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.     

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.     

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.     

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.     

Sequence diversity of pistil S-proteins associated with gametophytic self-incompatibility in Nicotiana alata

Summary In order to study the extent and nature of differences among various S-allele-associated proteins in N. alata, we carried out comparative studies of seven such proteins. We first isolated and sequenced cDNA clones for the S_z-, S_F11-, S₁-, and S_a-alleles, and then we compared the deduced amino acid sequences both of these four S-proteins and of three previously published S₂-, S₃-, and S₆-proteins. This comparison revealed (1) an average homology of 53.8% among the seven proteins and (2) two homology classes, with S_z and S_F11 in one class and S₁, S₂, S₃, and S₆ in the other class. There are 60 conserved residues, including 9 cysteines. Of the 144 variable residues, 50 were identified as hypervariable based on a calculation of their Similarity Indices. Although conserved, variable, and hypervariable residues are dispersed throughout the protein, some are clustered to form five conserved, five hypervariable, and a number of variable regions. Those variable sites which contain residues conserved within one class of S-proteins but different between classes might provide a clue to the evolutionary relationship of these two classes of S-proteins. The hypervariable residues, which account for sequence variability, may contribute to allelic specificity.     

Selective inhibition of the growth of incompatible pollen tubes by S-protein in the Japanese pear

The S-allele-associated proteins (S-proteins) in the styles of the Japanese pear (Pyrus serotina Rehd. var. culta Rehd.) were purified by cation exchange chromatography. Their inhibitory action on the growth of incompatible pollen tubes (pollen tubes bearing the same S- allele as in the style from which the S-proteins were prepared) was characterized in vitro. Germination and tube growth of self-pollen (pollen from the same cultivar from which the S-proteins were prepared) decreased dose-dependently when the S-protein was added to the medium. Tube length was reduced to 10% that of compatible pollen tubes (pollen tubes bearing the S-allele different from that in the style from which the S-proteins were prepared) at 1.5 μg μl¹. S-proteins from Shinsui (S ₄ S ₅ ) also inhibited growth of cross-incompatible Kosui (S ₄ S ₅ ) pollen tubes, but not of compatible Chojuro (S ₂ S ₃ ) pollen tubes. After inactivation of RNase of the S- protein, the inhibitory action of the S-protein disappeared. These results indicate that the S-protein acts directly to inhibit growth of incompatible pollen tubes in Japanese pear styles, and that the RNase activity of the protein is essential for the biological function. However, small amounts of proteins that co-migrated with the S-protein may also play some roles in the inhibition. This is the first report on the selective inhibitory action of S-proteins in Rosaceae. Received: 11 April 2000 / Revision accepted: 28 September 2000     

RNase X2, a pistil-specific ribonuclease from Petunia inflata,shares sequence similarity with solanaceous S proteins

Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S₃ and S₆ proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.     

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.     

Expression of two S-ribonucleases of Petunia inflata using baculovirus expression system.

We have previously shown that three Petunia inflata S-proteins, products of the multiallelic S-gene of the self-incompatibility system, are ribonucleases. Here we report the expression of cDNAs for two of these S-proteins using the baculovirus expression system. S2- and S3-proteins were found in both supernatants and lysates of Spodoptera frugiperda cells infected with recombinant baculoviruses. Both recombinant S-proteins contained glycosylated (25 kD) and nonglycosylated (23 kD) forms. Recombinant S2- and S3-proteins were purified from insect cell cultures, and the amino-terminal sequences determined from glycosylated S2- and S3-proteins indicated that the leader peptide encoded by each cDNA was correctly removed. Both glycosylated and nonglycosylated forms of S2- and S3-proteins exhibited ribonuclease activity.     

RNase 1 genes from the family Sciuridae define a novel rodent ribonuclease cluster

The RNase A ribonucleases are a complex group of functionally diverse secretory proteins with conserved enzymatic activity. We have identified novel RNase 1 genes from four species of squirrel (order Rodentia, family Sciuridae). Squirrel RNase 1 genes encode typical RNase A ribonucleases, each with eight cysteines, a conserved CKXXNTF signature motif, and a canonical His¹²-Lys⁴¹-His¹¹⁹ catalytic triad. Two alleles encode Callosciurus prevostii RNase 1, which include a Ser¹⁸?Pro, analogous to the sequence polymorphisms found among the RNase 1 duplications in the genome of Rattus exulans. Interestingly, although the squirrel RNase 1 genes are closely related to one another (77–95% amino acid sequence identity), the cluster as a whole is distinct and divergent from the clusters including RNase 1 genes from other rodent species. We examined the specific sites at which Sciuridae RNase 1s diverge from Muridae/Cricetidae RNase 1s and determined that the divergent sites are located on the external surface, with complete sparing of the catalytic crevice. The full significance of these findings awaits a more complete understanding of biological role of mammalian RNase 1s.     

Molecular cloning of two Solanum chacoense S-alleles and a hypothesis concerning their evolution

The polymerase chain reaction (PCR) has been used to clone two S-alleles (S13 and S14) from Solanum chacoense. The two alleles do not cross-hybridize on genomic Southern blots or on northern blots using stylar RNA. Although the S14 message was not detected in a stylar cDNA library prepared from mature flowers, a full-length copy of the S13 coding sequence was isolated by screening with the PCR fragment. We have analysed the sequences of the S13 cDNA and the S14 PCR fragment (60% of the mature protein coding sequence) in the context of S-RNase evolution, and propose that random point mutations may be sufficient to generate new S-alleles. Based on a phylogenetic tree composed of RNase sequences containing the conserved RNase motifs HGLWP and KHGXC, we suggest that gametophytic self-incompatibility genes are RNase genes that have acquired a new function in the gametophytic self-incompatibility system early in the evolution of flowering plants.     

RNase T2 genes from rice and the evolution of secretory ribonucleases in plants

The plant RNase T2 family is divided into two different subfamilies. S-RNases are involved in rejection of self-pollen during the establishment of self-incompatibility in three plant families. S-like RNases, on the other hand, are not involved in self-incompatibility, and although gene expression studies point to a role in plant defense and phosphate recycling, their biological roles are less well understood. Although S-RNases have been subjects of many phylogenetic studies, few have included an extensive analysis of S-like RNases, and genome-wide analyses to determine the number of S-like RNases in fully sequenced plant genomes are missing. We characterized the eight RNase T2 genes present in the Oryza sativa genome; and we also identified the full complement of RNase T2 genes present in other fully sequenced plant genomes. Phylogenetics and gene expression analyses identified two classes among the S-like RNase subfamily. Class I genes show tissue specificity and stress regulation. Inactivation of RNase activity has occurred repeatedly throughout evolution. On the other hand, Class II seems to have conserved more ancestral characteristics; and, unlike other S-like RNases, genes in this class are conserved in all plant species analyzed and most are constitutively expressed. Our results suggest that gene duplication resulted in high diversification of Class I genes. Many of these genes are differentially expressed in response to stress, and we propose that protein characteristics, such as the increase in basic residues can have a defense role independent of RNase activity. On the other hand, constitutive expression and phylogenetic conservation suggest that Class II S-like RNases may have a housekeeping role.     

Mouse eosinophil-associated ribonucleases: a unique subfamily expressed during hematopoiesis

A unique family of ribonucleases was identified by exhaustive screening of genomic and cDNA libraries using a probe derived from a gene encoding a ribonuclease stored in the mouse eosinophil secondary granule. This family contains at least 13 genes, which encode ribonucleases, and two potential pseudogenes. The conserved sequence identity among these genes (∼70%), as well as the isolation/purification of these ribonucleases from eosinophil secondary granules, has led us to conclude that these genes form a unique clade in the mouse that we have identified as the Ear (Eosinophil-associated ribonuclease) gene family. Analyses of the nucleotide substitutions that have occurred among these ribonuclease genes reveal that duplication events within this family have been episodic, occurring within three unique periods during the past 18 × 10⁶ years. Moreover, comparisons of non-synonymous (K_a) vs. synonymous (K_s) rates of nucleotide substitution show that although these genes conserve residues necessary for RNase activity, selective evolutionary pressure(s) exist such that acquired amino acid changes appear to be advantageous. The selective advantage of these amino acid changes is currently unclear, but the occurrence of this phenomenon in both the mouse and the human highlights the importance of these changes for Ear and, therefore, eosinophil effector function(s). Received: 25 October 2000 / Accepted: 18 December 2000     

Microinjected ribonuclease A as a probe for lysosomal pathways of intracellular protein degradation

There are multiple pathways of intracellular protein degradation, and molecular determinants within proteins appear to target them for particular pathways of breakdown. We use red cell-mediated microinjection to introduce radiolabeled proteins into cultured human fibroblasts in order to follow their catabolism. A well-characterized protein, bovine pancreatic ribonuclease A (RNase A), is localized initially in the cytosol of cells after microinjection, but it is subsequently taken up and degraded by lysosomes. This lysosomal pathway of proteolysis is subject to regulation in that RNase A is taken up and degraded by lysosomes at twice the rate when serum is omitted from the culture medium. Subtilisin cleaves RNase A between residues 20 and 21, and the separated fragments are termed RNase S-peptide (residues 1–20) and RNase S-protein (residues 21–124). Microinjected RNase S-protein is degraded in a serum-independent manner, while RNase S-peptide microinjected alone shows a twofold increase in degradation in response to serum withdrawal. Furthermore, covalent linkage of S-peptide to other proteins prior to microinjection causes degradation of the conjugate to become serum responsive. These results show that recognition of RNase A and certain other proteins for enhanced lysosomal degradation during serum withdrawal is based on some feature of the amino-terminal 20 amino acids. The entire S-peptide is not required for enhanced lysosomal degradation during serum withdrawal because degradation of certain fragments is also responsive to serum. We have identified the essential region to be within residues 7–11 of RNase S-peptide (Lys-Phe-Glu-Arg-Gln; KFERQ). To determine whether related peptides exist in cellular proteins, we raised antibodies to the pentapeptide. Affinity-purified antibodies to KFERQ specifically precipitate 25–35% of cellular proteins, and these proteins are preferentially degraded in response to serum withdrawal. Computer analyses of known protein sequences indicate that proteins degraded by lysosomes at an enhanced rate in response to serum withdrawal contain peptide regions related, but not identical, to KFERQ. We suggest two possible peptide motifs related to KFERQ and speculate about possible mechanisms of selective delivery of proteins to lysosomes based on such peptide regions.     

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.     

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.     

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.