Self-incompatibility Mechanism Research Articles

Variation in floral organs in wild populations of Brassica rapa L.

In the hermaphroditic plants, floral morphological traits can significantly affect reproductive system by favouring the deposit of self or cross pollen. In the Brassica species, the self-incompatibility mechanism is undoubtedly the main cause of their allogamy; however, floral organs variation can modulate or reinforce the cross pollination level of the plants. In this study, we investigated floral variation among wild populations of Brassica rapa L. from Algeria. We estimated 20 floral traits, including sepals, petals, pistil, stamens and nectaries as well as ovules production. Floral parameters were measured using stereomicroscope. Each flower's ovule number and stigma-anther separation were also quantified under dissecting microscope. We found that all the flower parameters vary among populations or among individuals, especially stigma surface, nectary surface and stigma-anther separation. These three parameters have a coefficient of variation greater than 18,9%. Moreover, the individual variation is different according to the populations. These results suggest that floral morphology may affect pollination efficiency and as a consequence seed production in B. rapa wild populations but with different contributions according to the floral traits.

Molecular mechanisms underlying the participation of Ribonuclease T2 gene into self-incompatibility of Citrus grandis var. Shatianyu Hort.

The RIB (ribonuclease T2) of Citrus grandis var. Shatianyu Hort involved in self-incompatibility (SI) mechanism was identified by prokaryotic expression. RT-qPCR results showed that the expression level of RIB in self pollinated stigma is significantly higher than that in cross pollinated stigma. A vector for prokaryotic expression of RIB was constructed after codon-optimization, and the recombinant protein was induced and purified. In vitro pollen germination test indicated that the RIB protein markedly inhibited pollen germination and pollen tube growth. The result is helpful for better understanding of the molecular mechanism underlying the SI in C. grandis.

Late-acting self-incompatibility: a barrier to self-fertilization in sunnhemp (Crotalaria juncea L.)

In search of causal factors underlying perceived allogamy in sunnhemp (Crotalaria juncea L.), we investigated the breeding system and pollen–pistil interaction. Floral biology study in the species established Fabaceae type hermaphrodite zygomorphic flower with dimorphic anthers. Study of the breeding system revealed preferential success of allogamy despite delayed autonomous selfing mechanism characteristically prevailing in the species. Pollen–pistil interaction study in selfed and crossed pistils revealed significant differences in pollen tube growth rates inside the pistil. Further, in self-pollinated pistils callose formation observed at and around nucellus cells suggesting the late-acting type of self-incompatibility mechanism in the species. To the best of our knowledge, this is the first scientific communication underscoring the prevalence of self-incompatibility mechanism in sunnhemp.

Simulation-based modeling of wild blueberry pollination

The high variability of wild (lowbush) blueberry plants in spatial and genetic structure, in combination with bee foraging behavior varying between species, and the complexity of these factors interacting over time and space, are major obstacles to understanding of pollination dynamics subject to environmental change. The bottom-up modeling paradigm provides an ideal approach to bridging the gap between known mechanisms of individual organisms and unknown spatial–temporal dynamics of pollination at the field scale. By linking empirical data to stochastically-based ecological process modeling, we present a spatially-explicit agent-based simulation model that enables exploration of how various factors, including plant spatial arrangements, outcrossing and self-pollination, bee species compositions and weather conditions, in isolation and combination, affect pollination efficiency throughout a blueberry bloom season. The firmly validated open-source model is a useful tool for hypothesis testing and theory development for wild blueberry pollination researches. Sensitivity analysis suggested that fruit set and resulting measures of productivity such as fruit mass and viable seeds per fruit were sensitive to parameterization of blueberry genotype or clone size and the amount of blueberry plant cover in a field. Fruit set due to pollen compatibility was sensitive to ovule number per flower and foraging bee density. Simulation experiments allowed us to compare bee pollination efficiencies at the bee taxon population level (honey bees, bumble bees, and native solitary bees), the effect of foraging distance from bee nest or colony site on fruit set, and test whether the mechanism of gametophytic self-incompatibility (pre- vs. post-zygotic decision making by the plant) in wild blueberry pollination at the field level matters in estimating yield.

Comparative Proteomics Analyses of Pollination Response in Endangered Orchid Species Dendrobium Chrysanthum.

Pollination is a crucial stage in plant reproductive process. The self-compatibility (SC) and self-incompatibility (SI) mechanisms determined the plant genetic diversity and species survival. D. chrysanthum is a highly valued ornamental and traditional herbal orchid in Asia but has been declared endangered. The sexual reproduction in D. chrysanthum relies on the compatibility of pollination. To provide a better understanding of the mechanism of pollination, the differentially expressed proteins (DEP) between the self-pollination (SP) and cross-pollination (CP) pistil of D. chrysanthum were investigated using proteomic approaches—two-dimensional electrophoresis (2-DE) coupled with tandem mass spectrometry technique. A total of 54 DEP spots were identified in the two-dimensional electrophoresis (2-DE) maps between the SP and CP. Gene ontology analysis revealed an array of proteins belonging to following different functional categories: metabolic process (8.94%), response to stimulus (5.69%), biosynthetic process (4.07%), protein folding (3.25%) and transport (3.25%). Identification of these DEPs at the early response stage of pollination will hopefully provide new insights in the mechanism of pollination response and help for the conservation of the orchid species.

Use of Domain-Swapping to Identify Candidate Amino Acids Involved in Differential Interactions between Two Allelic Variants of Type-1 S-Locus F-Box Protein and S3-RNase in Petunia inflata.

Petunia inflata possesses a self-incompatibility (SI) mechanism, which involves S-RNase and multiple S-locus F-box (SLF) genes at the polymorphic S-locus. For a given S-haplotype, each SLF is thought to interact with some of its non-self S-RNases, but not with its self S-RNase. In this work, we studied an allelic pair of SLF1, S2-SLF1 and S3-SLF1, which differ in 44 amino acids and show differential interactions with S3-RNase. We first used an in vivo transgenic assay to determine whether four chimeric proteins of S2-SLF1 and S3-SLF1, each with one of the three functional domains swapped, interact with S3-RNase. The results narrowed the candidate amino acids for specific interaction of S2-SLF1 with S3-RNase to the 16 in domain FD3. We then examined seven additional chimeric proteins by dividing FD3 into two subdomains and four mini-domains (A, B, C and D). The results further narrowed the candidate amino acids to four in mini-domain A and four in mini-domain D. Molecular modeling of interactions between S3-RNase and S2-SLF1 revealed that three of these eight are at the interaction surface, and all three are conserved in S1-SLF1 and S6a-SLF1, both of which interact with S3-RNase based on the in vivo transgenic assay. Three of the chimeric proteins were used for the in vivo transgenic assay to determine whether FD3 alone contains the amino acids required for S2-SLF1 to interact with S7-RNase and S13-RNase. The results revealed the diversity and complexity of interactions between SLF proteins and S-RNases.

Geographical Distribution, Botanical Description and Self-Incompatibility Mechanism of Genus Stevia

Stevia rebaudiana Bertoni, popularly known as ‘candy leaf’, is a sweet native herb of Paraguay. It became economically important for its significant contribution to the sugar and beverage industry throughout the world. This plant has been known to contain a calorie-free natural sugar in its leaves, which is an alternative to other artificially produced sugar substitutes. Stevia is conventionally propagated through seed and cutting, owing to its self-incompatibility, insufficient pollinator activity, and poor seed set, which results in the origination of heterozygous plants with varying concentration of glucosides in leaves, with low multiplication rate. This article compiles the literatures and depicts an overview on the geographical distribution, morphological, reproductive and cytological features, along with incompatibility mechanism of Stevia that would assist researchers to explore further and genetically refine this potential herb with immense medicinal importance.

The evolutionary history of plant T2/S-type ribonucleases.

A growing number of T2/S-RNases are being discovered in plant genomes. Members of this protein family have a variety of known functions, but the vast majority are still uncharacterized. We present data and analyses of phylogenetic relationships among T2/S-RNases, and pay special attention to the group that contains the female component of the most widespread system of self-incompatibility in flowering plants. The returned emphasis on the initially identified component of this mechanism yields important conjectures about its evolutionary context. First, we find that the clade involved in self-rejection (class III) is found exclusively in core eudicots, while the remaining clades contain members from other vascular plants. Second, certain features, such as intron patterns, isoelectric point, and conserved amino acid regions, help differentiate S-RNases, which are necessary for expression of self-incompatibility, from other T2/S-RNase family members. Third, we devise and present a set of approaches to clarify new S-RNase candidates from existing genome assemblies. We use genomic features to identify putative functional and relictual S-loci in genomes of plants with unknown mechanisms of self-incompatibility. The widespread occurrence of possible relicts suggests that the loss of functional self-incompatibility may leave traces long after the fact, and that this manner of molecular fossil-like data could be an important source of information about the history and distribution of both RNase-based and other mechanisms of self-incompatibility. Finally, we release a public resource intended to aid the search for S-locus RNases, and help provide increasingly detailed information about their taxonomic distribution.

Stigmatic limitations on reproductive success in a paleotropical tree: causes and consequences.

Success in reproduction is subject to the successful initiation as well as successful completion of a chain of consecutive events starting from flower formation and ending with viable seed production. A pivotal role in this chain is played by the stigma which is the seat of pollen recognition and initiation of pollen-pistil interaction. An interesting feature of the family Bignoniaceae is the presence of thigmosensitive stigmas which open, close and re-open in response to touch. Kigelia pinnata bears a touch sensitive stigma and is a low fruit setter in Jammu and Kashmir (India). One possible reason might be pollen limitation coupled with reported self-incompatibility. However, not much is known about the mechanism of self-incompatibility in K. pinnata or of the role of its thigmotropic stigma. Carefully designed manual pollination experiments along with critical field observations revealed naturally deposited pollen load to be too low to cause permanent closure of stigma lobes. A strong relationship exists between the threshold pollen load on stigma, its permanent closure and fertilization. Of the various pollination treatments undertaken, fruits were formed only in open and manual cross pollinations when ∼9200 pollen of legitimate type is deposited on the stigma. Thigmosensitivity further limited the opportunity for the deposition of optimum pollen loads. Although, frequented by as many as seven different visitors, an average of 8 bats per night are available for as many as 30 trees which reflects a baseline deficiency of effective pollinators. This limitation in pollen and pollinator availability affects pollination success of this species and ends up with low fruit set. Fluorescence microscopy reveals successful germination and tube growth of pollen grains of both self and cross type but fruit-set is 100 % in cross-pollinated pistils only. Despite slower rates of ovule penetration and evidence of delayed fertilization, absolutely no fruit initiation occurs in self-pollinated pistils. This strongly points towards self incompatibility being late acting.

Genetic Diversity and Population Structure of Varronia curassavica: A Medicinal Polyploid Species in a Threatened Ecosystem.

Varronia curassavica is an important medicinal species associated with the restinga, one of the most threatened coastal ecosystems of the Atlantic Forest. These circumstances call for studies aimed at estimating effective population size and gene flow to improve conservation efforts. Hence, the present study aimed to characterize the genetic diversity, ploidy level, and population structure of this species in different areas of restinga using microsatellites. Varronia curassavica was characterized as an autotetraploid, with high genetic variability, low divergence, and no significant fixation indices, indicating the absence of, or reduced, inbreeding and genetic drift in the study area. About 44% of the alleles occurred at low frequency in adults of all populations and 41% in the progenies evaluated. Gene flow was high, consistent with outcrossing species with high dispersal capacity (Nm = 4.87). The results showed no tendency toward isolation by distance. The estimated effective size indicates that the populations studied have the potential to ensure conservation of the species in the long term. The genetic variability and population structure of V. curassavica, as determined in this study, could form the foundation for activities directed toward the sustainable use of this resource and its conservation. Even though the restinga ecosystem has suffered dramatic reductions in area, this study provides evidence that this species is resilient to anthropogenic threats to its genetic integrity, since it is a polyploid with self-incompatibility mechanisms that contribute to maintaining high genetic diversity in an panmictic meta-population along the coast of Santa Catarina.

Reproductive Strategy of the Polyploid Species Varronia curassavica Jacq. in Restinga Environment.

This study aimed to elucidate the breeding strategies of Varronia curassavica, an important medicinal species associated with Brazilian restinga. This was accomplished by combining phenological and genetic data. Every 2 weeks over a period of 2 years, we measured flowering and fruiting phenology to evaluate the activity and intensity of phenophases (n = 60). We evaluated the mating system, pollen ovule ratio and genotypes from progeny and mother plants using 8 nuclear microsatellite loci. We observed flowering and fruiting of V. curassavica at low intensity throughout the entire year, but with 2 distinct peaks, one of which was seasonal, corresponding to the period of gradual increase of temperature and photoperiod. Overlapping of flowering and fruiting strategies favors gene flow among different groups of individuals and between populations by attraction of fauna throughout the year. Analysis of the mating system indicates that V. curassavica is a typical outcrossed species (t^ = 0.98; pollen/ovule ratio = 7087.50). Combining phenology with genetic studies improved our understanding of the reproductive strategies of this species. The typical outcrossing system of V. curassavica reflects the existence of functional self-incompatibility mechanisms still unaffected by changes in genetic balance by polyploidy.

Evidence for post-zygotic self-incompatibility in Handroanthus impetiginosus (Bignoniaceae).

Late-acting self-incompatibility (LSI) has been defined as a genetically controlled self-sterility mechanism that prevents seed set by selfing, despite normal pollen tube growth and ovule penetration in self-pollinated pistils. In species of the Bignoniaceae with LSI, such as Handroanthus impetiginosus, the selfed pistils are characterized by a marked delay in ovule penetration, fertilization, and endosperm initiation, followed by uniform pistil abscission. This highlights the contentious possibility of a post-zygotic self-incompatibility system. However, previous studies were unable to confirm fusion of the sperm and egg cell nuclei in selfed ovules. In the present study, the cytology of the embryo sac, double fertilization, and pistil longevity was investigated in H. impetiginosus using comparative nuclei microspectrofluorometry of DAPI-stained sections of self- vs. unpollinated pistils. Differences in both pistil longevity and ovary size between self- and unpollinated flowers at the time of pistil abscission were significant. Zygotes with double the DNA content in their nuclei relative to unfertilized egg cell nuclei were verified in selfed ovules from the first day after pollination onward, and G1 karyogamy appeared to have occurred. Our cytological analysis clearly indicates that ovules of self-pollinated pistils in H. impetiginosus are fertilized before pistil abscission but no embryogenesis initiation occurs, which strongly supports the idea of a post-zygotic self-incompatibility mechanism.

Fine-scale spatial genetic structure and gene flow in Acrocomia aculeata (Arecaceae): Analysis in an overlapping generation

This study aimed to assess the fine-scale spatial genetic structure (SGS) and gene flow among the developmental stages of Acrocomia aculeata (Arecaceae) in a natural population of the Brazilian savannah. The study was conducted in a 4.5 ha area using a population census composed of 72 adult and 144 juvenile plants. Six-hundred embryo samples were analyzed to investigate the gene flow of plant progenies. All individuals were genotyped using seven microsatellite loci. The microsatellite loci generated 10.1 alleles on average. Observed heterozygosity (Ho) was lower than expected heterozygosity (He) in all stages of development. The mean Ho and He values were 0.496 and 0.688, respectively. The A. aculeata population presented high genetic diversity despite its significant positive fixation index (0.279, p = 0.001). Significant SGS was observed for adults at the 2-m distance class and for juveniles at the 2 and 4-m distance classes. Average pollen flow distance was 105 m. High multilocus outcrossing rate was observed (1.2), indicating that the species is preferentially allogamous with evidence of self-incompatibility mechanisms. The A. aculeata population presents high potential for in situ and ex situ genetic conservation and germplasm collection.

HT proteins contribute to S-RNase-independent pollen rejection in Solanum.

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self-incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S-RNase and HT protein function in a pistil-side IRB that causes rejection of pollen from self-compatible (SC) red/orange-fruited species in the tomato clade. However, S-RNase-independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S-RNase-independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S-RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S-RNase, and then used RNAi to test whether they also function in S-RNase-independent pollen rejection. Suppressing HT expression in SC S.pennellii LA0716 allows S.lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S.arcanum LA2157 allows S.lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S-RNase-dependent and S-RNase-independent pollen rejection. The results support the view that overall compatibility results from multiple pollen-pistil interactions with additive effects.

Evaluation of F1 Hybrid Developed Thought Self-incompatibility Mechanism in Radish

Evaluation of F1 Hybrid Developed Thought Self-incompatibility Mechanism in Radish

A simple, high-throughput modeling approach reveals insights into the mechanism of gametophytic self-incompatibility.

Specificity in the GSI response results from the S-haplotype-specific molecular interaction of S-locus F-box (SLF/SFB) and SRNase proteins in the self-incompatibility locus (S-locus). The answer to the question of how these two components of the S-locus (SRNase and SLF/SFB) interact has been gathered from several models. Since there is not enough evidence as to which one is the definitive model, none of them can be ruled out. Despite the identification of interacting protein elements, the mechanism by which SLF/SFB and SRNase interact to differently trigger the self-incompatibility among families and subfamilies remain uncertain. The high-throughput modeling approach demonstrates structural visions into the possible existence of a Collaborative Non-Self Recognition model in apple. These findings postulate several prospects for future investigation providing useful information to guide the implementation of breeding strategies.

Patterns of ROS Accumulation in the Stigmas of Angiosperms and Visions into Their Multi-Functionality in Plant Reproduction.

Accumulation of reactive oxygen species (ROS) in the stigma of several plant species has been investigated. Four developmental stages (unopened flower buds, recently opened flowers, dehiscent anthers, and flowers after fertilization) were analyzed by confocal laser scanning microscopy using the ROS-specific probe DCFH2-DA. In all plants scrutinized, the presence of ROS in the stigmas was detected at higher levels during those developmental phases considered “receptive” to pollen interaction. In addition, these molecules were also present at early (unopened flower) or later (post-fertilization) stages, by following differential patterns depending on the different species. The biological significance of the presence ROS may differ between these stages, including defense functions, signaling and senescence. Pollen-stigma signaling is likely involved in the different mechanisms of self-incompatibility in these plants. The study also register a general decrease in the presence of ROS in the stigmas upon pollination, when NO is supposedly produced in an active manner by pollen grains. Finally, the distribution of ROS in primitive Angiosperms of the genus Magnolia was determined. The production of such chemical species in these plants was several orders of magnitude higher than in the remaining species evoking a massive displacement toward the defense function. This might indicate that signaling functions of ROS/NO in the stigma evolved later, as fine tune likely involved in specialized interactions like self-incompatibility.

A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass.

The grass family (Poaceae), the fourth largest family of flowering plants, encompasses the most economically important cereal, forage, and energy crops, and exhibits a unique gametophytic self-incompatibility (SI) mechanism that is controlled by at least two multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. Here, we report a fine-mapping approach to identify the male component of the S-locus in perennial ryegrass (Lolium perenne L.) and provide multiple evidence that a domain of unknown function 247 (DUF247) gene is involved in its determination. Using a total of 10,177 individuals from seven different mapping populations segregating for S, we narrowed the S-locus to a genomic region containing eight genes, the closest recombinant marker mapping at a distance of 0.016 cM. Of the eight genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium temulentum L.), whereas all of the self-incompatible species of the Festuca-Lolium complex were predicted to encode functional proteins. Our results represent a major step forward toward understanding the gametophytic SI system in one of the most important plant families and will enable the identification of additional components interacting with the S-locus.

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

Breaking linkage between mating compatibility factors: Tetrapolarity in Microbotryum.

Linkage of genes determining separate self-incompatibility mechanisms is a general expectation of sexual eukaryotes that helps to resolve conflicts between reproductive assurance and recombination. However, in some organisms, multiple loci are required to be heterozygous in offspring while segregating independently in meiosis. This condition, termed "tetrapolarity" in basidiomycete fungi, originated in the ancestor to that phylum, and there have been multiple reports of subsequent transitions to "bipolarity" (i.e., linkage of separate mating factors). In the genus Microbotryum, we present the first report of the breaking of linkage between two haploid self-incompatibility factors and derivation of a tetrapolar breeding system. This breaking of linkage is associated with major alteration of genome structure, with the compatibility factors residing on separate mating-type chromosome pairs, reduced in size but retaining the structural dimorphism characteristic for regions of recombination suppression. The challenge to reproductive assurance from unlinked compatibility factors may be overcome by the automictic mating system in Microbotryum (i.e., mating among products of the same meiosis). As a curious outcome, this linkage transition and its effects upon outcrossing compatibility rates may reinforce automixis as a mating system. These observations contribute to understanding mating systems and linkage as fundamental principles of sexual life cycles, with potential impacts on conventional wisdom regarding mating-type evolution.