1. INTRODUCTION - University of Iowa 2013-judged.pdf · generations: the haploid gametophyte and...
Transcript of 1. INTRODUCTION - University of Iowa 2013-judged.pdf · generations: the haploid gametophyte and...
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-
TATTAATGTTGCNAYNTTYGA, Reverse primer: GTTCCAGCATAATAACTCCATAAGAATANACRTCNCC)
with the following program: 5min at 950C, (30sec at 95
0C, 30sec at 50-60
0C, 30sec at 72
0C) for 39
cycles, 5min at 720C. The PCR product was cloned into a TOPOII® vector (Invitrogen) and
transformed into TOP10 F’ E. Coli cells (Invitrogen). Minipreps were performed with a Qiaogen
Plasmid Kit and the resulting plasmids were sequenced with T7 promoter primers on an ABI 3730
DNA Analyzer (Applied Biosystems) at the Carver Center for Genomics (University of Iowa).
Synchronized Fertilization
Nine days after germination, plates with gametophytes were inverted before the gametophytes are
sexually mature to prevent fertilization. When the gametophytes are sexually mature (12d after
germination), water was added into the culture plates to facilitate fertilization and the
gametophytes were collected at various time points (see the result section), flash frozen in liquid
nitrogen then stored at -700C.
RNA extraction and RT-PCR analysis
Total RNA extraction was performed as described in Corlde et al., 2012. Briefly, frozen tissues
were ground to a fine powder and total RNA was extracted using the guanidinium thiocyanate and
acid phenol method. The RNA pellet was dissolved in fresh, deionized formamide, heated to 700C
for 10min in the presence of 0.7M NaCl and 1% CTAB, and then extracted twice with
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chloroform. The RNA pellet was dissolved in DEPC treated water and 500ng of total RNA was
used for first strand cDNA synthesis using Superscript III Reverse Transcriptase (Invitrogen) and
random hexamer primers. RT-PCR was performed with the following program: 5min at 950C,
(30sec at 950C, 30sec at 58
0C, 30sec at 72
0C) for 35 cycles, 5min at 72
0C.
RT-PCR quantification
RT-PCR quantification was done using ImageJ 1.43r software (http://rsb.info.nih.gov/ij/)
(Abramoff et al., 2004). Briefly, after calibration, gel lanes were selected and plots were created
for each gel lane. Plot peaks, which represent each distinct PCR band within a gel lane, were
specified and the area beneath each peak was calculated. For each gel lane, the ratio of control
(UBQ11) area to experimental (CrMSP1) area was calculated. Graphs were created using
Microsoft Excel for Windows 2007 (Microsoft Corporation).
In-situ hybridization analysis
Probe synthesis
Plasmids were linearized overnight with either BamHI (NEB) or XbaI (NEB). Gel electrophoresis
was performed to verify complete linearization. 1µg of purified, linearized plasmids were used in
probe synthesis with T7 or SP6 RNA polymerases (Stratagene) and DIG RNA labeling mix
(Roche) to generate antisense and sense RNA probes. RNA probes were precipitated with 3M
LiCl to remove enzymes and DNA, and then resuspended in DEPC-treated water, then quantified
with the NanoDrop 1000 (Thermo Scientific).
Whole mount in situ hybridization
C. richardii gametophytes were fixed under vacuum for 10min in ice cold formaldehyde, acetic
acid and alcohol (FAA), and then fixed overnight at 40C in fresh FAA, stored in 75% Ethanol at -
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200C. For prehybridization, gametophytes were moved through a 3-step ethanol series in PBS pH
7.5, 20mins for each wash, permeabilized in 0.2M HCl for 20mins, neutralized with 2x washes in
PBS, and treated with 0.1mM Proteinase K for 10min at room temperature. Proteinase K
digestion was stopped with washes in PBS and glycine , then gametophytes were post-fixed in 4%
paraformaldehyde for 20min at room temperature. After a 90min prehybridization at 550C in
hybridization solution (6x SSC, 3% SDS, 50% formamide, 0.1mg/ml tRNA), probe hybridization
was performed at 550C for 15-18 hours with 1ng/µl denatured probe. Post hybridization washes
were performed with 0.2x SSC and 2x SSC, then RNase digestion was performed with 10µg/ml
RNase A in 2x SSC at 370C for 30min. Gametophytes were then washed with SSC and Tris-
buffered saline (TBS pH 7.5), incubated for 2 hours in 1% blocking solution (Roche), then 2
hours in a 1/1000 dilution of anti-DIG AP fab fragments (Roche) in 1% blocking solution.
Detection was performed with 1x color substrate solution (NBT/BCIP, Roche) in detection buffer
(100mM Tris-base pH 9.5 and 150mM NaCl) overnight. Gametophytes were mounted in 50%
glycerol and photos were taken using a Leica DM LB microscope.
Section in-situ hybridization
Samples were fixed in ice-cold FAA at 40C overnight, then dehydrated through a series of
ethanol, and embedded in paraplast in molds. The paraplast blocks were mounted on the
microtome and sections of 6-12μm thick were made and placed on poly-L-lysine coated slides.
Sections can be stores at 40C for several months. For in situ hybridization, sections were
transferred to a xylene to remove the wax and rehydrated in a series of ethanol at room
temperature. Hybridization was performed on slides at 550C for 16-20 hours, with the same
hybridization buffer as in whole mount, and the appropriate sense and antisense probes. Post
hybridization and detection were done as described in whole mount in situ hybridization above.
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3. RESULTS AND DISCUSSION
CrMSP1 is the homolog of Arabidopsis EMS1
Since the C. richardii genome is not sequenced, candidate gene approach has been used to
identify genes of interests. Using this approach, I was able to clone the fern MSP1 gene,
specifically the kinase domain of this gene, with degenerate primers. NCBI BLAST showed that
the CrMSP1 sequence is 78% identical to Arabidopsis EXS/EMS1, with an Evalue = 1e-108 (fig1.b).
Phylogram created by CLUSTALW program shows the close relationship between the fern MSP1
and the moss Physcomitrella homolog, as well as the evolution of this gene among the plant
kingdom (fig1c). However, since the CrMSP1 I have cloned is not complete (lacking the 5’
sequence upstream of the kinase domain), a longer sequence will obtain a more precise
phylogram, and further confirm the identity of this fern MSP1.
CrMSP1 is expressed during sporogenesis
Using Genevestigator program (www.genevestigator.com), a web-based database that contains
annotated database of public Affymetrix microarray experiments, the Arabidopsis EMS1/EXS
expression was examined in different plant tissues. AtEMS1/EXS expresses highly in the
inflorescence, especially in the ovules where the embryo sac is (fig2). This gene is also present in
the stamen where the anther is produced, although at a much lower level than in the ovules. This
result is consistent with previous studies of EMS1/EXS in Arabidopsis, as it was shown to regulate
anther formation (Zhao et al., 2002; Canales et al., 2002).
Compare to the expression pattern of EMS1/EXS in Arabidopsis, CrMSP1 is significantly induced
in the tip of the leaves, where sporogenesis occurs (fig3.e). This result is interesting because
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unlike angiosperms whose sporogenesis (and gametogenesis) occurs inside the flower, ferns form
the spores at the underside of the mature leaves. Fern sporogenesis occurs at the young part (the
tip) of a mature fern leaf, but not in young leaves, or in the base of the leaves where sporogenesis
is over. Therefore, the RT-PCR result indicates that CrMSP1 is involved in sporogenesis.
Tissues were also embedded and sectioned for in situ hybridization analysis to examine the
temporal and spatial expression of CrMSP1. It is clear that CrMSP1 is expressed in the spore
mother cells in the developing sporangia (fig3. a-d) suggesting a special role in these cells. To
understand the role it plays, it is necessary to study the biological function of the CrMSP1. I will
do this by knocking down or overexpressing the gene and then examine the effects in
sporogenesis, gamete formation, and embryogenesis (explained below) in C. richardii.
CrMSP1 expression is induced in the embryo and gametophyte after fertilization
EMS1/EXS is expressed in early developing embryos and homozygous ems1/exs embryos are
lethal (Canales et al., 2002). These findings indicate that this gene is also required for embryo
development in Arabidopsis. However, it is unclear whether EMS1/EXS acts alone or with other
genes to regulate embryo development.
Using whole mount in situ hybridization analysis (developed by me and another graduate student
in the lab) for C. richardii gametophytes, I detected the expression of CrMSP1 before and after
fertilization. For this experiment, it is necessary to obtain embryos at the same developing stages.
To achieve, I synchronized the fertilization in a population of mature gametophytes (described in
the Methodology). CrMSP1 is highly induced not only in the embryo (fig.4d) but also in the
whole gametophyte (fig.4b) at 72h post fertilization, but not in the gametophyte (fig.4a) or the
archegonia (harboring the egg cell) (fig.4c) before fertilization, or when sense probe was used
(data not show). RT-PCR using RNA extracted from gametophytes at various time points after
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fertilization further confirms the in situ hybridization result. High level of CrMSP1 expression
was detected at 24h post fertilization but not before fertilization (fig5). These results are consistent
with the expression pattern of the Arabidopsis homolog, EMS1/EXS, and indicate a role of
CrMSP1 in fern embryo development.
Is CrMSP1 involved in asexual reproduction in C. richardii?
The central question of my research is how asexual reproduction in C. richardii is controlled. As
described above, the expression of CrMSP1 increases in both sporogenesis and embryo
development, and this expression pattern is consistent with the Arabidopsis and rice homologs.
Loss of function mutations in this gene causes abnormal gamete formation in Arabidopsis and
rice. The next obvious experiment is to alter the expression of CrMSP1 in C. richarii and see
whether sporogenesis and embryo development will be affected. In order to do this I have
developed an Agrobacterium-mediated transformation protocol for knock-down and
overexpression of CrMSP1 in C. richardii. In addition to abnormality in sexual reproduction, we
will also carefully examine an increased rate of apogamy (the asexual reproduction of embryo
without fertilization).
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4. FIGURES
Scheme 1. Alternation of generations and
asexual reproductions in the fern
Ceratopteris richardii
Scheme 2. Candidate genes. All genes are involved in
Arabidopsis sexual reproduction steps as indicated except
CLF, BBM (also LEC1) who are involved in somatic
embryogenesis. Green represents sporophyte and blue
gametophyte. Genes with asterisks have not been cloned
from C. richardii.
Fig 1. C. richardii MSP1 (CrMSP1) is the fern homolog of the Arabidopsis MSP1 gene. a) Structure of the
AtMSP1 protein with the three different domains: leucine-rich repeat, transmembrane and kinase domains. b)
Sequence alignment by NCBI BLAST program shows the similarity between CrMSP1 and AtMSP1 in the
kinase domain (Evalue = 1e-108). c) Phylogram of MSP1 in the plant kingdom created by CLUSTALW
a
) c
) b
)
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Fig 5. CrMSP1expression is significantly induced in the
gametophytes after fertilization. a) RT-PCR result using CrMSP1
or UBQ11 (positive control) primers for gametophytes at various
time points after fertilization. b) Quantification of three separate
RT-PCR replicates using ImageJ program, bars indicate standard
errors.
0 12 24 48 72 96
Time after fertilization (h)
Rel
ativ
e m
RN
A l
evel
Time after fertilization (h)
0 12 24 48 72 96
CrMSP1
UBQ11
a
b
Fig2. In-silico analysis of microarray
database from
www.genesvestigator.com shows the
expression of AtMSP1 in the female
gametophyte (red box), numbers on the
right indicate the numbers of
microarrays analyzed.
Fig 3. CrMSP1 is expressed during sporogenesis in C. richardii. a-d) fern
leaf cross section in situ hybridization with CrMSP1 antisense probes (a &
c), or sense probe (b & d); c & d are higher magnification of insert in a &
b, respectively; e) RT-PCR result using CrMSP1 or UBQ11 (positive
control) primers for different tissue types (young leaves, tip of the leaves
and base of the leaves). Bars
Fig 4. Whole-mount in situ of CrMSP1. 12-d-
old gametophyte before (a) and 72 h after (b)
fertilization. (c), (d) are archegonia in (a) and
(b). Red arrows point to archegonia, green
arrows point to the egg in (c) and developing
embryo in (d). Note lack of staining in
archegonia. Bar=0.5 mm in a,b. Bar=0.05 mm
in c,d.
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