Characterization of Ribonuclease Activity of Three S-Allele-Associated Proteins of Petunia inflata

Three S-allele-associated proteins (S-proteins) of Petunia inflata, a species with gametophytic self-incompatibility, were previously found to share sequence similarity with two fungal ribonucleases, RNase T₂ and RNase Rh. In this study, the S-proteins from P. inflata plants of S₁S₂ and S₂S₃ genotypes were purified to homogeneity by gel filtration and cation-exchange chromatography, and their enzymatic properties were characterized. The three S-proteins (S₁, S₂, and S₃), with pairwise sequence identity ranging from 73.1 to 80.5%, were similar in most of the enzymatic properties characterized. The ribonuclease activity had a pH optimum of 7.0 and a temperature optimum of 50°C. Diethylpyrocarbonate at 1 millimolar almost completely abolished the ribonuclease activity; cupric sulfate and zinc sulfate at 1 millimolar reduced the ribonuclease activity of the three S-proteins by 50 to 75%. EDTA and RNasin had no inhibitory effect. All three S-proteins hydrolyzed polycytidylic acid preferentially, but varied in their nucleolytic activity toward polyadenylic acid and polyuridylic acid.     

Primary structural features of the self-incompatibility protein in solanaceae

Summary We present a sequence comparison of 12 S-allele-associated proteins from three solanaceous species with gametophytic self-incompatibility: Nicotiana alata, Petunia inflata, and Solanum chacoense. The allelic variants of the S-protein exhibit a very high degree of sequence diversity consistent with their function as recognition molecules. We identify 41 perfectly conserved residues, 18 of which are also conserved in two fungal ribonucleases, RNase T₂ and RNase Rh. The residues conserved in both the S-proteins and the ribonucleases include two histidines essential for catalysis, four cysteines involved in disulfide bridges, and hydrophobic residues probably involved in the core structure of the proteins. This conservation between the two ribonucleases and the 12 divergent S-proteins confirms the previously recognized similarity between 3 more closely related N. alata S-proteins and these ribonucleases, and argues strongly for the functional importance of the ribonuclease activity of the S-protein in self-incompatibility. We also identify the 19 most variable residues, which are the prime candidates for the S-allele-specificity determinant. Twelve of these nineteen residues are clustered in two regions of hypervariability, designated HVa and HVb, which are also the most prominent hydrophilic regions of the S-protein. We suggest that these two regions might form parts of the putative pollen recognition site. Identification of these structural features forms a foundation for the study of the molecular basis of self-recognition and the biochemical mechanism of inhibition of self-pollen tube growth.On sabbatical leave from Biotechnology Center, General Foods USA, Tarrytown, NY 10591, USA     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S ₂, encoded by the S ₂ allele, with the expected features of the Sp gene was identified. AhSLF-S ₂ is located 9 kb downstream of S ₂ RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S ₂ are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S ₂ is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

Sequence of an S-protein of Lycopersicon peruvianum and comparison with other solanaceous S-proteins

Summary cDNA clones for an S-allele, designated S₅, of the self-incompatibility locus (S-locus) of Lycopersicon peruvianum have been isolated by probing a pistil cDNA library with cDNAs for S-alleles of Petunia inflata and Solanum chacoense. The longest S₅-cDNA is 869 bp and contains an open reading frame of 217 amino acids. An alignment of the deduced amino acid sequence of S₅-protein with that of the 18 S-proteins from five other solanaceous species is presented. Sequence comparison further refines the primary structural features of the S-proteins previously revealed from comparison of subsets of these sequences. Based on this comparison and evidence presented elsewhere, it is proposed that accumulation of point mutations, and not intragenic recombination, is responsible for the generation of new allelic specificities.     

Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins

Summary We have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.     

Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense

Summary We have isolated and sequenced cDNAs for S₂- and S₃-alleles of the self-incompatibility locus (S-locus) in Solanum chacoense Bitt., a wild potato species displaying gametophytic self-incompatibility. The S₂-and S₃-alleles encode pistil-specific proteins of 30 kDa and 31 kDa, respectively, which were previously identified based on cosegregation with their respective alleles in genetic crosses. The amino acid sequence homology between the S₂- and S₃-proteins is 41.5%. This high degree of sequence variability between alleles is a distinctive feature of the S-gene system. Of the 31 amino acid residues which were previously found to be conserved among three Nicotiana alata S-proteins (S₂, S₃, and S₆) and two fungal ribonucleases (R Nase T₂ and R Nase Rh), 27 are also conserved in the S₂- and S₃-proteins of S. chacoense. These residues include two histidines implicated in the active site of the R Nase T₂, six cysteines, four of which form disulfide bonds in R Nase T₂, and hydrophobic residues which might form the core structure of the protein. The finding that these residues are conserved among S-proteins with very divergent sequences suggests a functional role for the ribonuclease activity of the S-protein in gametophytic self-incompatibility.     

Pollen S–locus F–box proteins of Petunia involved in S–RNase‐based self‐incompatibility are themselves subject to ubiquitin‐mediated degradation

Many flowering plants show self‐incompatibility, an intra‐specific reproductive barrier by which pistils reject self‐pollen to prevent inbreeding and accept non‐self pollen to promote out‐crossing. In Petunia, the polymorphic S–locus determines self/non‐self recognition. The locus contains a gene encoding an S–RNase, which controls pistil specificity, and multiple S‐locus F‐box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F‐box) complex that is responsible for mediating degradation of non‐self S‐RNase(s), with which the SLF interacts, via the ubiquitin–26S proteasome pathway. A complete set of SLFs is required to detoxify all non‐self S‐RNases to allow cross‐compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin–26S proteasome pathway, and identify an 18 amino acid sequence in the C‐terminal region of S₂‐SLF1 (SLF1 of S₂ haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S₂ haplotype and S₃ haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S₂‐SLF1 stabilized the protein but abolished its function in self‐incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self‐incompatibility.     

Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum

Summary Allelic complexity is a key feature of self-incompatibility (S) loci in gametophytic plants. We describe in this report the allelic diversity and gene structure of the S locus in Solanum tuberosum revealed by the isolation and characterization of genomic and cDNA clones encoding S-associated major pistil proteins from three alleles (S ₁, S _r1, S ₂). Genomic clones encoding the S₁ and S₂ proteins provide evidence for a simple gene structure: Two exons are separated by a small intron of 113 (S ₁) and 117 by (S ₂). Protein sequences deduced from cDNA clones encoding S₁ and S_r1 proteins show 95% homology. 15 of the 25 residues that differ between these S ₁and S _r1alleles are clustered in a short hypervariable protein segment (amino acid positions 44–68), which corresponds in the genomic clones to DNA sequences flanking the single intron. In contrast, these alleles are only 66% homologous to the S ₂allele, with the residues that differ between the alleles being scattered throughout the sequence. DNA crosshybridization experiments identify a minimum of three classes of potato S alleles: one class contains the alleles S ₁, S _r1and S ₃, the second class S ₂and an allele of the cultivar Roxy, and the third class contains at present only S ₄. It is proposed that these classes reflect the origin of the S alleles from a few ancestral S sequence types.     

Identification of pistil-specific proteins associated with three self-incompatibility alleles in Solanum chacoense

Summary Pistil proteins associated with three different S-alleles of the self-incompatibility locus (S locus) in Solanum chacoense have been identified which cosegregated with their respective S alleles in a series of genetic crosses involving six S. chacoense plants, their F₁ progeny, and backcrosses. The molecular weights of these three S-allele-associated proteins, designated S₁ S₂, and S₃, were 29 kDa, 30 kDa, and 31 kDa, respectively. They were all basic proteins with a similar pI of approximately 8.6. They have been found only in the stigma and style of the pistil where their maximum synthesis was reached at one day before anthesis. Their rate of synthesis in both self- and cross-pollinated pistils was the same as that in the unpollinated pistil until 2 days after pollination.On sabbatical leave from Laboratoire de Genetique et Physiologie du Developpement des Plantes, C.N.R.S., F-91190 Gifsur-Yvette, France     

The expression of a potato (Solanum tuberosum) S-RNase gene in Nicotiana tabacum pollen

Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S ₂ -RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S ₂ RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the -glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S ₂ -RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S ₂ polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S ₂ -RNase protein in pollen.     

S-alleles are retained and expressed in a self-compatible cultivar of Petunia hybrida.

Summary We identified two S-allele-associated proteins (S-proteins) in a self-compatible cultivar of Petunia hybrida based on their segregation in F₁ hybrids between P. hybrida and its self-incompatible relative, Petunia inflata (with S₂S₂ genotype), and in selfed progeny of P. hybrida. These two S-proteins, designated S_x-protein (24 kDa) and S_o- protein (31 kDa), are pistil specific, and their expression follows a temporal and spatial pattern similar to that of S-proteins characterized in self-incompatible solanaceous species. Their amino-terminal sequences also share a high degree of similarity with those of solanaceous S-proteins. Selfing of P. hybrida yielded plants with S_oS_o, SxSo, and S_xS_x genotypes in an approximately 1:2:1 ratio, indicating that the S_x- and S_o-alleles, though expressed in the pistil, failed to elicit a self-incompatibility response. The S₂-allele of P. inflata is expressed in all the F₁ hybrids, rendering them capable of rejecting pollen bearing the S₂-allele. The S_o-allele is not functional in the F1 hybrids, because all the F₁ progeny with S₂S_o genotype are self-compatible. However, in F₁ hybrids with S₂S_x genotype, approximately half are self-incompatible and half are self-compatible, indicating that the function of the S_x-allele depends on the genetic background. These results strongly suggest that the presence of functional S-alleles alone is not sufficient for expression of a self-incompatibility phenotype, and reaffirm the multigenic nature of gametophytic self-incompatibility suggested by earlier genetic studies.     

A genetic map of the Nicotiana alata S locus that includes three pollen-expressed genes

The S locus of solanaceous plants includes separate genes that control the self-incompatibility phenotype of the pistil and of the pollen. The gene controlling the self-incompatibility phenotype of the pistil encodes an extracellular ribonuclease, the S-RNase. The gene(s) controlling the self-incompatibility phenotype of pollen (the pollen-S gene) has yet to be identified. As part of a long-term strategy to clone the pollen-S gene by chromosome walking, a detailed map of the region near the S locus of Nicotiana alata was generated using a total of 251 F₂ plants. The map spans an interval of approximately 2.6 cM and contains five markers as well as the S-RNase gene. Two markers were detected with heterologous probes that also detect sequences linked to the S locus of Solanum tuberosum and the homologous region of the Lycopersicon genome. Three markers were identified by differential display using N. alata pollen RNA as a template. One of these markers is a pollen-expressed sequence, 48A, which detects a polymorphic marker no more than 0.5 cM from the S locus. RNA blot analysis indicates that the 48A gene is expressed primarily during pollen development after the completion of meiosis and is therefore a candidate for the pollen-S gene. The utility of these markers and the possible involvement of 48A in the molecular mechanism of self- incompatibility are discussed. Received: 28 June 1999 / Accepted: 24 September 1999     

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.     

Phosphorylation of style S-RNases by Ca2+-dependent protein kinases from pollen tubes

Solanaceous plants with gametophytic self-incompatibility produce ribonucleases in the transmitting tract of the style that interact with self-pollen and inhibit its growth. These ribonucleases are a series of allelic products of the S-locus, which controls self-incompatibility. Little is known about the pollen components involved in this interaction or whether a signal transduction pathway is activated during the self-incompatibility response. We have partially purified a soluble protein kinase from pollen tubes of Nicotiana alata that phosphorylates the self-incompatibility RNases (S-RNases) from N. alata but not Lycopersicon peruvianum. The soluble protein kinase (Nak-1) has several features shared by the calcium-dependent protein kinase (CDPK) class of plant protein kinases, including substrate specificity, calcium dependence, inhibition by the calmodulin antagonist calmidazolium, and cross-reaction with monoclonal antibodies raised to a CDPK from soybean. Phosphorylation of S ₂-RNase by Nak-1 is restricted to serine residues, but the site(s) of phosphorylation has not been determined and there is no evidence for allele-specific phosphorylation. The microsomal fraction from pollen tubes also phosphorylates S-RNases and this activity may be associated with proteins of M_r60 K and 69 K that cross-react with the monoclonal antibody to the soybean CDPK. These results are discussed in the context of the involvement of phosphorylation in other self-incompatibility systems.     

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S‐RNase activity

In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxh_SS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys₁₅₅‐Cys₁₈₅ disulphide bond of S_C10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection.     

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species (∼10–15 million years ago). Nonsynonymous substitutions per site (d _N) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (d _N / d _S) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense. Received: 8 April 1999 / Accepted: 22 June 1999     

Self-compatibility in aLycopersicon peruvianum variant (LA2157) is associated with a lack of style S-RNase activity

A series of crosses between a naturally-occurring self-compatible accession ofLycopersicon peruvianum and a closely-related self-incompatible accession were used to demonstrate that the mutation to self-compatibility is located at the S-locus. Progeny of the crosses contain abundant style proteins of about 30 kDa that segregate with the S₆and S₇-alleles from the SI parent and the S_c-allele from the SC parent. The S₆and S₇-associated proteins have ribonuclease activity whereas the S_c-associated protein is not an active ribonuclease. This finding indicates that S-RNases are determinants of self-incompatibility in the style and that the ribonuclease activity is essential for their function.     

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles

Genomic clones encoding the S ₂- and S ₆-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S ₂- and S ₆-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S ₆-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.     

Analysis of the Tissue-SpecificS RNase gene promoter associated with self-incompatibility in tomato (Lycopersicon peruvianum L.)

To understand the expression pattern of theS RNase gene in the floral tissues associated with self-incompatibility (SI), promoter region of S₁₁ RNase gene was serially deleted and fused GUS. Five chimeric constructs containing a deleted promoter region of the S₁₁ RNase gene were constructed, and introduced intoNicotiana tabacum using Agrobacterium-mediated transformation. Northern blot analysis revealed that the GUS gene was expressed in the style, anther, and developing pollen of all stages in each transgenic tobacco plant The developing pollen expressed the same amount of GUS mRNA in all stages in transgenic tobacco plants. In addition, histochemical analysis showed GUS gene expression in vascular bundle, endothecium, stomium, and tapetum cells during pollen development in transgenic plants. From these results, it is speculated that SI ofLycopersicon peruvianum may occur through the interaction ofS RNase expressed in both style and pollen tissues.