Biosynthesis of glycoproteins involved in the pollen-stigma interaction of incompatibility in developing flowers of Brassica oleracea L.

De-novo synthesis of the S-allele-specific glycoproteins of Brassica oleracea is demonstrated in stigmas at different developmental stages. Excised stigmas incorporate ¹⁴C-labeled amino acids into their S-glycoproteins early in development and before the self-incompatibility response is acquired, but the rate of synthesis accelerates prior to anthesis, resulting in the accumulation of high levels of the S-glycoproteins in the stigma and coinciding with the acquisition of the pollen-stigma incompatibility response. Since the self-compatible and self-incompatible zones of developing inflorescences are very sharply delineated, a threshold quantity of S-glycoproteins appears to be critical for the onset of self-incompatibility. Incorporation experiments in which [^35Smethionine was applied to intact stigma surfaces indicate that the papillae are the main sites of synthesis of the S-specific glycoproteins.Abbreviations IEF isoelectric focusing - SC self-compatibility - SDS sodium dodecyl sulfate - SI self-incompatibility     

SLR1 function is dispensable for both self-incompatible rejection and self-compatible pollination processes inBrassica

TheSLR1 gene inBrassica is related both in DNA sequence and in pattern of expression to theS-locus glycoprotein (SLG) gene involved in the self-incompatibility mechanism which recognises and arrests the germination of self pollen. However,SLR1 shows minimal allelic variation and is expressed in both self-incompatible and compatibleBrassica lines and in related, self-compatible cruciferous plants. The function of the SLR1 protein is unknown. TheSLR1 gene was specifically ablated in self-incompatible and self-compatibleBrassica plants byAgrobacterium-mediated transformation with an antisense construct. Primary transformants and homozygous T2 progeny of both self-incompatibleB. oleracea and self-compatibleB. napus recipients were found to exhibit normal pollination responses despite having no detectable SLR1 glycoprotein. This shows that the high, wild-type level of SLR1 protein is not required to sustain the self-incompatibility reaction, nor is it necessary for successful intra-specific cross-pollination between compatible lines.     

Use of the polymerase chain reaction to isolate an S-locus glycoprotein cDNA introgressed from Brassica campestris into B. napus ssp. oleifera.

Summary A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.     

Variation in the breeding system of two sympatricDombeya species on La Réunion island

The high frequency of dioecy on oceanic islands has attracted a great deal of attention in plant evolution. In this study we quantify variation in the breeding system of two sympatricDombeya species on La Réunion island. BothDombeya species are cryptically dioecious, each sex retaining non-functional morphological structures of the other sex. One species is strictly dioecious, whilst the other shows leaky dioecy, with 8/13 males producing fruit during two years of study. Males that set fruit did so following outcrossing but not after self-pollination despite the growth of self-pollen tubes in the style. This suggests the occurrence of a late-acting self-incompatibility system. Several morphologically intermediate individuals are recognized and suggested to be a result of interspecific hybridization. These purported hybrids are hermaphrodite and self-compatible, consistent with a complementary breakdown in dioecy and self-compatibility.     

Reproductive biology of the Andean-disjunct genusRetanilla (Rhamnaceae)

Various aspects of the reproductive biology of the Andean-disjunct genusRetanilla (DC.) Brongn. were studied in populations ofR. trinervia andR. ephedra located in central Chile and ofR. patagonica in southern Argentina. Flowering extends from -austral- winter (early spring inR. patagonica) to early summer. The small, white, entomophilous flowers are incompletely protandrous and last four to five days. A weak pleasant odor along with nectar and pollen attract thirty-seven diurnal insect species, ten of which are probable pollinators: these include the honey bee, eight species of solitary bees pertaining toAnthophoridae (1 sp.),Colletidae (3 spp.) andHalictidae (4 spp.), and a nemestrinid fly. Although flower morphology and individual flower phenology do not fully prevent self-pollination (and geitonogamy can easily take place), the level of autogamy is low. Therefore, some self-incompatibility mechanism seems operative inRetanilla. Fruit set of open-pollinated flowers is extremely low, with a maximal value of c. 3% inR. patagonica. In this species, ripe fruits contain on average 1.37 viable seeds. Predispersal (maternal) reproductive success (percent ovules becoming viable seeds) is 2.3%. For medium to full-sized individuals this corresponds to c. 4600 potential offspring per year. As presently known,Retanilla is a reproductively uniform group, in which the Andean disjunction seems to have exerted no particular impact, which is consistent with the view that pollination generalization exerts some stabilizing influence on floral morphology and other reproductive traits.Retanilla expresses a basic rhamnacean set of traits (including protandry and self-incompatibility) showing also high pollen production and secondary pollen presentation. The latter two traits seem characteristic of theRetanilla-Trevoa clade, suggesting that a trend to increased male effort and, perhaps, dependence upon polleneating has evolved within the tribe Colletieae.     

Flower variation and breeding systems in theCistaceae

Fourteen common southern Spanish species in theCistaceae were examined for quantitative variations in floral traits. These included the diameter of the corolla, number and size of anthers, number of ovules, and the size and shape of the gynoecium. The variables were in most instances positively correlated, species with larger flowers having more and larger anthers, more ovules, etc. Shrubs were observed to possess the largest, annuals the smallest, and subshrubs medium-sized flowers. Detailed observations of flower structure and function in a shrub (Cistus salvifolius), a spring-annual (Tuberaria guttata) and a winter-annual (T. inconspicua), revealed substantial variations in the breeding system. WhileC. salvifolius (and probably most woody species in this family) presents self-incompatibility, annual species ofTuberaria are self-compatible. In the studied population,T. inconspicua plants bore only reduced, cleistogamous flowers with no sign of a corolla.T. guttata has chasmogamous flowers that can facultatively self-pollinate.     

Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae

Japanese pear (Pyrus serotina Rehd.) exhibits gametophytic self-incompatibility. Following our previous findings that basic ribonucleases in the styles of Japanese pear are associated with self-incompatibility genes (S-RNases), stylar proteins with high pI values were analyzed by two-dimensional gel electrophoresis further to characterize S-RNases. A group of basic proteins of about 30 kDa associated with self-incompatibility genes were identified. These proteins contained sugar chains which reacted with concanavalin A and wheat germ agglutinin, and thus were designated as S-glycoproteins of Japanese pear. The fact that the S-glycoprotein was expressed at a much lower level in a self-compatible mutant than in the original variety suggested a role of S-glycoproteins in mediating self-incompatibility of Japanese pear. Immunoblot analysis indicated that S-glycoproteins are identical to previously identified S-RNases. The S-glycoproteins were predominantly expressed in the style, in the ovary in trace amounts, and not in leaf, pollen or germinated pollen. The N-terminal amino acid sequences of the S-glycoproteins showed homology not only with each other but also with those of the S-allele-associated proteins from plants of the family Solanaceae at levels of about 30–50%.     

Self-incompatibility reactions and compatible pollen-tube growth are retained with in vitro pollinations of sugar beet,Beta vulgaris L.

Summary It is shown that in vitro pollination can be used in future studies of the time course of pollen-tube development and analysis of self-incompatibility in sugar beet, Beta vulgaris L. Upon selfng a self-incompatible genotype showed the same incompatibility response after both in vitro and in vivo pollinations. No differences between cross-compatible and self-compatible pollentube growth were observed. The pollen-tube rejection occurred whether or not the pollen was prehydrated. RNA staining with Acridine Orange showed that there was less cellular RNA in the pistil tissue from in vitro-pollinated flowers. Nevertheless, pollen-tube growth and the self-incompatibility response were similar after in vivo and in vitro pollinations.     

The molecular biology of self-incompatibility systems in flowering plants

Self-incompatibility is a common mechanism by which flowering plants can exert some control over the process of fertilization. Typically, the self-incompatibility response involves the recognition and rejection of self-incompatible pollen which leads to a block in self-fertilization and, as a consequence, promotes outcrossing. In recent years, considerable progress has been made in the molecular understanding of several self-incompatibility systems. Interestingly, a common mechanism for self-incompatibility is not employed by all flowering plants, but in fact quite diverse mechanisms have been recruited for the rejection of self-incompatible pollen. In this review, the recent advances in the self-incompatibility systems of the Solanaceae, Papaveraceae, and Brassicaceae will be described as well as some of the molecular work that is emerging for the Poaceae and the heteromorphic self-incompatibility systems.     

Effects of grazing disturbance on the reproduction of a perennial herb, Cypella herbertii (Lindl.) Herb. (Iridaceae)

A recent study in a natural grassland in Argentina revealed that grazing strongly reduced the available pollinators for a population of Cypella herbertii. Comparing grazed and fenced areas (exclosures) we tested the hypothesis that self-incompatibility would be selected against in a pollinator-poor environment leading to changes in breeding system. No differences in breeding system were found between populations from inside and outside exclosures. However, we detected: (i) C. herbertii clearly exhibits a mixed-mating system but has a remarkably low spontaneous fruit formation; (ii) this species though highly self-compatible expresses a significant decay at seed set stage when self-pollinated. We conclude that grazing impact on pollinators together with C. herbertiis high dependence on pollinators should be considered when assessing the risk of extinction of this species.     

Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea

In Brassica, the S-locus glycoprotein (SLG) gene has been strongly implicated in the self-incompatibility reaction. Several alleles of this locus have been sequenced, and accordingly grouped as class I (corresponding to dominant S-alleles) and class II (recessive). We recently showed that a self-compatible (Sc) line of Brassica oleracea expressed a class II-like SLG (SLG-Sc) gene. Here, we report that the SLG-Sc glycoprotein is electrophoretically and immunochemically very similar to the recessive SLG-S15 glycoprotein, and is similarly expressed in stigmatic papillae. Moreover, by seed yield analysis, we observe that both alleles are associated with a self-compatibility response, in contrast with the other known recessive S haplotypes (S2 and S5). By genomic DNA blot analysis, we show the existence of molecular homologies between the Sc and S15 haplotypes, but demonstrate that they are not identical. On the other hand, we also report that the S2 haplotype expresses very low amounts of SLG glycoproteins, although it exhibits a self-incompatible phenotype. These results strongly question the precise role of the SLG gene in the molecular mechanisms that control the self-incompatibility reaction of Brassica.     

Enzyme polymorphism inCitrullus lanatus andC. colocynthis in Israel and Sinai

Electrophoretic and morphological variation was studied in 13 cultivars ofC. lanatus and 31 accessions ofC. colocynthis from Israel. Twelve enzyme systems were assayed, representing 19 loci. We found 12 commercially grown cultivars to be monomorphic at all loci. OneC. lanatus accession collected from Israel is highly polymorphic and carries alleles ofC. colocynthis; this accession is probably a representative of a locally cultivated land race grown by Bedouins for animal feed. Over a range of 500 km two forms ofC. colocynthis were identified: one which grows along the coastal plains of the Mediterranean and the other in the arid Negev and Sinal deserts. A high level of electrophoretic and morphological divergence was found between plants of the two regions, whereas within the ecotypes little variation was observed.     

Isolation of S-alleles from a wild population of Brassica campestris L. at Balcesme,Turkey and their characterization by S-glycoproteins

Summary S-alleles of self-incompatibility were isolated from a wild population of Brassica campestris growing at Balcesme, Turkey. Out of 88 plants observed, 73 were self-incompatible and 4 were self-compatible. In certain families, selfed progenies from a self-incompatible plant segregated into fewer than three incompatibility classes, which is consistent with a one-locus sporophytic genetic control of self-incompatibility. Out of 25 combinations of S-alleles tested, dominance interactions were observed in 6 of them on the pollen side and on 5 of them on the stigma side. The 35 S-homozygotes thus isolated consisted of 18 independent S-alleles. The number of S-alleles in this population was estimated to be more than 30. The S-locus glycoproteins (SLGs) corresponding to the respective S-alleles were identified by iso-electric focusing (IEF)-gel immunoblotting with a polyclonal antiserum against SLG⁸. SLGs in a stigma were generally composed of several bands, one major and a few minor ones, whose molecular weight was similar to each other, and the major and minor bands were heritable in correlation with each other. SDS-PAGE analysis of SLGs differentiated a few juxtaposed bands between 50 and 60 kDa, and the variations in these bands were considered to be due to differences in the number of polysaccharide residues. General features of the variation of S-genes and their SLGs between the populations in Balcesme, Turkey and Oguni, Japan, were comparatively similar to one another, despite the different surroundings and history of these populations.     

Breeding system and pollination of selected orchids of the genus Chloraea (Orchidaceae) from central Chile

The breeding system determines different ways whereby seeds will be produced, and the degree of dependency of plants on pollinators for seed set. The genus Chloraea (Orchidaceae) has its main center of diversity in southern South America. There is only poor knowledge concerning its breeding system and pollination. We determined the breeding system of C. crispa, C. chrysantha, C. galeata, and two color forms of C. bletioides (yellow- and white-flowered forms). None of the species in this study produced fruits through apomixis or autogamy, thereby indicating a complete dependency on pollen and pollinators. Geitogamy did not differ significantly with respect to xenogamy excepting in the yellow-flowered form of C. bletioides. Thus, the indexes of self-incompatibility for the white- and yellow-flowered forms of C. bletioides, C. galeata, C. crispa, and C. chrysantha, were 1.00, 0.56, 0.82, 1.09, and 0.81, respectively; indicating that, excluding the yellow-flowered form of C. bletioides which must be regarded as partially self-incompatible, all orchids assessed are totally self-compatible plants. Natural fruiting in the yellow-flowered C. bletioides, C. chrysantha and C. galeata was high, in spite of being nectarless orchids, since the availability of pollinators under natural conditions seemingly resulted unlimited. However, no pollinator was observed visiting C. chrysantha and C. galeata, whereas the yellow-flowered form of C. bletioides was visited by hymenopterans and coleopterans. At contrast, reproductive success of the white-flowered form of C. bletioides and C. crispa was pollen limited, the former being visited by hymenopterans, dipterans, and colepterans; and the latter by two hymenopterans.     

Cloning and sequencing of cDNAs encoding two SSS

A defective S-allele, S ₀, and a functional S-allele, S _x, have previously been found to be retained in an F₁ hybrid of a self-compatible commercial cultivar of Petunia hybrida. Pistil proteins associated with these two alleles have also been identified. Their amino-terminal sequences have been found to share a high degree of similarity with those of S-proteins characterized from self-incompatible solanaceous species. Here we report the isolation and sequencing of cDNAs encoding S ₀- and S _x-proteins. Their deduced amino acid sequences contain all the consensus primary structural features of S-proteins from self-incompatible solanaceous species. Both proteins also have ribonuclease activity. The implications of these findings are discussed in relation to the presumed function of the S-protein in the self-incompatibility interaction.     

Breeding system of the annual Cruciferae, <Emphasis Type="Italic">Arabidopsis kamchatica</Emphasis> subsp. <Emphasis Type="Italic">kawasakiana</Emphasis>

The breeding system of an annual Cruciferae, Arabidopsis kamchatica subsp. kawasakiana, was studied in three natural populations. We applied four experimental treatments, open pollination, bagging, emasculation + bagging, and emasculation + hand-pollination + bagging. None of the emasculated flowers with bags produced fruits but we observed high fruit sets in the other three treatments. The results confirmed that A. kamchatica subsp. kawasakiana is a self-compatible, non-apomictic species that can produce seeds through auto-pollination. Considering the life cycle as an annual, increased reproductive assurance through auto-pollination should be critical for the maintenance of populations of A. kamchatica subsp. kawasakiana.     

Variation and evolution inLinum tenuifolium (Linaceae)

The phenetic variation inL. tenuifolium s.l. was assessed using multivariate analyses of 27 characters found to be variable within the species. These results are discussed and interpreted in the context of the reproductive biology, chromosome number and ecological responses of the taxa. Evidence suggests that both the self-compatible and tetraploid races were derived independently from a self-incompatible, diploid type. Although no taxonomic scheme is compatible with all of the evidence presented, the study supports a recent decision to recognize four taxa at subspecific level.     

The reproductive biology of the genusBaldellia (Alismataceae)

The reproductive biology of the genusBaldellia was studied under cultivation and in nature.B. ranunculoides subsp.repens is self-incompatible whereasB. ranunculoides subsp.ranunculoides andB. alpestris are self-compatible. Bud-pollination occurs in theB. alpestris. Self-incompatibility correlates with the production of fewer, larger, more showy flowers, higher pollen production (but reduced pollen grain size), fewer carpels and smaller seeds. Resource allocation to sexual reproduction is inversely correlated with the degree of vegetative growth.B. ranunculoides subsp.repens produces more ramets than subsp.ranunculoides, which reproduces mainly by seeds.B. alpestris is intermediate between the two since it is self-compatible, produces fewer, larger seed than subsp.ranunculoides and is also able to propagate by vegetative means.     

MULTIPLE ORIGINS OF SELF-COMPATIBILITY IN LINANTHUS SECTION LEPTOSIPHON (POLEMONIACEAE): PHYLOGENETIC EVIDENCE FROM INTERNAL-TRANSCRIBED-SPACER SEQUENCE DATA

Phylogenetic reconstruction based on sequence variation in the internal transcribed spacer (ITS) region of rDNA was used to investigate the evolutionary dynamics of homomorphic self-incompatibility in Linanthus section Leptosiphon (Polemoniaceae), a group of annual plant species. Hand-pollination experiments revealed that five species were self-incompatible and four were self-compatible. Optimization of breeding systems onto the tree resulting from maximum-likelihood analysis, with no assumptions made about the ancestral condition, indicated that self-incompatibility has been lost four times in this section. An alternative tree rearrangement conforming to the hypothesis of three losses of self-incompatibility did not have a significantly lower likelihood than the maximum-likelihood tree as determined by a paired-sites test, but all rearrangements resulting in fewer than three losses were statistically rejected. Linanthus bicolor, a selfing species, was found to be polyphyletic, with populations from different geographic regions occurring in three well-supported clades. Morphological similarity in these distinct lineages is likely to have resulted from convergent evolution of traits associated with self-fertilization. Selection for reproductive assurance is hypothesized to have played an important role in the recurrent transformations from self-incompatibility to selfing in this group of annual species.     

Quantitatively determined self-incompatibility

Summary It has been claimed that Borage (Borago officInalis L.) has a multifactorial self-incompatibility system. Such systems may have a high level of ineffective pollination, and we show that this is the case in borage. The ranking of seed set from highest to lowest is as follows: bee-pollination; natural pollination in the absence of bees; artificial cross-pollination between unrelated plants; artificial cross-pollination between related plants; artificial self-pollination. In diallel crosses, significant parental effects were detected but no consistent patterns of seed set, which suggest a simple self-incompatibility system, were detected. The level of outcrossing with natural pollination was very variable but greater than 50%. Thus, there appears to be no straightforward self-incompatibility system in borage.