible Species

In self-incompatible (SI) plants, the S locus acts to prevent growth of self-pollen and thus promotes outcrossing within the species. Interspecific crosses between SI and self-compatible (SC) species often show unilateral incompatibility that follows the SI x SC rule: SI species reject pollen from SC species, but the reciprocal crosses are usually compatible. The general validity of the SI x SC rule suggests a link between SI and interspecific pollen rejection; however, this link has been questioned because of a number of exceptions to the rule. To clarify the role of the S locus in interspecific pollen rejection, we transformed several Nicotiana species and hybrids with genes encoding SA2 or SC10 RNase from SI N. alata. Compatibility phenotypes in the transgenic plants were tested using pollen from three SC species showing unilateral incompatibility with N. alata. S RNase was implicated in rejecting pollen from all three species. Rejection of N. plumbaginifolia pollen was similar to S allele-specific pollen rejection, showing a requirement for both S RNase and other genetic factors from N. alata. In contrast, S RNase-dependent rejection of N. glutinosa and N. tabacum pollen proceeded without these additional factors. N. alata also rejects pollen from the latter two species through an S RNase-independent mechanism. Our results implicate the S locus in all three systems, but it is clear that multiple mechanisms contribute to interspecific pollen rejection.     

G蛋白调节剂和花柱S-核糖核酸酶对花粉管生长及其钙离子浓度的影响

以‘丰水’和‘幸水’梨花柱及花粉为试材,用激光共聚焦显微技术,研究了离体条件下G蛋白活性调节剂和花柱S-RNA酶对花粉管生长及其游离Ca~(2 )浓度的影响。结果表明:G蛋白激活剂CTX可促进花粉管生长,且可解除花柱S-RNA酶对自身花粉管生长的抑制作用;G蛋白抑制荆PTX和花柱S-RNA酶共同处理使异体的花粉管生长受到抑制。CTX处理使花粉管尖端区的[Ca~(2 )]_i明显升高,花柱S-RNA酶处理引起自身花粉管尖端区的[Ca~(2 )]_i梯度消失;CTX和花柱S-RNA酶共同处理则使自身花粉管内的[Ca~(2 )J_i表现出两者单独处理时的综合特征;而花柱S-RNA酶和PTX共同处理后,异体的花粉管内[Ca~(2 )]_i表现出先升高后下降的趋势。     

Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.)

Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S(33) and S(34), that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S(35), plus the presence of two previously identified sweet cherry S-haplotypes, S(14) and S(16) are described here. Genetic segregation data demonstrated that the S(16)-, S(33)-, S(34)-, and S(35)-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that 'hetero-allelic' pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.     

S-RNase complexes and pollen rejection

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.     

On the origin of self-incompatibility haplotypes: transition through self-compatible intermediates

Self-incompatibility (SI) in flowering plants entails the inhibition of fertilization by pollen that express specificities in common with the pistil. In species of the Solanaceae, Rosaceae, and Scrophulariaceae, the inhibiting factor is an extracellular ribonuclease (S-RNase) secreted by stylar tissue. A distinct but as yet unknown gene (provisionally called pollen-S) appears to determine the specific S-RNase from which a pollen tube accepts inhibition. The S-RNase gene and pollen-S segregate with the classically defined S-locus. The origin of a new specificity appears to require, at minimum, mutations in both genes. We explore the conditions under which new specificities may arise from an intermediate state of loss of self-recognition. Our evolutionary analysis of mutations that affect either pistil or pollen specificity indicates that natural selection favors mutations in pollen-S that reduce the set of pistils from which the pollen accepts inhibition and disfavors mutations in the S-RNase gene that cause the nonreciprocal acceptance of pollen specificities. We describe the range of parameters (rate of receipt of self-pollen and relative viability of inbred offspring) that permits the generation of a succession of new specificities. This evolutionary pathway begins with the partial breakdown of SI upon the appearance of a mutation in pollen-S that frees pollen from inhibition by any S-RNase presently in the population and ends with the restoration of SI by a mutation in the S-RNase gene that enables pistils to reject the new pollen type.     

A functional study of stylar hydroxyproline-rich glycoproteins during pollen tube growth

Class III pistil-specific extensin-like proteins (PELPIII) are chimeric hydroxyproline-rich glycoproteins with properties of both extensins and arabinogalactan proteins. The abundance and specific localization of PELPIII in the intercellular matrix (IM) of tobacco (Nicotiana tabacum) stylar transmitting tissue, and translocation of PELPIII from the IM into the pollen tube wall after pollination, presume the biological function of these glycoproteins to be related to plant reproduction. Here we show that in in vitro assays the translocation of PELPIII is specifically directed to the callose inner wall of the pollen tubes, indicating that protein transfer is not dependent on the physiological conditions of the transmitting tract. We designed a set of experiments to elucidate the biological function of PELPIII in the stylar IM. To study the function of the specific interaction between PELPIII proteins and the pollen tube wall, one of the PELPIII proteins (MG15) was ectopically expressed in pollen tubes and targeted to the tube wall. We also generated transgenic tobacco plants in which PELPIII proteins were silenced. In vitro bioassays were performed to test the influence of purified PELPIII on pollen tube growth, as compared to tobacco transmitting tissue-specific proteins (TTS) that were previously shown to stimulate pollen tube growth. The various tests described for activity of PELPIII proteins all gave consistent and mutually affirmative results: the biological function of PELPIII proteins is not directly related to pollen tube growth. These data show that similar stylar glycoproteins may act very differently on pollen tubes.     

A pollen tube growth-promoting arabinogalactan protein from nicotiana alata is similar to the tobacco TTS protein

Upon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi-in vivo culture system. It has been reported that the self-incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP-diagnostic reagent beta-glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi-in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix-bound sub-population of the entire spectrum of N. alata TTS proteins.     

Molecular analysis of two Chinese pear (Pyrus bretschneideri Rehd.) spontaneous self-compatible mutants, Yan Zhuang and Jin Zhui

Yan Zhuang and Jin Zhui are spontaneous bud mutants of Chinese pear ( Pyrus bretschneideri Rehd.) from Ya Li. Both fruit set rate and seed number after self-pollination, together with pollen tube growth, prove that Yan Zhuang and Jin Zhui are self-compatible. The fruit set rate and seed number after cross-pollination suggest that the self-compatibility of Yan Zhuang and Jin Zhui may be due to natural mutations of the stylar S allele and pollen S allele, respectively. PCR amplification of the S-RNase gene in self-pollinated progeny of Yan Zhuang and Jin Zhui show that they contain point mutations in the stylar S₂₁ allele and pollen S₃₄ allele, respectively. The cDNA sequence of the Yan Zhuang stylar S-RNase gene revealed that the 182nd nucleotide of the S₂₁-RNase (cDNA) sequence had been substituted resulting in a Gly to Val mutation, and this might affect the stability of the S-RNase. In addition, Western blotting showed that one Yan Zhuang stylar S-RNase was absent and the expression level of another S-RNase protein was decreased compared to Ya Li. Therefore, we suggest that the self-compatibility of Yan Zhuang is caused by a point mutation in an S₂₁-RNase nucleotide.     

Effects of RNases on rejection of pollen from Nicotiana tabacum and N. plumbaginifolia

S-RNases are implicated in both inter- and intra-specific pollen rejection in Nicotiana. At least two mechanisms contribute to S-RNase dependent rejection of pollen from self compatilble species such as Nicotiana plumbaginifolia and N. tabacum. Cloned S-RNases from self incompatible N. alata expressed in styles of self compatible N. tabacum cause rejection of N. tabacum pollen through a factor-independent mechanism. However, rejection of N. plumbaginifolia pollen occurs only when S-RNases are expressed in conjunction with non-S-RNase factors from N. alata (factor-dependent pollen rejection). Here, we compared the pollen rejection activity of four RNases in these two systems. Recombinant RNase expression levels in the factor-dependent N. plumbaginifolia system were insufficient to cause pollen rejection. However, three S-RNases were active in the factor-independent N. tabacum pollen rejection system. This system shows the broadest specificity of any system so far examined. However, RNaseI from E. coli could not cause N. tabacum pollen rejection. Thus, RNase activity alone is not sufficient for pollen rejection. Our results suggest that S-RNases are specially adapted to function in pollen rejection. Received: 15 December 2000 / Accepted: 1 May 2001     

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.     

A time course of GFP expression and mRNA stability in pollen tubes following compatible and incompatible pollinations in Solanum chacoense

The self-incompatibility (SI) reaction in the Solanaceae involves molecular recognition of stylar haplotypes by pollen and is mediated by the S-locus from which a stylar-localized S-RNase and several pollen-localized F-box proteins are expressed. S-RNase activity has been previously shown to be essential for the SI reaction, leading to the hypothesis that pollen rejection in incompatible crosses is due to degradation of pollen RNA. We used pollen expressing the fluorescent marker GFP, driven by the LAT52 promoter, to monitor the accumulation of mRNA and protein in pollen after compatible and incompatible pollinations. We find that GFP mRNA and protein gradually accumulate in pollen tubes until at least 18-h post-pollination and, up to this time, are only slightly more abundant in compatible compared with incompatible crosses. However, between 18- and 24-h post-pollination, pollen tube GFP mRNA and protein levels show a dramatic increase in compatible crosses and either remain constant or decrease in incompatible crosses. In contrast to these molecular correlates, the growth rates of compatible and incompatible pollen tubes begin to differ after 6-h post-pollination. We interpret the changes in growth rate at 6-h post-pollination as the previously described transition from autotrophic to heterotrophic growth. Thus, while pollen rejection is generally considered to result from the cytotoxic effects of S-RNase activity, this time course reveals that a difference in the growth rate of compatible and incompatible pollen appears prior to any marked effects on at least some types of pollen RNA.     

A novel thioredoxin h is secreted in Nicotiana alata and reduces S-RNase in vitro

Thioredoxins type h are classified into three subgroups. The subgroup II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollen-pistil interaction processes or particularly in S-RNase-based self-incompatibility.     

Molecular analysis of the conserved C4 region of the S<Subscript>11</Subscript>-RNase of <Emphasis Type="Italic">Solanum chacoense</Emphasis>

The stylar component to gametophytic self-incompatibility in Solanaceae is an S-RNase. Its primary structure has a characteristic pattern of two hypervariable regions, involved in pollen recognition, and five constant regions. Two of the latter (C2 and C3) constitute the active site, while the highly hydrophobic C1 and C5 are believed to be involved in protein stability. We analyzed the role of the C4 region by site-directed mutagenesis. A GGGG mutant, in which the four charged residues in the C4 region were replaced with glycine, did not accumulate the protein to detectable levels in styles, suggestive of a role in protein stability. A R115G mutant, in which a charged amino acid was eliminated to reduce the potential binding affinity, had no effect on the pollen rejection phenotype. This suggests the C4 does not interact with partners such as potential pollen tube receptors facilitating S-RNase uptake. Finally, a K113R mutant replaced a potential ubiquitination target with arginine. However, this RNase acted as the wild type in both incompatible and compatible crosses. The latter crosses rule out the role of the conserved C4 lysine in ubiquitination.     

Compatibility and incompatibility in S-RNase-based systems

Background: S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity. SCOPE: The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility. CONCLUSIONS: Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI.