*The Field Museum, Department of Zoology, Chicago;
Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs
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Abstract |
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Introduction |
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The molecular basis of the gametophytic self-incompatibility mechanism has been well studied in the Solanaceae. In this family the most abundant protein in the style during the self-incompatibility response is a glycoprotein (Kehyr-Pour and Pernes 1985
) with ribonuclease (RNase) activity (McClure et al. 1989
). In fact, the self-incompatibility response will not occur if the molecule lacks RNase activity (Huang et al. 1994
). Thus, the term S-RNase refers to the characteristic protein associated with the self-incompatibility response.
An S-RNase is also involved with the self-incompatibility response in the other three groups of eudicots in which the molecular basis of gametophytic self-incompatibility has been documented, i.e., Rosaceae (Sassa et al. 1996
), Scrophulariaceae (Xue et al. 1996
), and Campanulaceae (Stephenson et al. 1992
), although the sequence of the gene encoding the S-RNase protein in the Campanulaceae has yet to be determined. Neither the Papaveraceae nor the Poaceae, the only other families with gametophytic self-incompatibility to have been examined at the molecular level, rely on an S-RNase in the self-incompatibility response (Franklin-Tong et al. 1993
; Li et al. 1997
).
Stigmatic glycoproteins associated with the self-incompatibility response in Papaveraceae have been isolated and cloned (Foote et al. 1994
). The sequence shows no similarity to the S-RNase protein, but it does show some similarities to the SLG and SRK genes involved in the self-incompatibility response of Brassica (Walker et al. 1996
). Furthermore, the self-incompatibility response in Papaver involves a highly complex series of events, including changes in calcium ion concentration, phosphorylation of specific proteins, transcription of specific genes within pollen tubes, and DNA fragmentation within nuclei of pollen tubes (Jordan, Franklin, and Franklin-Tong 2000
; Snowman et al. 2000
).
Unlike the other systems, gametophytic self-incompatibility in grasses is controlled by two unlinked multiallelic loci, S and Z. Recent molecular work indicates that protein kinases, similar to those in Brassica, and a thioredoxin play some role in self-incompatibility. Furthermore, the thioredoxin-like gene appears to be located about 1 cm from the S locus. Products of the Z locus have yet to be identified (Li et al. 1997
; Baumann et al. 2000
).
The evidence demonstrating the involvement of different molecules in self-incompatibility in the Poaceae, the Papaveraceae, and the families with S-RNases argues that gametophytic self-incompatibility likely has multiple origins. It is reasonable to conclude that there are at least three separate origins of gametophytic self-incompatibility in angiosperms. Is it possible, however, that the gametophytic self-incompatibility system of the Rosaceae, Solanaceae, and Scrophulariaceae has only one origin? If so, it would suggest that gametophytic self-incompatibility in all eudicots (Nandi, Chase, and Endress 1998
) had a single origin (Holsinger and Steinbachs 1997
).
Other studies have considered this question (Sassa et al. 1996
; Xue et al. 1996
; Richman, Broothaerts, and Kohn 1997
; Ushijima et al. 1998
; Igic and Kohn 2001
), examining the relationships among the S-RNases in eudicots and among the structurally related S-like RNases. Sassa et al. (1996)
first demonstrated that the rosaceous and solanaceous S-RNases each formed a monophyletic clade. With the addition of three S-RNase sequences from Antirrhinum, Xue et al. (1996)
suggested that all S-RNases formed a monophyletic clade, sharing a common ancestor, but more recent studies found very weak bootstrap support for the nodes uniting all S-RNases (Richman, Broothaerts, and Kohn 1997
; Ushijima et al. 1998
). Previous studies included only a small number of S-like RNases (e.g., 7 in Richman, Broothaerts, and Kohn [1997
]; 14 in Ushijima et al. [1998
]). As a result, these studies had only a limited ability to distinguish between single and multiple origins of S-RNasemediated gametophytic self-incompatibility. Igic and Kohn (2001)
improved on previous studies by increasing taxon sampling for the S-like group of sequences in addition to using maximum likelihood for tree reconstruction.
As with Igic and Kohn (2001)
, we independently took advantage of the substantial amount of new sequence information that has become available and assembled the most extensive sample of S-RNases and S-like RNases yet analyzed. Using more sophisticated alignment tools and methods of phylogenetic analysis, we demonstrate that S-RNasemediated gametophytic self-incompatibility is ancestral in the eudicots.
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Materials and Methods |
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Sequences Analyzed
We collected 72 amino acid sequences from PFAM 5.3 (Bateman et al. 2000
), for which there existed a corresponding nucleotide sequence in GenBank (version 117) (Benson et al. 2000
). After the selection process, our data set consisted of 49 S-RNases from Rosaceae, Solanaceae, and Scrophulariaceae, 21 S-like RNases from flowering plants, and two fungal RNases. The final data set includes the following organisms (abbreviated name in alignment: Genbank ID).
Fungal RNases
Aspergillus (rnt2-aspor: g133241), Rhizopus(rnrh-rhini: g133233).
S-like I
Nicotiana (rn-ngr2: g5902454), Arabidopsis (rns2-arath: g289210), Calystegium (c-sepium: g7288208).
S-like II
Arabidopsis (arath: g4262171; rns3-arath: g1173105; i64-arath: g5080798; rns1-arath: g561998; i65-arath: g5080799), Pyrus (rnpyrpyr: g1526417), Cicer (cicer: g3860325), Nicotiana (rne-nical: g532754; rn-ngr3: g5902456), Solanum (rnle-lyces: g1710615; rnlx-lyces: g1710616), Zinnia (rnze2-zele: g2148018; rnze1-zele: g2148017), Hordeum (hvulgare: g7435265), Nelumbo (nelumbo: g168740), Zea (kin1-zea: g1698670).
Luffa S-like
Luffa (lc1-luffa: g976231; lc2-luffa: g976233).
Solanaceae S-RNases
Petunia (sb1-pethyb: g4586870; sv-pethyb: g6706722; sxpethyb: g169248; s3-pethyb: g463993; sb2-pethyb: g4586872; s1-pethyb: g463991; sxb-pethyb: g169250; s1-petint: g169242; s3-petint: g169244), Nicotiana (rnsb-nical: g2696960; s7-nical: g533129; s6b-nical: g482815; rns2-nical: g133234; sa2-nical: g1184096; rns-nical: g2696958; nicsyl: g2578426), Solanum (s12-solch: g5919069; s11-solch: g548222; s14-solch: g7110526; s11-lperu: g1002594; s12-lperu: g1478373; s3-lperu: g2894088; rns2-soltu: g21576).
Scrophulariaceae S-RNases
Antirrhinum (rns2-anthi: g2500572; rns5-anthi: g1405428; rns4-anthi: g2500573).
Rosaceae S-RNases
Malus/Pyrus cladeMalus (s26-mdom: g2407178; s9-mdom: g642045; sd-mdom: g7229073; s3-mdom: g643447; s2-mdom: g643445; s27-mdom: g2407180; s24-mdom: g2407182; sh-mdom: g7229075; sg-mdom: g4587109; sf-mdom: g1018987; stmtrans: g7212796), Pyrus (s7-pyrpyr: g3434963; s1-pyrpyr: g3434939; s6-pyrpyr: g3434961; s3-pyrpyr: g7384768; s5-pyrpyr: g1772448; s2-pyrpyr: g4850324). Prunus cladePrunus (s6-pavium: g4115488; s1-pavium: g5763515; s4-pavium: g5763517; s3-pavium: g4115490; sb-pdulcis: g3927877; sc-pdulcis: g3927879).
Sequence Analysis
We first used MULTICLUSTAL (Yuan et al. 1999
) to align the amino acid sequences. MULTICLUSTAL uses ClustalW (Thompson, Higgins, and Gibson 1994
) and Boxshade iteratively to identify a set of alignment parameters that produce a high-scoring alignment. By identifying optimal ClustalW settings, we ensure that our alignment is objective and not dependent on the subjectivity that is associated with the manual alignment process. Furthermore, by reporting our optimal parameter settings, other researchers can independently evaluate our analysis. For this data set the ClustalW parameters are as followspairwise gap open penalty: 4; pairwise gap extension penalty: 0.1; pairwise matrix: ID; multiple gap open penalty: 20; multiple gap extension penalty: 0.1; multiple matrix: PAM. We then used mrtrans (Pearson 1992
) to align the DNA sequences, codon by codon, to the amino acid alignment.
Previous studies examining the primary and secondary structure of the RNase molecule have demonstrated that there are five highly conserved regions with two hypervariable regions (Green 1994
). A signal peptide lies at the N-terminus. We found it difficult to obtain a reliable alignment in the signal peptide region and in the set of residues found after the fifth conserved region. Whereas we aligned the amino acid sequences and corresponding DNA sequences along the entire length of the protein, for the phylogenetic analysis we truncated the sequences at both ends; the part of the sequence used starts at the beginning of the shortest signal peptide (arath sequence) and ends at the end of the shortest protein (sb1_pethyb sequence).
We used the Akaike Information Criterion in Modeltest (Posada and Crandall 1998
) to determine the best-fit model of evolution in a likelihood framework. In so doing, we ensure that the model employed in tree reconstruction has some statistical justification. For tree reconstruction the Bayesian methodology we use (Huelsenbeck and Ronquist 2002
) incorporates realistic models of DNA sequence evolution, and it allows rapid and accurate assessment of the reliability of the phylogenetic estimates we obtain. We used MrBayes (Huelsenbeck and Ronquist 2002
), a Bayesian tree estimation program, on the aligned DNA sequences, with the generalized time reversible (GTR) model of sequence evolution (Tava
e 1986
) including both among-site rate variation and invariable sites (see online version for MrBayes script file).
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Results and Discussion |
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The similarity between Luffa S-like alleles and Rosaceae S alleles should not be surprising. Previous studies have demonstrated a decrease in RNase activity in self-compatible plants in members of both the Solanaceae (no RNase activity) (Royo et al. 1994
) and the Rosaceae (only moderate activity) (Sassa, Hirano, and Ikehashi 1992
). Additionally, the extracellular ribonuclease RNase X2, found in the pistils of Petunia inflata, shows similar enzymatic properties to S alleles even though the rxn2 locus is not linked to the S locus, nor does the protein play any role in the self-incompatibility response. Furthermore, a genealogical analysis of RNase X2 places it into a clade containing Solanaceae S alleles, suggesting that it too is associated with duplication and the acquisition of a new function (Lee, Singh, and Kao 1992
).
Although self-incompatibility has evolved independently at least 21 times in angiosperms (see online version for details), our results strongly suggest that S-RNasemediated gametophytic self-incompatibility evolved only once in the ancestor to extant eudicots. At least three additional lines of evidence are needed to provide a strong test of this hypothesis: (1) The molecular details of gametophytic self-incompatibility must be elucidated in other eudicots. Our hypothesis predicts that the stylar response will be mediated by an S-RNase. (2) New S-RNase sequences, whether from families already surveyed or from other eudicot families with gametophytic self-incompatibility, must be obtained. Our hypothesis predicts that they will be part of the clade including Solanaceae, Rosaceae, and Scrophulariaceae S-RNase sequences. (3) New S-like RNases from flowering plants must be obtained. Our hypothesis predicts that they will fall outside the S-RNase clade unless they are associated with loss of gametophytic self-incompatibility.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Keywords: S-RNase
gametophytic self-incompatibility
molecular evolution
Address for correspondence and reprints: J. E. Steinbachs, The Center for Genomics and Bioinformatics, Indiana University, Jordan Hall, Bloomington, Indiana 47405-3700.E-mail: jen@compbiology.org
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References |
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Bateman A., E. Birney, R. Durbin, S. R. Eddy, K. L. Howe, E. L. L. Sonnhammer, 2000 The Pfam protein families database Nucleic Acids Res 28:263-266
Baumann U., J. Juttner, X. Bian, P. Langridge, 2000 Self-incompatibility in the grasses Ann. Bot 85: (Suppl. A) 203-209
Benson D. A., I. Karsch-Mizrachi, D. J. Lipman, J. Ostell, B. A. Rapp, D. L. Wheeler, 2000 GenBank Nucleic Acids Res 28:15-18
Darwin C., 1877 The various contrivances by which orchids are fertilized by insects John Murray, London
Foote H. C. C., J. P. Ride, V. E. Franklin-Tong, E. A. Walker, M. J. Lawrence, F. C. H. Franklin, 1994 Cloning and expression of a distinctive class of self-incompatibility S gene from Papaver rhoeas L Proc. Natl. Acad. Sci. USA 91:2265-2269[Abstract]
Franklin-Tong V. E., J. P. Ride, N. D. Read, A. J. Trewavas, F. C. H. Franklin, 1993 The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium Plant J 4:163-177[ISI]
Green P. J., 1994 The ribonucleases of higher plants Annu. Rev. Plant Phys. Plant Mol. Biol 45:421-445[ISI]
Holsinger K. E., J. E. Steinbachs, 1997 Mating systems and evolution in flowering plants Pp. 223248 in K. Iwatsuki and P. H. Raven, eds. Evolution and diversification of land plants. Springer-Verlag, Tokyo
Huang S., H. S. Lee, B. Karunanandaa, T. H. Kao, 1994 Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen Plant Cell 6:1021-1028
Huelsenbeck J. P., F. R. Ronquist, 2002 MRBAYES: Bayesian inference of phylogenetic trees Bioinformatics 17:754-755
Igic B., J. R. Kohn, 2001 Evolutionary relationships among self-incompatibility RNases Proc. Natl. Acad. Sci. USA 98:13167-13171
Jordan N. D., F. C. H. Franklin, V. E. Franklin-Tong, 2000 Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas Plant J 23:471-479[ISI][Medline]
Kehyr-Pour A., J. Pernes, 1985 A new S-allele and specific S-proteins associated with two S-alleles in Nicotiana alata Pp. 191196 in D. L. Mulcahy, G. B. Mulcahy, and E. Ottaviano, eds. Biotechnology and ecology of pollen. Proceedings of the International Conference on the Biotechnology and Ecology of Pollen; 911 July, 1985; University of Massachusetts, Amherst. Springer-Verlag, New York
Lee H. S., A. Singh, T. H. Kao, 1992 RNase X2, a pistil-specific ribonuclease from Petunia inflata, shares sequence similarity with solanaceous S proteins Plant Mol. Biol 20:1131-1141[ISI][Medline]
Li X., N. Paech, J. Nield, D. Hayman, P. Langridge, 1997 Self-incompatibility in the grasses: evolutionary relationship of the S gene from Phalaris coerulescens to homologous sequences in other grasses Plant Mol. Biol 34:223-232[ISI][Medline]
McClure B. A., V. Haring, P. R. Ebert, M. A. Anderson, R. J. Simpson, F. Sakiyama, A. E. Clarke, 1989 Style self-incompatibility gene products of Nicotiana alata are ribonucleases Nature 342:955-957[ISI][Medline]
Nandi O. I., M. W. Chase, P. K. Endress, 1998 A combined cladistic analysis of angiosperms using rbcL and non-molecular data sets Ann. Mo. Bot. Garden 85:137-212
Pearson W. R., 1992 mrtrans University of Virginia, Charlottesville
Posada D., K. A. Crandall, 1998 Modeltest: testing the model of DNA substitution Bioinformatics 14:817-818[Abstract]
Richman A. D., W. Broothaerts, J. R. Kohn, 1997 Self-incompatibility RNases from three plant families: homology or convergence? Am. J. Bot 84:912-917[Abstract]
Royo J., C. Kunz, Y. Kowyama, M. Anderson, A. E. Clarke, E. Newbigin, 1994 Loss of a histidine residue at the active site of S-locus ribonuclease is associated with self-compatibility in Lycopersicon peruvianum Proc. Natl. Acad. Sci. USA 91:6511-6514[Abstract]
Sassa H., H. Hirano, H. Ikehashi, 1992 Self-incompatibility-related RNases in styles of Japanese Pear (Pyrus serotina Rehd) Plant Cell Physiol 33:811-814[ISI]
Sassa H., T. Nishio, Y. Kowyama, H. Hirano, T. Koba, H. Ikehashi, 1996 Self-incompatibility (S) alleles of the Rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily Mol. Gen. Genet 250:547-557[ISI][Medline]
Schopfer C. R., M. E. Nasrallah, J. B. Nasrallah, 1999 The male determinant of self-incompatibility in Brassica Science 286:1697-1700
Snowman B. N., A. Geitmann, S. R. Clarke, C. J. Staiger, F. C. H. Franklin, A. M. C. Emons, V. E. Franklin-Tong, 2000 Signalling and the cytoskeleton of pollen tubes of Papaver rhoeas Ann. Bot 85: (Suppl. A) 49-57
Stephenson A. G., J. A. Winsor, T. E. Richardson, A. Singh, T. H. Kao, 1992 Effects of style age on the performance of self and cross pollen in Campanula rapunculoides Pp. 117121 in E. Ottaviano, D. L. Mulcahy, M. S. Gorla, and G. B. Mulcahy, eds. Angiosperm pollen and ovules. Springer-Verlag, New York
Tavae S., 1986 Some probabilistic and statistical problems in the analysis of DNA sequences Pp. 5786 in R. M. Miura, ed. Some mathematical questions in biologyDNA sequence analysis. American Mathematical Society, Providence, RI
Thompson J. D., D. G. Higgins, T. Gibson, 1994 CLUSTAL W: improving the sensitivity of progressive multiple alignment through sequence weighting, position-specific gap penalties and weight matrix choices Nucleic Acids Res 22:4673-4680[Abstract]
Ushijima K., H. Sassa, R. Tao, H. Yamane, A. M. Dandekar, T. M. Gradziel, H. Hirano, 1998 Cloning and characterization of cDNAs encoding S-RNases from almond (Prunus dulcis): primary structural features and sequence diversity of the S-RNases in Rosaceae Mol. Gen. Genet 260:261-268[ISI][Medline]
Walker E. A., J. P. Ride, S. Kurup, V. E. Franklin-Tong, M. J. Lawrence, F. C. H. Franklin, 1996 Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations Plant Mol. Biol 30:983-994[ISI][Medline]
Xue Y., R. Carpenter, H. G. Dickinson, E. S. Coen, 1996 Origin of allelic diversity in Antirrhinum S locus RNases Plant Cell 8:805-814
Yuan J., A. Amend, J. Borkowski, R. DeMarco, W. Bailey, Y. Liu, G. Xie, R. Blevins, 1999 MULTICLUSTAL: a systematic method for surveying Clustal W alignment parameters Bioinformatics 15:862-863