Identification of regions in which positive selection may operate in S-RNase of Rosaceae: Implication for S-allele-specific recognition sites in S-RNase

A stylar S-RNase is associated with gametophytic self-incompatibility in the Rosaceae, Solanaceae, and Scrophulariaceae. This S-RNase is responsible for S-allele-specific recognition in the self-incompatible reaction, but how it functions in specific discrimination is not clear. Window analysis of the numbers of synonymous (d_S) and non-synonymous (d_N) substitutions in rosaceous S-RNases detected four regions with an excess of d_N over d_S in which positive selection may operate (PS regions). The topology of the secondary structure of the S-RNases predicted by the PHD method is very similar to that of fungal RNase Rh whose tertiary structure is known. When the sequences of S-RNases are aligned with the sequence of RNase Rh based on the predicted secondary structures, the four PS regions correspond to two surface sites on the tertiary structure of RNase Rh. These findings suggest that in S-RNases the PS regions also form two sites and are candidates for the recognition sites for S-allele-specific discrimination.     

Identification and characterization of components of a putative petunia S-locus F-box-containing E3 ligase complex involved in S-RNase-based self-incompatibility

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF₂. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S₁-, S₂-, and S₃-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S₃-RNase is not degraded to any significant extent; however, deglycosylated S₃-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown.     

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.     

Glycosylation of S-RNases may influence pollen rejection thresholds in Solanum chacoense

A survey of Solanum chacoense plants expressing an authenticS₁₁-RNase transgene identified a line with partial compatibilityto S₁₁ pollen. By comparing fruit set to the S-RNase levelsdetermined immunologically in single styles, the minimum levelof S₁₁-RNase required for full rejection of S₁₁ pollen was estimatedto be 18 ng per style. The S₁₁-RNase threshold levels are thusconsiderably lower than those previously reported for the S₁₂-RNase.Interestingly, these two allelic S-RNases differ dramaticallyin the extent of glycosylation, with the number of glycosylationsites varying from one (S₁₁-RNase) to four (S₁₂-RNase). It issuggested that reduced glycosylation of the S₁₁-RNase may berelated to the lower threshold for pollen rejection. Key words: Gametophytic self-incompatibility, glycosylation, pistil-by-pistil analysis, S-RNase, Solanum chacoense, threshold Received 13 August 2007; Revised 27 November 2007 Accepted 30 November 2007     

Ectopic expression of S-RNase of <Emphasis Type="Italic">Petunia inflata</Emphasis> in pollen results in its sequestration and non-cytotoxic function

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature “S-RNase”, the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S₂-RNase, S₃-RNase and non-glycosylated S₃-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin–myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.     

The position of the disulfide bonds in human plasma α2HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma α₂HS-glycoprotein were determined. α₂HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α₂HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.     

Characterization of the flanking regions of the S-RNase genes of Japanese pear (Pyrus serotina) and apple (Malus×domestica)

Genomic sequences of the self-incompatibility genes, the S-RNase genes, from two rosaceous species, Japanese pear and apple, were characterized. Genomic Southern blot and sequencing of a 4.5-kb genomic clone showed that the S⁴-RNase gene of Japanese pear is surrounded by repetitive sequences as in the case of the S-RNase genes of solanaceous species. The flanking regions of the S²- and S^f-RNase genes of apple were also cloned and sequenced. The 5′ flanking regions of the three alleles bore no similarity with those of the solanaceous S-RNase genes, although the position and sequence of the putative TATA box were conserved. The putative promoter regions of the Japanese pear S⁴- and apple S^f-RNase genes shared a stretch of about 200 bp with 80% sequence identity. However, this sequence was not present in the S²-RNase gene of apple, and thus it may reflect a close relationship between the S⁴- and S^f-RNase genes rather than a cis-element important in regulating gene expression. Despite the uniform pattern of expression of the rosaceous S-RNase genes, sequence motifs conserved in the 5′ flanking regions of the three alleles were not found, implying that the cis-element controlling pistil specific gene expression also locates at the intragenic region or upstream of the analyzed promoter region.     

Action of the Style Product of the Self-Incompatibility Gene of Nicotiana alata (S-RNase) on in Vitro-Grown Pollen Tubes

The products of the S-locus expressed in female tissues of Nicotiana alata are ribonucleases (S-RNases). The arrest of growth of incompatible pollen tubes in styles may result from entry of the S-RNase into the pollen tube and degradation of pollen tube RNA. We investigated the action of isolated S-RNases on pollen tubes grown in vitro and found that S-RNase is taken up by the pollen without substantial alteration. The S-RNases inhibit incorporation of exogenously added radioactive amino acids into protein by the germinated pollen. The S-RNases also inhibit in vitro translation of pollen tube RNA in a wheat germ cell-free extract. We found no evidence for a specific mRNA substrate for the S-RNases, which implies that if RNase activity is involved in the control of self-incompatibility, allelic specificity is more likely to depend on the selective uptake of S-RNases into pollen tubes or their selective activation or inactivation by pollen factors, rather than cleavage of a specific substrate. Heat treating S2-RNase largely destroys its RNase activity but increases its inhibitory effect on in vitro pollen tube growth. This effect is not due to an increased uptake of S2-RNase by the pollen but is associated with a greatly enhanced accumulation of S2-RNase on the outer surface of the pollen grains.     

Cloning and characterization of cDNAs encoding S-RNases from almond (Prunus dulcis): primary structural features and sequence diversity of the S-RNases in Rosaceae

cDNAs encoding three S-RNases of almond (Prunus dulcis), which belongs to the family Rosaceae, were cloned and sequenced. The comparison of amino acid sequences between the S-RNases of almond and those of other rosaceous species showed that the amino acid sequences of the rosaceous S-RNases are highly divergent, and intra-subfamilial similarities are higher than inter-subfamilial similarities. Twelve amino acid sequences of the rosaceous S-RNases were aligned to characterize their primary structural features. In spite of?their high level of diversification, the rosaceous S-RNases were found to have five conserved regions, C1, C2, C3, C5, and RC4 which is Rosaceae-specific conserved region. Many variable sites fall into one region, named RHV. RHV is located at a similar position to that of the hypervariable region a (HVa) of the solanaceous S-RNases, and is assumed to be involved in recognizing S-specificity of pollen. On the other hand, the region corresponding to another solanaceous hypervariable region (HVb) was not variable in the rosaceous S-RNases. In the phylogenetic tree of the T2/S type RNase, the rosaceous S-RNase fall into two subfamily-specific groups (Amygdaloideae and Maloideae). The results of sequence comparisons and phylogenetic analysis imply that the present S-RNases of Rosaceae have diverged again relatively recently, after the divergence of subfamilies.     

Petunia hybrida S-proteins: ribonuclease activity and the role of their glycan side chains in self-incompatibility

Summary Self-incompatibility in flowering plants is controlled by the S-gene, encoding stylar S (allele-specific) glycoproteins. In addition to three previously characterized Petunia hybrida S-proteins, we identified by N-terminal sequence analysis another stylar S-protein, co-segregating with the S _b-allele. Purified S-proteins reveal biological activity, as is demonstrated for two of them by the allele-specific inhibition of pollen tube growth in vitro. Moreover, the four isolated S-proteins are ribonucleases (S-RNases). Specific activities vary from 30 (S₁) to 1000 (S₂) units per min per mg protein. We attempted to investigate the functionality of the carbohydrate portion of the S-RNases. Deglycosylation studies with the enzyme peptide-N-glycosidase F (PNGase F) reveals differences in the number of N-linked glycan chains present on the four S-RNases. Variability in the extent of glycosylation accounts for most of the molecular weight differences observed among these proteins. By amino acid sequencing, the positions of two of the three N-glycosylation sites on the S₂-RNase could be located near the N-terminus. Enzymic removal of the glycan side chains has no effect on the RNase activity of native S-RNases. This suggests another role of the glycan moiety in the self-incompatibility mechanism.     

The RHV region of S-RNase in the European pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>) is not required for the determination of specific pollen rejection

In the gametophytic self-incompatibility system, growth of self-pollen tubes in the style is inhibited in a haplotype-specific manner by S-RNase. The mechanism by which S-RNase confers its specificity is unknown. However, a hypervariable region (RHV in Rosaceae and HVa-HVb in Solanaceae) that differs among the many cloned S-RNase alleles has been proposed to be involved in conferring the S-haplotype specificity of the S-RNase. Region swapping experiments between S-RNases and crystallography of the enzyme support this assumption. However, the deduced amino acid sequences of Sn-RNase and Si-RNase alleles from the European pear (Pyrus communis) were recently found to have an identical RHV. In the present study it is shown that Sn-RNase does not prevent fertilization by Si-pollen haplotype, thus presenting a case in which RHV is not required for the determination of specific pollen rejection by S-RNase, and implying that other regions in the enzyme may be sufficient for this specificity.     

Functional characterization of two chimeric proteins between a <Emphasis Type="Italic">Petunia inflata</Emphasis><Emphasis Type="Italic">S</Emphasis>-locus F-box protein,PiSLF<Subscript>2</Subscript>, and a PiSLF-like protein,PiSLFLb-S<Subscript>2</Subscript>

Self-incompatible solanaceous species possess the S-RNase and SLF (S-locus F-box) genes at the highly polymorphic S-locus, and their products mediate S-haplotype-specific rejection of pollen tubes in the style. After a pollen tube grows into the style, the S-RNases produced in the style are taken up; however, only self S-RNase (product of the matching S-haplotype) can inhibit the subsequent growth of the pollen tube. Based on the finding that non-self interactions between PiSLF (Petunia inflata SLF) and S-RNase are stronger than self-interactions, and based on the biochemical properties of PiSLF, we previously proposed that a PiSLF preferentially interacts with its non-self S-RNases to mediate their ubiquitination and degradation, thereby only allowing self S-RNase to exert its cytotoxic function. We further divided PiSLF into three potential Functional Domains (FDs), FD1-FD3, based on sequence comparison of PiSLF and PiSLF-like proteins, and based on S-RNase-binding properties of these proteins and various truncated forms of PiSLF₂ (S ₂ allelic variant of PiSLF). In this work, we examined the in vivo function of FD2, which we proposed to be responsible for strong, general interactions between PiSLF and S-RNase. We swapped FD2 of PiSLF₂ with the corresponding region of PiSLFLb-S₂ (S ₂ allelic variant of a PiSLF-like protein), and expressed GFP-fused chimeric proteins, named b-2-b and 2-b-2, in S ₂ S ₃ transgenic plants. We showed that neither chimeric protein retained the SI function of PiSLF₂, suggesting that FD2 is necessary, but not sufficient, for the function of PiSLF. Moreover, since we previously found that b-2-b and 2-b-2 only interacted with S₃-RNase ~50 and ~30%, respectively, as strongly as did PiSLF₂ in vitro, their inability to function as PiSLF₂ is also consistent with our model predicating on strong interaction between a PiSLF and its non-self S-RNases as part of the biochemical basis for S-haplotype-specific rejection of pollen tubes.     

Basic RNases of wild almond (Prunus webbii): cloning and characterization of six new S-RNase and one "non-S RNase" genes

In order to investigate the S-RNase allele structure of a Prunus webbii population from the Montenegrin region of the Balkans, we analyzed 10 Prunus webbii accessions. We detected 10 different S-RNase allelic variants and obtained the nucleotide sequences for six S-RNases. The BLAST analysis showed that these six sequences were new Prunus webbii S-RNase alleles. It also revealed that one of sequenced alleles, S(9)-RNase, coded for an amino acid sequence identical to that for Prunus dulcis S(14)-RNase, except for a single conservative amino acid replacement in the signal peptide region. Another, S(3)-RNase, was shown to differ by only three amino acid residues from Prunus salicina Se-RNase. The allele S(7)-RNase was found to be inactive by stylar protein isoelectric focusing followed by RNase-specific staining, but the reason for the inactivity was not at the coding sequence level. Further, in five of the 10 analyzed accessions, we detected the presence of one active basic RNase (marked PW(1)) that did not amplify with S-RNase-specific DNA primers. However, it was amplified with primers designed from the PA1 RNase nucleotide sequence (basic "non-S RNase" of Prunus avium) and the obtained sequence showed high homology (80%) with the PA1 allele. Although homologs of PA1 "non-S RNases" have been reported in four other Prunus species, this is the first recorded homolog in Prunus webbii. The evolutionary implications of the data are discussed.     

Recognition of S-RNases by an S locus F-box like protein and an S haplotype-specific F-box like protein in the Prunus-specific self-incompatibility system

Key message

S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry.

Abstract

Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1–3 (SLFL1–3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS^{1, 6}-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS⁶-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.

     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

Molecular cloning of the self-incompatibility genes S1 and S3 from almond (Prunus dulcis cv. Ferragnès)

Almond (Prunus dulcis) displays gametophytic self-incompatibility. In the work reported here, we cloned two novel S-RNase genes from almond cultivar Ferragnès (genotype S1S3) using PCR. The S1-RNase gene has the same coding region as the Sb gene cloned from almond cultivated in the USA; however, their introns are different in sequence. S1 was cloned and sequenced from six different cultivars originating in Europe. The full-length of the S3-RNase gene was cloned using two primers corresponding to the start and stop codons contexts. Two introns are present in the S3 gene, unique among the S-RNase genes. Sequence-specific PCR was performed to confirm that the two cloned genes co-segregate with the S-locus using progenies of a controlled cross between Tuono (S1Sf) and Ferragnès (S1S3). Based on the structural differences of S- and S-like RNase genes, we discuss the evolutionary relationship between the two groups of RNase genes. Received: 18 February 2001 / Accepted: 26 June 2001     

Sequence comparison of the 5’ flanking regions of Japanese pear (Pyrus pyrifolia) S-RNases associated with gametophytic self-incompatibility

Genomic clones of 2.8 kb, 4.3 kb and 6.5 kb for the S₂-, S₃- and S₅-RNases of Japanese pear(Pyrus pyrifolia), respectively, were isolated and sequenced. Comparison of the 5’-flanking regions of these genes with the same region of the S₄-RNase gene indicated that a highly similar region of approximately 200 bp exists in the regions just upstream of the putative TATA boxes of the four Japanese pear S-RNase genes. This suggests the presence of cis-regulatory element(s) in this region. Received: 5 October 2000 / Revision accepted: 2 January 2001     

Self-Incompatibility in Petunia inflata: The Relationship between a Self-Incompatibility Locus F-Box Protein and Its Non-Self S-RNases

The highly polymorphic S (for self-incompatibility) locus regulates self-incompatibility in Petunia inflata; the S-RNase regulates pistil specificity, and multiple S-locus F-box (SLF) genes regulate pollen specificity. The collaborative non-self recognition model predicts that, for any S-haplotype, an unknown number of SLFs collectively recognize all non-self S-RNases to mediate their ubiquitination and degradation. Using a gain-of-function assay, we examined the relationships between S₂-SLF1 (for S₂-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S₂-SLF1 interacts with S₇- and S₁₃-RNases, and the previously identified S₁- and S₃-RNases, but not with S₅- or S₁₁-RNase. An artificial microRNA expressed by the S₂-SLF1 promoter, but not by the vegetative cell-specific promoter, Late Anther Tomato 52, suppressed expression of S₂-SLF1 in S₂ pollen, suggesting that SLF1 is specific to the generative cell. The S₂ pollen with S₂-SLF1 suppressed was compatible with S₃-, S₅-, S₇-, S₁₁-, and S₁₃-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S₅- and S₁₁-RNases and suggesting that S₂-SLF1 is not the only SLF in S₂ pollen that interacts with S₃-, S₇-, and S₁₃-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF.