CrMSP1, a candidate gene involved in asexual reproduction in the fern Ceratopteris richardii Linh T. Bui, Erin E. Irish, Chi-Lien Cheng 1. INTRODUCTION All land plants complete the life cycle through an alternation between two different generations: the haploid gametophyte and the diploid sporophyte. Sporophytes undergo meiosis to produce haploid spores, which then develop into multicellular gametophytes. These haploid gametophytes contain male and female gametes, which will fuse during fertilization to restore the ploidy in the zygote, the first cell of the sporophyte generation. Both the spores and gametophytes of angiosperms are ephemeral, embedded deep in the flower, and dependent on the sporophyte for nutrients and support. In contrast, the two generations in ferns are free-living entities. Fern spores once mature are shed from the sporophytes into the environment where, upon germination, will develop into photosynthetic gametophytes, from which gametes are produced. In addition to sexual reproduction pathway via meiosis and fertilization, approximately 400 species from 40 angiosperm families have evolved the ability to reproduce asexually bypassing both meiosis and fertilization through multiple pathways collectively called apomixis (Nogler, 1984; Carman, 1997). In ferns, some species can undergo asexual reproduction through the two separate pathways: apogamy and aposopry (Scheme 1). In nature, approximately 10% of fern species are obligatory apogamous; in these plants, sporophytes arise directly from gametophytes lacking functional archegonia or antheridia (Steil, 1939; Bell, 1992). During sporogenesis, a compensatory mechanism of forming restitution nuclei acts to give rise to diplospores with the same chromosome numbers as the apogamous sporophytes (Walker, 1979). Apospory can also occur in ferns, but rarely in nature (Walker, 1979). In apospory, diploid
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CrMSP1, a candidate gene involved in asexual reproduction in the fern Ceratopteris richardii
Linh T. Bui, Erin E. Irish, Chi-Lien Cheng
1. INTRODUCTION
All land plants complete the life cycle through an alternation between two different
generations: the haploid gametophyte and the diploid sporophyte. Sporophytes undergo meiosis to
produce haploid spores, which then develop into multicellular gametophytes. These haploid
gametophytes contain male and female gametes, which will fuse during fertilization to restore the
ploidy in the zygote, the first cell of the sporophyte generation. Both the spores and gametophytes
of angiosperms are ephemeral, embedded deep in the flower, and dependent on the sporophyte for
nutrients and support. In contrast, the two generations in ferns are free-living entities. Fern spores
once mature are shed from the sporophytes into the environment where, upon germination, will
develop into photosynthetic gametophytes, from which gametes are produced.
In addition to sexual reproduction pathway via meiosis and fertilization, approximately
400 species from 40 angiosperm families have evolved the ability to reproduce asexually
bypassing both meiosis and fertilization through multiple pathways collectively called apomixis
(Nogler, 1984; Carman, 1997). In ferns, some species can undergo asexual reproduction through
the two separate pathways: apogamy and aposopry (Scheme 1). In nature, approximately 10% of
fern species are obligatory apogamous; in these plants, sporophytes arise directly from
gametophytes lacking functional archegonia or antheridia (Steil, 1939; Bell, 1992). During
sporogenesis, a compensatory mechanism of forming restitution nuclei acts to give rise to
diplospores with the same chromosome numbers as the apogamous sporophytes (Walker, 1979).
Apospory can also occur in ferns, but rarely in nature (Walker, 1979). In apospory, diploid
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gametophytes are produced from somatic cells of sporophytes in the absence of meiosis. Both
processes can be induced in the laboratory with the sexual fern Ceratopteris richardii simply by
altering the level of sugar supplement in the growth media, and in the case of apogamy, by also
preventing fertilization (Cordle et al., 2007). Glucose at 2.5% (w/v) was optimal for the induction
of apogamy (Cordle et al., 2007) and medium with no sugar or 0.5% (w/v) glucose supplement
was used in apospory induction (Munroe and Sussex, 1969; De Young et al., 1997; Cordle et al.,
2011; Bui et al., 2012) in C. richardii.
Here, I use the model fern C. richardii to study how these two asexual reproduction
pathways are controlled. My long-term goal is to identify the key genes that play controlling roles
in determining the two fern asexual reproduction pathways, apogamy and apospory. The central
hypothesis for this research is that a group of conserved genes control sexual reproduction
in ferns and angiosperms, and an altered regulation of a subset of these genes will lead to
apogamy and apospory in C. richardii (Scheme2).
Currently, genes that control asexual reproduction in angiosperms have not been
identified, but seem to be restricted to one or a few dominant loci in the apomictic plant species
examined so far (Ozias-Akins and Van Dijk, 2007; Okada et al., 2011). Albertini et al. (2005)
proposed that specific genes are activated, modulated or silenced in the primary steps of sexual
reproduction to ensure the formation of functional embryo sac from meiotic spores or apomeiotic
cells. In contrast, genes involved in the development of both male and female gametophytes are
beginning to come to light, they include EXCESS MICROSPOROCYTES 1/ EXTRA
SPOROGENOUS CELLS (EMS1/EXS) and TAPETUM DETERMINANT (TPD1) genes which
function together as part of an intercellular signaling mechanism regulating sporogenic cell fate
(Jia et al., 2008); SPOROCYTELESS (SPL) and WUSCHEL (WUS) act on megasporemother cell
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differentiation (Sundaresan and Alandete-Saez, 2010); the DYAD/SWITCH1 (SWI1) genes are
required in the subsequent meiosis of the megasporemother cell (Mercier et al., 2001). There are
also genes that, when ectopically expressed will cause somatic embryogenesis from vegetative
tissues, similar to apomixis phenotypes, but it is still unknown if these genes actually involve in
apomixis. Among these genes, BABYBOOM (BBM) is a transcription factor that is expressed in
developing embryos and seeds (Boutilier et al., 2002); SOMATIC EMBRYOGENESIS
RECEPTOR KINASE 1 (SERK1) is expressed in nucellar tissue during meiosis, and in developing
embryo sac and zygotic embryo (Schmidt et al., 1997; Hecht et al., 2002). The LEAFY
COTYLEDON 1 (LEC1) regulates embryo development and is normally expressed in both
morphogenesis and maturation phases of embryogenesis in seeds (Braybrook and Harada, 2008).
Additionally, there are genes with recessive mutations have been identified, such as
FERTILIZATION INDEPENDENT SEED 1 and 2 (FIS1, FIS2), both are members of the PCR2
complex. These mutations cause the initiation of embryo development in the absence of
fertilization, hence comparable to asexual reproduction (Chaudhurry et al., 1997). Most
interestingly, a loss of function mutation in another gene member of the PCR2 complex, CURLY
LEAF (CLF), has been shown to cause apogamy in the moss P. patens (Okano et al., 2009),
suggesting that apogamy in moss and apomixis in angiosperms share genetic components, even
though these plants are evolutionarily distant (Cordle, University of Iowa PhD dissertation, 2011).
The rice MULTIPLE SPOROCYTE 1 (MSP1) was characterized by Nonomura et al.
(2003) as a master regulator that controls early sporogenic development. This gene encodes a
Leu-rich repeat receptor-like protein kinase (fig1. a), and loss of function mutation results in an
excessive number of both male and female sporocytes (Nonomura et al., 2003). In situ
hybridization showed that MSP1 is expressed in surrounding cells of both male and female
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sporocytes. The Arabidopsis homolog of this gene, EXS/EMS1, plays similar role in anther
development and also in embryo development (Zhao et al., 2002; Canales et al., 2002). It has
been shown in Arabidopsis that EXS/EMS1 acts together with another gene (TPD1) in restricting
surrounding cells undergoing meiosis and becoming the microsporocytes (Jia et al., 2008).
However, whether MSP1/ EXS/EMS1 is involved in asexual reproduction in angiosperms and
whether its homolog exists and plays a similar role in ferns and other lower plants are not yet
known. In this talk, I will focus on presenting the preliminary results on MSP1, one of the
candidate genes that I have cloned from C. richardii. Hereafter, the fern MSP1 will be called
CrMSP1. In situ hybridization and RT-PCR analysis showed that fern MSP1 expresses in both
sporogenesis and embryo development, similar to its homologs in Arabidopsis and rice.
Therefore, this gene is a promising candidate gene to study both sexual and asexual reproduction
pathways in C. richardii.
2. METHODOLOGY
Plant Material and Growth Condition
C. richardii plants used in these experiments were of the wild-type genotypes Rn3 and Hnn
(Carolina Biological Supply, Burlington, NC). Spore germination and gametophyte culture
conditions were as described by Cordle et al. (2007). Spores were inoculated at a density of 150-
300 spores per plate containing a basal medium (BM) that is half-strength Murashige and Skoog
salts (MS) at pH 6.0 with 0.8% agarose. Gametophyte culture plates were maintained at 28°C in
humidity domes under 16-h light/8-h dark cycle. Light source was provided with Philips Agro-
Lite fluorescent bulbs (Philips Lightning Company, Somerset, NJ) at 90-100 M m-2
s-1
.
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Degenerate primer design, cloning and sequencing
NCBI BLAST was used to find homologous sequences of EMS1/EXS in the land plant database.
Then, CODEHOP program (Rose et al., 1998) was used to design degenerated primers from
CLUSTALW multiple sequence alignment results. Gradient PCR was used to amplify fern
sequences using appropriate degenerate primers (Forward primer: GAGAGAACCTCTGTC-