In addition to model system studies and synthetic engineering, prioritizing the discovery of naturally occurring apomixis-like mutants in crops would provide an immediate route to applied innovation in plant breeding. Apomixis is a remarkable form of asexual reproduction that enables plants to produce seeds without meiotic reduction or gametic fertilization in ovules. This process is mainly divided into two types: sporophytic and gametophytic apomixis, each following a distinct embryogenetic pathway with specific processes and characteristics of significant importance in plant biology. Apomixis provides insight into plant reproductive strategies, facilitating genetic stability and adaptation without reliance on sexual reproduction. An in-depth understanding of these pathways not only enriches knowledge in plant biology but also holds potential implications for advancing agricultural practices and conservation efforts. Yet current research on apomixis follows two main directions: (1) identifying and characterizing genes in natural model systems, and (2) engineering synthetic apomixis by introducing genes that mimic key components of asexual reproduction. Here, we sustain the screening and investigation of naturally occurring mutants that may arise in populations from spontaneous DNA changes (i.e., mutants that are not induced artificially through genetic engineering or by molecular or cellular biotechnological methods), with possible elements of apomixis directly in crop plant species. Such a perspective may lead to new opportunities to harness apomixis for crop improvement and the development of resilient varieties, as well as to accelerate the transfer and preservation of desirable plant traits.

Bitter vetch (Vicia ervilia (L.) Willd.), one of the Near Eastern founder crops, is an annual cleistogamous legume domesticated during the Neolithic period. Originally used for human consumption, over time it was replaced by other pulses and downgraded to a fodder crop. When coupled with a small degree of cross hybridization, cleistogamy confers evolutive plasticity to the plant species. The aim of the present work consisted in setting up optimal conditions to overcome the existing cross hybridization barriers in V. ervilia. Genotypes of Turkish origin, characterized by an erect growth habit were crossed with Italian counterparts characterized by high seed production. A detailed cyto-histological analysis of flower development was undertaken to determine the optimal stage for emasculation and manual cross. Ninety-eight crosses were carried out and the hybrid nature of the putative F1 progenies was assessed by SSR (simple sequence repeat) DNA markers. Fifty-five seeds were obtained of which only five gave rise to hybrid plants. Among these, only three turned out to be fertile and two of which generated a consistent number of F2 seeds whose plants were assessed in greenhouse for seed production and plant growth habit. Most of the evaluated traits showed mean values of the F2 plants intermediate between the two initial parents, confirming that intraspecific hybridization is not only possible but also useful to exploit the diversity confined in different bitter vetch populations.

Bacterial growing resistance to antibiotics poses a critical threat to global health. This study investigates, for the first time, the antibiofilm properties of Vicia ervilia agglutinin (VEA) from six different V. ervilia accessions against pathogenic bacteria, and the yeast Candida albicans. In the absence of antimicrobial properties, purified VEA significantly inhibited biofilm formation, both in Gram-positive and Gram-negative bacteria, but not in C. albicans. With an inhibitory concentration ranging from 100 to 500 µg/ml, the VEA antibiofilm activity was more relevant against the Gram-positive bacteria Streptococcus aureus and Staphylococcus epidermidis, whose biofilm was reduced up to 50

AbstractPaspalum notatum Flüggé is an economically important subtropical fodder grass that is widely used in the Americas. Here, we report a new chromosome-scale genome assembly and annotation of a diploid biotype collected in the center of origin of the species. Using Oxford Nanopore long reads, we generated a 557.81 Mb genome assembly (N50 = 56.1 Mb) with high gene completeness (BUSCO = 98.73%). Genome annotation identified 320 Mb (57.86%) of repetitive elements and 45,074 gene models, of which 36,079 have a high level of confidence. Further characterisation included the identification of 59 miRNA precursors together with their putative targets. The present work provides a comprehensive genomic resource for P. notatum improvement and a reference frame for functional and evolutionary research within the genus.

EDITORIAL article Front. Plant Sci., 14 February 2023Sec. Plant Breeding Volume 14 - 2023 | https://doi.org/10.3389/fpls.2023.1145208

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route-based on ORC3 manipulation-to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

In the past decades, the grasses of the Paspalum genus have emerged as a versatile model allowing evolutionary, genetic, molecular, and developmental studies on apomixis as well as successful breeding applications. The rise of such an archetypal system progressed through integrative phases, which were essential to draw conclusions based on solid standards. Here, we review the steps adopted in Paspalum to establish the current body of knowledge on apomixis and provide model breeding programs for other agronomically important apomictic crops. In particular, we discuss the need for previous detailed cytoembryological and cytogenetic germplasm characterization; the establishment of sexual and apomictic materials of identical ploidy level; the development of segregating populations useful for inheritance analysis, positional mapping, and epigenetic control studies; the development of omics data resources; the identification of key molecular pathways via comparative gene expression studies; the accurate molecular characterization of genomic loci governing apomixis; the in-depth functional analysis of selected candidate genes in apomictic and model species; the successful building of a sexual/apomictic combined breeding scheme.

Apomixissensu stricto(agamospermy) is asexual reproduction by seed. In angiosperms it represents an easy byway of life cycle renewal through gamete-like cells that give rise to maternal embryos without ploidy reduction (meiosis) and ploidy restitution (syngamy). The origin of apomixis still represents an unsolved problem, as it may be either evolved from sex or the other way around. This review deals with a reappraisal of the origin of apomixis in order to deepen knowledge on such asexual mode of reproduction which seems mainly lacking in the most basal angiosperm orders (i.e., Amborellales, Nymphaeales and Austrobaileyales, also known as ANA-grade), while it clearly occurs in different forms and variants in many unrelated families of monocots and eudicots. Overall findings strengthen the hypothesis that apomixis as a whole may have evolved multiple times in angiosperm evolution following different developmental pathways deviating to different extents from sexuality. Recent developments on the genetic control of apomixis in model species are also presented and adequately discussed in order to shed additional light on the antagonist theories of gain- and loss-of-function over sexuality.

Hypericum perforatum L. (2n = 4x = 32) is an attractive model system for the study of aposporous apomixis. The earliest phenotypic features of aposporous apomixis in this species are the mitotic formation of unreduced embryo sacs from a somatic cell of the ovule nucellus and the avoidance of meiosis. In this research we addressed gene expression variation in sexual and apomictic plants, by focusing on the ovule nucellus, which is the cellular domain primarily involved into the differentiation of meiocyte precursors and aposporous embryo sacs, at a pre-meiotic developmental stage. Gene expression analyses performed by RNAseq identified 396 differentially expressed genes and 1834 transcripts displaying phenotype-specific expression. Furthermore, the sequencing and assembly of the genome from a diploid sexual accession allowed the annotation of a 50 kb sequence portion located upstream the HAPPY locus and to address the extent to which single transcripts were assembled in multiple variants and their co-expression levels. About one third of identified DEGs and phenotype-specific transcripts were associated to transcript variants with alternative expression patterns. Additionally, considering DEGs and phenotype-specific transcript, the co-expression level was estimated in about two transcripts per locus. Our gene expression study shows massive differences in the expression of several genes encoding for transposable elements. Transcriptional differences in the ovule nucellus and pistil terminal developmental stages were also found for subset of genes encoding for potentially interacting proteins involved in pre-mRNA splicing. Furthermore, the sexual and aposporous ovule transcriptomes were characterized by differential expression in genes operating in RNA silencing, RNA-mediated DNA methylation (RdDM) and histone and chromatin modifications. These findings are consistent with a role of these processes in regulating cell fate determination in the ovule, as indicated by forward genetic studies in sexual model species. The association between aposporous apomixis, pre-mRNA splicing and DNA methylation mediated by sRNAs, which is supported by expression data and by the enrichment in GO terms related to these processes, is consistent with the massive differential expression of multiple transposon-related sequences observed in ovules collected from both sexual and aposporous apomictic accessions. Overall, our data suggest that phenotypic expression of aposporous apomixis is concomitant with the modulation of key genes involved in the two interconnected processes: RNA splicing and RNA-directed DNA methylation.

The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses.

Bitter vetch (Vicia ervilia (L.) Willd.), a grain legume crop well adapted in marginal soils, has mainly been used for animal feed. Nowadays, bitter vetch seeds in feed formulations are replaced by other protein sources such as soybean meal. However, in the context of sustainable economic development, it may be beneficial to enhance the cultivation of bitter vetch landraces in marginal areas. Fifty-six bitter vetch accessions of different provenance were preliminarily characterised by microsatellite DNA analysis to discriminate landraces suitable for specific and restricted environments. Twenty-two landraces of two genetically different groups were then selected for further characterisation by agro-morphological analyses. Being late-flowering with a seed yield of up to 3–4 t ha–1 in experimental field conditions, with neither chemical nor water input, these plants will be valuable material for long-term study to develop new cultivars adapted for seed production under organic agricultural systems in Southern Europe. The seeds of these 22 landraces were also evaluated, with positive results, as partial replacement of soybean in rabbit diets.

In angiosperms, there are two pathways of reproduction through seeds: sexual, or amphimictic, and asexual, or apomictic. The essential feature of apomixis is that an embryo in an ovule is formed autonomously. It may form from a cell of the nucellus or integuments in an otherwise sexual ovule, a process referred to as adventitious embryony. Alternatively, the embryo may form by parthenogenesis from an unreduced egg that forms in an unreduced embryo sac. The latter may form from an ameiotic megasporocyte, in which case it is referred to as diplospory, or from a cell of the nucellus or integument, in which case it is referred to as apospory. Progeny of apomictic plants are generally identical to the mother plant. Apomixis has been seen over the years as either a gain- or loss-of-function over sexuality, implying that the latter is the default condition. Here, we consider an additional point of view, that apomixis may be anciently polyphenic with sex and that both reproductive phenisms involve anciently canalized components of complex molecular processes. This polyphenism viewpoint suggests that apomixis fails to occur in obligately sexual eukaryotes because genetic or epigenetic modifications have silenced the primitive sex apomixis switch and/or disrupted molecular capacities for apomixis. In eukaryotes where sex and apomixis are clearly polyphenic, apomixis exponentially drives clonal fecundity during reproductively favorable conditions, while stress induces sex for stress-tolerant spore or egg formation. The latter often guarantees species survival during environmentally harsh seasons.

Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the development of clonal progenies genetically identical to the mother plant. Here we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize cytosine methylation patterns occurring in florets of sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. From twelve polymorphic MSAP-derived sequences, one (PN_6.6, later renamed PN_SCD1) was selected due to its relevant annotation and differential representation in apomictic and sexual floral transcriptome libraries. PN_SCD1 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and acts as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN_SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are expressed in P. notatum florets and upregulated in apomictic plants. Our results disclosed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis.

Apomixis is a clonal mode of reproduction via seeds, which results from the failure of meiosis and fertilization in the sexual female reproductive pathway. In previous transcriptomic surveys, we identified a mitogen-activated protein kinase kinase kinase (N46) displaying differential representation in florets of sexual and apomictic Paspalum notatum genotypes. Here, we retrieved and characterized the N46 full cDNA sequence from sexual and apomictic floral transcriptomes. Phylogenetic analyses showed that N46 was a member of the YODA family, which was re-named QUI-GON JINN (QGJ). Differential expression in florets of sexual and apomictic plants was confirmed by qPCR. In situ hybridization experiments revealed expression in the nucellus of aposporous plants’ ovules, which was absent in sexual plants. RNAi inhibition of QGJ expression in two apomictic genotypes resulted in significantly reduced rates of aposporous embryo sac formation, with respect to the level detected in wild type aposporous plants and transformation controls. The QGJ locus segregated independently of apospory. However, a probe derived from a related long non-coding RNA sequence (PN_LNC_QGJ) revealed RFLP bands cosegregating with the Paspalum apospory-controlling region (ACR). PN_LNC_QGJ is expressed in florets of apomictic plants only. Our results indicate that the activity of QGJ in the nucellus of apomictic plants is necessary to form non-reduced embryo sacs and that a long non-coding sequence with regulatory potential is similar to sequences located within the ACR.

ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes.

Tobacco, Nicotiana tabacum , is an industrial crop traditionally used for manufacturing cigarettes. However, due to restriction of European subsidies, an alternative use of tobacco is needed, such as biofuel or biomass. Tobacco is indeed an oilseed crop with an oil yield ranging from 30 to 40 % of seed dry weight. Yet, there is still little information on the cultivation of tobacco for seed oil production. Here, we tested tobacco cultivation as an oilseed crop in Central Italy, where flue-cured tobacco is traditionally cultivated, with an additional trial in Northest Italy. We used the cultivar Solaris, selected for its reduced size and elevated number of flowers, and tested its adaptability to different field management practices in 2013–2014. Results show that in Central Italy fields, at the beginning of ripening, tobacco plants showed on the average a 94–105 cm height and a stem diameter between 1.3–1.6 cm; they had 123–151 capsules and each plant produced on average from 31 to 34 g of seeds. Seed production was evaluated to be 1.1–1.8 t/ha, with an oil yield up to 0.59 t/ha. In Northest Italy, two seed harvests determined a total seed yield of 4.5 t/ha, from which 1.48 t oil/ha could be obtained. The cultivar Solaris was extremely adaptable in terms of morphological parameters and seed yield to different management practices as well as climatic conditions. This study shows for the first time the cultivation feasibility in Italy of a small-size tobacco variety selected for high seed production. With a further optimization of the cultivation protocol to increase the oil yield and to use the by-products, tobacco can really become a novel industrial crop providing renewable sources for both biofuel and biomass as well.

Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.

Olea europaea subsp. cuspidata, belonging to the olive complex, is largely distributed in Asia and Africa. In this research, reproductive structures were studied at both structural and functional levels in Asian and African cuspidata accessions with the aim to broaden the knowledge about the floral biology of this subspecies. Morpho-structural observations showed that the cuspidata flower is very similar to the europaea one. Nevertheless, some biometric parameters (length of pollen, pistil, ovule, and embryo sac) significantly differed between the two subspecies. Furthermore, numerous and interesting differences were observed between Asian and African cuspidata accessions for the morphology of stigma papillate cells, degree of secondary cell wall thickenings of the endothecial cells, persistence of the tapetum, ovule and embryo sac length. At functional level, main differences were in the synchrony/asynchrony of the microsporogenesis, percentage of male meiotic abnormalities and sterile pollen, development of ovules. The influence of the growth environment on these features is discussed. (C) 2016 Elsevier GmbH. All rights reserved.

To enrich the source of germplasm of cultivated olive ( Olea europaea subsp. europaea L.), inter-subspecific hybrid plants have been produced by experimental crosses between several varieties of cultivated olive and Asian and African accessions of the wild related subspecies cuspidata . Germination of putative hybrid seeds was enhanced by using in vitro embryo culture. The genetic make-up of germinated seedlings was assayed with the aid of both AFLP and SSR molecular markers and their hybrid nature was proved by the presence of male-specific alleles in their molecular patterns. Most of the parent specific alleles showed segregation among F 1 progenies indicating high heterozygosity content of the parental lines. The majority of the hybrids derived from crosses in which an African accession of cuspidata was used as female parent. The overall morphological aspect of hybrids resembled that of the female parent. The production of inter-subspecific hybrid plants in Olea is discussed in relation to the genetic improvement of cultivated olive.

Apomixis, a clonal plant reproduction by seeds, is controlled in Paspalum spp. by a single locus which is blocked in terms of recombination. Partial sequence analysis of the apomixis locus revealed structural features of heterochromatin, namely the presence of repetitive elements, gene degeneration, and de-regulation. To test the epigenetic control of apomixis, a study on the distribution of cytosine methylation at the apomixis locus and the effect of artificial DNA demethylation on the mode of reproduction was undertaken in two apomictic Paspalum species. The 5-methylcytosine distribution in the apomixis-controlling genomic region was studied in P. simplex by methylation-sensitive restriction fragment length polymorphism (RFLP) analysis and in P. notatum by fluorescene in situ hybridization (FISH). The effect of DNA demethylation was studied on the mode of reproduction of P. simplex by progeny test analysis of apomictic plants treated with the demethylating agent 5'-azacytidine. A high level of cytosine methylation was detected at the apomixis-controlling genomic region in both species. By analysing a total of 374 open pollination progeny, it was found that artificial demethylation had little or no effect on apospory, whereas it induced a significant depression of parthenogenesis. The results suggested that factors controlling repression of parthenogenesis might be inactivated in apomictic Paspalum by DNA methylation.