Homomorphic self-incompatibility, with stylar rejection site, in Luehea grandiflora Mart. and Zucc. (Tiliaceae-Malvaceae s.l.)

The breeding system of Luehea grandiflora (Tiliaceae-Malvaceae s.l.) was investigated using hand pollinations and fluorescence microscopy studies of pollen tube growth. Although selfed flowers persisted for some 10 days, our study indicates that L. grandiflora is self-incompatible, with self pollen tube inhibition in the upper style, as occurs in many taxa with homomorphic, gametophytic self-incompatibility (GSI). L. grandiflora is only the second species reported within the Malvales with homomorphic stylar inhibition. This result is discussed within the context of a report for self-compatibility in this species, and we also consider the phylogenetic implications for the occurrence of GSI in the family Malvaceae s.l.

The effect of different levels of inbreeding on self-incompatibility and inbreeding depression in pineapple

A high level of heterozygosity in domesticated pineapple is one of the main obstacles hindering the efficient development of new varieties. Selfing has been proposed as a strategy to minimise this heterozygosity through the development of parentals with a greater level of homozygosity. Selfing and a range of lesser levels of inbreeding were evaluated for their effects on seed production and inbreeding depression in the early growth of seedlings. Selfing produced few seeds, and very few viable seedlings. The paternal backcross and several half-sib combinations exhibited minimal effects on seed development or early growth and in some cases were similar to the outcross. Sibcrosses were generally unsuccessful. The highest inbreeding coefficient that was not associated with severe inbreeding depression was approximately 0.25. The effect of inbreeding depression and the level of homozygosity on several quantitative traits including those related to fruit quality within the inbred populations, is now being assessed.

Effects of self-, chase and mixed self/cross-pollinations on pistil longevity and fruit set in Ceiba species (Bombacaceae) with late-acting self-incompatibility

Background and aims Late-acting self-incompatibility (LSI). in which selfed flowers fail to form fruits despite apparently successful growth of the pollen tubes to the ovules, is a contentious and still poorly understood phenomenon. Some studies have indicated pollen tube-pistil interactions, and major gene control. Others favour an early acting inbreeding depression explanation.Methods Experimental pollinations, including selfs (in a subsample of which the style was cut before pollen tubes reached the ovary), chase self/cross-pollinations, crosses, and mixed self/cross-pollinations were used to study floral/pistil longevity and effect on fruit set and seed yield in two Ceiba species known to have LSI.Results Self-pollinations, including those with a cut style, had extended floral longevity compared with unpollinated flowers. Chase pollinations in which cross-pollen was applied up to 3 h after selfing set fruits, but with reduced seed set compared with crosses. Those with cross-pollen applied at 4 and 8 h after self-pollination all failed to set fruits. Flowers subjected to 1 : 1 and 2 : 1 self/cross-pollinations all produced fruits but again with a significantly lower seed set compared with crosses.Conclusions Extended floral longevity initiated with self-pollen tubes growing in the style indicates some kind of pollen tube-pistil interaction. Fruit set only in chase pollinations up to 3 h implies that self-pollen tubes either grow more slowly in the style or penetrate ovules more slowly on arrival at the ovary compared with cross-tubes. This agrees with previous observations indicating that the incidence of penetrated ovules is initially lower in selfed compared with crossed pistils. However, the low seed yield from mixed pollinations indicates that self- and cross-pollen tubes arrive at the ovary and penetrate ovules more or less simultaneously. Possible explanations for these discordant results are discussed. (C) 2004 Annals of Botany Company.

Floral biology and late-acting self-incompatibility in Jacaranda racemosa (Bignoniaceae)

Breeding-system studies have been conducted with 38 of the approximately 800 species of Bignoniaceae, and self-incompatibility was found in 31 of these. In species for which the site of self-incompatibility barrier was studied, self-pollinated flowers consistently failed to develop into fruits, even though pollen tubes grew down to the ovary and penetrated most of the ovules. In this study, we have investigated the. oral biology and the breeding system in Jacaranda racemosa Chamisso, with hand-pollination experiments and the histology of post-pollination events. Flower anthesis lasted 1-3 days, and although the frequency of flower visitation was extremely low, natural pollination seemed to be effected mainly by medium-sized bees. Because the conspicuous staminodium favours eventual pollination by small bees, a possible role of the staminodium in the increase of potential pollinators is suggested. Hand-pollinations indicated that J. racemosa is a self-sterile species. Histological analysis of post-pollination events indicated the occurrence of a kind of late-acting self-incompatibility in which the processes of ovule penetration, fertilisation and endosperm initiation were slower in selfed than in crossed pistils. Until the time of self-pollinated pistil abscission, no signs of endosperm malfunction or proembryo development were observed in selfed pistils. Therefore, inbreeding depression is an unlikely explanation for self-sterility in J. racemosa.

The pollen determinant of self-incompatibility in Brassica campestris

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLG (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S9 haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S9 haplotype applied to papillar cells of S9 stigmas, but not of S8 stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.

Structural Analysis and Molecular Model of a Self-Incompatibility RNase from Wild Tomato1

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

How flowering plants discriminate between self and non-self pollen to prevent inbreeding.

Flowering plants have evolved various genetic mechanisms to circumvent the tendency for self-fertilization created by the close proximity of male and female reproductive organs in a bisexual flower. One such mechanism is gametophytic self-incompatibility, which allows the female reproductive organ, the pistil, to distinguish between self pollen and non-self pollen; self pollen is rejected, whereas non-self pollen is accepted for fertilization. The Solanaceae family has been used as a model to study the molecular and biochemical basis of self/non-self-recognition and self-rejection. Discrimination of self and non-self pollen by the pistil is controlled by a single polymorphic locus, the S locus. The protein products of S alleles in the pistil, S proteins, were initially identified based on their cosegregation with S alleles. S proteins have recently been shown to indeed control the ability of the pistil to recognize and reject self pollen. S proteins are also RNases, and the RNase activity has been shown to be essential for rejection of self pollen, suggesting that the biochemical mechanism of self-rejection involves the cytotoxic action of the RNase activity. S proteins contain various numbers of N-linked glycans, but the carbohydrate moiety has been shown not to be required for the function of S proteins, suggesting that the S allele specificity determinant of S proteins lies in the amino acid sequence. The male component in self-incompatibility interactions, the pollen S gene, has not yet been identified. The possible nature of the pollen S gene product and the possible mechanism by which allele-specific rejection of pollen is accomplished are discussed.

Destinée des S-RNases dans les tubes polliniques lors des croisements compatibles et incompatibles

L’auto-incompatibilité (AI) est la capacité génétiquement déterminée d’une plante fertile de rejeter son propre pollen. Chez les Solanacées l’AI dépend des éléments d’un locus fort complexe (locus S) multigénique. L’élément du locus-S exprimé dans le pistil est une ribonucléase (S-RNase) dont le rôle est de dégrader l’ARN chez le pollen self, tandis que l’élément du locus S exprimé dans le pollen est un ensemble de protéines du type F-box, qui sont normalement impliquées dans la dégradation des protéines. Cependant, comment les S-RNases self restent actives lors des croisements incompatibles et comment les S-RNases non-self sont inactivées lors des croisements compatibles ce n’est encore pas clair. Un modèle propose que les S-RNases non-self soient dégradées lors des croisements compatibles. Un autre modèle propose que toutes les S-RNases, self et non-self, soient d'abord séquestrées à l’intérieur d’une vacuole, et elles y resteraient lors des croisements compatibles. Lors de croisements incompatibles, par contre, elles seraient relâchées dans le cytoplasme, où elles pourront exercer leur action cytotoxique. Notre étude tente de répondre à ces questions. Notamment, nous cherchons à mettre en évidence la localisation vacuolaire et/ou cytoplasmique des S-RNases et leur concentration par immunolocalisation, en utilisant un anticorps ciblant la S11-RNase de Solanum chacoense et la microcopie électronique à transmission. Nos résultats montrent que la densité de marquage observée pour les S-RNases cytoplasmiques est significativement plus haute dans les tubes incompatibles que dans ceux compatibles ce qui nous indique que pour qu’un tube pollinique soit compatible il doit contenir une faible densité de S-RNase cytoplasmique.

Destins des S-RNases et interactions moléculaires dans le tube pollinique dans le cadre de l’auto-incompatibilité gamétophytique chez Solanum chacoense

L’auto-incompatibilité (AI) est une barrière reproductive prézygotique qui permet aux pistils d’une fleur de rejeter leur propre pollen. Les systèmes d’AI peuvent prévenir l’autofertilisation et ainsi limiter l’inbreeding. Dans l’AI gamétophytique, le génotype du pollen détermine son propre phénotype d’incompatibilité, et dans ce système, les déterminants mâles et femelles de l’AI sont codés par un locus multigénique et multi-allélique désigné le locus S. Chez les Solanaceae, le déterminant femelle de l’AI est une glycoprotéine stylaire extracellulaire fortement polymorphique possédant une activité ribonucléase et désignée S-RNase. Les S-RNases montrent un patron caractéristique de deux régions hypervariables (HVa et HVb), responsables de leur détermination allélique, et cinq régions hautement conservées (C1 à C5) impliquées dans l’activité catalytique ou la stabilisation structurelle de ces protéines. Dans ce travail, nous avons investigué plusieurs caractéristiques des S-RNases et identifié un nouveau ligand potentiel aux S-RNases chez Solanum chacoense. L’objectif de notre première étude était l’élucidation du rôle de la région C4 des S-RNases. Afin de tester l’hypothèse selon laquelle la région C4 serait impliquée dans le repliement ou la stabilité des S-RNases, nous avons généré un mutant dans lequel les quatre résidus chargés présents en région C4 furent remplacés par des résidus glycine. Cette protéine mutante ne s’accumulant pas à des niveaux détectables, la région C4 semble bien avoir un rôle structurel. Afin de vérifier si C4 est impliquée dans une liaison avec une autre protéine, nous avons généré le mutant R115G, dans lequel un acide aminé chargé fût éliminé afin de réduire les affinités de liaison dans cette région. Ce mutant n’affectant pas le phénotype de rejet pollinique, il est peu probable que la région C4 soit impliquée dans la liaison des S-RNases avec un ligand ou leur pénétration à l’intérieur des tubes polliniques. Enfin, le mutant K113R, dans lequel le seul résidu lysine conservé parmi toutes les S-RNases fût remplacé par un résidu arginine, fût généré afin de vérifier si cette lysine était un site potentiel d’ubiquitination des S-RNases. Toutefois, la dégradation des S-RNases ne fût pas inhibée. Ces résultats indiquent que C4 joue probablement un rôle structurel de stabilisation des S-RNases. Dans une seconde étude, nous avons analysé le rôle de la glycosylation des S-RNases, dont un site, en région C2, est conservé parmi toutes les S-RNases. Afin d’évaluer la possibilité que les sucres conjugués constituent une cible potentielle d’ubiquitination, nous avons généré une S11-RNase dont l‘unique site de glycosylation en C2 fût éliminé. Ce mutant se comporte de manière semblable à une S11-RNase de type sauvage, démontrant que l’absence de glycosylation ne confère pas un phénotype de rejet constitutif du pollen. Afin de déterminer si l’introduction d’un sucre dans la région HVa de la S11-RNase pourrait affecter le rejet pollinique, nous avons généré un second mutant comportant un site additionnel de glycosylation dans la région HVa et une troisième construction qui comporte elle aussi ce nouveau site mais dont le site en région C2 fût éliminé. Le mutant comportant deux sites de glycosylation se comporte de manière semblable à une S11-RNase de type sauvage mais, de manière surprenante, le mutant uniquement glycosylé en région HVa peut aussi rejeter le pollen d’haplotype S13. Nous proposons que la forme non glycosylée de ce mutant constitue un allèle à double spécificité, semblable à un autre allèle à double spécificité préalablement décrit. Il est intéressant de noter que puisque ce phénotype n’est pas observé dans le mutant comportant deux sites de glycosylation, cela suggère que les S-RNases ne sont pas déglycosylées à l’intérieur du pollen. Dans la dernière étude, nous avons réalisé plusieurs expériences d’interactions protéine-protéine afin d’identifier de potentiels interactants polliniques avec les S-RNases. Nous avons démontré que eEF1A, un composant de la machinerie de traduction chez les eucaryotes, peut lier une S11-RNase immobilisée sur résine concanavaline A. Des analyses de type pull-down utilisant la protéine eEF1A de S. chacoense étiquetée avec GST confirment cette interaction. Nous avons aussi montré que la liaison, préalablement constatée, entre eEF1A et l’actine est stimulée en présence de la S11-RNase, bien que cette dernière ne puisse directement lier l’actine. Enfin, nous avons constaté que dans les tubes polliniques incompatibles, l’actine adopte une structure agrégée qui co-localise avec les S-RNases. Ces résultats suggèrent que la liaison entre eEF1A et les S-RNases pourrait constituer un potentiel lien fonctionnel entre les S-RNases et l’altération du cytosquelette d’actine observée lors des réactions d’AI. Par ailleurs, si cette liaison est en mesure de titrer les S-RNases disponibles à l’intérieur du tube pollinique, ce mécanisme pourrait expliquer pourquoi des quantités minimales ou « seuils » de S-RNases sont nécessaires au déclenchement des réactions d’AI.

Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

Regulation of S-Like Ribonuclease Levels in Arabidopsis. Antisense Inhibition of RNS1 or RNS2 Elevates Anthocyanin Accumulation1

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T2 ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.