Journal of Experimental Botany, Vol. 60, No. 4, pp. 1069–1081, 2009 doi:10.1093/jxb/erp024

DARWIN REVIEW Darwin’s foundation for investigating self-incompatibility and the progress toward a physiological model for S-RNase-based SI

Bruce McClure* Division of Biochemistry, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 65211-7310, USA

Received 31 October 2008; Revised 20 January 2009; Accepted 22 January 2009

Abstract Charles Darwin made extensive observations of the pollination biology of a wide variety of plants. He carefully documented the consequences of self-pollination and described species that were self-sterile but that could easily be crossed with other plants of the same species. He believed that compatibility was controlled by the ‘mutual action’ of pollen and pistil contents. A genetic model for self-sterility was developed in the early 1900s based on studies of the compatibility relationships among, what are now referred to as, self-incompatible (SI) Nicotiana species. Today, it is believed that SI in these species is controlled by an interaction between S-RNases produced in the pistil and F-box proteins expressed in pollen and, moreover, that this S-RNase-based SI system is shared by a great diversity of other plant species. Current research is aimed at understanding how the mutual actions of these S-gene products function in the physiological context of pollen tube growth.

Key words: Darwin, self-incompatibility, SFB, SLF, S-RNase.

The 200th anniversary of Darwin’s birth and the 150th hypotheses and extend his studies. Darwin’s insight not- anniversary of the publication of Origin of species is a good withstanding, the modern understanding of inheritance—in time to reflect on Darwin’s broad contributions. Darwin’s terms of the unit of transmission, the gene, its chemical family was definitely on the winning side of the industrial nature, the processes of gene expression, and the genomic revolution; he was a grandson of Josiah Wedgwood who and population-level contexts of genes—represents dramatic developed the famous line of manufactured pottery. Given advances that would surely have thrilled him. Thus, this the importance of Darwin’s ideas to modern biology, it is review will also highlight current SI research; insights that a little frightening to consider how contingent they were on this author would share with him, if that were possible. The the whims of history. Nevertheless, it is fortunate that an reader is referred to de Nettancourt’s (1977, 2001) classic intellect of Darwin’s quality ended up in the circumstances monographs and the recent volume edited by Franklin- to travel the world observing and collecting, and then to Tong (2008a) for more in-depth treatments. spend a lifetime in further reflection and experimentation. This review will recall Darwin’s work on self- and cross- pollination, in particular, on what is now called self- incompatibility (SI). The focus will be on progress toward Darwin on heterostyly and self-sterility understanding S-RNase-based SI. Darwin’s own words will Darwin had a very keen interest—expressed on several be used to convey his thoughts and his fascination with levels—in plant reproduction. He published two editions on many of the same questions that ignite students today. The pollination of orchids (Darwin 1862, 1877a), but the works insights are often uncanny, and researchers interested in most relevant here are The different forms of flowers on population-level studies still find it productive to test his plants of the same species, published in 1877, and The effects

* To whom correspondence should be addressed. E-mail: [email protected] ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 1070 | McClure of cross and self-fertilisation in the vegetable kingdom, first published in 1876 with a second edition in 1878. He was well aware of earlier works (especially by Ko¨lreuter, Mu¨ller, and Scott) that described instances of what we now refer to as SI. An early paper on self-sterility by EM East includes a useful review of this early work (East and Park, 1917). Darwin’s later work shows that he was definitely excited by the variety and the subtlety of plant reproduction. There is hardly anything more wonderful in nature than the sensitiveness of the sexual elements to external influences, and the delicacy of their affinities..We see how sensitive the sexual elements of those plants must be, which are com- pletely sterile with their own pollen, but are fertile with that of any other individual of the same species. (Darwin, 1878; Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 p. 467) In The different forms of flowers, Darwin discusses many instances where a single species elaborates alternative floral Fig. 1. Legitimate and illegitimate pollination in Primula. The figure morphologies, but plants that display, what is now referred is reproduced from Darwin (1877b). Reproduced with permission to as, heteromorphic SI capture the most attention. Hetero- from John van Wyhe, ed., The complete work of Charles Darwin styly in Primula and its relatives is the best known system. online (http://darwin-online.org.uk/). His passion for comprehensive, meticulous observation and experimentation is clearly expressed. Darwin describes the differences between floral morphs in great detail, including On a more theoretical level, Darwin recognized the pollen size and appearance, organ positioning and size, and importance of restricted mating and described it as a mech- stigma papillae. He experimentally probes the function anism for dividing a population into distinct ‘bodies’ of of organ positioning, but he finds, importantly, that it is compatible partners. not sufficient to ensure that crossing occurs only between (W)e have here a case to which no parallel exists in the morphs: vegetable or, indeed, in the animal kingdom. The individual plants of the present species, and as we shall see of several It follows from the position of the organs that if the proboscis other species of Primula, are divided into two sets or bodies, of a dead humble-bee, or a thick bristle or rough needle, be which cannot be called distinct sexes, for both are hermaph- pushed down the corolla, first of one form and then of the rodites; yet they are to a certain extent sexually distinct, for other, as an insect would do in visiting the two forms growing they require reciprocal union for perfect fertility. (Darwin, mingled together, pollen from the long-stamened form 1877b; p. 28) adheres round the base of the object, and is left with certainty on the stigma of the long-styled form; whilst pollen from the He also understood that mating was subject to natural short stamens of the long-styled form adheres a little way selection and that heterostyly had emerged independently in above the extremity of the object, and some is generally left on different plant lineages at different times. Furthermore, he the stigma of the other form. In accordance with this conducted experiments showing the benefit of outcrossing observation I found that the two kinds of pollen, which could and the ‘evil’ of selfing in many species. He perceived that easily be recognised under the microscope, adhered in this the benefit of outcrossing within a species was sufficient to manner to the proboscides of the two species of humble-bees select for emergence of incompatibility systems. Barret and and of the moth, which were caught visiting the flowers; but Shore (2008) emphasize that a more developed view takes some small grains were mingled with the larger grains round account of different benefits of heterostyly per se versus the base of the proboscis, and conversely some large grains intramorph incompatibility; the former reduces pollen waste with the small grains near the extremity of the proboscis. Thus pollen will be regularly carried from the one form to the by facilitating its placement on receptive stigmas while other, and they will reciprocally fertilise one another. . The incompatibility prevents inbreeding and contributes more several foregoing facts led me to try the effects of the two to maternal fitness. kinds of pollen on the stigmas of the two forms. (Darwin, We may feel sure that plants have been rendered hetero- 1877b;p.24) styled to ensure cross-fertilisation, for we now know that His experiments revealed that intermorph (i.e. legitimate) a cross between the distinct individuals of the same species is pollinations were fully fertile while intramorph (i.e. illegiti- highly important for the vigour and fertility of the offspring. (Darwin, 1877b; p. 258) mate) pollinations set few seed. Figure 1, reproduced from Darwin’s book, shows these relationships diagrammatically; (W)e may confidently believe that this has been effected in this figure has been so frequently imitated by subsequent order that cross-fertilisation should be assured. For the full generations of scholars that any student of pollination will and legitimate fertilisation of these plants pollen from the recognize it. one form must be applied to the stigma of another.. There Progress toward a model for S-RNase-based SI | 1071

is reason to believe that the sterility of these unions has not same result been confirmed by several subsequent trials. been specially acquired, but follows as an incidental result (Darwin, 1878; p. 188) from the sexual elements of the two or three forms having been adapted to act on one another in a particular manner, It is clear that Darwin found self-sterility noteworthy and so that any other kind of union is inefficient, like that appreciated its potential significance. However, lacking between distinct species. (Darwin, 1877b; p. 345) a genetic framework for understanding self-sterility, he seems, at times, to conflate self-sterility due to environmen- Here, and in other places, Darwin seems to suggest that tal circumstances with, what is now regarded as, genetically intramorph incompatibility must arise from interactions controlled SI. between the constituents of pollen and pistils. This observa- tion seems especially prescient since he did not understand We know that self-fertilised seedlings are inferior in many how traits are transmitted between generations. It implies respects to those from a cross; and as with plants in a state of nature pollen from the same flower can hardly fail to be that he understood that, however inheritance occurred, it often left by insects or by the wind on the stigma, it seems at was the basis for not only overt morphological variation, first sight highly probable that self-sterility has been but also for compositional (or, as we would say today, gradually acquired through natural selection in order to Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 molecular) variation. This insight anticipates the rich prevent self-fertilisation..Nevertheless, the belief that self- molecular-level studies possible today. It is noteworthy that sterility is a quality which has been gradually acquired for he also suggests a fundamental similarity between intra- and the special purpose of preventing self-fertilisation must, interspecific mechanisms, at least in so far as these arise I believe, be rejected. (Darwin, 1878; p. 345) from the ‘constituents’ of pollen and pistils. In this observation, Darwin seems to make a distinction We must therefore look to the appearance of inner or hidden between intramorph incompatibility in heterostylous species constitutional differences between the individuals of a vary- and self-sterility. He clearly regards the former as a response ing species, of such a nature that the male element of one set to natural selection, but he has a less settled view about self- is enabled to act efficiently only on the female element of sterility, as exemplified by this quote. This is understandable another set..But what the nature of the inner constitutional since factors such as nutrient availability can affect fertility differences may be between the sets or forms of the same and probably explain lack of seed set in some of Darwin’s varying species, or between distinct species, is quite un- crosses. Also, while different mating types are easily known. (Darwin, 1877b; p. 267) recognized in heterostylous species, this is not so for the SI While The different forms of flowers on plants of the same species Darwin described in Cross and self-fertilisation; species deals extensively with heterostyly, and Darwin incompatible or partially compatible crosses probably were definitely regarded intramorph incompatibility as an essen- also observed, particularly if Darwin’s plants contained S tial aspect of such systems, he also investigated self-sterility a relatively small number of -haplotypes. Nevertheless, his distinction has little appeal to modern readers trained to among species that do not display distinct floral morpholo- explain biological phenomena in terms of biochemistry and gies. In The effects of cross and self-fertilisation in the genetics; modern research into intramorph incompatibility vegetable kingdom, Darwin describes exhaustive experiments proceeds from the same basic assumptions as research into with >50 species that provide overwhelming support for his SI (Barrett and Shore, 2008). Furthermore, modern views of conclusion ‘that generally cross-fertilisation is beneficial, SI are largely shaped by studies of model species specifically and self-fertilisation is often injurious’ (Darwin, 1878). selected for their robust response. Darwin’s plants, as well Several of the species investigated were fully or partially as many wild species, are not so well behaved (Good-Avila ‘self-sterile’ including species still studied today. Regarding et al., 2008). Still, his persistent study of self-sterile species Petunia violacea, Dingy purple variety, Darwin noted: suggests that he certainly did not always attribute the (P)rotected flowers with their own pollen placed on the phenomenon to environmental effects and that he appreci- stigma never yielded nearly a full complement of seed; whilst ated the significance of specific mechanisms that control those left uncovered produced fine capsules, showing that pollination. Darwin’s study of Reseda odorata, common pollen from other plants must have been brought to them, mignonette, provides an interesting example. probably by moths. Plants growing vigorously and flowering Plants of the common mignonette were raised from pur- in pots in the greenhouse, never yielded a single capsule; and chased seed, and several of them were placed under separate this may be attributed, at least in chief part, to the exclusion nets. Of these some became loaded with spontaneously self- of moths. fertilised capsules; others produced a few, and others not Six flowers on a plant covered by a net were crossed with a single one. It must not be supposed that these latter plants pollen from a distinct plant and produced six capsules, produced no seed because their stigmas did not receive any containing by weight 4.44 grains of seed. Six other flowers pollen, for they were repeatedly fertilised with pollen from were fertilised with their own pollen and produced only the same plant with no effect; but they were perfectly fertile three capsules, containing only 1.49 grains weight of seed. with pollen from any other plant. (Darwin, 1878; p. 119) From this it follows that an equal number of crossed and self-fertilised capsules would have contained seeds by weight Darwin’s R. odorata materials may have been segregating as 100 to 67. I should not have thought the proportional for SI, although he would not have understood it in this contents of so few capsules worth giving, had not nearly the way. Notwithstanding, it can be inferred that Darwin 1072 | McClure understood the basic behaviour of species like this in the When individuals of any two classes are crossed, two classes same way SI is understood today and also appreciated the appear in approximately equal numbers, but the class to implications of such a system, as suggested by the following which the female parent belongs is never represented. observations in Cross and self-fertilisation: X3Y gives Y and Z Z3X gives X and Y Y3X gives X and Z Y3Z gives X and Z It is an extraordinary fact that with many species, flowers X3Z gives Y and Z Z3Y gives X and Y fertilised with their own pollen are either absolutely or in At first sight one might suppose that these results, differing some degree sterile; if fertilised with pollen from another as they do from any hitherto obtained in genetic work, are flower on the same plant, they are sometimes, though rarely, due to a novel type of gamete distribution; yet such a little more fertile; if fertilised with pollen from another a supposition is rendered unlikely by two facts. Morpholog- individual or variety of the same species, they are fully ical characters in these varieties appear to be transmitted in fertile; but if with pollen from a distinct species, they are the normal Mendelian manner; furthermore the ratios in sterile in all possible degrees, until utter sterility is reached. which the classes appear here are strikingly like those We thus have a long series with absolute sterility at the two expected when F1, monohybrids are backcrossed with pure ends;—at one end due to the sexual elements not having recessives.

been sufficiently differentiated, and at the other end to their To be brief, we believe that the inheritance in these cases is Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 having been differentiated in too great a degree, or in some of the ordinary type, but that there are physiological peculiar manner. limitations to the opportunity for fertilization. The fertilisation of one of the higher plants depends, in the Assume that in these populations there are three allelo-

first place, on the mutual action of the pollen-grains and the morphs, S1,S2 and S3; and that Class X ¼ S1S3, Class stigmatic secretion or tissues, and afterwards on the mutual Y ¼ S1S2, Class Z ¼ S2S3. Assume further that a plant action of the contents of the pollen-grains and ovules. Both affords stimulus only to pollen which bears sterility factors actions, judging from the increased fertility of the parent- other than its own. Thus a Class Z plant (S2S3) affords plants and from the increased powers of growth in the proper stimulus only to gametes carrying factors other than

offspring, are favoured by some degree of differentiation in S2 or S3. In other words, of the three factors present in these the elements which interact and unite so as to form a new various populations, the Z plants afford stimulus only to S1 being. (Darwin, 1878; p. 455) gametes. (East and Mangelsdorf, 1925) Darwin’s insights are uncanny. He anticipates the modern Later, East and Yarnell (1929) reported 16 S-alleles in view to the extent that we think about mechanisms Nicotiana. Even today, the diversity of S-alleles is a subject controlling pollination in terms of the ‘constitution’ of the of much research in theoretical and population biology, but, pollen and pistil. We also appreciate the overall function of at the time, this must have seemed like a wild excursion into such systems as avoiding deleterious effects from inbreeding new territory. The nature of the gene was still controversial, and interspecific crosses. A genetic framework is clearly and the existence of so many S-alleles contributed to missing from Darwin’s work, however. With an under- the debate. Importantly, each S-allele behaved as a discrete standing of inheritance and with a greatly developed entity that could be passed unchanged from one generation understanding of the ‘constitution’ of cells (i.e. biochemis- to the next. East argued, in a spirited and entertaining try), mechanisms controlling pollination are now conceived way, that this is not compatible with a quantitative gene of as a seamless continuum from the molecular to the concept. population level. It is natural to speculate as to the bearing of these facts, if any, on the nature of the gene, and in particular on GOLDSCHMIDT’S quantitative theory of heredity. We Early genetic studies of SI have isolated 16 allelomorphs of the S factor (including Sf). They are all point mutations since they all behave as Botanists working at the end of the 19th century articulated allelomorphs to each other and since the crossover values of the basic rules of Mendelian inheritance. In the first decades 3 of them with the corolla-color factor C are approximately of the 20th century, inheritance was linked to chromosomes, the same. .. Now, it may be that each of these 16 and genetic maps were being developed. However, the allelomorphs is charactized by a pollen-tube growth-rate of nature of the gene was also under debate; many traits did velocity different from all others.. not seem to be inherited simply. Studies of the presence of On the other hand, one may assume that each S allelo- self-sterility revealed Mendelian inheritance. For example, morph differs from the next in order by a definite quantum Compton (1913) reinvestigated R. odorata and found that expressed in reaction velocity and may speculate as to what self-pollination of some self-fertile plants yields self-fertile conclusions follow, if any, in connection with our concep- and self-sterile plants in a 3:1 ratio. East and Park (1917) tion of the gene. To put the matter most simply: Does made the important observation that the term ‘self-sterility’ a postulate of the above type lead to the conclusion that the different velocities of the reactions directed by the is not really apropos. By analysing large families of various allelomorphs of a single gene are due to the Nicotiana forgetiana3N. alata hybrids over five generations, presence of different quantities of the gene? Arguments in they showed that the families consisted of classes that were favor of such a conclusion have been presented with much intrasterile but interfertile. One of East’s later studies vigor and clarity in the various writings of GOLDSCHMIDT. revealed that the classes were produced according to specific The writers have only admiration for the brilliant work of rules. the Berlin geneticist and certainly do not wish to be Progress toward a model for S-RNase-based SI | 1073

numbered among those who say that his position is ‘neither pollination specificity. Studies of SI also took on signifi- sound genetics nor sound physiology’, (GOLDSCHMIDT cance as an example of plant cell–cell communication. The 1928); yet they are of the opinion that a sympathetic and single-locus genetics of SI, with its implied simplicity at the receptive agnosticism in regard to his quantitative theory of underlying biochemical level, seemed to offer both a good gene action is at present the wisest course to take. (East and prospect for success and an entry point for molecular-level Yarnell, 1929) studies. The approaches used to identify S-gene products East’s works from this period, especially the passage were based on the biological and genetic foundations laid quoted above from East and Mangelsdorf (1925), can be previously. said to mark the beginning of modern thinking about SI. Broadly, the stigma and style function to support the Although he continued to use the term self-sterility, East growth of desirable pollen and prevent the growth of definitively showed that the system is better characterized as undesirable pollen. Interactions between the pollen and a genetically controlled mechanism for differentiating pistil are mediated by the extracellular matrix (ECM) of families into intrasterile, interfertile compatibility classes both partners, and signals travel in both directions. The that could be understood as distinct S-genotypes. In ECM contains lipids, carbohydrates, and glycoproteins. summary, the ‘Nicotiana type’ of SI was found to be Thus, the problem has been framed as identifying S-specific Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 controlled by a series of alleles of a single locus, designated products in the ECM. the S-locus. Plants with two S-alleles in common are Standing on the shoulders of a giant like East makes incompatible. When plants sharing one allele are crossed, identifying S-genes easy; they are the genes that determine only the pollen with the S-allele that is distinct from the S-specific pollen rejection (Fig. 2). Identifying S-gene pistil can effect fertilization. Since it is the S-genotype or, as products is another matter. Bredemeijer and Blass (1981) it is currently understood, the S-haplotype, of the male first correctly identified S-glycoproteins in N. alata. They gametophyte that determines compatibility, this type of SI analysed pistil protein extracts and identified bands associ- is now called gametophytic SI. ated with specific S-alleles. Earlier, Pandy (1967) had used Clearly, with these advances, compatibility between a similar approach and identified isozymes associated with classes was conceived as being controlled at the physiolog- S-alleles, but they did not prove to be products of the ical level. Although no mechanism was postulated, the S-locus. The different results from these two studies physiology was thought of as directly controlling the foreshadowed a lingering problem in SI studies: SI favours opportunity for fertilization (East and Mangelsdorf, 1925). outcrossing and, thus, maintains a high level of poly- This conclusion can be seen as a dramatic realization of morphism (especially in genes closely linked to the S-locus) Darwin’s notion, quoted earlier, that pollination could be that complicates genetic analysis. Modern researchers do controlled by ‘mutual action of the pollen-grains and the not generally have the wherewithal for studies such as East’s stigmatic secretion or tissues’ (Darwin, 1878). Physiological that involve tens of thousands of crosses (East and control over compatibility occurs against the background of Park, 1917). siphonogamy; that is, the stigma and style provide an Anderson et al. (1986) cloned the first S-glycoprotein; environment for pollen to germinate and for pollen tubes they obtained the sequence from protein isolated from the to grow toward the ovary and carry the sperm cells that will pistil ECM and then used this information to identify the N. fertilize the egg and central cells. Thus, today, it is believed alata S2-glycoprotein cDNA. McClure et al. (1989) showed that Darwin’s ‘mutual action’ is best regarded as a series of that S-glycoproteins possess RNase activity, and, hence- interactions in the stigma, style, and ovary that must occur forth, these proteins have been known as S-RNases. successfully for fertilization to occur. The challenge for Cloning the first S-RNase was a breakthrough that opened understanding SI at the physiological level came to be the door to characterizing the structure and expression of S- understood in terms of identifying the products of the S- gene products and laid the groundwork for understanding locus genes. In the Nicotiana system, it took 60 years to the physiological basis for pollen rejection that Darwin meet this challenge. Species with similar SI systems are now sensed, but could not grasp, a century earlier. Early studies studied throughout the Solanaceae, Rosaceae, and Planta- showed that S-RNase expression coincided with the de- ginaceae. SI in Papaver has an entirely different mechanism, velopmental onset of SI and that expression was restricted although the genetics are identical (Franklin-Tong, 2008b), to cells forming the path from the stigma to the ovary and many other distinct SI systems have been described in (Anderson et al., 1986; Cornish et al., 1987). Sequence other families (deNettancourt, 1977, 2001; Franklin-Tong, analysis showed that S-RNases possess secretion signals, 2008a). and immunolocalization studies provided direct evidence that they are deposited in the ECM (Anderson, 1989). The abundance of S-RNase in the ECM was studied by protein Identification and characterization of purification and immunological techniques, and both S-genes in S-RNase-based SI systems methods support concentration estimates near 1 mM (Jahnen et al., 1989). S-RNase transcripts are also A major goal of the last quarter century has been to identify extremely abundant and display very long poly(A) tails the genes that control SI, characterize their products, and (McClure et al., 1993). All S-RNases also possess N-linked place them in a physiological framework that accounts for glycans (Woodward et al., 1989), and Oxley et al. (1996, 1074 | McClure

or some other function in SI. Requirement for high-level expression is clear (Clark et al., 1990; Murfett et al., 1994, 1995), but it is still not known why so much S-RNase is needed. Many experiments characterizing S-RNases relied on the ability to manipulate cloned genes in vitro and to reintro- duce them into transgenic plants. These experiments were challenging because of the extraordinary expression levels of S-RNases. However, experiments in Nicotiana, Petunia, and Solanum showed that transformation of a cloned S-RNase into a new genetic background is sufficient to cause rejection of the corresponding pollen S-haplotype (Huang et al., 1994; Lee et al., 1994; Murfett et al., 1994, 1995; Matton et al., 1997, 1999; Beecher and McClure, 1999). For example, expressing SA2-RNase from N. alata causes re- Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 jection of SA2-pollen (Murfett et al., 1994). Similarly, suppressing S-RNase expression prevents S-specific pollen rejection (Lee et al., 1994; Murfett et al., 1995). These experiments are important because they definitively show that the S-RNase protein contains all the information needed to determine S-specificity in the pistil. In a classic experiment, Huang et al. (1994) expressed catalytically inactive S3-RNase in P. inflata and showed that it does not cause S-specific pollen rejection. This finding provided direct evidence that RNase activity is required for pollen rejection. Further experiments addressed how S-specificity is encoded in the S-RNase sequence. Sequence comparisons showed that S-RNases contain five conserved regions, C1– Fig. 2. Crossing relationships define S-genes. In Nicotiana-type, C5, that altogether comprise ;40 residues (Ioerger et al., or other gametophytic SI systems, pollen is rejected when the S- 1991; Ishimizu et al., 1998). Histidine residues implicated in haplotype of haploid pollen is matched by at least one of the two catalysis are located in C2 and C3 (Ishimizu et al., 1995). S-haplotypes in the diploid pistil. Thus, a cross is fully compatible Structural studies show that C1, C2, and C5 form three (Full) when parents have no shared S-haplotypes, half-compatible strands of b-sheet at the protein core; one of the catalytic (1/2) when parents share one S-haplotype, or incompatible (–) histidines projects from C3 toward one face of the sheet, when they share no S-haplotypes. However, since pollen is usually and C4 contributes hydrophobic residues that interact with in excess, a half-compatible pollination often results in full seed set the other side of the sheet (Ida et al., 2001). However, and, in practice, cannot be distinguished from a fully compatible sequence variation is the most striking feature of S-RNases. pollination. (A) Progeny of a fully compatible cross S1S23S3S4 are Even in the conserved regions, there are only a small shown. After East and Park (1917) the classes of intrasterile, number of highly conserved residues, and the rest of the interfertile progeny are understood as having the same S- protein (i.e. ;160 residues) is subject to variation (Ishimizu haplotypes [e.g. the S1S3 class is intrasterile (–), but fertile with et al., 1995). Domain swap experiments tested whether other classes (1/2 or Full)]. (B) Progeny of a forced-cross. SI is a specific S-RNase region determines S-specificity. Zurek developmentally controlled and often can be overcome by et al. (1997) made a series of nine chimeric constructs pollinating immature buds. S-heterozygotes reject pollen from all exchanging sequences between N. alata SA2- and SC10- siblings, while homozygotes are interfertile with 75% of their RNases. Although all these chimeric proteins were active siblings. The two classes of homozygotes, S S and S S , are 1 1 2 2 RNases, none was able to cause rejection of either SA2-or intrasterile and interfertile. When available, force-cross families SC10-pollen. Similar results were obtained with two con- provide an efficient route to identifying S-genes because of the structs in Petunia (Kao and McCubbin, 1996). These results smaller number of progeny classes and the simple behaviour of suggest that residues that determine S-specificity are not homozygotes. restricted to a particular region of S-RNase. Nevertheless, sequence analyses suggest that two regions, often referred to as HVa and HVb, are especially variable (Ioerger et al., 1998) have determined the structure of several glycans 1991). Experiments in Solanum showed that exchanging just present on S-RNases from N. alata. Although it is clear four of these residues between two highly similar proteins is that N-glycans are not required for S-specific recognition sufficient to change S-RNase specificity (Matton et al., per se (Karunanandaa et al., 1994), it is possible that 1997). Thus, these regions are important for pollen recogni- glycosylation facilitates secretion, solubility in the ECM, tion but are not sufficient in all cases. Progress toward a model for S-RNase-based SI | 1075

Non-S-specific factors also contribute to SI on the pistil carrier to the client (Moon et al., 2004; Smalle and Vierstra, side. Unlinked modifiers of S-RNase-based SI have been 2004). The polyubiquitylated client is subject to degradation noted in numerous studies (Anderson and de Winton, 1931; by the 26S proteasome. Although the Antirrhinum S-linked East, 1932; Martin, 1961, 1968; Ai et al., 1991; Bernatzky F-box protein gene did not, at first, appear to be sufficiently et al., 1995), but only two have been cloned. The HT-B gene polymorphic to determine S-specificity, later studies from N. alata encodes a 101 residue asparagine-rich protein strongly support that F-box proteins do function as pollen expressed in the pistil (McClure et al., 1999). Suppression of S genes in S-RNase-based SI. These genes are referred to as HT-B in Nicotiana and Solanum interferes with S-specific S-locus F-box (SLF) or S-locus F-box (SFB) in different pollen rejection (McClure et al., 1999; O’Brien et al., 2002). species. Strong evidence implicating F-box proteins in SI Although the exact function of HT-B is not known, was obtained in Prunus species with relatively compact immunolocalization experiments show that pollen tube genomes (reviewed in McClure, 2004). Sijacic et al. (2004) uptake of S-RNase occurs normally in plants with undetect- definitively showed that the SLF2 gene from P. inflata has able HT-B protein (Goldraij et al., 2006). Thus, HT-B is an S-specific effect on pollination, providing direct evidence required only after S-RNase has entered the pollen tube. In that this gene determines S-specificity. An Antirrhinum SLF

Solanum, two very similar genes, HT-A and HT-B, have gene has also been implicated in SI, but S-specificity could Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 been identified, but RNA interference (RNAi) results only not be directly tested (Qiao et al., 2004a). implicate HT-B in SI (O’Brien et al., 2002). Further studies SLF proteins usually show less sequence polymorphism in the tomato clade of Solanum suggest that mutations in than S-RNases. A recent summary showed non-synonomous HT-B genes might have been important in the transition substitution rates of 0.14–0.51 for S-RNases from Antirrhi- from SI to self-compatability (SC) (Kondo et al., 2002a, b). num, Petunia,andPrunus compared with 0.01–0.11 for SLF Other HT-family genes have been identified in Petunia and or SFB (Newbigin et al., 2008). SLF sequences from Nicotiana, and sequence analysis of the larger family Antirrhinum are especially noteworthy for their lack of suggests similarity to a class of membrane-associated sequence polymorphism. This is surprising not only because glycine-rich proteins (Sassa and Hirano, 2006; Kondo and a certain level of polymorphism is expected of recognition McClure, 2008). Interestingly, HT-B is selectively degraded proteins, but also because of the contrast with S-RNase after compatible pollinations in N. alata (Goldraij et al., sequences. The results are difficult to reconcile with the 2006). strong expectation for a shared evolutionary history for The 120 kDa glycoprotein (120K), an abundant arabino- pairs of pollen and pistil specificity genes in a given S- galactan protein present in the N. alata pistil ECM (Lind haplotype. Petunia SLF genes show more polymorphism et al., 1994), has also been implicated in SI (Hancock et al., than Antirrhinum sequences, and, as noted previously, these 2005). 120K is an S-RNase-binding protein (Cruz-Garcia genes have been directly implicated in SI. Prunus SFB genes et al., 2005) that is taken up by pollen tubes growing in the display a degree of polymorphism that is closer to S-RNase. transmitting tract’s ECM (Lind et al., 1996; Goldraij et al., The presence of multiple SLF-orSFB-like genes linked to 2006). S-RNase is taken up normally in 120K-suppressed S-RNase is a serious complication. Wheeler and Newbigin plants, suggesting that, as with HT-B, 120K plays a role in (2007) found 10 F-box protein genes linked to S-RNase in a later step in the SI response. Yeast two-hybrid (Y2H) N. alata, seven of which were expressed in pollen. It is not experiments identified pollen proteins that interact with clear which, if any, of these genes is pollen S. Further, 120K, one of which, interestingly, is NaSBP1 (N. alata phylogenetic analyses of Antirrhinum and Petunia sequences S-RNase-binding protein 1), a protein first identified in show that SLF genes do not display all the features P. hybrida that interacts with S-RNase (Sims and Ordanic, expected of pollen S genes (Wheeler and Newbigin, 2007; 2001) and the S-locus F-box protein (Hua and Kao, 2006). Newbigin et al., 2008), and yet the evidence at the molecular 120K also interacts with a pollen-specific C2-domain- level seems incontrovertible (Sijacic et al., 2004). In containing protein, NaPCCP (Lee et al., 2008), which may contrast, phylogenetic analyses of Prunus SFB genes do have a role in transporting proteins from the pistil’s ECM conform to expectations (Newbigin et al., 2008). Together, to the pollen tube’s endomembrane compartments. the results suggest that there is still much to learn about the NaPCCP’s C-terminal domain binds to 120K, while the C2 role of SLF and SFB genes in SI. domain specifically binds to phosphatidylinositol-3-phos- Y2H and in vitro studies show that SLF proteins bind to phate (PIP3; CB Lee et al., 2009). S-RNase as well as to a variety of pollen proteins. Qiao The determinant of S-specificity on the pollen side is et al. (2004b) reported Y2H and pull-down experiments referred to as pollen S. Unlike S-RNase, which was showing that the C-terminal portion of AhSLF-S2 binds to identified using protein-based approaches, pollen S was S-RNase in a non-S-specific fashion. Subsequent studies identified by searching for pollen-expressed genes linked to identified a pollen-specific Skp1-like protein, AhSSK1, that the S-RNase gene. Lai et al. (2002) were the first to identify binds to AhSLF proteins and also interacts with Cullin-1 an F-box protein gene linked to S-RNase. F-box proteins (Huang et al., 2006). The authors suggest that AhSSK1, are best known for their functions in the ubiquitin– Cullin-1, and AhSLF proteins function together in an proteasome system. In this context, they usually function to SCF-like complex. Somewhat different results have been bind a client protein to an SCF (Skp1–Cullin–F-box) E3 obtained with Petunia SLF proteins. Hua and Kao (2006) ubiquitin ligase complex that transfers ubiquitin from an E2 specifically tested three P. inflata Skp1 homologues for 1076 | McClure interaction with PiSLF2, but reported no interaction in inhibition based on S-RNase-based cytotoxicity emerged: Y2H. This is an extremely interesting result given that the S-RNases are secreted into the ECM by pistil cells and F-box specifically functions to bind an F-box protein to taken up by growing pollen tubes, and incompatibility is a Skp1-like protein (Bai et al., 1996). Nevertheless, Hua and explained by a cytotoxic effect of RNA degradation (Gray Kao (2006) reported that, instead, PiSLF proteins bind to et al., 1991). PhSBP1, a protein also reported to interact with S-RNase, The cytotoxic model provided a neat explanation for 120K, and certain transcription factors (Sims and Ordanic, incompatibility and, consequently, focus shifted to under- 2001; O’Brien, 2004; Ben-Naim et al., 2007; Lee et al., standing how pollen tubes evade cytotoxic S-RNase in 2008). SBP1 proteins contain RING domains and are compatible pollinations. Two alternatives were suggested: thought to act as E3 ubiquitin ligases (Sims and Ordanic, pollen S might function as an S-specific S-RNase receptor 2001; Hua and Kao, 2006; Hua et al., 2008). Hua and Kao that provides for uptake of only self S-RNase; or S-RNase (2006) also reported that PiSBP1 binds to a cullin protein uptake could be non-specific, and pollen S could function to and proposed that it forms an SLF–SBP1–Cullin complex inhibit non-self S-RNase (Thompson and Kirch, 1992). The that functions in SI. Other studies suggest that non-self former hypothesis received little support, but the hypothesis

S-RNases bind to SLF more productively than self S-RNases that pollen S functions to inhibit the action of S-RNase Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 (Hua et al., 2007). SLF proteins are divided into three stimulated much research. For example, mutational studies domains, FD1, FD2, and FD3. FD2 is a putative non- in Nicotiana showed that all pollen-part mutants could be S-specific S-RNase-binding domain (SBD), and putative explained as duplications of all or parts of the S-locus (Golz recognition regions are present in the flanking FD1 and et al., 2001). The absence of deletion mutants was inter- FD3 domains. The authors proposed that, in incompatible preted as evidence that pollen S is an essential gene and, in pollinations, FD2 binds all S-RNases but that self-inter- turn, that it functions to inhibit S-RNase. Studies of actions (e.g. S1-RNase interacting with PiSLF1) prevent breakdown of SI in tetraploids of solanaceous species were a productive complex from forming. A non-self interaction also consistent with pollen S acting as an inhibitor. A (e.g. S1-RNase with PiSLF2) is proposed to be stable, tetraploid such as S1S1S2S2 is typically SC. The defect is on allowing ubiquitylation and subsequent degradation of the pollen side since it rejects pollen from SI S1S2 diploids, non-self S-RNase in compatible pollinations (Hua et al., but the reciprocal pollination is compatible. Significantly, 2008). Thus, there is evidence that SLF proteins interact only heteroallelic pollen (i.e. diploid S1S2 pollen) shows SI with other pollen proteins and with S-RNase. However, breakdown; S1S1 and S2S2 diploid pollen are rejected there is conflicting evidence about whether these pollen normally. This is sometimes referred to as the heteroallelic proteins form an SCF-like complex or, a novel SLF–SBP1– pollen (HAP) effect; it is thought to reflect an inhibitory Cullin complex. action of pollen S where the pollen S alleles provide cross- Darwin recognized that the constitution of the pollen and protection against S-RNases. For example, pollen S1 pistil control pollination. Many of the constituents required inhibits the action of all S-RNases except S1-RNase, and for S-RNase-based SI have been identified: S-RNase, SLF/ pollen S2 inhibits all S-RNases except S2-RNase. However, SFB, 120K, HT-B, and perhaps SSK1, Cullin, and SBP-1. different results have been obtained in Prunus species that S-RNase and SLF determine S-specificity, so their interac- also possess S-RNase-based SI. For example, an SC mutant tion ultimately controls compatibility. The complexes in Prunus avium possesses a deletion of the pollen S gene, formed with pollen proteins and their potential interactions known as SFB in this species (Sonneveld et al., 2005). with pistil factors such as HT-B and 120K are central to Furthermore, the HAP effect does not occur in tetraploid models of the physiology of SI. cherry, Prunus cerasus (Hauck et al., 2006). Some authors suggest that these conflicting results reflect different un- derlying SI mechanisms between Prunus and solanaceous Physiological models for S-RNase-based SI species (Hauck et al., 2006). However, neither system is sufficiently understood at the biochemical level to test this The discovery that the pistil-side S-specificity determinants hypothesis properly. are S-RNases was a turning point in the field, as it Preferential degradation of non-self S-RNase in compat- suggested a physiological basis for pollen rejection. ible pollen tubes could explain resistance to its cytotoxic Enzymes that attack RNA function as cytotoxins in effects (Fig. 3). This is a logical hypothesis since, as organisms as diverse as bacteria and plants. Therefore, it is described in the previous section, there is evidence that reasonable to hypothesize that S-RNases have a cytotoxic SLF proteins participate in ubiquitin ligase complexes. In effect on incompatible pollen tubes (McClure, 1989, 1990; the S-RNase degradation model for S-RNase-based SI, Gray, 1991). Experimental evidence for cytotoxicity was either an SCFSLF (i.e. in Antirrhinum, Huang et al., 2006) provided from tracer experiments showing that pollen or a PiSLF–PiSBP1–PiCUL1-G (i.e. in Petunia, Hua et al., rRNA is degraded after incompatible, but not compatible, 2008) complex provide for ubiquitylation and subsequent pollinations (McClure et al., 1990). Further experiments proteasomal degradation of non-self S-RNases in compati- showed that S-RNases can enter pollen tubes where they ble pollen tubes. Hua and Kao (2007) provided support for can act as effective translational inhibitors (Gray et al., this proposal by showing that non-self S-RNases might 1991). Thus, a physiological mechanism for pollen tube indeed bind to SLF proteins more tightly than self Progress toward a model for S-RNase-based SI | 1077 Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 Fig. 3. Models for S-RNase-based SI. Top: compartmentalization model. S-RNase (SRN) is taken up by endocytosis and transported in the pollen tube endomembrane system. Most S-RNase is known to be transported to the vacuole (lower). By analogy with the cytotoxin ricin, some S-RNase might be transported to the ER (upper), where it could exit the endomembrane system and interact with SLF. Bottom: S-RNase degradation model. S-RNase enters the cytoplasm where it interacts with SLF. Non-self S-RNase is ubiquitylated and degraded in a compatible pollination.

S-RNases. Finally, in vitro experiments with extracts show effective mechanism to prevent its cytotoxicity, and release that S-RNase can be degraded in pollen tube extracts, of S-RNase from the endomembrane system could account although degradation is not S specific (Hua et al., 2006). for pollen tube rejection. It has already been mentioned that Thus, many results are consistent with a non-self S-RNase HT-B, a pistil protein required for pollen rejection, is degradation model to explain how compatible pollen tubes selectively degraded in compatible pollen tubes. Goldraij evade S-RNase cytotoxicity. et al. (2006) speculated that HT-B might have a role in S- Studies of the uptake and fate of S-RNase in pollen tubes RNase release, although there are many other possibilities. led to formulation of an alternative model, one that The S-RNase–SLF interaction may somehow stabilize HT- explains compatibility as arising from compartmentalization B in incompatible pollinations indirectly. For example, in of S-RNase. The compartmentalization model consists of compatible pollinations, HT-B could be degraded by a pollen three main steps: non-S-specific uptake of S-RNase into the protein that, in an incompatible pollination, is destabilized by endomembrane system, an S-specific step that affects HT-B a self S-RNase–SLF interaction (Goldraij et al.,2006).The stability, and release of S-RNase in incompatible pollina- compartmentalization model is compatible with breakdown tions. In this model, compatibility is regarded as a default of SI in tetraploids if a biochemical interaction between condition arising from sequestration of S-RNase into allelic SLF proteins interferes with self-recognition. One a vacuole where it cannot attack RNA in the cytoplasm. possibility is that SLF proteins form inactive multimers, as Incompatibility actively interferes with the default mecha- has been previously proposed (Luu et al.,2001).For nisms pollen tubes use to escape S-RNase cytotoxicity; example, in an S1S2 diploid pollen tube, an inactive SLF1– ultimately, incompatibility leads to release of S-RNase from SLF2 dimer might be incapable of inactivating the pollen the endomembrane system. Luu et al. (2000) presented protein that degrades HT-B, causing the pollen tube to revert immunolocalization results showing non-S-specific uptake to the default condition of compatibility. of S-RNase in Solanum chacoense. They reported S-RNase in the pollen tube cytoplasm, but their fixation methods were not designed for optimum membrane preservation, Prospects and specific compartment markers were not used. Later, Goldraij et al. (2006) investigated S-RNase and 120K There are unresolved issues in both the S-RNase degrada- uptake in N. alata pollen tubes using immunolocalization tion and compartmentalization models. The S-RNase deg- and confocal microscopy. In compatible pollen tubes, the radation model does not easily accommodate a role for the results showed S-RNase present in the lumen of pollen non-S-specific factors required for SI in Nicotiana, Petunia, vacuoles and 120K present in the lumen and the surround- and Solanum (Anderson and de Winton, 1931; East, 1932; ing membrane. Two additional markers, vacuolar pyro- Martin, 1961, 1968; Ai et al., 1991; Bernatzky et al., 1995). phosphatase and aleurain, confirmed the identity of the Some of the molecular-level details of this model are S-RNase-containing vacuolar compartment. Late-stage in- awkward. For example, the ‘common domains’ in compatible pollen tubes, in contrast, did not show S-RNase S-RNases, usually referred to as conserved regions C1–C5 compartmentalization, but rather a signal distributed (Ioerger et al., 1991), are located in the protein interior (Ida throughout the pollen tube. Clearly, compartmentalization et al., 2001) where it is unlikely they could interact with an of S-RNase away from pollen tube cytoplasm would be an SBD on SLF. All other S-RNase regions are subject to 1078 | McClure sequence variation, making it difficult to envisage how with incompatible pollinations (Goldraij et al., 2006). Thus, numerous non-self S-RNases would always be positioned its stability is regulated by the S-RNase–SLF interaction. to interact preferentially with a given SLF protein. In vitro As discussed above, it is possible that this occurs by action studies point to direct binding between S-RNase, SLF, and of a pollen protein whose stability is regulated in SI. auxiliary pollen proteins, and also provide evidence for However, no data confirm this hypothesis. It is also possible ubiquitylation (Qiao et al., 2004b; Hua and Kao, 2006, 2008; that regulation of HT-B stability could be less direct. Huang et al., 2006; Hua et al., 2007). Further studies show The S-RNase degradation and the compartmentalization that S-RNase is subject to degradation by the 26S protea- models make different inferences about the relationship some in pollen extracts. However, further research is needed between the S-specificity-determining S-RNase–SLF inter- to provide a firm connection between these results. S-RNase action and the ability of pollen to prevent the cytotoxic degradation in vitro is not S-specific, which could be action of S-RNase. In the S-RNase degradation model, non- interpreted as evidence that SLF is not involved (Hua and self S-RNase is degraded as a direct result of this interaction. Kao, 2006). It might also be significant that glycosylated In the compartmentalization model, inhibition arises by S-RNase isolated from pistils is not degraded by pollen anon-S-specific mechanism that sequesters S-RNase into extracts (Hua and Kao, 2006). In yeast and mammals, PNG a vacuole where it cannot interact with its substrate, and the Downloaded from https://academic.oup.com/jxb/article/60/4/1069/568810 by guest on 01 October 2021 (peptide:N-glycanase) associates with the 26S proteasome via S-RNase–SLF interaction interferes with this process. Thus, HR23B, where it assists with glycoprotein degradation the degradation model predicts that SLF is an essential gene, (Katiyar et al.,2004;Liet al., 2005). Thus, the possibility and this is consistent with interpretations of mutagenesis that the non-S-specific degradation of S-RNase in pollen experiments in solanaceous species (Golz et al.,2001).Inthe tube extracts is due to a process entirely separate from SI has compartmentalization model, SLF causes pollen rejection so not been eliminated. Similarly, SBP1–SLF binding should be is not essential, and this is consistent with identification of interpreted cautiously. SBP1 is expressed in almost all tissues pollen S deletion mutants in Prunus (Sonneveld et al.,2005). (Sims and Ordanic, 2001; O’Brien, 2004; Lee, 2008), and it Nevertheless, the models are not irreconcilable. Pollen tubes appears to bind to a variety of client proteins. Several of its might possess a multilayered resistance to S-RNase where clients are implicated in SI [i.e. S-RNase, SLF, and 120K most non-self S-RNase is rendered harmless by compartmen- (Sims and Ordanic, 2001; Hua and Kao, 2006; Lee et al., talization but a small amount is degraded. 2008)], but some are not (Ben-Naim et al., 2007; Lee et al., Darwin clearly showed keen insight; he inferred that 2008). This observation in no way excludes a role for SBP1– pollination is controlled by mutual action between constit- SLF–S-RNase interactions in SI, but it highlights the need uents of the pollen and pistil. Yet, he did not understand for experiments that distinguish between S-specific processes genetics as we do, and East’s genetic model for SI might be and other cellular mechanisms. said to have laid the groundwork for all subsequent The compartmentalization model also has deficiencies. molecular-level studies. Using increasingly subtle tools of Localization studies in Antirrhinum show that SLF is modern biochemistry, molecular biology, and cellular localized in the cytoplasm, with some concentrations imaging, modern researchers have identified several key observed near the endoplasmic reticulum (ER) (Wang and pollen and pistil constituents and proposed ‘mutual actions’ Xue, 2005). Thus, S-RNase must move to the cytoplasm to that could control pollination. For better or worse, this is interact with SLF. To date, no data address how this what we have to show for a century and a half. We can only occurs. One speculation is that transport of S-RNase may hope our progress will be judged as useful in another be similar to that of the cytotoxin ricin (McClure, 2006). century and a half. Ricin is a binary ribosome-inactivating cytotoxin synthe- sized in cotyledons of Ricinus communis and sequestered in protein storage vacuoles. It enters mammalian cells by Acknowledgements endocytosis, and, like S-RNase, most ricin is sequestered in I thank Melody Kroll for editorial assistance and help with the vacuole. However, a small portion of ricin moves the figures. I also thank Professor John van Wyhe for through the Golgi and then to the ER by retrograde permission to use Fig. 1 and his work on the Darwin Online transport (van Deurs et al., 1988). The cytotoxic ricin A project (http://darwin-online.org.uk/). Work in the author’s chain is thought to exit the ER using the ER-associated laboratory is supported by US National Science Founda- degradation (ERAD) system (Wesche et al., 1999). During tion grants IOB 0614962 and DBI 0605200. exit, it is acted upon by PNG, and only a small portion of the ricin A chain taken up by the cell evades degradation to exert its cytotoxic effect. Ricin provides a model for References movement of a cytotoxin from luminal compartments in the endomembrane system to the cytoplasm, and it is Ai Y, Kron E, Kao T-H. 1991. S-alleles are retained and expressed in possible that S-RNase follows a similar pathway (Fig. 3). a self-compatible cultivar of Petunia hybrida. Molecular and General The precise role of factors such as HT-B and, in particular, Genetics 230, 353–358. the mechanism of HT-B degradation in compatible pollen Anderson E, de Winton D. 1931. The genetic analysis of an unusual tubes are not yet explained. Results clearly show that HT-B relationship between self-sterility and self-fertility in Nicotiana. Annals stability is decreased in compatible pollinations compared of the Missouri Botanical Garden 18, 97–116. Progress toward a model for S-RNase-based SI | 1079

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