T REPRO N DU LA C The International Journal of Plant Reproductive Biology 9(2) Jul., 2017, pp.153-169 P T I F V O E

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S H Is sex irreplaceable? Towards the molecular regulation of apomixis T Vladimir Brukhin1, 2 1 Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University 41 Sredniy Prospekt, Vasilievsky Island, Saint Petersburg 199004, Russia 2Department of Plant Embryology, Komarov Botanical Institute RAS, Prof. Popov str. 2, Saint Petersburg 197376, Russia e-mail: [email protected] Received: 03.05.2017; Revised: 15.05.2017; Accepted & Published online: 01.06.2017 ABSTRACT Apomixis, defined as the asexual plant reproduction through seeds that results in the production of genetically uniform progeny and a natural way of cloning. Currently there are more than 400 plant species known to use apomixis as a strategy for their propagation. The primary fundamental aspects of apomixis are the bypassing of meiosis and parthenogenetic development of the embryo without fertilization. Apomixis attracts special attention because of its potential value for agriculture, as it could be harnessed for plant breeding programs enabling the permanent fixation of heterosis in crop plants. A better understanding of the molecular and genetic regulation of apomixis is important for developmental and evolutionary perspectives but also for implementation of engineering of apomixis traits into agricultural crop plants. Despite apomixis is considered as one of the key technologies for the improving agriculture, but currently how genetic and molecular regulation of this important trait occurs is not fully known. Recent information on the biology of apomixis and genes and genetic loci associated with the regulation of different components of apomixis is provided in the present review. Keywords : apomixis, sexual reproduction, asexual reproduction, heterosis, fixation of hybrid vigour.

Sexual reproduction provides an infinite variety of reproduction during which some steps are lost reduced, genetic variants upon which adaptive selection acts in the deregulated or asynchronised (Koltunow AM 1993, course of evolution in wild species and provides material for Grossniklaus et al. 1998, Grossniklaus et al. 2002, the improvement of crop quality in agriculture. The drawback Grossniklaus 2001). of sexual propagation is the segregation of advantageous traits Thus, apomictic and sexual reproduction is in subsequent generations, such that progeny can lose the developmentally and evolutionarily closely related processes advantageous gene combinations of their ancestors. However, sharing many regulatory components. The ability to produce some plants can also reproduce asexually through the seed maternal clones and therefore to fix useful traits in subsequent alone (without fertilization), a process known as apomixis generations of various crop plants could streamline agricultural (Fig. 1). breeding strategies based on apomixis. Back in the 1930s, the More than 400 plant species representing almost 40 Russian geneticists Georgy Karpechenko and Sergey Navashin families are known to reproduce through apomixis (Nogler realized the enormous potential of apomixis in fixing hybrid 1984, Carman 1995, 1997). The recent studies report the vigour in plants. Since then, many other possible uses of following numbers for different types of apomixis: apomixis in agriculture have been proposed (Grossniklaus et al. adventitious embryony was recorded in 148 genera, apospory 2002, Jefferson 1994) and its importance for sustainability and in 110 genera, and diplospory in 68 genera (Hojsgaard et al. food security has been recognized (Toennissen 2001). A better 2014). It was shown that apomixis - is one of the main understanding of the molecular basis of apomixis is crucial for reproductive strategies along with the sexual propagation. increase in yield, for sustainable production of food, feed, There is a positive correlation between apomixis occurrences biomaterials, fibres, and biofuels. Unfortunately, among major and taxonomic diversity and biogeography. Apomixis is crop cultivars no natural apomicts have been found (Savidan taxonomically widespread; it occurs at all taxonomic levels. It 2000). Most of the known apomictic plants are facultative was also found that all major families have apomict- apomicts, i.e. they can switch their developmental program containing plant forms (Hojsgaard et al. 2014). The term from asexual to sexual route back and forth. Further, all natural “apomixis” was coined by the German scientist Hans Winkler apomicts known to date are polyploids and this makes it difficult in early 20th century to describe asexual reproduction that to study the genetics of apomixis. Though the molecular and replaces sexual reproduction in plants. Apomictic embryos genetic basis underlying apomixis regulation is poorly arise not as a result of fusion of male and female gametes as understood (Grossniklaus 2001, Bicknell and Koltunow 2004, occurs during sexual reproduction but clonally from the Koltunow and Grossniklaus 2003, Rodriguez-Leal and Vielle- maternal tissues of ovules. It is believed that apomixis evolved Calzada 2012).The potential of apomixis for plant breeding has independently in several taxa from sexual ancestors (van Dijk attracted a huge interest in elucidating the mechanisms of its and Vijverberg 2005, Hand et al. 2015). Apomixis could be regulation. The main thrust is focused on a better understanding considered as a developmental short-circuit of sexual of the molecular basis of apomixis as a key ‘enabling 154 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.153-169

Fig.1–Schematic representation of sexual and apomictic development. Left side shows the sexual pathway and sporophytic apomixis (adventive embryony). In this pathway, a 7-celled, 8-nucleate mature embryo sac with egg cell and synergid cells and three antipodal cells develops. Two polar nuclei fuse to form the diploid nucleus of the central cell. After double fertilization, one sperm fuses with the egg cell forming the diploid zygote, whereas the second sperm fertilizes the central cell producing the first nuclei of triploid endosperm. Synergids participate in the perception of the sperm cells and burst after fertilisation. The mature seed consists of the diploid embryo, the triploid endosperm (nourishing tissue) and remnants of the maternal sporophytic nucellar and integumental tissues form the seed coat. An adventive embryo may develop from the nucellar or inner integumental sporophytic tissues and develop alongside the sexual embryo. The Right part of the figure shows gametophytic apomixis. Meiosis is bypassed and consequently reduction is avoided. Embryo sac development is initiated from an unreduced diplospore (diplospory) or from an aposporic initial cell (apospory). The embryo develops parthenogenetically from the unreduced egg cell while endosperm forms either autonomously or through fertilisation of the central cell (in case of pseudogamy). The mature apomictic seed contains the embryo with a relative ploidy level of 2n, while the endosperm could be of variable ploidy but usually not less than 4n (adopted from Brukhin 2017). 2017 Is sex irreplaceable? Towards the molecular regulation of apomixis 155

technology’ of immense benefit to agriculture, because it could apomixis) (Fig. 1). In sporophytic apomixis (also known as facilitate the permanent fixation of favourable genotypes in adventive embryony), activation of nucellar or integumental crop plants. The genetic control of apomixis and its application initial cells takes place followed by the somatic embryo in agriculture will allow the fixing of any heterozygous development directly from either nucellus or integument genotypes leading to simpler and faster breeding programs. respectively (Naumova 1993). The developing embryo is still This will greatly facilitate the development of superior crop dependent on the presence of a sexually derived endosperm. varieties better adapted to changing environmental conditions, Somatic embryos of sporophytic origin often develop without the disadvantages of genetic recombination occurring alongside the sexual embryo in a seed. Therefore, where during sexual reproduction. The application of apomixis in sporophytic apomixis takes place, species are often agriculture will therefore, result in accelerated breeding characterized by polyembryony, for example, in citrus (García programs, a significant reduction in production costs, and et al. 1999). However, the adventive embryo is usually more increase in yields resulting from the wide application of competitive and it suppresses the sexual embryo at the seed. technology able to fix heterosis. In short, genetic resources can Sporophytic apomixis is one of the most common types of be exploited far more efficiently using apomixis than with apomixis in nature. It occurs sporadically throughout the sexual crop plants. Researchers who study apomixis regulation phylogenetic tree of flowering plants. Most typically it could adhere to the two basic strategies: 1) identification of the genes be found in trees, shrubs, and perennial grasses that grow in causing apomixis in the apomictic plants, and 2) the attempt to tropics and subtropics, and many of these possess high activate apomictic pathways in sexual plants, thereby causing numbers of chromosomes and are polyploids. Sporophytic some components of apomixis (Hand and Koltunow 2014). apomixis can be used as a taxonomic characteristic for some Because apomixis is a short-circuited variation of sexual species and in certain genera (Sarcacocca, Poncirus, Citrus, propagation and many genes that regulate the latter are also Evonymus). To date, sporophytic apomixis has been described crucial for controlling of the apomictic pathways, it makes sense in more than 250 species belonging to 57 families of flowering to first consider the consecutive stages of sexual seed formation. plants, and includes a number of economically valuable plants DEVELOPMENTAL FEATURES OF APOMIXIS VS Citrus, Mangifera, Ribes, Beta, Alnus, and others (Naumova SEXUAL REPRODUCTION—Apomictic reproduction 1993, Naumova and Vielle-Calzada 2001). Hojsgaard et al. might be considered as a modified and reduced sexual process, reported that sporophytic apomixis has been found in plants during which a functional female gametophyte develops but from 148 genera. Sporophytic apomixis has not sexual gene assortment and recombination are precluded. One comprehensively been studied at the molecular level. of the hallmarks of apomictic reproduction is a developmental However, the genetic nature of sporophytic apomixis is rather heterochrony (Grossniklaus 2001). Developmental aspects of complex (Hand and Koltunow 2014). Gametophytic apomixis apomixis and varieties of apomictic scenarios have been very is characterized by ameiotic initiation of the diploid egg cell well described before (Nogler 1984, Koltunow AM 1993, and further parthenogenetic embryo development from this Gustafsson A 1947a, Gustafsson A 1947b, Carman et al.1991, egg cell. The gametophyte can arise directly from the nucellar Asker and Jerling 1992, Naumova 1993, Peacock et al. 1993, cell (apospory) resembling a megaspore. Also the embryo sac Chaudhury et al.1998, Spillane et al. 2004, Hörandl et al. can originate from a MMC that either bypasses meiosis or 2007) even though the molecular aspects of apomixis undergoes through an aberrant meiosis without chromosome regulation are still poorly understood. The concept of segregation resulting in the formation of an unreduced apomixis is currently interpreted more broadly by the majority megaspore or a dyad of megaspores, instead of four haploid of scientists and includes not only a method of a sporophyte megaspores (diplospory) (Fig.1). Usually, only one formation, but also all processes associated with apomictic megaspore participates in embryo sac formation, while the seed development. other degenerates, though bisporic development sometimes In contrast to sexual reproduction, apomixis can occur in takes place, when both megaspores contribute to the a variety of ways. However, all forms of apomixis share the development of the embryo sac (Ixeris-type embryo sac). A following features: i) a lack of meiosis during embryo sac characteristic feature of the megasporocyte, which undergoes formation (apomeiosis); ii) development of the embryo an abnormal meiosis, is the lack of callose deposition around without fertilization (parthenogenesis); iii) adequate the cell membrane. In contrast, the callose envelope around endosperms formation either via fertilization with the sperm the megaspore mother cell is formed continuously during the cell (pseudogamous) or independently without development of the normal, sexual Polygonum-type embryo fertilization(autonomous).Traditionally, two mechanisms of sac. The callose test can be used to identify diplospory in apomixis can be distinguished, based on the way of apomictic angiosperms (Nogler 1984, Rodkiewicz 1970, Carman et al. embryo development 1) via the female gamethophyte 1991, Leblanc et al. 1995, Naumova and Willemse 1995. (embryo sac) (gametophytic apomixis) or 2) from sporophytic Diplospory (Fig. 1) has been identified among members of cells of the ovule (nucellar or integumental cells) (sporophytic different species from the families Asteraceae, Solanaceae, 156 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.153-169

Rosaceae, Poaceae, Brassicaceae, including Taraxacum reason for this may be related to the nonstandard diploid ratios officinale (dandelion), Boechera spp., Erigeron annuus of the parthenogenetic embryo and autonomous endosperm (butterweed), and Tripsacum dactyloides. It has also been e.g. (2n: 4n), but also because of imprinting. At the early stages found in crop plants – Manihot esculenta (cassava) and of embryo and endosperm-development, many gene alleles Sorghum bicilor. The diplospory has been found in plants are silenced (imprinted) depending on their parental origin. In belonging to 68 genera (Hojsgaard et al. 2014). In the United order for endosperm development to be completed States special research programs were initiated for the successfully, a certain ratio of maternal and paternal genomes comprehensive study of the reproductive processes and should be included (dosage effect). Any deviation in the apomixis in Sorghum bicilor and Tripsacum (Bradly et al. dosage will result in the arrest of endosperm development and 2007). Apospory (Fig. 1) is a type of apomixis where embryo seed abortion. Low seed productivity because of endosperm sac development initiates from a nucellar cell that occupies a death is typical for many apomicts (Naumova et al. 2001, position where the MMC should be located (aposporous Kojima and Nagato 1992, Naumova 1997). However, initial). In the ovule several such initials can arise and as a apomictic plants have developed various strategies to secure consequence several aposporous embryo sacs could develop. viable endosperm formation (Curtis and Grossniklaus 2008). Sometimes within an ovule both the aposporic embryo sac and Due to the bypassing of meiosis and fertilization in apomixis sexual embryo sac, which arises from the megaspore as a development, the alternation of generations (sporophyte – result of meiosis, can develop at the same time. The aposporic gametophyte transition) does not occur. Apomixis is gametophyte is usually more competitive compared to the ultimately a genetically controlled reproductive system. meiotic one. Development of aposporic embryo sacs mainly GENETIC CONTROL OF SEXUAL AND APOMICTIC follows the Hieracium- and Panicum- type (Rutishauser REPRODUCTION—Gregor Mendel was apparently the 1969). The Hieracium-type embryo sac is formed from first scientist faced with the peculiarities of apomictic aposporous initials as a result of the three successive mitotic genetics. He made several attempts to confirm his discovery of divisions. This type of embryo sac is bipolar containing seven the basic laws of genetics with other plant species besides peas cells and eight nuclei, which is morphologically similar to the (Pisum sativum). To this end, he conducted a series of Polygonum-type embryo sac. The Panicum-type embryo sac experiments on the crossing of species from the genus is produced as a result of two consecutive mitoses of Hieracium (Asteraceae), commonly known as hawkweed. A aposporous initial. At maturity the Panicum-type embryo sac tragic disappointment awaited him: the experimental results is monopolar and doesn’t contain the antipodes. Interestingly, he obtained with peas were not confirmed on hawkweed. The in Pennisetum ciliare the apomictic embryo sac develops likely reason was that the mode of reproduction for Hieracium according to the Panicum-type, whereas sexual mega has features that were not yet known to science (reproduction gametophytes of Pennisetum are formed by the typical through apomixis and parthenogenesis). These features were Polygonum-type. Apospory has been described in different not considered in the crossing methods used by Mendel. In the groups of flowering plants belonging to 110 genera ((Hojsgaard et al. 2014). It is most common among end, the great scholar himself lost faith that he had made the representatives of such families as Asteraceae, Poaceae, discovery. As mentioned above, apomixis was only Rosaceae. In a number of other families apospory occurs discovered later in early 20thcentury (Kerk et al. 2003).To date, sporadically in individual species (Adoxaceae, Boraginaceae, study of the genetic nature of apomixis is carried out on both Brassicaceae, Chenopodiaceae, Cyrillaceae, Cucurbitaceae, apomictic species, as well as on sexual mutant plants, which Globulariaceae, Myrtaceae, Ochnaceae, Polygonaceae, display certain components of apomixis. However, until now Ranunculaceae, Rutaceae, Taccaceae, Urticaceae). Most there have been no fully apomictic mutants isolated. Crossings apomictic plant forms are polyploid, and it has been of apomictic individuals as pollen donors with sexual plants as hypothesized that polyploidy in apomictic forms originated as maternal individuals showed that apomixis is inherited as a a result of hybridisation and genome duplications (Carman dominant trait. Earlier it was believed that apomixis is 1997). The only known apomictic plant, which has a set of inherited as a single dominant locus in most apomictic plants. diploid chromosomes, is Boechera spp. (Naumova et al. However, later genetic studies found that different loci control 2001). A wide variety of the ploidy ratios have been identified the developmental components of apomixis, i.e. avoidance of between the embryo and endosperm of developing seeds in meiosis, parthenogenetic embryo formation, and independent populations of apomictic Boechera holboellii (3:9, 3:6, 2:6, endosperm development. It was also revealed that in most 2:4, 2.6: 6, 4:6, 3:9, 3:6, 6:9). This is fundamentally different species apomictic loci exhibit a suppressed recombination from the standard ploidy ratios between embryo and (Grossniklaus 2001). Most of the gametophytically acting endosperm (2n:3n) in sexual plants (Naumova 2008). genes and genes involved in inducing various apomixis traits Endosperm formation in aposporous embryo sacs via polar have been identified through gene trap and enhancer nuclei fusion and without fertilization is impossible. The trapdetections in large-scale genetic screens (Grossniklaus 2017 Is sex irreplaceable? Towards the molecular regulation of apomixis 157

2001, Brukhin et al. 2005, 2011). More recently, powerful GENES ASSOCIATED WITH APOMIXIS TRAITS— laser assisted micro dissection (LAM) combined with The genes involved in gametophytic apomixis are considered microarray validation allowed the identification of differential here, as they are the most interesting genes to study the gene expression at the level of a single cell (Kerk et al. 2003, phenomenon of apomixis and are also likely to be important Day et al. 2005, Johnston et al. 2007, Wuest et al. 2010, for creating technologies for bioengineering of apomixis. As Schmidt et al. 2012, Florez Rueda et al. 2016).These tools mentioned above, the main features of gametophytic apomixis drastically improved our knowledge about genes specifically are the lack of reduction of spores (apomeiosis), the formation expressed in the cells of sexual and apomictic embryo sacs. of unreduced egg cells, development of the parthenogenetic Nowadays with the advent of the post-genomic era, next embryo without fertilization, and, in some cases, an generation sequencing technologies have highlighted new independent development of the endosperm. avenues in gene discovery, including the genes responsible for Megasporogenesis is a complex, multi staged process in megasporogenesis, megagametogenesis, parthenogenesis and which the determination of the megaspore mother cell, independent endosperm development. For example a genomic meiosis, and differentiation of the functional megaspore take and transcriptomic resource was developed recently for place. All these processes are controlled by certain genes and Hieracium spp. (Asteraceae). The resource includes sexual, gene families or loci that were identified in different model or apomictic and mutant apomict plants exhibiting reversion to agricultural crop plants through gene knockouts, differential display techniques, and through large scale screens of sexual reproduction (Rabiger et al. 2016). It has been shown mutants. that apomicts develop additional female gametogenic cells that suppress the sexual pathway in ovules. Comparisons of 1. Genes and mutants affecting megasporogenesis— whole ovary transcriptomes from mutant apomicts, relative to Until the 1990s, very few genes had been reported to the parental apomict, revealed that differentially expressed be involved in the implementation of the developmental genes were enriched for processes involved in small RNA decision to induce an unreduced cell of the ovule to enter biogenesis and chromatin silencing. In our research, we have meiosis and initiate megagametogenesis. In the mid-1990s, sequenced and assembled the whole genomes of three species megasporogenesis-defective mutant (msd) was isolated in of Boechera based on the Illumina and Pacific Bioscience Arabidopsis (Schneitz et al. 1997). This mutant does not form platforms. An attempt has been made to annotate genomes and a gametophyte, although the ovule develops normally. The mutation also affects microsporogenesis and is likely find differences between sexual and apomictic accessions of responsible for the avoidance of meiosis. In most Boechera. The species B. divaricarpa includes both apomictic angiosperms, only a single archesporial cell and consequent and sexual accessions. Accession ES517 was chosen, because megaspore mother cell forms in an ovule. However, in maize it is diploid (Schranz et al. 2005) and produces 98-100% seeds multiplearchegonial cells1 (mac1) mutant, up to 21 with a 2:6 embryo: endosperm ratio (Aliyu et al. 2010), megasporocytes per ovule develop (Sheridan et al. 1996).The indicative of efficient apomeiosis, parthenogenesis of the phenotype of mac1 resembles apospory when several unreduced (2n) egg, and pseudogamous fertilization of the aposporic initials might be formed around the megaspore central cell with unreduced (2n) pollen. B. divaricarpa is mother cell. It was later revealed, however, that as in case with particularly well suited as a reference species for apomixis for the msd mutant, mac1 causes abnormal pollen formation as several reasons. First, apomixis usually occurs in polyploid well (Sheridan et al. 1999). Meiosis is a universal mechanism perennial species, while diploid species of the same genus for the majority of eukaryotes. Many key player genes reproduce sexually (Grossniklaus 2001). In the B. holboellii involved in meiosis were discovered in yeast. Among the complex, however, several diploid accessions with a high master genes that regulate the early stages of meiosis is IME1. penetrance of apomixis have been identified. Second, IME1 regulates genes that are expressed at the early stages of genomic resources for both ancestral species are available meiosis (Kassir et al. 1988, Kawaguchi et al. 1992). To (Phytozome; Sequence Read Archive [SRA] at NCBI), activate IME1 and make it functional, it has to be facilitating comparative genomic analyses. Third, apomictic phosphorylated by RIM11 (Bowdish et al. 1994), which is a B. divaricarpa has been the focus of genetic, cytological, and type of trigger gene that helps initiate meiosis. Homologs of ecological studies (Schranz et al. 2005, Sharbel and Mitchell- RIM11 were eventually found in rice (Jefferson and Nugroho Olds 2001, Kantama et al. 2007,Vogel et al. 2007), due to its 1998) and Arabidopsis (Grossniklaus 2001), and all these close relatedness to the model plant Arabidopsis thaliana. The contained conservative protein kinase domains. RIM11 might use of RNAseq data obtained from different plant organs, such be one of the candidate genes to manipulate in order to bypass as flower, silique, leaf, stem, and root will improve the meiosis for bioengineering of apomixis. A number of mutants annotation quality and also reveal the difference in gene participating in the switching of cell meiotic programs to the expression in these organs. Let’s consider some genes and mitotic ones have been isolated in maize. Among these mutants relevant to apomixis development. mutants is ameiotic 1(am1) (Palmer 1971, Goiubovskaya et 158 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.153-169

al. 1992, Golubovskaya et al. 1993, 1997). In am1, meiosis the megaspore mother cell (Podio et al.2014). This suggests doesn’t occur in the megasporocyte and is replaced by mitotic SERK may play a role in specifying an embryo sac fate to divisions. A similar phenotype was observed in Arabidopsis somatic cells. Another gene that was isolated from Poa with the switch1 (swi1) mutant (Mercier et al. 2001). pratensis is APOSTART. Similarly to PpSERK, the expression Orthologs of SWI1 have been recently found in natural of APOSTART differs in apomictic and sexual genotypes apomicts from Boechera species (Sezer et al. 2016). The SWI1 (Albertini et al. 2005). APOSTART contains a lipid-binding cDNAs were obtained from both apomictic and sexual domain and is involved in meiosis. APOSTART was Boechera young flower buds. The predicted proteins contain a potentially associated with apomixis, and its transcripts were phospholipase C domain and a nuclear localization signal. It detectable specifically in aposporic initials and embryo sacs. It was shown that SWI1-like sequences were common across has been suggested that APOSTART may be related to program plant families, regardless of the presence of a sexual or cell death (PCD) in the non-functional megaspores and apomictic reproduction system. However, the expression nucellar cell degeneration events that permit enlargement of levels of SWI1 mRNA in young unopened flower buds in maturing embryo sacs. Orthologs of APOSTART have been apomictic Boechera plants were relatively high in comparison also found in Arabidopsis. Functional characterisation of with the sexual Boechera plants. Another mutant in maize these genes in Arabidopsis showed their involvement in where the first meiotic division is substituted to mitosis is embryo sac and embryo development (Barcaccia and absence of first division (afd) (Goubovskaya 1979). The Albertini 2013). A novel gene that was directly involved in scenario of the reversion of meiosis to mitosis resembles the controlling entry into the second meiotic division, was restitutional division at meiosis I during apomixis. In the discovered and characterized in Arabidopsis known as mutants elongate1 (el1) from maize (Barrell and Grossniklaus omission of second division (OSD) (Cromer et al. 2012). A 2005) and triploid inducer (tri) from barley (Finch and mutant in this gene, omission of second division 1 (osd1), in Bennett 1979) disruption of the second meiotic division combination with two other recessive mutant genes, Atspo11- resembles the diplospory. Similarly, in the Arabidopsis mutant 1,and Atrec8, results in deregulation of meiosis and its dyad, the diploid embryo sac forms via a dyad rather than a replacement by mitosis in crossings. The triple mutant tetrad of megaspores by the disruption of meiosis (Siddiqi et mimicking the apomeiosis phenotype is called MiMe (Mitosis al.2000, Ravi et al. 2008. To get clonal offsprings, it is very instead of Meiosis) (D’Erfurth et al. 2009). Atspo11-1 and important to avoid recombination during cell division. DNA Atrec8 lead to a mitotic-like first meiotic division, such that meiotic recombinase 1 (DMC1) encodes an early plants with these mutants express an apomeiosispheno type in recombination protein that is essential for meiotic synapsis, which meiosis is completely replaced by a mitotic-like and it also plays a critical role in achieving the correct division. The osd1 mutation prevents the second meiotic chromosome segregation and increasing genetic diversity. division from taking place. Both the MiMe and dyad mutants DMC1 has been identified in many eukaryotic organisms. have been used to engineer apomixis in Arabidopsis Homologs of DMC1 have also been revealed in some (Marimuthu et al. 2011). Both MiMe and dyad mutants flowering plants, in particular Lilium longiflorum (Kobayashi produce diploid clonal gametes. After crossing these mutants et al. 1994) and Arabidopsis (Klimyuk and Jones 1997, Zhao with a strain, whose chromosomes are engineered to be et al. 2014, Pradillo et al. 2014). It was found that in eliminated after fertilization (Ravi and Chan 2010) the authors Arabidopsis AtDMC1 affects meiosis and is required for obtained up to 34% of the diploid progeny that were clones of bivalent formation. In meiocytes of the Arabidopsis dmc1 their maternal parent, as the paternal genome had been mutant, univalents rather than bivalents were observed eliminated, demonstrating that first-generation cloned plants (Pradillo et al. 2012). Other interesting candidate genes can be cloned again (Marimuthu et al. 2011). While the inducing apospory include the gene that plays a fundamental outcome of these crossings is similar to apomixis, crossings role in somatic embryogenesis of angiosperms and also in are still required. The authors claimed that clonal reproduction apomixis, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE through seeds can be achieved in a sexual plant by KINASE (SERK). In the apomictic plant, Poa pratensis, manipulating two to four conserved genes. Comparative gene PpSERK is expressed in cells around the megaspore mother expression studies in the aposporous grass Brachiaria cell, at the region where apospory initial cells are formed brizantha through sequencing of expressed sequence tag (Albertini et al. 2004, Albertini et al. 2005. It was also found (EST) libraries allowed the identification of three genes that PnSERK2 from the SERK family, revealed in the specifically expressed in the region where the aposporous apomictic plant Paspalum notatum, displayed a spatially and initial cells appear. These were genes encoding a MADS-box temporally altered expression pattern in ovules of the transcription factor (BbrizAGL6), a helicase (BbrizHelic), and apomictic genotype with respect to the sexual one. a stress induced protein (BbrizSti1) (Silveira et al. 2012, Specifically, it is expressed in nucellar cells of the apomictic Guimarães et al. 2013). Involvement of stress-related genes in genotype during meiosis, but in the sexual genotype only in the induction of aposporous initial cells was also noted in 2017 Is sex irreplaceable? Towards the molecular regulation of apomixis 159

Hieracium praealtum (Okada et al. 2013). A number of genes megaspore does not proceed with megagametogenesis and that regulate the specification of ovule cells, aborts the development at the one-nucleate stage (Christensen megasporogenesis, and embryo sac formation have an et al. 1998). In the mutant prolifera (prl), proper cytokinesis epigenetic nature. Mutations in ARGONAUTE (AGO) genes doesn’t occur and embryo sac is arrested at an early stage of lead to an increase in the number of cells capable of initiating development (Springer et al. 1995). PROLIFERA (PRL) is the embryo sac (Rabiger et al. 2016, Olmedo-Monfil 2010, essential for DNA replication and it is expressed in the Singh et al.2011). AGO proteins bind small interfering RNA developing megaspore mother cells (Holding and Springer (siRNA) and have endonuclease activity against the mRNA 2002). The mutant indeterminate gametophyte (ig) in maize that is complementary to the fragments of the bound siRNAs. showed no fixed amount of eggs and polar nuclei in the central Also, these proteins contribute to RNA dependent DNA cell, rather the numbers for both of these features was methylation (Meister 2013). Mutant ago9 isolated in indeterminate (Kermicle 1994). It was also found that the Arabidopsis formed multiple cells within a single ovule that indeterminate gametophyte1 gene of maize encodes LOB developed in the embryo sacs. The phenotype of the mutant domain protein required for embryo sac and leaf development was very much like apospory (Olmedo-Monfil 2010). The (Evans 2007). The other important gene affecting phenotype of another AGO mutant, ago104, isolated in maize, megagemetogenesis is Slow Walker1 (SWA1) gene in resembles diplospory where a megaspore mother cell Arabidopsis (Shi et al. 2005). This gene encodes an essential undergoes mitosis instead of meiosis, producing diploid WD40 protein that provides nuclear division and organization gametes (Singh et al. 2011). It was found that AGO104 during embryo sac formation. In the swa1 mutant, the proteins are accumulated in the ovule cells around the MMC, progression of the mitotic division cycles of the female thereby possibly suppressing somatic activity of the MMC gametophyte is disrupted, leading to the arrest of embryo sac and facilitating a developmental switch of the programs from development at early stages. Knockout of SWA1 gene led to mitosis to meiosis. An additional AGO mutant isolated in rice, the accumulation of unprocessed 18S pre-rRNA (Shi et al. named MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), 2005). These data suggest that SWA1promotes rRNA was specifically expressed within the ovule cells and was biogenesis essential for cell cycle progression during the necessary for the beginning of meiosis and sporogenesis in female gametophyte development. Previously described six rice (Nonomura et al. 2007). Arabidopsis mutants in genes female gametophytic mutants of Arabidopsis isolated in a involved in siRNA regulation pathway rna-dependent rna gene trap insertional mutagenesis screen (Brukhin et al. 2011). polymerase 6 (rdr6) and suppressor of gene silencing 3 (sgs3) These mutants were classified into mitotic class (kupalo and closely resembled the apospory phenotype of ago9 mutants in astlik); karyogamy class (amon and apis); degeneration of Arabidopsis, where multiple cells have the potential to central cell, egg cell, and synergid class (yarilo); and mitotically form the embryo sacs within a single ovule gametophytic maternal-post-fertilization defect class (Olmedo-Monfil 2010). (didilia), in accordance with the scenario of embryo sac 2. Genes and mutants affecting megagametogenesis— (megagametophyte) arrest. These mutants had disruptions in The functional megaspore, altered MMC, or apospory initial multiple genes: PTM1-like protein (kupalo); F-box family cell can initiate embryo sac development by a few rounds of protein, cellulose synthase-like protein, AAA-type ATPase mitotic divisions. Until the early 1990’sonly one family protein, RPN3a subunit of 26S proteasome, and gametophytic factor1 (gf1) mutant was described Embryo Defective 2719 (astlik); AIR9 auxin-induced in root (Christensen et al. 1997, Redei 1965). However, during the cultures (yarillo); and TATC-like protein, Unfertilized last two decades a large number of genes and mutants involved Embryo Sac3 (didilia). We subsequently concluded that the in embryo sac (megagametophyte) development have been mutations revealed pleiotropic effects in gametophyte arrest isolated in Arabidopsis and in other plants (Hand and and were often associated with chromosomal rearrangements. Koltunow 2014, Brukhin et al. 2005, Brukhin et al. 2011). Mutation in the gene of histidine kinase CK I1 caused a block Many megagametophyte acting genes were isolated in in megagametogenesis and led to female gametophytic mutants showing abnormalities in mitotic progression, polar lethality in Arabidopsis (Hejátko et al. 2003, Deng et al. 2010. nuclei fusion (karyogamy), fertilization and cell degeneration The expression of CK I1 starts at the beginning of female (Brukhin et al. 2011). These mutants are characterised by non- gametophyte development and continues until fertilization. Mendelian segregation and semi-sterility or reduced fertility These results suggest that the developing megagametophyte (Grossniklaus 2001, Brukhin et al. 2005, Brukhin et al. 2011, remains sensitive to the signals recognized by CK I1 Feldmann et al. 1997). Consequently, they are highly relevant throughout all development until fertilization and provides for studying apomixis because they demonstrate heterochrony evidence that expression of a sensor-like molecule in the during the megasporogenesis and embryo sac development, gametophyte is crucial for specific processes during embryo both of which are features intrinsic to apomixis. In the mutants sac development (Hejátko et al. 2003). Plant hormones such as female gametophyte2 (fem2) and fem3, the functional auxin and cytokinin play an important role in embryo sac 160 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.153-169

initiation and further formation. The auxin efflux carrier genes previously been shown in animals that calcium ions are from the PIN family participate in the establishment and essential for egg cell activation. Injection of calcium in eggs of regulation of auxin levels in ovule cells, a necessary condition sea urchin and mice was sufficient to induce parthenogenetic for the successful initiation of germ cells and the embryo sac. development of the egg cell (Steinhardt and Eppel 1974, Down regulation of PIN1 severely affects female Uranga et al.1996). Parthenogenetic egg activation is a key gametophyte development and leads to the arrest of the component of plant apomixis that may occur both embryo sacs at the mono- or binuclear stages (Ceccato et al. spontaneously and after induction. In the 1970s and 1980s, the 2013). In the mutant nomega of Arabidopsis, embryo sac high frequency of haploid production in barley plants development is arrested at the two nucleate stage (Kwee and homozygous for haploid initiator (hap) and in the Salmon Sundaresan 2003). The NOMEGA gene product has high wheat was reported (Grossniklaus 2001). The homozygous homology to the APC6/CDC16 subunit of the anaphase- mutation in hap results in formation of 30% haploid embryos promoting complex (APC/C) and is involved in post- (Hagberg and Hagberg 1980, Asker et al. 1983). In this system metaphase events (separation of chromosomes and the sperm cell fertilize the egg whereas the central cell is cytokinesis). The APC/C complex functions as an E3 fertilized normally, producing a viable endosperm. Later on it ubiquitin ligase in the ubiquitin-proteasome degradation was shown that Salmon wheat lines can produce up to 90%of pathway that performs proteolysis of the regulatory proteins parthenogenetic haploids (Matzk 1996). The nuclear factor of during cell cycle progression. It was found that in nomega parthenogenesis in the Salmon wheat system has been mutants, the embryo sac regulatory protein cyclin B is not genetically determined and the Ptg gene was found as an degraded and the cell is arrested at telophase. An interesting inducer of parthenogenesis (Matzk et al. 1997, Matzk et al. phenotype has been demonstrated in Arabidopsis with the 2005). Salmon wheat is a unique system for the study of mutant gene retinoblastoma-related 1 (rbr1) (Ebel et al. parthenogenesis at the molecular and genetic level. Genes 2004). Three mutant alleles of the gene for the Arabidopsis participating in activation of the unfertilized egg cell and retinoblastoma-related protein, RBR1 were observed to be somatic cells to reprogram these cells for embryo-zygotic gametophytic lethal. The unfertilized mutant fails to arrest development has been identified in both apomictic plants and mitosis in the developing embryo sac, which results in in sexual mutants. The gene BABY BOOM (BBM) was excessive nuclear proliferation. Numerous nuclei appear at the identified using subtractive hybridization of Brassica napus. micropylar end of the embryo sac, while the central cell BBM showed similarity to the AP2/ERF family of nucleus is able to produce an autonomous endosperm transcription factors and is expressed preferentially in development reminiscent of fertilization independent seed developing embryos and seeds (Boutilier et al. 2002). Ectopic (fis) mutants. Thus, RBR1 controls the cell cycle in the course expression of BBM in Arabidopsis and Brassica led to the of gametogenesis and also represses an autonomous spontaneous formation of somatic embryos and cotyledon- endosperm development. It was later found that RBR1 is like structures in seedlings. Sequencing of the apospory- involved in the regulation of imprinted genes. Along with specific genomic region (ASGR) in the apomictic plants MSI1, it represses the expression of MET1. This in turn Pennisetum and Cenchrus showed that form 40 protein coding activates expression of the imprinted genes FIS2 and FWA fragments identified in this region had a sequence similarity to (Jullien et al. 2008. Other genes with possible roles in the rice BBM gene. Therefore, BBM could be one of the epigenetic regulation of megagametogenesis and apomixis are candidate genes that have a strong potential to induce and DMT102 and DMT103 (Garcia-Aguilar et al. 2010). promote embryo development during apomixis (Conner et al. Comparing reproductive development of sexual maize with 2008) [127]. Other candidate genes that initiate apomictic hybrid maize-Tripsacum, six loci were identified to embryogenesis and are essential for embryo development are have been down regulated in ovules of apomictic plants. It has Leafy Cotyledon 1 (LEC1), LEC2, and FUSCA3 (FUS3) (Gaj also been found that inactivation of two DNA methyl et al. 2005, Wang and Perry 2013).Each of these genes encode transferase genes in maize DMT102 and DMT103 induced an a B3 domain transcription factor. LEC1 also encodes a apomictic-like phenotype with development of diploid CCAAT box-binding factor. Mutants in genes prematurely multiple embryo sacs within an ovule (Garcia-Aguilar et al. cease embryogenesis programs and switch to the seedling 2010). This indicates that germ cell fate specification in programs. Ectopic expression of these genes induces apomictic development might involve methyl trasferases and embryogenesis in seedlings. FUS3 also upregulates the requires an epigenetic regulation through a modification of the ABSCISIC ACID INSENSITIVE3-Like (ABI3-Like) chromatin state. transcription factor that is involved in the transition from 3. Genes and mutants affecting embryogenesis—The embryo to seedling development (Wang and Perry 2013). The most important features of apomixis are a fertilization- MULTICOPY SUPPRESSOR OF IRA1 (MSI1) gene encodes independent embryo and endosperm development. In the first WD 40 domain protein MSI1 belonging to an evolutionarily instance it is crucial to find out the events that cause activation conserved protein family. MSI1-like proteins are present in of the egg cell and initiation of embryogenesis. It has various protein complexes, including the epigenetic 2017 Is sex irreplaceable? Towards the molecular regulation of apomixis 161

regulatory Polycomb repressive complex 2 (PRC2) (Köhler et from the second embryo in the seed (Sargant 1900). Some al. 2003). It was shown that single mutations in MSI1 are able genes that are involved in the control of fertilization- to initiate parthenogenetic development of the embryo in independent development of endosperm and/or embryo have Arabidopsis thaliana from egg cells produced by meiosis been isolated from sexual mutant plants; these genes have also (Guitton and Berger 2005). These parthenogenetic haploid been described. These include the genes MEDEA(MEA) msi1 embryos turned out to be nonviable. Expression of the (Grossniklaus et al.1998, Schmidt et al. 2013), molecular markers and polarity similar to diploid wild-type FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) Arabidopsis embryos produced by fertilization proved the (Ohad et al. 1999), and FERTILIZATION-INDEPENDENT maternal contribution to the early patterning of these embryos. SEED2 (FIS2) (Chaudhury et al. 1997, Luo et al. 1999). These Thus, the genes studied in sexual organisms can be used as genes are associated with the epigenetic regulatory Polycomb clues to study induced parthenogenetic embryogenesis in repressive complex 2 (PRC2). PRC2 is conserved among most apomictic systems and also for providing candidate genes for of eukaryotes. The function of PRC2 is silencing of gene engineering the components of apomixis. Finally, an expression via trimethylation of histone H3 at lysine 27 important event for apomixis is the development of a viable (H3K27me3). The fertilization-independent seed (FIS) PRC2 endosperm, which can occur either through an independent or complex (FIS-PRC2) consists of MEDEA (MEA), FIS2, pseudogamous development. FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), 4. Genes and mutants affecting endospermogenesis— and the already-mentioned MULTICOPY SUPPRESSOR OF As mentioned above, endosperm is a complex nourishing IRA1 (MSI1). Loss of function of any of these genes results in tissue that originates as a result of double fertilization. In a phenotypes of fertilization-independent seed development in normal wild type sexual seed, endosperm is usually triploid, Arabidopsis and, in case of MSI1 mutants, parthenogenetic while the embryo is diploid. Successful completion of seed embryo initiation (Guitton and Berger 2005, Schmidt et al. development depends on a balance between the contribution 2013). Furthermore, a few genes/mutations epistatic to the of maternal and paternal genomes into the embryo. The mea mutant have been identified, such as DEMETER (DME), endosperm is crucial to this process, because some genes may which is mainly expressed in the central cell of embryo sac and be imprinted according to parental origin. Proper development is required for maternal allele expression of MEA (Choi et al. of the endosperm is strictly limited by a ratio of maternal to 2002), and GLAUCE (GLC) (Ngo 2007). The glc mutation paternal genomes 2m: 1p (Grossniklaus 2001, Birchler 1993). suppresses autonomous endosperm development observed in However, in Arabidopsis and in some other species deviation the fertilization-independent seed (fis), and glc central cells from this ratio is possible, such as in inter ploidy crosses, e.g. appear to be unfertilized. Summarizing the discussion of these between diploid and tetraploid species, produce viable seeds results, we can conclude that although a large number of (Grossniklaus et al. 1998). Nonetheless, seed size is strictly mutants isolated in sexual plants are currently available and dependent on parent-of-origin effects in inter ploidy crosses despite many candidate genes involved in the induction of (Scott et al. 1998). It has also been reported that apomictic different components of apomixes have been described, the species do not show significant reduction in fertility because regulation of these processes in the natural apomicts is much of their relaxed constraints on imprinting and/or because their more complex, involving various modifiers and epigenetic mechanisms of fertilization have been modified (Grossniklaus mechanisms that cause desynchronization and circumvention 2001). The former assertion might be supported by the of the meiotic events, a relaxed requirement for imprinting, observation that the endosperm in apomictic species can be of and that contribute to the fertilization-independent embryo different ploidy level (Johri et al. 1992). Nevertheless, and endosperm development. Even though previous imprinting may be a serious obstacle to the introduction of techniques of “wet biology” in the pre-genomic era have apomixis into sexual crop plants. Sexual seed is the product of enormously promoted our knowledge of apomixis, they were a heterogeneous formation, consisting of the tissues with three not yet able to solve the “mystery” of apomixis and create genomes - diploid maternal sporophytic, diploid embryo efficient tools for its engineering in agricultural crop plants. sporophytic and triploid endosperm genomes. The interaction With the advent of the post-genomic era emergence of systems between all these tissues is rather complex and still poorly biology, the availability of new tools such as genotyping by understood. In contrast, an apomictic seed consists of tissues sequencing, and bioinformatics pipelines for analyses of with one (in case of autonomous endosperm development) or species lacking reference genomes (Worthington et al. 2016) with two (in case of pseudogamous endosperm) genomes. might drastically streamline our knowledge on apomixis Despite the functional differences, there is a significant regulation. overlap in the gene expression and genetic regulation of GENETIC LOCI RELATED TO APOMICTIC embryo and endosperm development, which is consistent with TRAITS—Previous generations of geneticists considered the hypothesis of the evolutionary relationship of the embryo apomixis as a single dominant locus. Since the first attempts to and endosperm, according to which the endosperm is derived identify apomixis loci using map-based cloning approaches, 162 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.153-169

geneticists faced the challenging fact that molecular markers not with parthenogenesis, showing that these two components presumably associated with apomixis and that are spread over of apomixis are independently controlled (Schallau et al. a large region in sexual counterparts, are strictly co-segregated 2010). The authors showed that part of the HAPPY sequence is together (Ozias-Akins et al.1998, Pessino et al.1998, homologous to the Arabidopsis thaliana gene ARI7 encoding Grimanelli et al. 1998, Noyes and Rieseberg 2000, Pupilli et the ring finger protein ARIADNE7-like E3 ligase (HpARI), al.2001). The distortion of the recombination frequency at which is involved in various regulatory processes, including apomixis loci was due to the fact that recombination around ubiquitin-mediated protein degradation. In this study, these loci is suppressed (Grossniklaus 2001). Further studies apospory was inherited as a dominant simplex gene at the of recombination in suppressed regions and analysis of tetraploid level. One of the four HpARI alleles within the gamma-deletion mutants revealed that in a number of plant tetraploid apomict is truncated compared to sexual plants. The species avoidance of meiosis, parthenogenesis, and truncated HpARI gene may act in a dominant-negative fertilization-independent endosperm development are fashion, interacting with the three remaining intact alleles. controlled by independent loci. For example, two independent Thus, altered expression of the truncated HpARI allele loci have been identified that control diplospory and probably results in switching to apomictic development. parthenogenesis in species of Taraxacum and Erigeron Components related to the ubiquitin-mediated proteasome (Noyes and Rieseberg 2000, van Dijk et al. 1999). It has also pathway have been earlier reported to prevail at the been reported that in species of Hypericum, Poa and aposporous initial cells when compared to somatic cells of the Cenchrus, two unlinked loci are responsible for apospory and ovule (Okada et al. 2013). That makes this pathway a parthenogenesis (Albertini et al. 2001, Schallau et al. 2010, candidate for involvement in aposporous initial cell Conner et al. 2013). In Hieracium praealtum (hawkweed), origination and growth. Recently the locus APOLLO (for two loci relevant to apomixis were revealed in a study using apomixis-linked locus) was identified in plants of the genus gamma deletion mutants. The first locus is called LOSS OF Boechera. Transcripts of the gene Aspartate Glutamate APOMEIOSIS (LOA) and it is associated with apospory, while Aspartate Aspartate histidine exonuclease, located within the the second locus, which is responsible for fertilization- locus, are down-regulated in sexual ovules when they enter independent seed development, is called LOSS OF meiosis and are up-regulated in apomeiotic ovules at the same PARTHENOGENESIS (LOP) (Hand et al. 2015, Catanach stage of development (Corral et al. 2013). APOLLO has both 2006, Koltunow et al. 2011, Shirasawa et al. 2015). Deletion “apoalleles” and “sexalleles”, which differ in their of either LOA or LOP locus results in reversion to the sexual polymorphisms and which encode different transcription pathway. Deletion of both loci leads to complete reversion to factors and home box proteins. All apomictic Boechera spp. sexual development (Koltunow et al. 2013). Moreover, accessions proved to be heterozygous for the APOLLO alleles apomictic forms of Hieracium are capable of forming (having at least one apoallele and one sex allele), while all fertilization-independent endosperm. It has been found that sexual genotypes were homozygous for sex alleles. Thus, this trait segregates independently of the other two apomictic biallelic inheritance of APOLLO suggests that the expression components as well (Ogawa et al. 2013). Along with of a single deregulated allele could induce the cascade of suppressed recombination around apomictic loci and the events leading to asexual female gamete formation in an resulting strong divergence of alleles at these loci, some apomictic plant. apomixis-related genomic regions have also been associated CONCLUSION with heterochromatin, extensive repetitive sequences or transposable elements (Calderini et al. 2006). For example, in Apomixis is a unique natural phenomenon in plant Pennisetum squamulatum, the apospory-specific genomic reproduction that results in clonal propagation through seed, region (ASGR) is located in a telomeric, heterochromatic, where the genotype of the embryo is identical to the genotype nonsynaptic region. This locus has been associated with of the maternal plant. Apomixis is obviously regulated hemizygosity because it was located at a single chromosome genetically, although its core genetic components have not (Worthington et al. 2016, Akiyama et al. 2005). Identified in been fully determined yet. Genes and loci that demonstrate an Paspalum, the hemizygous apomictic controlling locus involvement in the key components of apomixis, such (ACL) showed a strong rearrangement compared to its asapomeisois, parthenogenesis, and autonomous endosperm homolog in rice and it is associated with the presence of development have been identified in sexaul mutants transposable elements (Calderini et al. 2006). The ACL locus demonstrating features of apomixis or through the molecular also demonstrated a high level of suppression of comparison of natural apomictics with their sexual recombination. In Hypericum perforatum (St John’s wort) the counterpart. Apomixis seems to be superimposed on sexual HAPPY locus (for Hypericum APOSPORY) was isolated as a development rather than being a completely independent result of amplified fragment length polymorphism (AFLP) pathway, i.e. it apparently redirects the gametic potential of profiling. The HAPPY locus co-segregates with apospory but the germ cells via heterochronicity and alternation of the 2017 Is sex irreplaceable? Towards the molecular regulation of apomixis 163

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