Incompatibility and Incongruity in Wild and Cultivated Plants, pp. 141-145 (2013)
By Dreux de Nettancourt

3.5 Monofactorial Stylar GS1 with Multiple Alleles: the Nicotiana Type

3.5.7.1.2 Rosaceae. S ribonucleases have been found to operate in the SI systems of several species of Rosaceae (a family that is not related to the Solanaceae), including apples, Japanese pears, cherries (Hiratsuka 1992a, 1992b; Sassa et al. 1992, 1993, 1994, 1996; Broothaerts et al. 1995) and, more recently, almonds (Tao et al. 1997) and apricots (Burgos et al. 1998).

Sassa et al. (1992) were the first to establish that polymorphism in stylar ribonucleases of Japanese pears is associated with S genotypes and to describe a self-compatible mutant where stylar ribonuclease is barely detectable but is normally produced in the original variety from which the mutant developed as a sport. In 1993, they showed that the N-terminal sequences of the ribonuclease from Japanese pears are similar to those of the Solanaceae. Broothaerts et al. (1995; with apples) and Sassa et al. (1996; with apples and Japanese pears) isolated cDNA clones and deduced the AA sequences. The position of C4 in the Solanaceae is occupied by region RC4, which shares no homology with C4 (Ushijima et al. t998a). Other regions present striking similarities with the Solanaceae. These similarities are meaningful. In particular, throughout the two families, the conservation of the eight cysteine residues that form the four disulfide bridges of the S ribonucleases demonstrates the importance of these cross-links for the stabilization of the tertiary structure of the protein (Ishimizu et al. 1996). Using cryosections from apple pistils and specific antibodies against S proteins, Certal et al. (1999) showed that the S ribonucleases of apples are localized in the intercellular space of the transmitting tissue along the pollen-tube pathway in both the stigma and style. Non-S-specific intracellular labeling confined to one layer of the nucleus was observed in all ovary sections.

3.5.7.1.3 Scrophulariaceae. In the Scrophulariaceae, close relatives of the Solanaceae, research has concentrated on Anthirrhinum. Xue and co-workers (1996) characterized the cDNAs encoding polypeptides homologous to S ribonucleases. Through co-segregation studies with DNA gel blots probed with cONAs, determination of the period of gene expression at anthesis and the observation of sequence polymorphism typical for S allele, they demonstrated that these cDNAs are derived from functional S alleles. Region C4 is missing, and considerable inter-allelic diversity can be observed for regions Hva and Hvb.

3.5.7.2 What Are the Effects of S Ribonucleases on rRNA and mRNA?

It has been shown, through EM observations of ribosomes and polysomes in pollen tubes formed after self-pollination in Datura suaveolens and N. alata, that S-RNases do not act as intracellular cytotoxins that degrade rRNA and perhaps mRNA. Walles and Han (1998), who undertook that study, found that there is no decrease over time in the number of bound ribosomes per unit of rough endoplasmic-reticulum membrane after incompatible pollination. They concluded that the substrate for S-RNases is probably far more specific than expected. The discovery implies that the effects of S-RNases on the physiology of incompatible pollen tubes may be more discrete than initially considered (Walles and Han 1998; Sect. 3.5.7.2). In her review of the different causes for the death of incompatible pollen tubes, Geitmann (1999) lists some of the reasons, essentially based on the presence of large quantities of ribosomes in incompatible tubes, that led her to reconsider the exact function of S ribonucleases. However, she agrees with Clarke and Newbigin (1993) that "it seems unlikely that the RNase function would have been so tightly conserved if it were not functional in SI." Indeed, as suggested by the observations of Lush and Clarke (Sects. 3.5.2.1, 3.5.7.3), the possibility also exists that the rRNAs degraded by specific S ribonucleases are, at least to some extent, replaced by the pollen tube through de novo synthesis. The final fate (death through bursting of its apex, no growth or slow growth) of the incompatible tube could be determined by the rate of replacement.

3.5.7.3 Are the Effects of Ribonucleases Irreversible?

Stylar grafts, which associate the top of a style (the scion) bearing known S alleles with the base of another style (the stock) having a different S genotype, have been used in the past for different purposes (Sect. 4.1.5). The technique was utilized recently by Lush and Clarke (1997) in a study of the behavior of incompatible pollen‑tube growth in N. alata (Fig. 3.20). Through the grafting of incompatible and compatible scions and stocks, they found that the tubes (which continue to grow very slowly in an incompatible scion) could revert to normal growth if they managed to cross the graft junction and reach compatible territory. Not all incompatible tubes are able to perform in this manner, because some burst open and others stop growing; however, a substantial proportion of them finally reach the ovary. The observations require very precise measurements of growth rate. They suggest that either S‑ribonuclease action is not instrumental in the expression of SI (Sect. 3.5.7.2) or — and this is more likely that pollen tubes have the ability to synthesize fresh rRNA when degraded RNA needs to be replaced, at least under SOS conditions. This second explanation does not agree with conclusions from McClure et al. (1990) and Mascarenhas (1993) that rRNA is not expressed in angiosperm pollen tubes. However, the explanation is supported by results from earlier work by Campbell and Ascher (1975), Tupý et al. (1977) and Bagni et al. (1981). A third possibility, resulting from the work of Walles and Han (1998), is that there is no such thing as a general degradation of rRNA in incompatible pollen tubes (Sect. 3.5.7.2).

Fig. 3.20. The growth of pollen tubes (genotype S6) in intact styles and in style grafts. Grafts were made 5 h after the pollination of styles. Scions were 6 mm long. Pollen-tube lengths were measured 24 h after grafting. Figures are the means of five pollinations or grafts. (Lush and Clarke 1997)

3.5.7.4 Why Pollen Tubes Are Not Inhibited in the Stigma

The responsibility probably lies on the side of the pollen tube, because McClure et al. (1993) clearly established that S ribonuclease purified from the stigma is indistinguishable (by SOS-PAGE, chromatographic behavior and ribonuclease activity) from S ribonuclease purified from the style. Furthermore, although the stigma transcripts are shorter and more heterogeneous that those of the style, sequence analyses of cloned cDNA showed that stigmatic and stylar S transcripts derive from the same gene. It is possible that ribonucleases cannot have access to the pollen or to pollen RNA before the tube has reached a particular length or developed a certain receptivity. Another explanation could be that the concentration of rRNA in the tube at the onset of pollen germination is sufficient to sustain protein synthesis during the first period of growth through the stigma.

3.5.7.5 What Determines the S Specificity of Stylar Ribonucleases?

S ribonucleases are glycoproteins, and their S specificity must lie in the carbohydrate moiety, the AA sequence or a combination of the two.

3.5.7.5.1 The Role of the Carbohydrate Moiety. Five potential N-glycosylation sites occur in the S ribonucleases of N. alata; these sites are present in variable numbers in different S alleles (five in S3; four in S2, S6, S7; one in S1...) and may play a role in S specificity (Oxley et al. 1996 for information and detailed descriptions of the structure of N-glycans on S alleles).

Taking into account the important role played by the glycan chains of many animal cell proteins in cell-cell recognition, Karunanandaa et al. (1994) studied the possibility that the glycan chain of the S3 ribonuclease of P. inflata is involved in self/non-self recognition. They replaced the codon for Asn-29 by a codon for Asp at the only potential N-glycosylation site of the S3 protein and introduced this mutated S3 gene into S1S2 plants via Agrobacterium-mediated transformation. Six transgenic plants produced a normal level of the non-glycosylated S3 protein and expressed the ability to reject S3 pollen completely (Sect. 4.2.4.2). In other words, the carbohydrate moiety of the S3 ribonuclease does not participate in the recognition or rejection of self pollen. The possibility, suggested by Oxley et al. (1996), that the lack of N-glycosylation had abolished the specificity of the SI reaction in the transgenic plant and allowed it to reject any pollen is unlikely because, in this case, one would not expect the non-glycosylated S3 allele to reject all pollen. Instead, it would act as an SC allele that accepts all pollen.

In a discussion of the role of the N-glycans on the S-ribonuclease of N. alata, Oxley et al. (1999) noted that all cDNA sequences of the S-RNase products of functional S alleles contain at least one potential N-glycosylation site, with site I conserved in all cases. While not dismissing the conclusions of the Karunanandaa group, they observe that site I is not present on the S-like ribonucleases that do not participate in SI (Green 1994). As a consequence, they maintain their opinion that site I is possibly involved in SI. Ishimizu and co-workers (1999) identified the structures of the N-glycosylalion sites of seven S-RNases in Pyrus pyrifolia (Rosaceae). The presence of N-acetylglucosamine and chitobiose in the putative recognition sites of the S-RNases led Ishimizu and co-workers to suggest that these sugar chains may interact with pollen S products.

The debate on the possible role of the glycan chains in pollen-pistil recognition is important, because the current efforts to identify the recognition determinants of S-ribonucleases through domain swapping and site-directed mutagenesis (Sect. 4.2.7.1) are based on the hypothesis that S specificity results from differences in the AA sequences of the S domains.

3.5.7.5.2 The Role of HV Regions. One naturally assumes that the two regions Hva and Hvb, which display a high degree of sequence diversity and are hydrophilic, are directly involved in the recognition interaction of S ribonucleases with pollen determinants. In order to assess this role, three sets of mosaic constructs among S ribonucleases have been realized. These constructs involved the swapping of HV regions in Petunia (Kao and McCubbin 1996), of the different domains of the protein in Nicotiana (Zurek et al. 1997) and of nucleotides in the HV regions of Solanum (Matton et al. 1997). These experiments (Sect. 4.2.7.1) led to diverging conclusions.

The work in Petunia and Nicotiana showed that the transfer of HV regions and domains between alleles resulted in the production of hybrid ribonucleases unable to cause the rejection of any of the two parental alleles. The sequences necessary for pollen recognition are apparently not confined only to the HV regions but are scattered throughout the entire ribonuclease. In contrast, the elimination (through an appropriate replacement of nucleotides) of differences between the HV regions of two closely related alleles of S. chacoense conferred the recognition capacity of the second allele to the modified alleles (Matton et al. 1997). In other words, it seems that the HV regions (and only they) determine the specificities of stylar ribonucleases.

While the controversy is not settled (Verica et al. 1999; Matton et al. 1999), it appears that at least two reasons can be advanced to account for the differences of results by the Montreal group and the two American universities. First, the two Solanum alleles compared by Matton and co-workers are nearly identical (with a variation of four AAs between the HV regions and six among the other regions). It is therefore likely that other parts of the ribonuclease sequence, possibly involved in the determination of S specificity, are identical in the two alleles. It is also possible, if only the HV regions control S specificity, that the swapping of HV regions in Petunia and Nicotiana interfere with their activities and functions.

3.5.8 S-Gene Products in Pollen Grains and Pollen-Pistil Recognition

3.5.8.1 The S-Ribonuclease Gene is Expressed in Developing Pollen Grains ...

It has been found, in conformity with the dimer hypothesis of Lewis (1965), that S-ribonuclease transcripts accumulate in young anthers and developing pollen grains.