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Chapter V
A review on apomeiosis in Poa pratensis L. and
Medicago sativa L. mutants
Emidio Albertini & Gianni Barcaccia
In contrast to sexual reproduction, apomixis bypasses meiosis and egg cell
fertilization. Gametophytic apomixis occurs with the parthenogenetic development of unreduced egg cells from apomeiotic embryo sacs originating
from a nucellar somatic cell (apospory) or a megaspore mother cell with no,
or modified, meiosis (diplospory). Apomeiosis, along with parthenogenesis, excludes segregation and recombination during meiosis and fertilization. Thus, understanding the genetic control and the molecular mechanisms underlying apomeiosis is critical for the comprehension of apomixis
as a whole. In this paper we review the available data on apospory in the
facultative apomictic species Kentucky bluegrass (Poa pratensis L.) and on
diplospory in reproductive mutants of the sexual species alfalfa (Medicago
sativa L.). Our recent acquisitions on candidate genes for apomeiosis are
reported and strategies for elucidating the inheritance of this trait by means
of genomics and expression studies are presented and discussed. In particular, experimental data focus on PpSERK and MsMob1 genes. We document that PpSERK transcripts are specifically expressed in the megaspore
mother cells of sexual genotypes and in the aposporic initials of sexual
genotypes, suggesting that PpSERK plays a role in early stages of embryo
sac development. Moreover, the altered expression of MsMob1 in ovules of
a reproductive mutant producing diplosporic eggs is reported, and the
implication of MsMob1 proteins in programmed cell death of meiotic
megaspores is also considered.
KEYWORDS: apospory, candidate genes, diplospory, gene expression study.
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INTRODUCTION
Sex is the queen of problems in evolutionary biology. Perhaps no other natural
phenomenon has attracted so much interest; certainly none has caused as much
confusion (Bell, 1982). One of the major problems which have plagued evolutionary biologists is understanding what factors lead to the maintenance of sex under
natural selection in biological populations? The easiest answer is that sex, through
genetic recombination at meiosis and genomic fusion by fertilization, allows genotype rearrangement, diversification and adaptation. But even the most dogmatic
interpretation of the meaning of sex can be challenged. For example, the existence
of apomixis (asexual reproduction through seed) can be an argument which questions the essentiality of sex. In apomictic plants, the embryo develops inside an
embryo sac formed from either a megaspore mother cell (diplospory) or somatic
cell of the nucellus (apospory). It is independent from fertilization and retains the
maternal genotype (Savidan, 2000; Grimanelli & al., 2001; Spillane & al., 2001).
Fertilization may be required for endosperm formation, which can either retain the
2:1 maternal-to-paternal genome ratio or completely lack a paternal genome equivalent (Nogler, 1984; Spillane & al., 2001; Tucker & al., 2003; Savidan, 2007; see
chapter 1).
The development of apomixis technology for plant breeding would lead to an
overall reduction in the costs and time associated with standard breeding practices
using sexual reproduction or vegetative propagation (Bicknell & Koltunow, 2004).
Moreover, farmers of the developing world would benefit from new, advanced,
highly-producing varieties developed from local varieties at low cost and set for
mechanized agricultural systems. The apomictic processes result in progeny that
are genetically exact copies of the mother plant.
Although many years of descriptive studies have provided a solid documentation of the types of apomictic processes that occur in a wide variety of plant
species, molecular studies aimed at understanding the basis of apomixis have failed
to shed more than a dim and wavering light on its central mystery, partly because
the majority of apomicts do not constitute agriculturally important crops and, with
few exceptions (e.g., Tripsacum and maize), do not have agriculturally important
relatives (Bicknell & Koltunow, 2004). Furthermore, the polyploid nature of most
apomicts hinders genetic mapping studies and the building up of populations for
reverse genetics. Even though apomixis can be influenced by environmental factors in some taxa (Mazzucato & al., 1996), it is generally accepted that apomixis is
under strong genetic control, at least for facultative forms (Nogler, 1984; Savidan,
1990; Asker & Jerling, 1992; Savidan, 2000; Bicknell & Koltunow, 2004).
If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets.
Until the gene(s) that promote and control apomixis are identified and understood
on the molecular level, this trait can only be introgressed into agricultural crops
through traditional breeding methods, most of which are slow and laborious and
require progeny tests for the selection of apomictic genotypes after each round of
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
95
backcrossing. Among the practical choices for minimizing the time and costs of
breeding programs, an easy method for early selection based on molecular markers appears to be the most promising and would entail: simple genetic inheritance
of the trait under selection, and the availability of molecular markers strongly associated with the trait. The available data on the apomictic reproduction indicate that
the first requirement is met.
In Kentucky bluegrass (Poa pratensis L.) (Matzk, 1991; Barcaccia & al.,
1998a; Albertini & al., 2001a) and several unrelated species including Panicum
maximum (Savidan, 1983), Pennisetum squamulatum (Ozias-Akins & al., 1993;
1998; Gustine & al., 1997; Roche & al., 1999), Brachiaria decumbens (Pessino &
al., 1997), and Tripsacum dactyloides (Leblanc & al., 1995), apomixis seems to be
under simple genetic control. Cyto-embryological and molecular analysis have
revealed that only a single or a few tightly clustered, dominant genes are required
for the genetic transmission of apospory and/or parthenogenesis, and that apomixis is a simply inherited trait (Matzk, 1991; Barcaccia & al., 1997a, 1998a), although
low levels of recombination between apospory and parthenogenesis is known to
occur (Albertini & al., 2001b). The development of an appropriate set of genetic
markers to enable selection for the apomixis trait in this group would be of great
practical interest, however the availability of plant material amenable to genetic
analysis and a lack of resources have hindered progress.
Defining the nature and genetic control of apomixis may be crucial for both
understanding the trait itself and better illustrating the meaning of sexuality. Gene
expression-based techniques combined with differential display methods have
helped to shed light on the apomixis mechanism, while gene expression studies of
Arabidopsis mutants and rice have led to the cloning and characterization of
apomixis-like genes: SPL (Schiefthaler & al., 1999), NZZ (Yang & al., 1999),
SERK (Schmidt & al., 1997; Hecht & al., 2001), LEC1, LEC2 and PKL (Ogas &
al., 1999), FIS1 (or MEA), FIS2 and FIS3 (or FIE) (Ohad & al., 1996; Grossniklaus
& Schneitz, 1998; Kiyosue & al., 1999; Luo & al., 1999; Vielle-Calzada & al.,
1999). Comparative gene expression studies have been carried out on the early
stages of apomictic and sexual embryo sac development in Panicum maximum
(Chen & al., 1999), Brachiaria species (Leblanc & al., 1997; Dusi, 2001;
Rodrigues & al., 2003), Pennisetum (Vielle-Calzada & al., 1996; Jessup & al.,
2003), Paspalum (Pessino & al., 2001), and Poa pratensis (Albertini & al., 2003),
including apomeiotic mutants of Medicago falcata (Barcaccia & al., 2001). Most
work has been based on subtractive hybridization techniques which have led to the
isolation of only a few genes to which, disappointingly, no clear function could be
assigned. Hybridization-based studies, even if negative in context, add support to
the proposal that sexual and apomictic developmental pathways differ primarily in
their ability to regulate common elements (Bicknell & Koltunow, 2004).
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APOSPORY IN THE APOMICT KENTUCKY BLUEGRASS
(POA PRATENSIS L.)
Kentucky bluegrass is a hardy, persistent, attractive forage and turf grass
adapted to a wide range of soils and climates (van Wijk, 1997). It has an extremely diverse breeding system which, besides clonal growth, can range naturally from
nearly obligate apomixis to complete sexuality (Mazzucato & al., 1995). Although
sexual forms are predominantly allogamous, apospory is stimulated under the
influence of foreign pollen, which is also required for endosperm development
(pseudogamy). Facultatively apomictic polyploids, acting as seed parents, can generate hybrids as well as an autonomously derived progeny via both somatic
embryogenesis and apomeiotic parthenogenesis. The combination of a pollen
recognition system and the aposporic nature of apomixis confers a strong ability to
hybridize with, and retain, alien genomes (Wedin & Huff, 1996) and so determine
high ploidy levels and unusual chromosome numbers (x = 7, 2n = 28–147,
Speckmann & van Dijk, 1972). The high polyploidy and the contrasting mode of
reproduction of P. pratensis should make it a model species for investigating
apomixis and for cloning candidate apomixis genes. The highly flexible reproductive mode of such complex systems should permit genotypes recombinant for features of apomixis to be isolated (Matzk & al., 2005). Recombinants should prove
of immense value in gene expression studies, as candidate genes can accurately be
correlated with quantitative variation in specific apomixis components, rather than
to the apomictic process as a whole. Regulation of apospory is generally considered to depend upon a few dominant or codominant genes (Asker & Jerling, 1992;
Koltunow & al., 1995) which allow a somatic nucellar cell to form an embryo sac
without meiosis, and an embryo to develop from an egg cell without fertilization.
Once apomictic genes initiate nucellar embryo development and the initial cell
forms and divides, the genes controlling embryo cell formation, structure and
embryo pattern formation are probably the same as those required for sexual
embryo development. A number of studies support the hypothesis that zygotic
embryogenesis and apomeiotic parthenogenesis follow similar pathways during
embryo and seed production (Bicknell & Koltunow, 2004; Albertini & al., 2004),
and apomixis would be more easily understood if the genes responsible for both
specific and differential expression patterns in embryo sac and embryo formation
were identified.
Taken together, the results in P. pratensis demonstrate that between two and
five distinct genetic factors, or systems, control apospory and parthenogenesis,
which may be developmentally uncoupled (Albertini & al., 2001a; 2001b; Matzk
& al., 2005). Some recombinant genotypes (i.e., parthenogenetic but meiotic) were
recovered by Barcaccia & al. (2000b) within experimental lines produced through
the in vitro rescue of autonomous embryos obtained after auxin-induced parthenocarpy. These findings suggested that parthenogenetic capacity is not uniquely
expressed by aposporic egg cells, and that egg cells can develop autonomously
irrespective of their cytological origin. Other recombinant types (i.e., aposporic
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
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but non-parthenogenetic) were isolated by Albertini & al. (2001b) within an F1
population of P. pratensis segregating for the mode of reproduction (Porceddu &
al., 2002). In particular, the results of a cytohistological investigation of apospory
and parthenogenesis showed that the F1 population stemming from a cross between
a sexual (non-aposporic and non-parthenogenetic) and an apomictic (aposporic and
parthenogenetic) parent segregated for both apospory and parthenogenesis, thus
indicating that the traits are both genetically dominant and heterozygous in the
apomictic parent. Apospory was detected in 18 of 38 analysed individuals,
although detection of the trait was not simple due to variation in the number of
embryo sacs and the timing of the appearance of aposporic initials. However,
apospory was scored in all parthenogenetic individuals and in two non-parthenogenetic genotypes (Albertini & al., 2001b).
These recombinants were used, together with apomictic and sexual genotypes,
in a cDNA-AFLP transcriptional profiling study which led to the isolation of more
than 2.000 messengers from developmentally staged inflorescences (Fig. 1), 179 of
which were differentially expressed in apomictic and sexual genotypes (Albertini
& al., 2004). Since the vast majority of genes expressed in florets differed in
expression timing rather than for presence vs. absence of specific transcripts,
zygotic embryogenesis and apomeiotic parthenogenesis probably share a similar
pathway. Moreover, the cDNA-AFLP investigation led to the isolation of a group
of genes putatively involved in signaling and trafficking events required during
sporogenesis, gametogenesis and embryogenesis in plants (Albertini & al., 2004).
These data are concordant with the idea that apomixis could reflect a deregulation
of the sexual reproductive program in time and space that leads to changes in the
cell fate and the omission of steps critical to sexual progression (Bicknell &
Koltunow, 2004). The resultant signaling changes from ovule malformation could
stimulate a parthenogenetic embryo-development program in a set of somatic cells
in a manner similar to stress-induced somatic embryogenesis (Mordhorst & al.,
1997; Bicknell & Koltunow, 2004). Some of the isolated ESTs (Albertini & al.,
2004) were thus characterized as possibly involved in a signaling/development
pathway.
In particular, one cDNA fragment (named PpRAB1) shared 63% identity with
a GTP-binding protein from Zea mays (Albertini & al., 2004). GTP-binding proteins play an important role in the regulation of basic processes in all living organisms such as signal transduction, translocation and cell-cycle regulation (SikoraBorgula & al., 2002). Another EST (named PpArmadillo-like, PpARM) showed
96% identity with a putative nuclear protein from Oryza sativa and 77% identity
with the CTNNBL1 (catenin caderin-associated protein β-like 1, Jabbour & al.,
2003) gene of Homo sapiens which encodes for a protein having predicted structural homology with β-catenin and other armadillo (arm) family proteins. A third
differentially expressed cDNA fragment, named PpAnkyrin-like Protein Kinase
(PpAPK) shared 71% identity with a probable Ankyrin protein kinase (RING finger homology) of Arabidopsis thaliana and 81% with an EST isolated from an
apomictic pistil library of Pennisetum ciliare (Jessup & al., 2003).
Fig. 1. Morphological appearance of inflorescences (upper panel) at
the developmental stages
collected as verified by
cyto-histologial investigations (lower panel):
A, pre-meiotic (MMC formed); B, meiotic (dyads
or triads); C–D, post-meiotic (functional and degenerating megaspores for
sexual genotypes and
aposporous initials and
degenerating megaspores for apomictic individuals); E–F, anthesis (one
or more embryo sacs formed) and of leaves (bar
= 40 µm). Abbreviations:
ae, aposporous embryo
sac; ai, initial cell of aposporous embryo sac; d,
degenerated megaspore;
dy, dyad; es, sexual embryo sac; fm, functional
megaspore; ii, inner integument; mmc, megaspore mother cell; oi, outer
integument; ow, ovary
wall. From Albertini & al.
2005 (© Am. Soc. Pl.
Biol.).
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Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
99
An additional EST showed a highly significant amino acid sequence similarity with a SERK family member of Arabidopsis thaliana (Albertini & al., 2005) and
was named PpSERK (Fig. 2). In apospory, a cell of the nucellus becomes an
“aposporic initial” and then develops into a non-reduced embryo sac which,
through parthenogenesis, gives rise to a viable embryo. How and why somatic cells
of the ovule change their developmental fate and gain embryogenetic potency is
not known (Fehér & al., 2003). Whereas the zygote, formed as a consequence of
egg cell fertilization, is clearly predetermined to follow its embryogenetic cell fate,
in other forms of plant embryogenesis (including apomixis) there is a transition
phase during which competent and embryogenetic cell types are formed (Fehér &
al., 2003). The transition phase is clearly very complex, but elucidation of its
underlying mechanisms should lead to a deeper understanding of the developmental strategy adopted by apomictic plants. The changing fate/acquisition of embryogenetic competence relies mainly on dedifferentiation, a process whereby existing
transcriptional and translational profiles are erased or altered so that the cell can set
Fig. 2. Organization of genomic PpSERK (A–B) and APOSTART (D–G) genes showing position and size of exons and introns and their deduced proteins (C and H). A,
PpSERK1 (AJ841698); B, PpSERK2 (AJ841697); D, APOSTART1 (AJ786392); E,
APOSTART2 (AJ786393); F, APOSTART-like At4g19040 (NP193639); G, Oryza sativa
APOSTART-like (AE017106).
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a new developmental program. Schmidt & al. (1997) hypothesized that while most
elements involved with the origin and target processes of cell-to-cell communication in early plant embryogenesis are lacking, the SERK gene may be a significant
component in the mechanism leading to the formation of plant cells destined to
become embryos. Tucker & al. (2003) studied the expression of the AtSERK1
homolog gene in Hieracium and found the pattern between sexual and apomictic
plants to be conserved.
The ISH data reported by Albertini & al. (2005) revealed that PpSERK is
expressed in the megaspore mother cell (MMC) of sexual genotypes, but not in that
of apomictic plants. In contrast, the strong signals detected in nucellar cells neighboring the MMC suggests that PpSERK is involved in embryo sac development
from nucellar cells (Fig. 3).
Lastly, one of the isolated ESTs from Albertini & al. (2004) was very similar
to an EST isolated from a pistil-specific cDNA library of apomictic Pennisetum ciliare (Jessup & al., 2003) and to a lipid-binding START domain-containing protein
from Arabidopsis. This P. pratensis cDNA fragment was named APOSTART (Fig.
2) because of its START domain and putative involvement in apomixis, and could
be of extreme interest because the human START domain was named after the discovery of the StAR gene involved in human congenital lipoid adrenal hyperplasia,
which is characterized by a marked impairment in the biosynthesis of adrenal and
gonadal steroid hormones. The clinical phenotype of this disease includes the onset
of profound adrenocortical insufficiency shortly after birth, hyperpigmentation due
Fig. 3. PpSERK (A–C) expression in longitudinal sections of flowers studied by in
situ hybridization (bars 40 µm). Sections were probed with digoxigenin-labelled
antisense (A, B) or sense (C) RNAs and viewed under a bright field that gives a purple label. A, longitudinal section of ovule of a sexual genotype containing the
megaspore mother cell (mmc) showing a strong hybridization signal; B, an apomictic genotype ovule where the hybridization signal was not detected in the
megaspore mother cell (mmc), but was present in two neighbor nucellar cells; C, no
signals were detected in the ovule sections hybridized with sense probes.
Abbreviations: mmc, megaspore mother cell; nc, nucellar cell; ii, inner integument;
oi, outer integument; ow, ovary wall. From Albertini & al., 2005 (© Am. Soc. Pl. Biol.).
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
101
to increased production of propiomelanocortin, elevated plasma renen activity as a
consequence of reduced aldosterone synthesis and male pseudo-hermaphroditism
resulting from deficient fetal testicular testosterone synthesis (Christenson &
Strauss, 2001). Albertini & al. (2005) showed that APOSTART1 and APOSTART2
are expressed exclusively in the inflorescences of P. pratensis. They also propose
that the APOSTART1 gene is involved in sporogenesis, a point supported by its
almost complete lack of expression in apomictic genotypes, while recombinant
genotypes were characterized by APOSTART1 expression levels 10-fold that of
apomictic genotypes but lower than that of strictly sexual genotypes, in accordance
with a 62% apospory ratio (Albertini & al., 2001b, 2005). Moreover, in situ
hybridization analysis revealed that APOSTART members are expressed during
both male and female meiosis in all micro- and mega-spores. Together, these data
suggest that APOSTART expression may also be related to PCD in P. pratensis,
which is involved in non-functional megaspores and degeneration of nucellar cells
which permit enlargement of maturing embryo sacs (Wu & Cheung, 2000). Search
of the Genevestigator database (Zimmermann & al., 2004) for the expression of the
APOSTART homolog of A. thaliana revealed that this gene is exclusively up-regulated during senescence, and is almost double that of any other hormone-treated or
stress-induced case. These data bring to mind the MMD1 (MALE MEIOCYTE
DEATH1) gene of Arabidopsis (Yang & al., 2003) which was shown to be involved
in the regulation of gene expression during meiosis. Furthermore, a mmd1 mutation triggers cell death in male meiocytes (Yang & al., 2003). The predicted localization of APOSTART in the mitochondrion membrane and its putative role in regulating mitochondrial membrane permeabilization makes APOSTART a prime candidate for apoptosis.
The above-reported genes (Albertini & al., 2004, 2005) are involved in cell-tocell interactions for both signaling pathways and hormone stimulation/production.
Hecht & al. (2001) proposed that AtSERK is a component of an embryogenesissignaling pathway, whereby competent cells may contain an inactive receptor
which is activated by the presence of a proper ligand to switch on the embryogenesis program. According to Hecht & al. (2001) the acquisition of embryogenic
competence in tissue cultures requires the presence of non-embryogenic cells that
produce and secrete molecules that can be perceived by other cells, which in turn,
express their own competence and develop into embryos (Pennel & al., 1992; de
Jong & al., 1993). It is thought that there is a point at which ovule nucellar cells act
as a sink for all nutrient sources, shunting nutrients from the dividing megaspore
mother cell to themselves. We have previously hypothesized that some differentially expressed genes between apomictic and sexual P. pratensis are differently
timed and have diverse localization of transcripts (Albertini & al., 2004). We propose that PpSERK activation in nucellar cells of apomictic genotypes is the switch
that channels embryo sac development and that it could redirect signaling gene
products to compartments other than their typical ones. The SERK-mediated signaling pathway may well, at some point, interact with the auxin/hormonal pathway
controlled by APOSTART, but PpSERK is certainly not an integral part of it.
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DIPLOSPORY IN REPRODUCTIVE MUTANTS OF ALFALFA
(MEDICAGO SATIVA L.)
The Medicago sativa-coerulea-falcata complex consists of several outcrossing
diploid (2n = 2x = 16) or tetraploid (2n = 4x = 32) subspecies which are interfertile and have the same karyotype (Barcaccia & al., 2003). The cultivated tetraploid
forms are characterized by tetrasomic inheritance, with multiple allelism, and pronounced inbreeding depression. Whereas normally reduced gametes largely predominate in diploids, restitutional 2n gametes (i.e., gametes with the somatic chromosome number) often occur and are capable of fertilization. In the cultivated
alfalfa complex, combinations of n and 2n gametes have led to the establishment
of new sexual polyploids which have proven useful for understanding evolutionary
processes, allowing germplasm transfer and cultivar improvement.
Investigations of the reproductive system have provided information on which
types of meiotic alterations are responsible for the production of unreduced egg
cells. Specific cyto-embryological methodologies, such as those based on stainclearing and sectioning, were a crucial requirement for analyzing the various steps
of sporogenesis and gametogenesis within both whole ovules and sectioned ovaries
(Stelly & al., 1984; Tavoletti & al., 1991; Barcaccia & al., 1996; 1997b). Analysis
of meiotic mutants revealed that the occurrence of 2n egg cells is due to cytological alterations genetically equivalent to first division restitution (FDR) and second
division restitution (SDR) mechanisms. The cytological mechanism of 2n egg formation in alfalfa was studied for the first time by Pfeiffer & Bingham (1983), who
showed that developmental sequences in the formation of n and 2n eggs were the
same throughout anaphase II but, after this phase, cytokinesis occurred only in the
micropylar dyad and not in the chalazal one (Fig. 4). Micropylar megaspores disintegrated leaving a functional 2n megaspore of the SDR type at the chalazal end.
Although 2n egg production was mainly associated with the absence of cytokinesis after telophase II (Tavoletti & al., 1991) or the failure of the second meiotic
division (Tavoletti, 1994), the omission of the first meiotic division was also documented (Tavoletti, 1994; Barcaccia & al., 1996). The latter abnormality was
shown to give rise to apomeiotic 2n megaspores of the FDR type (Tavoletti & al.,
1996a; Barcaccia & al., 1997b). It should be noted that cytological investigations
of megasporogenesis may reveal the presence of unreduced nuclei at early meiosis, but the distinction of apomeiotic mechanisms, such as the establishment of
either the meiotic restitution pathway or mitotic diplospory, can only be evidenced
using molecular methods (Koltunow, 1993). Apomeiotic 2n eggs produced through
diplospory are of interest because they should retain and transmit all (mitotic
diplospory) or most (meiotic diplospory) of the maternal genotype, whereas only a
small part is conserved and transferred in 2n eggs formed by second division restitution mechanisms.
Since an effective triploid block operates in alfalfa, interploid matings lead to
the elimination of almost all triploid embryos due to endosperm imbalances
(Veronesi & al., 1986), hence the number of seeds produced per flower pollinated
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
103
(i.e., seed set) in 2x–4x crosses gives a measure of the frequency of 2n eggs produced by the diploid parent on the female side. Several 2n egg producers were
selected from diploid natural populations of M. sativa subsp. coerulea and M. sativa subsp. falcata on the basis of seed set values in crosses with tetraploid parental
counterparts. Nucleolus diameter was shown to be a good marker to discriminate
between restitutional 2n and n megaspores or embryo sacs (Tavoletti, 1994;
Barcaccia & al., 1997b), being on average 1.6-fold greater (2.40 µm vs. 1.51 µm)
in apomeiotic than meiotic cells (Barcaccia & al., 1999a). However, only a concurrent examination of the nucleolus size, integument growth and cell appearance
can give a reliable estimate of diplospory in alfalfa (Fig. 4).
Apomixis as a whole has not been detected in the genus Medicago, but components of apomixis have been documented. The formation of unreduced eggs
through diplosporic apomeiosis would be an extremely interesting trait to have in
alfalfa (Tavoletti, 1994; Tavoletti & al., 1996a; Barcaccia & al., 1996, 1997b), as
is the induction of haploid parthenogenesis in tetraploid lines of alfalfa (Bingham,
Fig. 4. Schematic representation of the principal abnormalities of female meiosis in
alfalfa responsible for unreduced megaspore formation: A, normal megasporogenesis
leading to a functional chalazal n megaspore; B, reductional division of centromeres
with absence of cytokinesis after the second division in both chalazal and micropylar
cells giving rise to 2n second division restitution megaspores; C, formation of a 2n first
division restitution megaspore through diplosporic apomeiosis based upon the omisssion of reductional division followed by equational centromeric division.
Abbreviations: t, tetrad of normally reduced megaspores; dy, dyad of binucleated
megaspores; mmc, apomeiotic megaspore mother cell; ii, inner integuments; nu, unreduced nucleus. Bar = 20 µm.
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1971; Bingham & McCoy, 1979). Progress in assembling a functional system of
apomictic seed production in alfalfa was reviewed by Barcaccia & al. (2003).
A progeny test based on morphological traits and molecular markers has indicated that apomeiosis is not tightly associated with parthenogenesis in reproductive
mutants of diploid alfalfa, as the two traits are only rarely found together
(Barcaccia & al., 1997c). Gametophytic apomixis has also been shown to be
strongly correlated with the occurrence of hybridity and polyploidy. Although
numerous non-reductional meiotic mutants have been described in diploid forms of
sexual species, the expression of apomixis is restricted mostly to polyploid apomictic complexes (Asker & Jerling, 1992). Therefore, the introgression of the diplosporic apomeiosis mutation at the tetraploid level was attempted to induce somatic parthenogenesis through auxin-based treatments and wide-crosses with unrelated diploid materials.
While the formation of apomeiotic unreduced embryo sacs and eggs in alfalfa is known to be under genetic control (Barcaccia & al., 2000a), the autonomous
development of embryos by parthenogenesis seems to be “latent” in this species
(Bingham, 1971; Bingham & McCoy, 1979). If asexual reproduction occurs in
available diplosporic stocks, the degree of apomixis is too low to be of any use for
breeding purposes. The fact that both reduced and unreduced eggs are produced by
the same plant suggests that expression timing of the apomeiotic mutation during
megasporogenesis leads either to the normal or diplosporic pathway. Apomixis is
though to be controlled by a few tightly linked loci with dominant alleles leading
to both apomeiosis and parthenogenesis (Savidan, 2000; Grossniklaus & al., 2001),
and hence if the diplosporic mutation documented in alfalfa involves the genetic
factor responsible for gametophytic apomixis, one would expect that the unreduced
egg cell should have a propensity for autonomous development. Alternatively,
genetic independence of the two processes implies that there remain some possibilities for sexual reproductive variation. Diplospory and parthenogenesis are not
associated in diploid or tetraploid apomeiotic mutants, with the overall Mendelian
ratios observed in segregating progenies between plants showing a null or very low
degree of diplospory and plants scoring a moderate to high degree of diplospory
agree with the hypothesis of recessive, rather than dominant, genetic control
(Barcaccia & al., 1999a; 2000a).
In the last few years, information on the inheritance of apomeiosis in reproductive mutants of M. sativa has been gained using molecular markers. Alfalfa
genetic linkage maps were obtained by using RFLP markers alone (Brummer & al.,
1993; Tavoletti & al., 1996b; Brouwer & Osborn, 1999) or in combination with
RAPD markers (Kiss & al., 1993; Echt & al., 1994; Kalò & al., 2000) and SSR
markers (Diwan & al., 2000). Genome mapping in alfalfa would help to elucidate
the genetic inheritance and expression of genes involved in meiosis and gamete
formation, and for this purpose two linkage maps of a 2n egg mutant were constructed using a pseudo-testcross mapping strategy. The first map included only
RFLP marker loci arranged in eight major linkage groups (Tavoletti & al., 1996b)
whereas the second one was mainly based on PCR-derived marker loci (Barcaccia
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
105
& al., 1999b) and was later supplemented with retrotransposon markers (Porceddu
& al., 2002). The inheritance of 2n eggs was investigated by Barcaccia & al.
(1998b, 2000a) using distinct segregating populations obtained by crossing the
apomeiotic mutant of M. sativa subsp. falcata with wild-type plants of M. sativa
subsp. coerulea. The genetic capacity for 2n egg production was assessed by handpollinating the F1 progenies with tetraploid plants of M. sativa subsp. sativa. Seed
set in 2x–4x crosses was used to discriminate n from 2n egg producers: progeny
plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n egg producers. A bulked segregant
analysis using multi-locus PCR-derived markers was employed to identify a genetic linkage group related to the 2n egg trait using a two-way pseudo-testcross strategy, and enabled us to find a paternal Inter-SSR marker of 610 bp, located 9.8 cM
from a major gene, named tne (two-n-eggs), that putatively controls 2n eggs and to
detect a 30% recombination genomic window surrounding the target locus. Seven
additional molecular markers of maternal origin significantly co-segregated with
the trait under investigation (Barcaccia & al., 2000a). The minimum number of
QTLs controlling seed set in 2x–4x crosses was estimated by multiple regression
analysis: four maternal and three paternal independent molecular markers were
associated with the trait. A map of the chromosome regions carrying the minor
genes that influence the expression level of 2n eggs has been constructed using
selected RAPD and AFLP markers, and two of these genes are linked to previously mapped RFLP loci belonging to groups 1 and 8 (Fig. 5). Molecular and genetic
evidence supports the involvement of one major gene, named tne1, and the variation in the expressivity of this trait is thought to involve a few minor genes
(Barcaccia & al., 2000a).
Concerning the inheritance of apomeiosis in alfalfa, the continuous variation
in occurrence of functional unreduced eggs observed both in natural and experimental populations implies that the trait is not simply inherited, but is rather the
result of multiple genetic factors. However, the question of the genetic control and
regulation of SDR- and FDR-type 2n eggs remains unexplained. Various genes
could control different cytological mechanisms which all lead to 2n egg production, but with each having different implications for 2n egg genetic constitution.
However, the fact that in a given genotype (e.g., mutant TWO-N-EGG) normal
tetrads are usually present together with SDR dyads and FDR monads does not
necessarily imply that independent genetic systems are responsible for each type of
abnormality. In fact, it cannot be ruled out that the occurrence of either variant may
stem from differences in the expression timing of a single mutant allele during
megasporogenesis in a common genetic system (Barcaccia & al., 2000a).
In a functional genomics approach, an EST structurally homologous to the
MOB gene family members was isolated from the apomeiotic mutant TWO-N-EGG,
during an attempt to clone candidate genes for unreduced egg production in a differential display of mRNAs that combined cDNA-AFLP markers and bulked segregant analysis (Barcaccia & al., 2001). In this alfalfa mutant, the production of 2n
eggs is mostly associated with absence of the final cytokinesis, genetically equiva-
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lent to abnormal meiosis of the SDR-type (Pfeiffer & Bingham, 1983). An alternative mechanism is the formation of unreduced eggs through diplosporic apomeiosis
of the FDR-type (Tavoletti, 1994; Barcaccia & al., 1995). Our histological and
molecular data provided independent evidence that SDR (~43%) and FDR (~21%)
mechanisms are active in different ovules of a given ovary (Tavoletti & al., 1996b;
Barcaccia & al., 1997b). In particular, female sporogenic abnormalities such as the
absence of the post-meiotic cytokinesis and the failure of the first meiotic division
documented in the mutant TWO-N-EGG make it an appropriate tool for studying the
role of the alfalfa Mob1-like genes during plant reproduction.
Citterio & al. (2005; 2006) have recently reported the isolation and characterization of alfalfa Mob1-like genes, starting from the EST clone previously isolated
from the apomeiotic mutant. Mps-one-binder (Mob) proteins play an important
role in chromosome separation and cell plate formation in yeast, and certain data
suggest that MsMob1-like genes can also play a key role in the reproductive pathway of plants. Of the two Mob1-like genes isolated from the apomeiotic mutant,
one gene proved to be constitutively expressed while the other was expressed only
in flower buds during sporogenesis and gametogenesis (Citterio & al., 2005). For
Fig. 5. AFLP, RAPD and Inter-SSR linkage groups and QTLs related to normal egg and
2n egg formation in wild-type (2DI) and mutant TNE, as defined by bulked segregant
analysis and multiple regression; * markers that significantly co-segregated with the
2n-egg trait; § markers that exhibited a significant effect on 2n-egg production selected as independent by stepwise regression. Linkage group 1 of the wild-type contains
the 2n egg locus of which the recessive form (tne) carried by the mutant determines
2n egg production (modified from Barcaccia & al., 2000a).
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
107
the analysis of gene expression during reproduction in alfalfa wild-types and
apomeiotic mutants, a specific antisense riboprobe was designed for the Mob1-like
transcripts and a polyclonal antibody was raised against the Mob1-domain containing proteins. In situ mRNA localization as well as protein immunolocalization
demonstrated that Mob1-like genes are specifically expressed in degenerating
megaspores of normal ovules and in enlarged megaspore mother cells and embryo
sacs of apomeiotic ovules (Fig. 6). Gene products were also found in microspore
Fig. 6. Results of immunolocalization analysis performed in ovules of the alfalfa
apomeiotic mutant TNE using the anti-Mob1 protein polyclonal antibody (the yellow
to green stain represents the immunolocalization signal). Pistils were also counterstained with DAPI (blue to white staining). A, specificity of the antibody as revealed
by the spatial localization of the protein into one of the ovules of a given ovary; B,
ovules of the same ovary counterstained with DAPI; C, localization pattern of the
protein around and within an apomeiotic megaspore mother cell (for additional
details, see Citterio & al., 2005); D, the same ovule counterstained with DAPI.
Abbreviations: ow, ovary; ov, ovule; end, endothelium; mmc, megaspore mother
cell; nu, nucleus; ii, inner integuments; p, pollen grains. Bar = 30 µm.
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Albertini & Barcaccia
tetrads at the beginning of pollen development, as well as in the tapetum cells of
anthers undergoing programmed cell death to allow pollen dispersal at maturity.
The altered expression of Mob1-like genes in the reproductive tissues of a mutant
which produced apomeiotic eggs via the absence of cytokinesis was demonstrated,
and the participation of their transcripts and proteins in programmed cell death
within reproductive organs was also considered (Citterio & al., 2005).
As no further information is presently available, one can only speculate about
the role this gene plays in the restitutional mechanisms that lead to 2n megaspore
formation. Lack of post-meiotic cytokinesis (SDR) and diplospory (FDR) could
account for the disorganization of the skeletal structures responsible for phragmoplast development and for the defective spindle assembly, respectively. The cluster
of large grains formed in apomeiotic ovules that contain excessively enlarged
MMCs was never seen on normally shaped MMCs of meiotic ovules. In addition,
the lack of cytokinesis after normal meiosis in conjunction with the absence of signals in ovules that contain SDR-type dyads are consistent with a possible role of
Mob1-like proteins during megasporogenesis.
Several mutants with disturbed female meiosis are known in the Medicago sativa-coerulea-falcata complex. In these mutants normal gametes predominate but
unreduced egg cells occur and function in fertilization (Barcaccia & al., 2003).
Correct reductional cell division requires the precise co-ordination and execution of
certain pre-meiotic and meiotic events, including DNA replication, centrosome
duplication, spindle assembly, chromatid separation and cytokinesis. Failure or
improper timing of any of these events can lead to the formation of restitutional
nuclei and result in unreduced gametes, and the formation of unreduced embryo
sacs that contain egg cells with 2n chromosomes is common to several types of
apomixis (Savidan, 2000). The physical movements of chromosomes and cytokinesis are directed, respectively, by the spindle and the phragmoplast, and hence the
formation of 2n megaspores could depend on cytoskeletal alterations, particularly
protein defects that compromise the function and integrity of the spindle and phragmoplast microtubules. The mechanisms leading to 2n egg cells could involve failure in the cytokinetic machinery that normally separates nuclei and cytoplasm of the
meiocyte into four haploid spores. In normal meiosis, cytoskeletal arrays follow predictable spatial and temporal pathways (Baskin, 2000). Genualdo & al. (1998)
found changes in the configuration of MTs and MFs during meiosis II and cytokinesis that were correlated with 2n gamete formation in parallel spindle and premature cytokinesis in meiotic potato mutants, while abnormal sporogenesis seemed to
coincide with skeletal disruption in several meiotic corn mutants (divergent dv,
meiosis mei1, and male sterile ms). For instance, spindle pole abnormalities during
prometaphase are correlated with disorganized MT and MF arrays, and lead to
defective chromosome movement and disturbed chromosome segregation. Since
partial or total cytokinetic failure is associated with altered MT and MF dynamics,
cytoskeletal arrays must be crucial to ensuring correct completion of meiosis.
Further experiments are needed to both define the function of the Mob1-like
proteins in mitotic and meiotic assembly and cytoplasm partitioning, and to estab-
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
109
lish whether the apomeiotic or apoptotic fate of ovule cells is correlated with
MsMob1-like gene expression in alfalfa reproductive mutants.
DISCUSSION
The origin of apomixis is still a mystery as it is difficult to conceive how various deleterious traits such as apomeiosis and parthenogenesis can coevolve to produce a viable apomict (Mogie, 1992).
Apomeiosis in flowering plants is a rare phenomenon, except when it is coupled with parthenogenesis to yield gametophytic apomixis. In naturally occurring
sexual plants, the formation of unreduced embryo sacs is frequent only in interspecific hybrids, and it is considered a key step in the evolution of polyploid forms
(Ramsey & Schemske, 1998). In contrast, apomeiosis attains elevated regular
expression in naturally reproducing apomictic species (Noyes, 2005), and current
data suggest that apomeiosis may be functionally and genetically independent from
other aspects of apomixis (parthenogenesis, autonomous endosperm formation).
Apomeiosis via either diplospory or apospory is the starting, fundamental step
of apomixis without which parthenogenesis of egg cells does not result in exact
replicas (i.e., clones) of the mother plant. A lack of knowledge on the genetic and
molecular control of apomeiosis and its developmental relationship with parthenogenesis has hindered attempts to manipulate and transfer the apomictic trait beyond
natural sexual barriers. Many analyses have shown that apomeiosis is controlled by
a dominant mendelian factor typically found in a chromosome block not subjected
to recombination. However, little is known regarding the size and number of genes
located in these regions for most species, and suppressed recombination suggests
that apomeiosis is controlled by a single gene or a tightly linked gene complex.
In P. pratensis most evidence suggests that the dominant genetic factors for
apomixis are closely linked and inherited together, but rare events of segregation
of apospory from parthenogenesis are possible (Barcaccia & al., 2000b; Albertini
& al., 2001b; Matzk & al., 2005). In M. sativa mutants, diplospory is not developmentally coupled with parthenogenesis, and segregation patterns support a recessive genetic control (Barcaccia & al., 2000a; 2001). In these two species, expression of apomeiosis can vary from about 1% to almost 100%. Cytological data have
shown that both regular meiosis and apomeiotic mechanisms are active in different
ovaries of the same genotype (in P. pratensis) and also in different ovules of the
same ovary (in M. sativa). It has been proposed that an incomplete penetrance of
the trait and the involvement of a few modifiers able to regulate the expressivity of
target genes could account for variations in the percentage of unreduced egg cells
(Barcaccia & al., 2000a; Matzk & al., 2005).
Modifiers have been invoked to explain different developmental patterns (timing of initiation, position, developmental progression) of apomeiotic embryo sacs
in several apomicts (Koltunow & al., 2000). Since quantitative assessments of
apomeiosis are rarely performed, the capacity of the trait to vary among individu-
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als and the likely influence of genetic modifiers on the trait may be underappreciated (Noyes, 2005). As we accelerate towards elucidating the principal pathways
and genes involved in apomixis, the role of modifiers in the expression of apomeiosis will likely take on greater importance.
Differential screening of plants with contrasting modes of reproduction is considered one of the most powerful tools for identifying and isolating the genes
underlying the expression of apomixis. A differential display method based on
mRNA profiling techniques (e.g., DD-cDNA-AFLP) in combination with the
analysis of bulked segregants has demonstrated its effectiveness for detecting
expressed sequence tags (ESTs) and cloning candidate genes for aposporic apomixis in the facultative apomict P. pratensis (Albertini & al., 2004; 2005) as well as in
diplosporic mutants of the sexually reproducing M. sativa (Barcaccia & al., 2001;
Citterio & al., 2005). In the case of apomeiosis, such a method relies on pooling
cDNA subsets from reproductive organs at specific developmental stages of individuals sharing the same genetic background, but which show divergent classes for
the pattern of female sporogenesis and gametogenesis, and then screening for differentially-expressed genes between meiotic and apomeiotic genotypes using
AFLP markers. This approach enables the analysis of a large number of transcriptderived fragments, and increases the reliability of amplification based-gene expression analysis. Fine mapping of the genomic region carrying the apomixis determinants for apomeiosis and/or parthenogenesis is crucial for selecting differentially
expressed ESTs. A combination of transcriptomics and proteomics tools is also crucial for studying the temporal and spatial expression profiles of candidate genes
(Citterio & al., 2006).
Following this approach, we have cloned and characterized potentially interesting candidate genes for apomeiosis in P. pratensis and M. sativa (e.g., APOSTART and MOB, respectively). Now we are attempting insertional T-DNA mutagenesis and knockdown experiments based on RNA silencing and antisense technologies in model plant species in order to examine the function of our gene homologues.
Plant reproductive development is a plastic phenomenon, as is demonstrated in
nature by the diverse pathways that result in apomictic seed formation. Mutants
producing functional apomeiotic eggs cells have been isolated in alfalfa (two-n-egg
producer, tne), potato (premature cytokinesis, pc), barley (triploid inducer) and
maize (elongate, el). A better understanding of sexual reproduction in plants, such
as Arabidopsis and rice, should permit the identification of genes that would potentially allow engineering of apomixis in crop plants.
ACKNOWLEDGEMENTS
Part of the data reported and discussed in this work are the results of projects
supported by the Ministry of University, Research, Science and Technology.
Projects: “Genetic aspects of seed production: an integrated approach towards the
Chapter V • Apomeiosis in Poa pratensis L. and Medicago sativa L.
111
understanding of apomixis” (National coordinator: Mario Falcinelli) and “Isolation
and characterization of genes affecting sporogenesis and gametogenesis in
Medicago spp.” (National coordinator: Fabio Veronesi).
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