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Theses and Dissertations
2012
Genes involved in asexual sporophyte development
in Ceratopteris richardii and Arabidopsis thaliana
Angela Ruth Cordle
University of Iowa
Copyright 2012 Angela Ruth Cordle
This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/4598
Recommended Citation
Cordle, Angela Ruth. "Genes involved in asexual sporophyte development in Ceratopteris richardii and Arabidopsis thaliana." PhD
(Doctor of Philosophy) thesis, University of Iowa, 2012.
http://ir.uiowa.edu/etd/4598.
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GENES INVOLVED IN ASEXUAL SPOROPHYTE DEVELOPMENT IN
CERATOPTERIS RICHARDII AND ARABIDOPSIS THALIANA
by
Angela Ruth Cordle
An Abstract
Of a thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Biology
in the Graduate College of
The University of Iowa
May 2012
Thesis Supervisor: Associate Professor Chi-Lien Cheng
1
ABSTRACT
The life cycle of land plants involves cycling between two multicellular phases,
the haploid gametophyte and the diploid sporophtye. Asexual reproductive strategies, that
bypass meiosis and fertilization, have evolved in many plants representing diverse taxa
among the land plant phylogeny. Apogamy is an asexual reproductive strategy that
produces a sporophyte directly from a gametophyte cell without the usual ploidy change
associated with syngamy. The genes that drive the process of apogamy are almost
completely unknown. Knowledge of these genes and their functions will provide insight
into the evolution of asexual reproduction, and has the potential to provide insight into
the evolution aspect of the development of the sporophyte body and the alternation of
generations in land plants, two very important questions in plant science. My Ph. D.
research has focused on elucidating the genes that are involved in the process of
apogamy, and understanding the functions of the homologues of these genes in
angiosperms.
First, I induced apogamy experimentally from the semi-aquatic fern Ceratopteris
richardii by culturing them on 2.5% glucose. I discovered that C. richardii gametophytes
begin to become committed to the production of apogamous sporophytes after 10 days of
culture on glucose. I then created a normalized, subtracted cDNA library that represents
genes that increase in transcription in gametophytes during this commitment time. By
comparing the Gene Ontology terms mapped to this cDNA library with that of the
gametophyte transcriptome of the homosporous fern Pteridium aquilinum, I discovered
that the C. richardii apogamy library is enriched in genes that are involved in stress
response and metabolism. The C. richardii apogamy library also contains many
sequences whose homologues in Arabidopsis are specifically expressed or upregulated in
flower organs or seed structures, both of which are absent in ferns. One of these genes,
UNC93-like, is also expressed in the eggs of C. richardii gametophytes, as evidenced by
2
whole mount in situ hybridization. Functional egg cells are implicated as necessary for C.
richardii gametophytes to be induced to form apogamy.
In Arabidopsis, a homozygous T-DNA line for the AtUNC93-like gene, which
produces a partial AtUNC93-like transcript and likely retains partial functionality, has
vegetative and reproductive defects. Embryo abortion and male and female gametophyte
lethality contribute to a small seed set in these plants. However, reciprocal crosses
indicate that the mutant allele does not affect gametophyte function. Instead, it appears to
be a failure of the maternal plant to sustain the life of a percentage of the gametophytes
that it produces. Thus, the AtUNC93-like gene has functions that are necessary for normal
sporophyte vegetative growth and reproductive success, but is dispensable for the
function of gametophytes.
These studies have provided insight into the mechanisms that induce apogamy in
the fern C. richardii. The apogamy cDNA library will provide a valuable resource for
future investigations into the genes that are involved in apogamy.
Abstract Approved: ____________________________________
Thesis Supervisor
____________________________________
Title and Department
____________________________________
Date
GENES INVOLVED IN ASEXUAL SPOROPHYTE DEVELOPMENT IN
CERATOPTERIS RICHARDII AND ARABIDOPSIS THALIANA
by
Angela Ruth Cordle
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Biology
in the Graduate College of
The University of Iowa
May 2012
Thesis Supervisor: Associate Professor Chi-Lien Cheng
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Angela Ruth Cordle
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Biology at the May 2012 graduation.
Thesis Committee: ___________________________________
Chi-Lien Cheng, Thesis Supervisor
___________________________________
Erin E. Irish
___________________________________
Daniel Eberl
___________________________________
Jan Fassler
___________________________________
Jackie R. Bickenbach
To Mom, Dad, Craig, and Gracie
ii
ACKNOWLEDGMENTS
I would like to express my gratitude to the following people: My advisor Chi-Lien
Cheng for her patience, guidance and friendship. The current and past members of the
Cheng and Irish labs including: Joshua Strable, Setu Vora, Ben Beydler, Amelia Hurst,
and my good friend Linh Thuy Bui. My committee members including Jan Fassler, Dan
Eberl, Jackie Bickenbach for their helpful suggestions, and especially Erin Irish and ChiLien who conceived of this project and provided guidance and direction while allowing
me to mold it into a Ph. D. thesis.
Thanks to Doug Houston for useful technical advice regarding in situ
hybridization and RT-qPCR experiments, and also allowing me the use of his dissecting
microscope. Thanks to Gery Hehman for technical advice and assistance.
Finally, I thank my best friend and husband, Craig, who keeps me sane, and our
beautiful daughter, Gracie, who is my favorite person in this world.
iii
ABSTRACT
The life cycle of land plants involves cycling between two multicellular phases,
the haploid gametophyte and the diploid sporophtye. Asexual reproductive strategies, that
bypass meiosis and fertilization, have evolved in many plants representing diverse taxa
among the land plant phylogeny. Apogamy is an asexual reproductive strategy that
produces a sporophyte directly from a gametophyte cell without the usual ploidy change
associated with syngamy. The genes that drive the process of apogamy are almost
completely unknown. Knowledge of these genes and their functions will provide insight
into the evolution of asexual reproduction, and has the potential to provide insight into
the evolution aspect of the development of the sporophyte body and the alternation of
generations in land plants, two very important questions in plant science. My Ph. D.
research has focused on elucidating the genes that are involved in the process of
apogamy, and understanding the functions of the homologues of these genes in
angiosperms.
First, I induced apogamy experimentally from the semi-aquatic fern Ceratopteris
richardii by culturing them on 2.5% glucose. I discovered that C. richardii gametophytes
begin to become committed to the production of apogamous sporophytes after 10 days of
culture on glucose. I then created a normalized, subtracted cDNA library that represents
genes that increase in transcription in gametophytes during this commitment time. By
comparing the Gene Ontology terms mapped to this cDNA library with that of the
gametophyte transcriptome of the homosporous fern Pteridium aquilinum, I discovered
that the C. richardii apogamy library is enriched in genes that are involved in stress
response and metabolism. The C. richardii apogamy library also contains many
sequences whose homologues in Arabidopsis are specifically expressed or upregulated in
flower organs or seed structures, both of which are absent in ferns. One of these genes,
UNC93-like, is also expressed in the eggs of C. richardii gametophytes, as evidenced by
iv
whole mount in situ hybridization. Functional egg cells are implicated as necessary for C.
richardii gametophytes to be induced to form apogamy.
In Arabidopsis, a homozygous T-DNA line for the AtUNC93-like gene, which
produces a partial AtUNC93-like transcript and likely retains partial functionality, has
vegetative and reproductive defects. Embryo abortion and male and female gametophyte
lethality contribute to a small seed set in these plants. However, reciprocal crosses
indicate that the mutant allele does not affect gametophyte function. Instead, it appears to
be a failure of the maternal plant to sustain the life of a percentage of the gametophytes
that it produces. Thus, the AtUNC93-like gene has functions that are necessary for normal
sporophyte vegetative growth and reproductive success, but is dispensable for the
function of gametophytes.
These studies have provided insight into the mechanisms that induce apogamy in
the fern C. richardii. The apogamy cDNA library will provide a valuable resource for
future investigations into the genes that are involved in apogamy.
v
TABLE OF CONTENTS
LIST OF TABLES ...................................................................................................... viii
LIST OF FIGURES ........................................................................................................ix
CHAPTER
I.
INTRODUCTION ........................................................................................ .1
The Alternation of Generations Through Sexual and Asexual Means ............. 2
Sexual and Asexual Alternation of Generations in Ferns ................................ 7
Sexual and Asexual Alternation of Generations in Angiosperms .................. 10
Experimental Strategy .................................................................................. 16
II.
THE INDUCTION OF APOGAMY IN CERATOPTERIS RICHARDII ....... 18
Introduction ................................................................................................. 18
Materials and Methods ................................................................................. 21
Plants and Growth Conditions ................................................................ 21
Apogamy Induction Experiments ........................................................... 23
Histology................................................................................................ 23
Results ......................................................................................................... 25
C. richardii Gametophytes Can Be Induced to Form Apogamous
Sporophytes ............................................................................................ 25
C. richardii Gametophytes Become Committed to Apogamy After 12
Days of Induction ................................................................................... 35
Discussion.................................................................................................... 39
III.
GENES UPREGULATED DURING APOGAMY INDUCTION IN C.
RICHARDII ................................................................................................. 43
Introduction ................................................................................................. 43
Materials and Methods ................................................................................. 45
Plants and Growth Conditions ............................................................... 45
RNA extraction ..................................................................................... 45
Suppression Subtractive Hybridization .................................................. 45
Sequence Identification and Analysis .................................................... 46
Cloning, Sequencing, and RT-qPCR ...................................................... 47
Whole Mount in situ Hybridization ....................................................... 50
Results ......................................................................................................... 52
Strategy to Identify Genes Enriched During Apogamy Commitment ..... 52
Validation of the Apogamy Library ....................................................... 53
Composition of the Apogamy Library ................................................... 57
GO-slim Mapping and Comparative Analysis with the P. aquilinum
Gametophyte Transcriptome .................................................................. 63
in silico Expression Patterns of Apogamy Library Closest
Arabidopsis Homologues ....................................................................... 69
Whole Mount in situ Analyses in C. richardii gametophytes ................. 72
Discussion.................................................................................................... 78
IV.
THE UNC93-LIKE GENE IN ARABIDOPSIS AND C. RICHARDII........... 84
vi
Introduction ................................................................................................. 84
Materials and Methods ................................................................................. 88
Plant Materials and Growth Conditions ................................................. 88
Bioinformatics ....................................................................................... 89
Root Growth Assays, Fresh Tissue Weights and K+ in Apogamy .......... 90
Arabidopsis Seed Area and Seed Weight ............................................... 91
RT-PCR, 3’ RACE, PCR, and Degenerate PCR..................................... 91
Alexander Staining, Tissue Clearing, Optics and Photography............... 93
Results ......................................................................................................... 94
CrUNC93-like and AtUNC93-like Proteins are Conserved .................... 94
Vegetative Phenotypes of the Arabidopsis unc93-like Mutant ................ 99
Comparison of unc93-like Phenotypes with K+ Channel Mutants ........ 103
Reproductive Phenotypes of Arabidopsis unc93-like Mutant ............... 104
The Role of K+ in the Induction of Apogamy from C. richardii
Gametophytes...................................................................................... 111
Discussion.................................................................................................. 112
V.
SUMMARY AND FUTURE DIRECTIONS.............................................. 117
REFERENCES ............................................................................................................. 124
vii
LIST OF TABLES
Table
1.
Primer sequences for semi-quantitative and RT-qPCR analyses ............................ 48
2.
The 170 apogamy library clones identified with confidence .................................. 58
3.
Primers used for RT-PCR, 3’ RACE, PCR and degenerate PCR ........................... 93
viii
LIST OF FIGURES
Figure
1.
The dominant and reduced phases of the sexual life cycles of 3 representative
land plants .............................................................................................................. 3
2.
A phylogenetic tree depicting the relationships among extant land plants ................ 4
3.
Ovule development in Arabidopsis ....................................................................... 11
4.
The normal gametophyte and sporophyte forms of C. richardii ............................. 26
5.
Various apogamous sporophyte forms of C. richardii ........................................... 29
6.
The gametophyte-sporophyte junction in zygote-derived and apogamous plants ... 30
7.
Apogamous sporophytes have typical sporophyte features .................................... 31
8.
Confirmation of apogamy with ploidy comparisons .............................................. 33
9.
Quantitative determinations of ploidy levels by Hoechst dye intensity .................. 34
10.
Apogamy induction from C. richardii her mutant gametophytes ........................... 36
11.
Apogamy induction from C. richardii fem mutant gametophytes........................... 37
12.
Relative RT-PCR analyses of select sequences from the apogamy library ............ 54
13.
RT-qPCR analyses of select sequences from the apogamy library ......................... 55
14.
Top hit species and classifications ......................................................................... 64
15.
Distribution of plant GO-slim categories mapped to the 170 unigenes in the
apogamy cDNA library .......................................................................................... 66
16.
Apogamy library enriched GO-slim terms ............................................................. 68
17.
in silico expression patterns of Arabidopsis homologues of selected unigenes ....... 70
18.
Whole mount in situs of nucleolar essential protein and RNA recognition
motif clones ........................................................................................................... 73
19.
The BTB and FTSH protease whole mount in situs show preferential
expression in early embryo and gametophyte structures ........................................ 74
20.
UNC93-like whole mount in situs show staining in egg cells of 12-day
gametophytes ........................................................................................................ 75
ix
21.
UNC93-like protein alignments and Arabidopsis UNC93-like gene structure ........ 95
22.
Phenotypes of a homozygous T-DNA insertion mutant of AtUNC93-like .............. 97
23.
Number of lateral roots from plants grown on medium containing
varying concentrations of K+ ................................................................................ 98
24.
Fresh root and shoot weights of unc93-like and WT plants................................... 100
25.
Phenotypes of kco3 and tpk1 potassium channel mutants on 0 mM K+ differs
from unc93-like and WT plants ........................................................................... 102
26.
Seed sizes of unc93-like and WT plants ............................................................... 105
27.
unc93-like plants have fewer seeds in their mature siliques than WT plants ......... 106
28.
Examination of pollen viability in the unc93-like mutant ..................................... 107
29.
Significantly fewer seeds produced from crosses performed with females
heterozygous for the unc93-like mutant allele...................................................... 109
30.
The effect of K+ concentration on apogamy induction in C. richardii ................. 110
x
1
CHAPTER I
INTRODUCTION
The alternation of generations is the defining feature of the life cycle of land
plants. This life cycle delegates the major events of sexual reproduction, meiosis and
fertilization, to two distinct, multicellular bodies: the diploid sporophyte and the haploid
gametophyte. Meiosis in the sporophytic generation produces spores, which are the first
cells of the gametophyte generation. The gametophyte generation produces gametes
mitotically. Syngamy completes the life cycle by producing the zygote, the first cell of
the sporophytic generation. This life cycle differs from that of animals, in which the
products of meiosis do not execute a developmental program to form a haploid,
multicellular body, but rather differentiate directly into unicellular gametes.
There are also asexual reproductive strategies that have evolved in land plants that
bypass fertilization and meiosis. A type of asexual reproduction that is important in ferns
and angiosperms is the formation of an asexual sporophyte, from the cells of a
gametophyte or surrounding a gametophyte, to continue the life cycle of the plant without
syngamy. In this thesis I describe my investigations into the genes that trigger asexual
sporophyte development in the fern Ceratopteris richardii and investigate the expression
patterns and function of a subset of these genes in the angiosperm Arabidopsis thaliana.
The genes that trigger asexual reproduction in land plants are unknown. Knowledge of
the genes involved in this process has the potential to provide insight into the evolution
aspect of asexuality in land plants, the alternation of generations, and the land plant
sporophyte body. Knowledge of the genes involved in apogamy is also important in
2
agriculture, and could lead to the development of self-propagating superior hybrid
varieties of crop plants.
The Alternation of Generations Through Sexual and
Asexual Means
The extant descendants of the first plants to successfully colonize land comprise a
monophyletic group called the Embryophyta (Nikas and Kutchera, 2009).
“Embryophyte” refers to the fact that the developing sporophyte embryos are kept within
the walls of an archegonium, where they are protected and nourished until they can fend
for themselves (Walbot and Evans, 2003). Land plants have a heteromorphic alternation
of generations, in which the gametophyte and sporophyte are distinctly different in size,
morphology, and physiology. There are varying degrees of relative dominance of the
phases (Fig. 1) in the different clades of the land plant tree (Fig. 2). The ancestral state of
land plants, whether having a multicellular gametophyte, sporophyte, or both, is currently
in question (Bennici, 2008; Becker and Marin, 2009). It is known that the Chlorophyta
and streptophyte algae (green algae) are structurally and biochemically very closely
related to land plants (Lewis and McCourt, 2004), thus the life cycle of the green algae
sister taxon will be the most informative about the ancestral state of land plants (Haig,
2008; Niklas and Kutschera, 2009). Modern green algae include taxa in which
gametophyte, sporophyte, or both generations are multicellular (Lewis and McCourt
2004; Becker and Marin, 2009), and the specific taxon that is the most closely related to
embryophytes is unclear (Becker and Marin, 2009). However, currently, all sister taxon
candidates are charophycean green algae in which the dominant gametophyte phase is
3
Figure 1 The dominant and reduced phases of the sexual life cycles of 3 representative
land plants. The sporophyte phase of a) bryophytes, b) ferns, and c)
angiosperms. The gametophyte phase of d) bryophytes, e) ferns, and f)
angiosperms. The sizes of the gametophytes and sporophytes are not to scale.
4
Figure 2 A phylogenetic tree depicting the relationships among extant land plants.
Model plants representing different clades are in parentheses. All model
plants listed have sequenced genomes except the fern C. richardii. The
lengths of branches do not indicate distance. Redrawn from Palmer et al.,
2004.
5
multicellular and the sporophyte is unicellular (Karol et al., 2001; Turmel et al., 2006;
Qiu et al., 2006; Qiu, 2008). Thus, current data suggest that extant land plants evolved
from an ancestor that had a haploid multicellular generation, with meiosis proceeding
directly from the zygote (Niklas and Kutschera 2010). This scenario dictates that the
modern day land plant sporophyte generation is a derived condition that is the result of
delayed meiosis and the introduction of mitotic cellular divisions after the formation of
the zygote (Niklas and Kutschera 2010). In line with this, we see a transition from the
basal lineage of extant land plants, the bryophytes, that have a dominant gametophyte
generation and a tiny, dependant, ephemeral sporophyte, to the more derived state of
sporophyte dominance and the progressive reduction of the gametophytic phase of the
life cycle in higher land plants (Figure 1 and 2). Thus, all land plants engage in the
alternation of generations, splitting the mechanisms of meiosis and fertilization between
their two separate, multicellular bodies, but different clades differ in the dominance of the
two phases. This difference may be regarded fundamentally as a difference in the timing
of the process of meiosis (Niklas and Kutschera 2010).
Despite the evolutionary benefits of sexual reproduction through the production of
diverse progeny through meiotic recombination and syngamy, asexual reproductive
strategies have evolved among diverse taxa of both animals and plants. This suggests
that there are evolutionary benefits of asexuality, at least among some taxa in certain
environments. Some basic assumptions about the benefits of asexuality are based on the
costs of sexuality (Horandl 2009). The first are the costs of meiosis, in which favorable
combinations of gene alleles may be recombined. Second, among non-hermaphroditic
organisms the males do not reproduce. Finally, the time and energy that is associated with
6
meiosis and fertilization (Maynard Smith 1978; Bell 1982; Lewis 1987). In certain
circumstances, then, asexuality may be favored because it would maintain combinations
of gene alleles, circumvent the requirement for sperm, and allow escape from
environmental requirements necessary for successful fertilization, such as individual
density or, in the case of some plants, water films for the transport of flagellated sperm.
Therefore, asexual strategies may at least provide short-term advantages, allowing less
costly and quick colonization of certain environments.
Asexual reproductive strategies of land plants involve the circumvention of
fertilization combined with an abnormal meiosis to restore diploidy and continue the
asexual life cycle. In ferns and mosses, the process of forming an asexual sporophyte
from the cell or cells of a gametophyte is called apogamy. In seed plants, the various
processes that circumvent fertilization and meiosis to form asexual sporophytes are
collectively called apomixis (Asker and Jerling, 1992). Regardless of the asexual
reproductive strategy employed, the switch from the development of one generation to
the other remains, albeit in the absence of a change in ploidy. Thus, the genetic program
for determining the development of sporophyte or gametophyte can be uncoupled from
that for meiosis and fertilization.
Defining the genes involved in these asexual processes has the potential to
provide information regarding the evolution of asexual processes in land plants.
Additionally, comparing and contrasting the genes and the mechanisms that drive asexual
pathways in different plant taxa may provide insight into the evolution of the land plant
life cycle.
7
Sexual and Asexual Alternation of Generations in Ferns
The gametophyte and sporophyte generations of ferns are both free living and
photosynthetic (Figure 2). Although the gametophytes of different fern taxa take various
forms, there is a general similarity to their structures (Raghavan 1989). Male
gametophytes have determinate growth, forming only a three-dimensional cluster of
antheridia, the structures in which sperm develop. Hermaphrodites form a twodimensional prothallus, on which both archegonia, which contain egg cells, and
antheridia develop. When gametophytes come in contact with water, mature antheridia
swell and burst, releasing the flagellate sperm into an environment in which they can
swim to the open channels of mature archegonia and fertilize the egg. After fertilization,
the developing sporophyte remains attached to the gametophyte prothallus, drawing
nutrients from it, until the root system reaches soil.
Over a century ago it was noted that there were fern gametophytes in nature that
did not form archegonia, yet produced sporophytes without fertilization (Farlow 1874,
DeBary 1878). This process has now been termed apogamy. Apogamy, bypassing
fertilization, involves the development of a sporophyte directly from the vegetative cells
of a gametophyte. This obviously forms a sporophyte body that has the same
chromosome number as the gametophyte, and if the life cycle is to continue, a
mechanism is necessary to generate diploid spores of the next generation. There are 2
types of compensatory mechanisms to allow for the continuation of the life cycle for the
approximately 10% of obligatorily apogamous ferns in nature (Sheffield and Bell, 1987).
One involves a mitotic division that does not complete cytokinesis, and these cells
proceed through meiosis to generate spores (Walker, 1979). In this scheme, the only
8
diploid cell in the life cycle is that which immediately undergoes meiosis. The other
mechanism allowing for apogamous life cycle continuation involves a failed reductional
division (meiosis I), and a normal meiosis II when generating spores (Klekowski, 1979).
In this scheme, ploidy level (whether haploid or diploid) does not change in any cell of
either generation. Both schemes produce spores containing the same number of
chromosomes as the apogamous sporophyte. Gametophytes that develop from these
spores produce sporophytes apogamously to complete the life cycle. Because this life
cycle does not require a water film for sperm to swim for fertilization, it is thought to
have evolved as an adaptation to drier environments (White, 1979). For some
Chelianthoid ferns an apogamous life cycle allows life in xeric environments (Mickel,
1979), suggesting that the dry environment renders sperm motility moot, and over time
the habit of developing sperm becomes degraded. Ferns that are apogamous in nature
form nonfunctional gametes and are sterile. Some bear archegonia that are either
abortive, or fail to open a channel sufficient for sperm entry (Laird and Sheffield, 1986).
Not long after the discovery of ferns that reproduce apogamously in nature, the
ability to induce apogamy from the gametophytes of fern species that normally reproduce
sexually was recognized. This process is called “facultative” or “induced” apogamy and
it was first accomplished by the prolonged culture of homosporous fern gametophytes in
dry conditions that prevented the release of male gametes, or their mobility, so that
fertilization is prevented (Lang, 1897). More efficient methods for inducing apogamy
from fern gametophytes have been developed using axenic growth conditions in
laboratory and involve the exogenous supply of sugars or other factors (Dyer, 1979;
Raghavan, 1989; Bell, 1992).
9
In general, the factors that have been recognized as broadly apogamy-inducing in
axenic culture systems are light, hormones, and sugars (Raghavan, 1989). The addition of
high levels of sugars to the culture medium has been established as sufficient to induce
apogamy from many fern species, and different sugars have different optimal
concentrations for the induction of apogamy (Whittier and Steeves 1960, 1962; Whittier,
1964a; Mehra and Sulklyan, 1969; Kato, 1970; Padhya and Mehta, 1981; Kawakami et
al., 2003). There is also evidence that light intensity plays a role in the induction of
apogamy, because dark-grown gametophytes generally do not produce apogamous
sporophytes, even when grown on sugars (Kato, 1970; Whittier, 1976; Whittier, 2004).
Additionally, different wavelengths of light seem more effective at inducing apogamy
from some ferns, suggesting a role for light signaling, rather than just photosynthetic flux,
in apogamy (Whittier, 1964b; Whittier and Pratt, 1971). Plant hormones modulate plant
growth patterns, and may induce the production of roots or shoots from gametophyte
tissue that has formed a callus, or may actually increase the production of apogamous
sporophytes in culture, but these effects only occur when combined with growth on a
suitable carbohydrate. (Elmore and Whittier, 1973; Elmore and Whittier, 1975a, 1975b;
Whittier, 1966; Kato, 1970).
The phenomenon of apogamy has been thoroughly studied with respect to
physiology and development, but the genes that are involved in the process of apogamy
are largely unidentified. Recently the Physcomitrella patens CURLY LEAF homologue
(PpCLF) was identified as being involved in apogamy in this moss. PpCLF is a member
of the polycomb group complex (PCR2) and loss of this gene in P. patens causes
apogamous sporophytes to form from the gametophytes (Okano et al., 2009). Other
10
members of PCR2 in angiosperms cause parthenogenic development of egg cells or
autonomous endosperm development (Chaudhury et al., 1997; Guitton and Berger, 2005).
Sexual and Asexual Alternation of Generations in
Angiosperms
Seed development in angiosperms requires the execution of several sequential,
defined steps. The shoot apical meristem of the plant must first transition from the
vegetative phase to the reproductive phase of organ production. The floral meristem
produces the floral organs in which the male and female gametophytes (the anther and
carpels, respectively) are eventually formed after meiosis. The mature male gametophytes
are the three-celled pollen, containing two sperm cells and a generative cell, the latter
eventually growing into the pollen tube, upon deposition on a stigma, to deliver the two
sperm to the female gametophyte within the ovule. The ovules of angiosperm flowers are
the structures that give rise to the seed after fertilization (Fig 3). The ovule also is the
structure that gives rise to seeds derived from asexual reproduction. The mature ovule
contains the female gametophyte, also called the embryo sac, which is typically a sevencelled structure including the egg cell (Fig 3C, shaded gray) and the binucleate central
cell (Fig 3C). The development of the ovule begins with the ovule primordium, which has
three regions, the nucellus, chalaza, and funinculus (Fig A). The meiotic initial, the
megaspore mother cell, emerges from the nucellus, which divides to form the megaspore,
which is the meiotic product closest to the chalazal region, and 3 spores that eventually
degenerate (Fig 3B). The megaspore undergoes three rounds of mitosis to form an
embryo sac with 7 cells and 8 nuclei (Fig 3C). From the chalazal region, the outer and
11
Figure 3 Ovule development in Arabidopsis. (A) The ovule primordium emerges from
the placenta and differentiates into the funinculus (f), chalazal (c), and
nucellar (n) zones. (B) The primordia of outer (oi) and inner integuments
(ii) arise from the chalazal region and grow around the nucellar tissue,
eventually becoming the seed coat after fertilization. A megaspore mother
cell develops from the nucellar tissue (n) and undergoes meiosis. The
funinculus (f) is the vascularized stalk that connects the ovule to the
placenta. (C) A single megaspore mother cell (shaded gray), the meiotic
product closest to the chalazal region, divides three times to form the 7celled female gametophyte, the embryo sac (es), which contains the egg
cell (shaded gray). Redrawn from Park et al., 2005.
12
inner integument initials form (Fig 3B), which, during ovule development, grow around
the nucellus (Fig 3C), protecting and nourishing it and the developing embryo sac. After
fertilization the integuments develop into the seed coat. The funinculus is the
vascularized stalk that connects the ovule, and by extension the female gametophyte and
developing zygotic embryo, to the placenta (Figure 3C). Sexual reproduction proceeds
when a pollen tube delivers two sperm to the ovule. One sperm fertilizes the egg and the
other the binucleate central cell, forming the diploid zygote and the triploid endosperm,
respectively.
A functional, triploid endosperm is necessary for the normal development of a
zygotic embryo and apomictic embryos in many species. Although the majority of
angiosperms reproduce sexually to form seeds, approximately 400 species from 40
families of angiosperms have evolved the ability to reproduce via apomixis (Nogler,
1984; Carman, 1997). In fact, many apomictic plant taxa retain the ability to reproduce
sexually, often producing sexual and apomictic embryos on the same plant. This makes it
difficult to identify all plant taxa that are apomictic in nature, and the current count of
apomictic species is likely a conservative one (Nogler, 1984; Bicknell and Kolunow
2004; Plitman, 2002). The process of apomixis involves the bypassing of meiosis during
gamete formation, along with the absence of fertilization to form the embryo, instead
producing embryos, without paternal contribution, from somatic cells (Tucker and
Koltunow, 2009). The different types of apomixis differ in the cell that gives rise to the
asexual embryo.
Three types of apomixis have been described: adventitious embryony, diplospory,
and apospory. Adventitious embryony produces multiple asexual embryos spontaneously
13
from the diploid, sporophytic cells of the nucellus or integument. This form of apomixis
occurs alongside normal sexual reproduction (Asker and Jerling, 1992; Tucker and
Koltunow, 2009). An apomictic embryo may invade the embryo sac to utilize the
endosperm at the expense of the sexual embryo, or sexual and apomictic embryos may
coexist and share the same endosperm (Asker and Jerling, 1992). Either way, the survival
of the apomictic embryos appears to depend upon the successful development of a
sexually derived endosperm, possibly because of the nutrients or growth signals it confers
(Asker and Jerling, 1992; Koltunow et al., 1995; Tucker and Koltunow, 209).
In diplospory, the megaspore mother cell, which is normally destined to undergo
meiosis and form the first cell of the female gametophyte, fails to initiate or complete
meiosis. Instead, mitosis forms a diploid, 8-nucleate embryo sac. The diploid, egg-like
cell then initiates embryogenesis without fertilization. These apomictic embryos also
depend upon an endosperm for their survival, whether the endosperm develops
autonomously or arises from fertilization of the central cell (Tucker and Koltunow,
2009). Some species may also be facultative diplosporous apomicts, capable of sexual
reproduction when necessity or opportunity arises (Grimanelli et al., 1998; Schranz et al.,
2005).
In apospory, the megaspore mother cell typically initiates meiosis, which it may
or may not complete (Tucker and Koltunow, 2009). Meanwhile, in the vicinity of the
megaspore mother cell, one or multiple somatic cells differentiate into aposporous
initials, which then bypass meiosis, instead dividing mitotically to form a diploid embryo
sac (Tucker and Koltunow, 2009). As in diplospory, the diploid egg-like cell initiates
embryogenesis without fertilization, and the endosperm can be formed autonomously or
14
via fertilization. Additionally, in some aposporous apomicts for which sexual embryo sac
development proceeds normally, both sexual and clonal embryos may be found on a
single plant (Araujo et al., 2000; Araujo et al., 2005).
All forms of apomixis have three features in common: the generation of a cell
that has the capacity to form an embryo without first undergoing or completing meiosis,
the ability to produce an embryo without fertilization, and the formation of an
endosperm, either through fertilization or independent of fertilization, to assist in the
development of the apomictic embryo (Tucker and Koltunow, 2009).
The genes that control apomixis are unknown, but seem to be restricted to one or
a few dominant loci in the majority of apomictic plants so far examined (Ozias-Akins and
van Dijk, 2007). There are several genes that, when ectopically expressed, cause somatic
embryogenesis (SE) from vegetative tissues, similar to apomixis phenotypes, but it is
unknown whether these same genes are truly involved in apomixis. Genes with gain-offunction SE phenotypes when ectopically expressed include WUSCHEL (WUS),
BABYBOOM (BBM) AND SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1
(SERK1). WUS encodes a homeodomain protein normally involved in regulating stem
cell fate in the meristem, and recently has been found to be required for
megasporogenesis (Laux et al., 1996; Mayer et al., 1998; Lieber et al., 2011). BBM, a
transcription factor, is preferentially expressed in developing embryos and seeds
(Boutillier et al, 2002). SERK1 is normally expressed throughout nucellar tissue during
meiosis, and throughout the development of the embryo sac and zygotic embryo
(Schmidt et al., 1997; Hecht et al., 2001), and confers competence for embryonic
development. The LEAFY COTYLEDON 1 (LEC1) and LEAFY COTYLEDON2 (LEC2)
15
are master regulators of embryo development that are normally expressed during the
morphogenesis and maturation phases of embryogenesis in seeds (Lotan et al., 1998;
Stone et al., 2001; Braybrook and Harada, 2008). Recently, a role for RNA silencing has
been implicated in apomixis with the recovery of the ARGONAUTE104 gene in a screen
for mutations that form viable, unreduced female gametophytes, a phenotype that
resembles diplospory, in maize (Singh et al., 2011).
Recessive apomicitc genes, like FIS2, a member of the PCR2 complex that causes
the initiation of embryo and endosperm development without fertilization similar to
apomictic plants (Chaudhury et al., 1997) are also known. Mutations in a member of the
PCR2 complex causes apogamy in the moss P. patens, suggesting that the processes of
apogamy and somatic embryogenesis, if not apomixis, share genetic components, even
though these plants are evolutionarily quite distant (Fig. 2).
The obligate production of a haploid sporophyte from gametophyte cells is called
androgenesis, rather than apogamy, in angiosperms (Segui-Simarro, 2010). Androgenesis
occurs very rarely in angiosperms, and involves a process whereby egg fertilization
occurs, but the female nucleus is somehow inactivated and a haploid sporophyte develops
with only paternal contribution (Burke, 1962; Chase, 1969; Goodsell, 1961; SeguiSimaro, 2010). However, the production of apogamous sporophytes in angiosperms can
be accomplished through inductive culture of pollen or embryo sacs. These inductive
processes are called pollen embryogenesis or gynogenesis, depending on whether it is the
male or female gametophyte being induced (Segui-Simarro, 2010). The genes involved in
this inductive process are unknown, and inductive conditions vary, but, universally,
16
include stress treatment (Shariatpanahi et al., 2006). It seems that the pollen must first go
through a stress response, which in turn triggers the embryonic response.
Experimental Strategy
My experimental strategy for elucidating the genes that are involved in asexual
reproduction in land plants took advantage of the separate and independent generations of
ferns, along with their unique ability to be easily induced to apogamy using laboratory
culture conditions. I used Ceratopteris richardii, a fern with a normally diploid
sporophyte that is a model fern system for the study of plant developmental processes
(Hickock et al. 1995; Banks 1999; Chatterjee and Roux 2000). In chapter 2 of this thesis,
I describe the induction of apogamy in C. richardii with culture on high levels of
exogenous sugars, and describe the dramatic phenotypic changes of the gametophytes
grown on sugar, as well as the apogamous outgrowths eventually produced. I also define
a time period during which C. richardii gametophytes become committed to the
production of apogamous sporophytes.
In chapter 3, I describe the creation of a subtracted, normalized cDNA library,
called the “apogamy library” that represents genes that increase in expression in C.
richardii gametophytes grown on sugar, during the time that they are becoming
committed to apogamy. This chapter also includes the list of genes from the apogamy
library that were confidently identified using BLAST searches, including a broad
overview of the function of these genes through Gene Ontology (GO) assignments. The
GO summary of the apogamy library is compared to that of the gametophyte
transcriptome of a homosporous fern to define the gene expression landscape of the
unique genes during apogamy commitment. Also, the in silico anatomical expression
17
patterns of the Arabidopsis homologues of the apogamy library sequences are examined,
and the expression patterns of a subset of these genes is also examined in C. richardii
gametophytes undergoing apogamy induction.
In chapter 4, I describe the characterization of one of these genes, UNC93-like, in
Arabidopsis. This gene, when mutated, confers vegetative, root, and reproductive
abnormalities. The defects include small, dark green leaves and slow development, an
excess of lateral roots, and a reduced seed set due to embryo abortion and a failure of the
parent plants to maintain gametophyte viability. In chapter 5, I summarize the findings in
the thesis and discuss further lines of investigation that may be performed to more fully
understand the functions of the UNC93-like gene, as well as the other genes in the
apogamy library.
18
CHAPTER II
THE INDUCTION OF APOGAMY IN
CERATOPTERIS RICHARDII
Introduction
Both the sporophyte and gametophyte generations of land plants begin from a
single cell, the zygote and the spore, respectively. Yet, the two generations develop into
multicellular bodies that are remarkably different in morphology and habit. The
mechanisms that trigger these two developmental pathways are unknown. While meiosis
and fertilization normally precede the production of the zygote and spores, they are not
necessary for the induction of sporophytic and gametophytic developmental pathways.
This is evident from the development of sporophytes and gametophytes even in the life
cycles of plants that engage in asexual life cycles that circumvent fertilization, meiosis, or
both events altogether (Asker and Jerling, 1992; Tucker and Kolunow, 2009).
Apogamy is a phenomenon in which a sporophyte is produced directly from the
vegetative cells of a gametophyte without fertilization, thereby circumventing
chromosome doubling (For a review of nonsexual cycles in plants, see Bell, 1992). This
phenomenon has been well documented in ferns, and its existence provides evidence that
the signal that determines which developmental program will commence, sporophytic or
gametophytic, is not simply an assessment of ploidy by the single-celled zygote or spore.
Apogamy is obligate in some homosporous fern species whose gametophytes produce
nonfunctional gametes. To compensate for the lack of chromosome doubling, these
apogamous sporophytes form restitution nuclei during sporogenesis, either through an
19
incomplete mitotic division or a failed first meiotic division, thereby allowing the
production of spores with one set of chromosomes (Raghavan, 1989). In a process termed
‘‘induced’’ or ‘‘facultative’’ apogamy, several homosporous fern species that normally
produce sporophytes through syngamy have been induced to form apogamous
sporophytes through the manipulation of culture conditions. In general, sporophytes
formed through induced apogamy have abnormal morphology compared with zygotederived sporophytes or sporophytes formed through obligate apogamy (Lang, 1898; Steil,
1939, 1951; Whittier, 1962); however, they do display one or more of the hallmarks of
sporophyte development. These hallmarks, none of which are normally present in fern
gametophytes, include leaves, stems, roots, vascular tissue, and stomata.
The experimental conditions used to induce apogamy in ferns have already
provided valuable information about the signals that induce the sporophytic
developmental pathway. Two basic methods have been employed to induce apogamy in
ferns. The first simply involves the extended culture of fern gametophytes while
simultaneously preventing fertilization by carefully controlling water supply (Lang, 1898;
Stei,l 1939, 1951; Duncan, 1941). Apogamy induction in this manner has generally been
in combination with high light levels, suggesting that increased metabolic flux plays a
role in the process of apogamy. Culturing fern gametophytes on medium containing high
levels of exogenous sugars is sufficient to induce the apogamous production of
sporophytes in many fern species (Whittier and Steeves, 1960, 1962). Exogenous sugars
have also been found to induce the differentiation of sporophyte tissue from callus
derived from the fern Pteris cretica, whereas sugar deprivation induces gametophyte
differentiation from callus (Bristow, 1961). These studies suggest that sugar metabolism
20
and/or sugar signaling play a role in determining the gametophyte and sporophyte
developmental pathways in ferns.
The semi aquatic fern Ceratopteris richardii was chosen two decades ago in an
effort to develop this fern into a model system for studying developmental processes
(Hickock et al., 1995; Banks, 1999; Chatterjee and Roux, 2000). Two laboratory strains
of obligately apogamous Ceratopteris have been described, both of which produce
nonfunctional spermatozoids. One strain resulted from the aneuploid gametophyte of a
triploid hybrid and the other from a diploid hybrid between C. richardii and C.
pteridoides (Hickock, 1977, 1979). Here, I report that apogamy can be induced in the
diploid, normally syngamous C. richardii by sugar. Apogamy was confirmed by
measures of ploidy level and documented by the presence of the hallmarks of sporophytic
tissues. I also documented the variation in morphology of apogamous sporophytes in this
fern species. Most importantly, I have identified the minimum culture time that C.
richardii gametophytes require the presence of exogenous sugar for sugar-induced
apogamous sporophyte production. A differentiating cell in a gametophyte can follow
one of several developmental pathways: vegetative gametophyte, gamete, or vegetative
sporophyte (apogamy), depending on the developmental cues to which it responds.
Focusing on initiation of the sporophyte from apogamy rather than from a zygote
separates this direct switching between gametophyte and sporophyte from
embryogenesis. It has been proposed that the conditions for sporophytic growth of the
zygote have already been set in the mature egg (Bell, 1970). It has also been
demonstrated in the homosporous fern Pteridium aquilinum that mature eggs in which
RNA synthesis has been inhibited with thiouracil will, when egg and archegonium are
21
isolated from the prothallus, develop into structures whose morphologies are intermediate
between gametophyte and sporophyte (Jayasekera et al., 1972; Jatasekera and Bell,
1972). In our view, once the zygote is formed, the identity of the sporophyte generation
in normal alternation of generations has been determined. Therefore, the ability to
generate sporophytes from fern gametophytes through induced apogamy, albeit abnormal
in morphology, provides a tractable experimental system for understanding the gene
network that controls the switching from one generation to another.
Materials and Methods
Plants and Growth Conditions
The C. richardii plants used in these studies were of the mutant genotype
hermaphrodite (her) (Carolina Biological Supply, Burlington, NC) and feminization1
(fem1) (a gift from J. Banks, Purdue). C. richardii her mutants are self-fertile, but are
restricted to hermaphroditic development, because of their insensitivity to the maleinducing pheromone antheridiogen (Banks, 1994; Banks, 1997). Like other homosporous
ferns, C. richardii gametophytes develop into hermaphrodites in the absence of the maleinducing pheromone antheridiogen (Naf, 1979; Naf et al., 1975; Banks et al., 1993). The
hormone itself is secreted by hermaphroditic gametophytes when they are no longer
competent to respond to it (Banks et al., 1993). Thus, the first spores to germinate in a
wild-type population become hermaphrodites, and these same hermaphrodites induce the
development of male gametophytes from the surrounding germinating spores by the
secretion of antheridiogen into their surroundings. Fertilization results in the loss of the
gametophyte notch meristem function and subsequent gametophyte senescence. The
prevention of fertilization is therefore important both for the occurrence of apogamy and
22
for preventing sporophytes generated from fertilization. The absence of males in her
gametophyte cultures reduces the likelihood of fertilization. The fem1 spores produce
gametophytes that have archegonia but no antheridia, either in the presence or absence of
the male-inducing pheroemone antheridiogen (Banks, 1994; Banks, 1997). The complete
absence of sperm-producing structures from fem1 gametophytes ensures that all
sporophytic structures that appear from these gametophytes are apogamous.
Spore germination and gametophyte culture conditions were essentially as
described by Hickock et al. (1995). Spores were sterilized for 5 min in 3% sodium
hypochlorite with one drop of Tween 20, rinsed five times with sterile water, and imbibed
in water in the dark for 3 days at room temperature to synchronize germination. Spores
were inoculated at a density of 150–300 spores per 95 mm petri plate containing basal
medium (1% agarose supplemented with 0.5x Murashige and Skoog salts at pH 6) and,
where noted, rich medium (supplemented with 2.5% glucose). Medium supplemented
with 1%–9% sucrose or 0.5%–4% trehalose was also employed in initial experiments to
determine which sugar and at what concentration the best induction of apogamy is
produced in this fern. Gametophyte culture plates were maintained in humidity domes at
28°C with constant illumination under Philips Agro-Lite fluorescent bulbs (Philips
Lighting Company, Somerset, NJ) at 90–100 µM m2 s-1. Fifteen days after inoculation,
gametophyte culture plates were inverted to reduce the incidence of fertilization by
preventing water condensation on gametophytes when her mutant plants were used.
Fertilization did not occur before this time under our culture conditions. All
gametophytes were examined for the appearance of apogamous structures under sterile
conditions every other day for 54 days using a Leica Wild MC3 dissecting microscope.
23
Apogamy Induction Experiments
Sterilized, dark-treated her or fem spores were inoculated on petri plates either
with rich medium or with basal medium. Every 2 days after inoculation, gametophytes
growing on a rich medium plate were transferred in two cohorts, consisting of 120–169
gametophytes each, to fresh medium. The transfers were performed manually, with one
cohort transferred to rich medium and one cohort transferred to basal medium. This was
repeated every 2 days until day 16, the last day of transfer. In addition, every plate of
gametophytes was moved to fresh medium every 14 d after the original transfer to ensure
an adequate supply of nutrients at all times. In this manner, no gametophyte remained on
medium for longer than 2 weeks except those that were on rich medium for 16 days
before their first transfer.
Histology
For toluidine blue staining, four gametophytes, on which fertilization-derived
sporophytes were growing, and six gametophytes, on which apogamous sporophytes
were growing, were chosen randomly, fixed at 4°C overnight in FAA (10%
formaldehyde, 50% ethanol, and 5% acetic acid), and then processed through an alcohol
dehydration series and embedded in Paraplast Plus (Kendall, Mansfield, MA). Ninemicrometer sections were deparaffinized, hydrated, and stained for 10 min in 0.05%
toluidine blue (pH 6). Sections were then dehydrated and cleared in xylenes. Coverslips
were affixed with Permount (Fisher Chemical, Fair Lawn, NJ).
For chromosome squashes, whole gametophytes or sporophyte shoot and root tips
were fixed in 3:1 ethanol:acetic acid overnight. Samples were then rinsed in distilled
water, placed on a slide in a drop of saturated acetocarmine stain beneath a coverslip,
24
heated briefly over an open flame, and squashed. Digital images of chromosome squashes
were used to count chromosome numbers. Five chromosome counts of putatively
apogamous outgrowths were performed, four from shoot tips and one from a root tip. One
chromosome count each was made from squashes prepared from haploid gametophyte
notch meristems and diploid sporophyte root tips to confirm chromosome numbers.
Vascular tissue was imaged by bleaching unfixed samples in 95% ethanol for several
hours to remove chlorophyll and mounting bleached samples onto slides in glycerol.
For ploidy level determination using Hoechst dye, at least two putatively
apogamous structures from each cohort of gametophytes were cleared in 95% ethanol
overnight at 4°C. Tissue was then rinsed in water and immersed in 40 mg/mL Hoechst
33342 dye for 2.5 h, rinsed twice in distilled water, mounted on slides in glycerol, and
imaged under DAPI filtered UV light. To ensure uniformity of fluorescence comparisons,
Hoechst dye-stained samples were analyzed immediately after mounting, and compared
images were captured at the same gain and exposure. Fluorescence intensity was
quantified using ImageJ 1.37j software (http://rsb.info.nih.gov/ij/). The fluorescing nuclei
of three Hoechst images, representing three different experiments, each containing a
putatively apogamous tissue sample along with haploid and diploid controls, were
measured to obtain relative fluorescence intensities. The images were converted to 8-bit
gray scale and binarized using the intensity thresholding function. The selected
fluorescing nuclei were regions of interest. Mean gray values were then obtained from the
defined regions of interest and converted to relative intensities. Mean pixel intensities of
7 to 18 in-focus nuclei from each putatively apogamous, haploid standard, and diploid
standard were compared to quantify ploidy of the corresponding tissues. To view
25
stomata, cleared tissue samples were incubated overnight in 40 mg/mL Hoechst 33342
dye, rinsed in distilled water, and mounted on slides in glycerol; images were captured
through DAPI filter.
Chromosome squash images were recorded with a Zeiss Axioskop 20 microscope
with MC100 attachment camera system (Carl Zeiss, Thornwood, NY). Apogamous plants
were photographed with a Zeiss stereomicroscope SR and MC100 attachment camera
system. All other images were recorded using a Leica DMRBE upright microscope and
DC300F digital camera system (Leica Microsystems, Bannockburn, IL).
Results
C. richardii Gametophytes Can Be Induced
to Form Apogamous Sporophytes
Under optimum conditions (described in ‘‘Material and Methods’’), C. richardii
gametophytes reach sexual maturity after 12–15 d of culture. At this stage,
hermaphroditic gametophytes have several antheridia and archegonia (Fig. 4a). After the
gametophytes are flooded, spermatozoids are released from antheridia and are free to
swim into mature archegonia to fertilize the eggs. Connected at the region called the foot
to the senescing gametophyte, the embryonic sporophyte develops until the root and
shoot are established (Fig. 4b). The first-formed sporophyte fronds are sterile and simple
three lobed. Later-formed fronds are fertile and have a dissected morphology (Fig. 4c).
Sporangia, the structures within which spores are produced through meiosis, are formed
abaxially within the inrolled margins of fertile fronds.
The C. richardii hermaphroditic prothallus cells normally divide in only two
dimensions, producing a gametophyte that is one cell layer thick with a meristem
26
Figure 4 The normal gametophyte and sporophyte forms of C. richardii. a, An
hermaphroditic gametophyte grown on basal medium for 20 days is
sexually mature, with many antheridia (an) and several mature
archegonia (ar) located underneath the notch meristem. b, After
fertilization, a zygote-derived sporophyte (sp) emerges from the
senescing gametophyte (ga) until the root and shoot systems are
established. c, A 4-month-old dissected sporophyte with dissected, fertile
fronds. Black bars represent 1 mm, white bar represents 10 cm.
27
in the notch between the two lobes of the prothallus. In the absence of fertilization,
gametophyte notch meristem activity is maintained, and gametophyte growth will
continue indefinitely if supplied with sufficient nutrients. When grown on a medium that
lacks sugar (basal medium), the continued gametophyte growth takes the form of
additional prothalli that originate from the notch meristem and are one cell layer thick
(Fig. 5a). Extended gametophyte growth on a medium that contains sugars (rich medium)
also results in additional prothallus growth, but those prothalli are thickened and are
accompanied by a proliferation of ectopic rhizoids, seen as a transparent mass (Fig. 5b).
We grew her mutant gametophytes on agar-solidified medium, supplemented with
various concentrations of trehalose, sucrose, or glucose, to determine whether apogamy
could be induced by sugar in C. richardii. Whereas gametophytes were not viable on
concentrations of glucose at or above 4%, presumably because of osmotic effects, all
other sugar concentrations from 1% to 3% successfully induced the formation of
apogamous sporophytes (data not shown). We found that medium supplemented with
2.5% glucose was optimal for the induction of apogamy, with 10%–28% of gametophytes
per plate (150–300 gametophytes per plate) displaying apogamous sporophytes after 2
months of culture.
Apogamous sporophytes assumed various morphologies in C. richardii, none of
them identical to normal, zygote-derived sporophytes. The most common forms were
slightly lobed leaves (Fig. 5c) but with no accompanying roots as is the norm with
zygote-derived sporophytes (cf. Fig. 4b with Fig. 5c), outgrowths displaying intermediate
sporophyte-gametophyte morphology that were cylindrical (Fig. 5d) or flattened (Fig.
5e), and clusters of sporophyte leaves and leaf-like protrusions (Fig. 5f). Rarely (2 out of
28
249 total cases of sugar-induced apogamy) a solitary root appeared in the absence of any
visible leaf or shoot development (data not shown). Ramenta are one-cell-thick scales
that normally occur on the stem and petiole of zygote-derived fern sporophytes. Ramenta
accompanied apogamous sporophyte structures, appearing at the base or along cylindrical
outgrowths. Although they become brown and scale-like on mature C. richardii stems,
they initially appear translucent and accompany every leaf of young, zygote-derived
sporophytes (Fig. 5g). Ramenta also appeared in the absence of other sporophyte
structures, appearing in clusters or as a single apogamous ramentum (Fig. 5h).
The gametophyte-sporophyte junction differed between zygote-derived and
apogamous plants. Normal gametophyte-sporophyte junctions of C. richardii are
characterized by sporophyte haustorial cells that develop before the emergence of the first
leaf and contain extensive wall ingrowths (Duckett and Ligrone, 2003). Toluidine blue–
stained longitudinal sections of normal sporophyte-gametophyte junctions display these
wall ingrowths in a deep blue color (Fig. 6a). These ingrowths are unique and serve as an
easily identified marker for the junction of the gametophyte and the zygote-derived sporophyte. This boundary is not evident at the junction between apogamous sporophytes
and the gametophyte from which the sporophytes are generated. These junctions contain
no clear demarcations save a tangle of vascular tissue and occasional bits of ingrown cell
wall with no discernible pattern (Fig. 6b).
At the microscopic level, C. richardii apogamous sporophytes were observed to
possess stomata and vascular tissue; both are sporophyte-specific features. Stomata,
which were observed on 100% of zygote-derived sporophyte leaves (Fig. 7a), were also
29
Figure 5 Various apogamous sporophyte forms of C. richardii. Gametophyte
proliferation in the absence of fertilization results in additional twodimensional prothalli growth after 45 d on basal medium (a), while 45 d
on rich medium results in prothallus thickening (arrowhead) and rhizoid
proliferation (arrow) (b). Apogamy took various forms, including
slightly lobed leaves (c), cylindrical (d) or bladelike (e) growths that
were of intermediate gametophyte and sporophyte morphology, and
clusters of leaves and outgrowths (f). Ramenta (arrows) accompanied
every emerging leaf of young, zygote- derived sporophytes (g) and were
the most common apogamous structure seen, appearing alone (h) or in
conjuction with almost all other apogamous structures. Bar = 1 mm.
30
Figure 6 The gametophyte-sporophyte junction in zygote-derived and apogamous
plants. a, The junction of a gametophtye (ga) and zygote-derived
sporophyte (sp) is characterized by sporophyte haustorium cells with
extensive cell wall ingrowths (arrows). b, The typical apogamous junction
contains disorganized vascular tissue and some evidence of extra cell wall
deposits (arrow) but no defined boundary between the gametophyte (ga)
and apogamous sporophyte (sp). Bar = 0.05 mm.
31
Figure 7 Apogamous sporophytes have typical sporophyte features. Hoechst DNA
staining revealed the elongated nuclei of guard cells (arrow) flanking
stomata on the leaves of zygote-derived sporophytes (a) and apogamous
sporophytes (b). Vascular tissue, never present in normal gametophytes,
could be seen in zygote-derived sporophyte leaves (c) and apogamous
sporophyte leaves (d). Bar = 0.05 mm.
32
observed, albeit at a lower density, on 100% of apogamous sporophyte leaves and
apogamous structures with intermediate morphology (Fig. 7b). Vascular tissue, which exists in all zygote-derived sporophyte leaves (Fig. 7c), was seen in apogamous structures
with few exceptions (Fig. 7d).
If the sporophyte organs generated from gametophytes that have been grown on
rich medium are the product of apogamy, they should have the same (haploid) ploidy
level as gametophytes. The apogamous nature of sporophyte structures was verified by
Hoechst dye staining of DNA or chromosome counts. With Hoechst dye staining of
DNA, the ploidy level of putatively apogamous tissue samples could be determined by
comparing the intensity of nuclear fluorescence with the nuclear fluorescence of known
haploid (Fig. 8a) and diploid (Fig. 8b) standards. Mitotic chromosome squashes were also
performed where meristematic tissue was present. Comparisons of putative apogamous
tissue (Fig. 8c) with known standards (Fig. 8d, 8e) indicated ploidy levels. Approximate
chromosome counts were made of five squashes from five independent instances of
apogamy as well as one haploid and one diploid standard to verify chromosome numbers.
Approximate chromosome counts for putatively apogamous tissue varied from 35 to 39
chromosomes. The approximate haploid and diploid control counts were 36 and 64 (out
of the 78 chromosomes, only 64 were visible for counting), respectively. The relative
fluorescence intensities between the putatively apogamous, haploid, and diploid samples
in figure 8 are shown in figure 9. Similar results were obtained in two other experiments.
33
Figure 8 Confirmation of apogamy with ploidy comparisons. Hoechst DNA dye
intensity was used as a ploidy marker by comparing nuclear fluorescence
of putatively apogamous tissue (ap in a) with known haploid (b) and
diploid (a) standards. Chromosome squashes of mitotically dividing cells
were also used to confirm ploidy of the putatively apogamous (c) by
comparing to haploid (d) and diploid (e) standards. Ceratopteris richardii
haploid n=39, diploid n=78. Bar = 0.05 mm (a, b); bar = 0.01 mm (c–e).
34
Figure 9 Quantitative determinations of ploidy levels by Hoechst dye intensity.
The mean pixel intensity calculated from 7 to 18 in-focus nuclei
from each of putatively apogamous, haploid, and diploid samples
were compared, with the highest mean value set at 100. Bars
represent the relative fluorescence intensities. Vertical lines represent
standard deviation.
35
C. richardii Gametophytes Become Committed to
Apogamy after 12 Days of Induction
To determine when gametophytes become committed to apogamy by sugar
induction, we cultured gametophytes on rich medium and transferred a subset of these
gametophytes to basal medium every 2 days. As a positive control, cohorts of these
gametophytes were transferred from rich medium to rich medium every 2 days. Thus,
subsets of gametophytes, consisting of 120–169 gametophytes each, received the
apogamy induction signal for increasing lengths of time. We then monitored these
gametophyte populations for the production of apogamous sporophytes. In this way, we
could determine the time point at which gametophytes are committed to sugar-induced
apogamy development. Similar experiments were performed twice using the her mutant
and twice with the fem mutant (Fig. 11), and very similar results were obtained in all
three experiments, save the fact that much higher percentages of fem1 gametophytes
undergo apogamy. This may be explained in part by the fact that the experiment with the
fem mutant was conducted for 80 days, compared to 55 days for the her mutants,
allowing more time for the gametophytes to form apogamous sporophytes. In one
experiment, 54 days of continuous culture of the her mutant gametophytes on rich
medium exhibited 14%-20% apogamous sporophytes (Fig. 10a, open bars; Fig. 10b).
Surprisingly, gametophytes were found to produce a basal level of apogamous
sporophytes in the absence of sugar induction, with approximately 2% of gametophytes
displaying apogamous sporophytes after 54 d of growth on basal medium (Fig. 10a, lane
36
Figure 10 Apogamy induction from C. richardii her mutant gametophytes. Bar
graph (a) shows the percentage of each cohort of gametophytes that
displayed apogamy after 55 d. Filled bars indicate cohorts that were
transferred to basal medium; open bars indicate cohorts transferred
to rich medium. B indicates gametophytes cultured continuously on
basal medium. R indicates gametophytes cultured continuously in
rich medium. Line graphs illustrate the kinetics of the appearance of
apogamous sporophytes on rich medium (b) and basal medium (c).
Notice the different scales on the Y-axis in b and c.
37
Figure 11 Apogamy induction from C. richardii fem mutant gametophytes. Depicted
is the percentage of gametophytes that displayed apogamous structures
after 80 days of growth. “R” refers to those gametophytes cultured on
glucose for the entire 80 days, “B” were grown on basal fern medium for
80 days. Gametophytes were transferred from rich medium to basal
medium on the corresponding days after sowing. Graph represents two
identical experiments, and error bars depict standard deviation.
38
B; Fig. 10c, B). Although apogamy could be seen in gametophytes cultured without
sugar, its appearance was substantially slower compared with apogamy induced by sugar.
Gametophytes that were maintained on rich medium for 2–10 days also displayed a
similar low rate of apogamy, with 0%–2% of gametophytes displaying apogamy after 54
days of culture (Fig. 10a, lanes 2–10; Fig. 10c). When gametophytes were cultured on
rich medium for longer than 12 days, the percentage induced to apogamy rose
substantially when they were observed on day 54. Seven percent of gametophytes were
induced to apogamy after 12 days (Fig. 10a, lane 12) of culture on rich medium, and this
value rose to 12% after 16 days of culture (Fig. 10a, lanes 16; Fig. 10c).
Similar experiments were conducted with fem mutant gametophytes to examine
the timing of apogamy commitment in fern gametophytes that have no capacity for
sexual reproduction (Fig. 11). Differently, in this experiment, total apogamy counts were
not conducted periodically throughout the experiment, but tallied only at the end of an
80-day experiment. It was found that these gametophytes show a dramatic increase in the
commitment to apogamous sporophyte production after 10 days of culture on rich
medium (Fig. 11). Indeed, approximately 4 times as many fem gametophytes became
committed to the apogamy after 12 days of culture on rich medium compared to her
gametophytes. This difference may be attributed to the fact that the fem experimental
observations continued for 80 days compared to 54 days. Alternatively, it may be that fem
gametophytes are more competent for apogamy induction in general, perhaps indicating
an apogamy-suppressing role for antheridia and/or sperm. However, the basal level of
apogamy displayed from fem gametophytes is very similar to that displayed by the her
39
mutants (Fig. 10a, Fig. 11), which is evidence against the hypothesis that fem mutants are
generally more competent for induction to apogamy.
Discussion
Here I have documented and characterized the sugar induction of apogamy in the
fern C. richardii. The apogamous sporophytes of C. richardii take many forms, ranging
from intermediate between sporophyte and gametophyte in morphology to clusters of
sporophyte leaves. In agreement with previous descriptions of induced apogamy in other
fern species (Lang, 1898; Whittier and Steeves, 1960, 1962), none of these forms are
identical to normal zygote-derived sporophytes. Additionally, the gametophyteapogamous sporophyte junction lacks the extensive cell wall ingrowths that form in the
“foot” of normal C. richardii gametophyte-sporophyte junctions (Duckett and Ligrone,
2003). In sexual reproduction, these wall ingrowths have been described as facilitating
the transfer of nutrients from the gametophyte to the developing sporophyte (Gunning
and Pate, 1969; Khatoon, 1986; Frey et al., 1994). How the apogamous sporophytes in
this study are acquiring nutrients is unknown. Possibly, extensive cell wall ingrowths are
unnecessary for the transfer of sufficient nutrients for apogamous sporophyte growth. A
somewhat limited supply of nutrients through a poorly developed or non-existent
placental region may explain why the apogamous sporophytes generated in this study fail
to complete development and eventually die (data not shown). Alternatively, the
apogamous sporophytes may acquire nutrients directly from the agar medium without the
need for gametophytic transfer. This scenario may indicate that the failure of the
apogmaous sporophytes to continue development is a side effect of their haploid nature,
or the lack of a full complement of sporophytic development gene activities.
40
By removing exogenous sugar every other day during the induction period, we
determined that C. richardii gametophytes begin commitment to apogamy development
after growth for 12 d in the presence of sugar. At day 12, the gametophytes showed
neither visible signs of apogamy nor any morphological difference under dissecting
microscope compared with those grown on basal medium. However, the significantly
higher frequency of apogamy in gametophytes that have been grown on rich medium
observed at a later time indicates that a fundamental change at the molecular level has
taken place by this critical time point (between day 10 and 12). It is this change that
allows the gametophyte cells to commence, or to respond to the commencement of, a
sporophyte-specific developmental program.
The basal level of apogamy in the absence of exogenous sugars was surprising
because it had not been reported previously. The implications of this intriguing result are
exciting. The difference in kinetics of induced versus spontaneous apogamy suggests that
different mechanisms are employed. It is possible that another aspect, in addition to
exogenous sugar, of the culture conditions in our experiments was conducive to apogamy
induction in C. richardii. One possible factor is the light conditions under which these
gametophytes were grown, as high light intensity has been hypothesized to induce
apogamy in other fern species (Lang, 1898; Duncan, 1941). Previously, C. richardii
gametophyte cultures were grown under light intensities of 60–90 mM m2 s-1 (Hickock et
al., 1995), which is slightly lower than our light conditions (90–100 mM m2 s-1). No
matter what this factor may be, this result suggests a certain developmental plasticity of
the gametophyte cells. This plasticity allows a small percentage of the gametophytes to
produce apogamous sporophytes in conditions such as dryness that otherwise would not
41
allow for sexual production of sporophytes. The remarkable ability of infertile
Ceratopteris strains to modify their life cycles and compensate for the inability to form a
zygote (Hickock, 1977, 1979) is a testament to the developmental plasticity of this fern.
Before our study in C. richardii, apogamy was experimentally induced in several
species of homosporous ferns by culturing gametophytes on exogenous sugars (Whittier
and Steeves, 1962). In addition, apospory, the formation of gametophytes from the
vegetative tissue of sporophyte leaves can be induced in many ferns by culturing
sporophyte leaves on basal medium (Raghavan, 1989). These two opposing culture
conditions (exogenous sugar/no exogenous sugar) each induce an opposite alternative
pathway, sporophytic development from gametophyte tissues, or vice versa, in many fern
species (Whittier and Steeves, 1960, 1962; Raghavan, 1989). Moreover, sugar promotes
sporophyte regeneration and lack of sugar promotes gametophyte regeneration from
callus (Bristow, 1961). These results strongly point to the critical role of sugar, as
metabolite and/or as a signal, in determining the two alternate developmental pathways in
ferns. Sugar status, either through the sensing of metabolic flux or sugar signal
transduction, is known to coordinate molecular and environmental cues in the control of
plant growth and development (Sheen et al., 1999; Smeekens, 2000; Rolland et al., 2002;
Rosa et al., 2009; Hanson and Smeekins, 2009). Furthermore, there is an extensive
crosstalk between sugar-signaling pathways and the signaling pathways of ethylene,
cytokinin, auxin, and abscisic acid in Arabidopsis (Smeekens, 2000; Rolland et al. 2002;
Rosa et al., 2009). Some evidence shows that different hormone treatments, in
combination with exogenous sugars, can promote the formation of apogamous
sporophytes from fern gametophytes (Elmore and Whittier, 1973; Raghavan, 1989).
42
Despite the complex interplay between sugar signaling and hormonal signaling pathways,
modulating sugar levels allowed me to establish the minimum time C. richardii
gametophytes require an inductive cue to gain competence to undergo apogamy. The
identification of genes whose expression specifically increased during this time is
described in Chapter 3.
43
CHAPTER III
GENES UPREGULATED DURING APOGAMY
INDUCTION IN C. RICHARDII
Introduction
In the previous chapter and, Cordle et al., 2007, I showed that the homosporous
fern C. richardii can be efficiently induced to form apogamous structures by culturing
gametophytes on medium containing 2.5% glucose. C. richardii gametophytes also begin
to become committed to the production of apogamous gametophytes between 10 and 12
days of growth on this apogamy-inductive medium. An alternative way of explaining this
phenomenon would be that the gametophytes are not capable of responding to the
inductive signal until at least 10 days growth on the inductive medium. Regardless, the
identification of this response time provides an opportunity to identify genes that are
differentially expressed during the commitment, or induction, of apogamy. Genes
identified as differentially expressed during this time period may play an important role
in the induction of apogamy in this fern.
I used subtractive suppression hybridization (SSH; Diatchenko et al, 1996) to
identify the genes expressed during this time period. This method allows for the
comparison of two populations of mRNA and enables one to obtain clones that are
present in one population while absent in the other, while at the same time normalizing
the two populations, so that low copy transcripts will not be excluded from the library
clones obtained (Diatchenko et al., 1996). The SSH method has been used to construct
many cDNA libraries containing specifically expressed rare transcripts (for instance:
44
Jeong et al., 2004; Archambault and Stromvik, 2011; Landreville et al., 2011). The
enrichment can be as high as 5000 fold (Diatchenko et al., 1996).
In this chapter I describe how I utilized SSH to obtain cDNA clones that are
present in a population of mRNAs from C. richardii gametophytes grown on inductive
(rich) medium for 12 days, but absent or expressed at a low level from a population of
mRNAs isolated from gametophytes grown on rich medium for 9 days. In this way, I
cloned sequences that represent genes induced during the time period when C. richardii
gametophytes are responding to an apogamy-inducing signal, or are becoming committed
to the production of apogamy. I then mapped plant GO-slim terms to the identified genes
in the apogamy library, and compared them to those mapped to the entire gametophyte
transcriptome of Pteridium aquilinum, another homosporous fern (Der et al., 2011). I
found that the apogamy library is enriched in GO-slim terms that are associated with
stress response and metabolism, and includes several genes that are mapped to flower and
embryonic development processes in plants.
Additionally, I found that many of the Arabidopsis homologues of the apogamy
library genes are specifically expressed or upregulated in Arabidopsis flower organs and
seed structures, including embryos, based on in silico expression analyses. A subset of
these genes was examined via whole mount in situ for their expression profiles in C.
richardii gametophytes undergoing apogamy commitment, and 3 were identified as being
upregulated in the sexual structures or the early embryo.
45
Materials and Methods
Plants and Growth Conditions
C. richardii hermaphrodite mutant spores were sterilized in 3% sodium
hypochlorite and Tween 20, rinsed 5 times with sterile water, and plated on ½ strength
MS medium supplemented with 2.5% glucose. Plates were incubated at room
temperature in the dark for 5 days to synchronize spore germination, and then cultured as
previously described (Cordle et al., 2007).
RNA Extraction
Gametophytes cultured for 9 days and 12 days on rich medium were removed
from plates with sterile blades and flash frozen in liquid nitrogen. The frozen tissue was
ground to a fine powder and total RNA was extracted using the guanidinium thiocyanate
and acid phenol method (Chomczynski and Sacchi, 1987) with the following
modifications. Before precipitation with isopropanol, 0.4 volume of a high salt solution
(0.8M sodium citrate, 1.2M sodium chloride) was added to minimize the precipitation of
polysaccharides. The RNA pellet was dissolved in freshly deionized formamide, heated
to 70ºC for 10 minutes in the presence of 0.7M NaCl and 1% CTAB, and extracted twice
with chloroform to remove precipitated polysaccharides. After adding 1 volume of
DEPC-treated water, total RNA was recovered with standard ethanol precipitation.
mRNA was enriched from the total RNA with the Oligotex mRNA mini kit (Qiagen)
according to the manufacturer's instructions.
Suppression Subtractive Hybridization
The 12-day subtracted, normalized cDNA population was created according to the
PCR-Select cDNA Subtraction Kit instructions (BD Biosciences; Diatchenko, et al.,
46
2004), with minor modifications. First and second strand cDNA was created from 1µg of
mRNA from 9-day-old and 12-day-old gametophytes. The synthesis of both first and
second strands was monitored by incorporation of P-32 labeled dCTP in parallel
reactions. The cDNA was digested with RsaI (New England Biolabs), and then
precipitated with ammonium acetate and ethanol to remove unincorporated nucleotide
triphosphates and primers. Completion of RSAI digestion was verified by agarose gel
electrophoresis with pre- and post-digested cDNAs. Ligating equal proportions of 12-day
cDNA with two different adapters created the tester cDNA population. The ligation
reaction was performed overnight at 4° C with T4 DNA ligase (New England Biolabs).
Ligation efficiency was tested with PCR using C. richardii hexokinase gene-specific
primers along with adapter-specific primers. The 12-day tester cDNA populations were
then hybridized with an approximately 30x excess of 9-day driver cDNA to normalize
and enrich for differentially expressed sequences at day12. Another round of
normalization and enrichment with approximately 10x excess of 9-day driver cDNA was
then performed to create templates for PCR amplification of differentially expressed
sequences. Using primers specific for the two adapter sequences, two cycles of PCR, the
second with nested primers, were performed to amplify sequences that were present in
the 12-day population and absent in the 9-day population.
Sequence Identification and Analysis
Sequences were uploaded in FASTA format into the Blast2GO program (Conesa
et al., 2005; Conesa and Gotz, 2008) and were identified with BLASTx against the nonredundant NCBI protein database. Those sequences that could not be identified in this
manner were identified manually via BLASTx at the Joint Genome Institute or BLASTn
47
searches followed by BLASTx of best hits. Those sequences that were identified with an
E-value significance of less than 1E-3 were selected for further mapping and annotation
with the Blast2Go program. The Genevestigator program (Zimmerman et al., 2004;
Zimmerman et al., 2005) was utilized for in silico expression analysis of Arabidopsis
homologues. Choosing only those probe sets specific for the genes of interest, 6100 high
quality ATH1 22K array experiments were chosen to create a meta-profile. Gene
hierarchical clustering was performed to facilitate identifying genes with similar
expression profiles. The resulting heat map output was modified using Adobe Photoshop
CS4 (Adobe Systems Inc.).
Cloning, Sequencing, and RT-qPCR
Amplified sequences were cloned directly into the pCRII-TOPO dual promoter
vector (Invitrogen) and transformed into TOP10 F’ E. coli cells (Invitrogen). Clones were
arrayed into 96-well plates and stored as glycerol stocks. Minipreps were performed with
a Manual PerfectPrep Plasmid 96 Vac Kit (5 PRIME) 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).
For semi-quantitative RT-PCR analyses, C. richardii mutant gametophytes grown
on 2.5% glucose or no glucose medium for 8 to 13 days were flash frozen in liquid
nitrogen. Total RNA was extracted from these gametophytes as described above. Total
RNA purity and integrity were verified via A260/A280 and A260/A230 readings
(NanoDrop 2000, Thermo Scientific) and native gel electrophoresis, respectively.
Specific primers were designed based on apogamy library clone sequences, and in the
case of CrUBQ10, based on the publicly available sequence from a spore germination
48
Table 1 Primer sequences for semi-quantitative and RT-qPCR analyses
Sequence Name
Primer Sequence
Glycine Rich Protein LP
5’-CTCGCTGTTCTCTTCCACTTG-3’
Glycine Rich Protein RP
5’-AGGACCAGGAACATGAATGC-3’
P3F12 LP
5’-GTGCTCCCTCATCTCGTTTC-3’
P3F12 RP
5’-CAAACTCAGGCTGGCTTCTC-3’
UNC93-like LP
5’-CACTTCTGCTGCTAAAAGTTATGC-3’
UNC93-like RP
5’-CATGCAACCAAGAAATGTAAGG-3’
PHD Finger LP
5’-CGTTCTTTGATTCTCCCTCTG-3’
PHD Finger RP
5’-CAGGACGAAAACGGACATAG-3’
RNA Recog. Motif LP
5’-GGCGGATTCGGAGTTATTG-3’
RNA Recog. Motif RP
5’-GCTGAAAGATCATGGGGAAC-3’
Snf2-like LP
5’-AGGAGGCATACGGATGAAGA-3’
Snf2-like RP
5’-GCTTCTTGGGGAGGTTATGA-3’
Sugar Transporter LP
5’-TCTGCACAGTTTACCAAAATGG-3’
Sugar Transporter RP
5’-TGTCACAGAAGATTAGTGTCTCCAG-3’
Leunig-like LP
5’-GAGAGGTACGGCTGAACTGC-3’
Leunig-like RP
5’-CACAATGGAAACGCACAGTC-3’
Glycine Clvage Syst. H LP
5’-CCTGTGGGTGGTAAGGTGAT-3’
Glycine Clvage Syst. H RP
5’-GCCCCTCATAATCAGAAGCA-3’
Nucleolar Essential LP
5’-GAAACAGCCAGTCACAAAACATC-3’
Nucleolar Essential RP
5’-GCACCAACCACAAATACCATC-3’
RPN9 LP
5’-TGGGTTCAGCCAAGAGTTTT-3’
RPN9 RP
5’-AGACGGACAAGGGTTTTATGC-3’
HMG Protein LP
5’-TGCCTTTTTCGTGTTCCTTG-3’
HMG Protein RP
5’-AAAGCCAATATGACAGAGGTTCC-3’
BTB-domain Containing LP
5’-AGGTCCCTGGAACAACTGATT-3’
BTB-domain Containing RP
5’-CAGCAGCAAATGATTGTGAGT-3’
49
library (Salmi et al., 2005; Table 1). The exponential amplification range of all targets
was determined by removal of aliquots of PCR amplifications every 2 cycles followed by
gel electrophoresis and ethidium bromide staining. For each gene, the cycle at which
amplicons became visible with ethidium bromide staining (using 12-day gametophyte
cDNA as template) was used for subsequent PCR analyses. Both control and
experimental primers were used in the same PCR reaction tubes, and final PCR products
were run on 1% Agarose gels, stained with ethidium bromide and imaged under UV light.
Relative expression was determined using ImageJ 1.43r (Abramoff et al., 2004). Briefly,
after calibration, gel lanes were selected and plots 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 (UBQ10) area to
experimental area was calculated. The highest ratio was arbitrarily set as 1, and all other
ratios calculated relative to that. Graphs were created using Microsoft Excel for Mac
2004 (Microsoft corporation) and finished with Adobe Photoshop CS4 (Adobe Systems
Inc.).
For RT-qPCR, C. richardii hermaphrodite mutant gametophytes grown on 2.5%
glucose or no glucose medium for 9 to 12 days were flash frozen in liquid nitrogen. Total
RNA was extracted and its quality verified as described above. 1 µg of total RNA was
used to create first strand cDNA using 9-nucleotide random primers. 2 µL of a 1/10
dilution of the resulting cDNAs was used for qPCR reactions. Specific primers were
carefully designed based on the corresponding apogamy library clone sequences, and
tested for Tm, the amplification of predicted products, and the production of primer
dimers before qPCR analysis (Table 1). The primer concentration in the final qPCR
50
reactions was 1 µM. The qPCR experiments were executed using the LightCycler 480
SYBR Green I Master kit (Roche Applied Science) and the LightCycler 480 Real Time
PCR System (Roche Applied Science). The cycling parameters were a 10-minute
denaturing step at 95°C followed by 45 cycles of 10 seconds at 95°C, 20 seconds at 58° C
annealing, and 15 seconds at 72° C for extension, with a single fluorescence read at the
end of each extension. A melting curve analysis was performed, and analyzed using the
Tm calling software module to generate melting peaks at the end of each run to verify the
absence of significant primer dimers or other non-specific PCR products. Within each
plate of qPCRs, pairs were set manually and raw data was analyzed using Crossing point
(Cp) calculation and relative quantification using the advanced relative quantification
software modules. Cp calling was obtained by the second derivative max method and
standard curve settings were set to use the default PCR efficiency. The LightCycler 480
software calculated relative quantification automatically and the resulting data and bar
graphs were exported. The highest ratio for each gene/reference pair was arbitrarily set to
1, and the results for all other gene/reference pairs relative to that, and standard errors
were adjusted accordingly. The final bar graphs were created in Microsoft Excel for Mac
and were finished using Adobe Photoshop CS4. The data for this experiment is a
combination of 2 biological and 2 technical replicates.
Whole mount in situ Hybridization
For probe synthesis, plasmids were linearized overnight with BamHI (NEB) or
XbaI (NEB). Complete linearization was verified with gel electrophoresis. 1 µg of
purified, linear plasmid was subsequently used in probe synthesis with T7 (Stratagene) or
SP6 (Stratagene) RNA polymerases and DIG RNA labeling mix (Roche) to generate
51
antisense and sense probes. Probes were precipitated with 3M LiCl to remove enzyme
and template, and after resuspension in DEPC H 2 O, quantified with the NanoDrop 1000
(Thermo Scientific).
C. richardii gametophytes were fixed under vacuum for 10 minutes in ice cold
formaldehyde, acetic acid, and alcohol (FAA), then fixed overnight at 4°C in fresh FAA,
and then stored at -20°C in 75% ethanol. For all steps, gametophytes were kept in small
baskets made from 0.2 mL tubes for PCR, cut in half, with 70-micron nylon mesh glued
to the bottom. These baskets facilitated the movement of gametophytes through the
various treatments and washes, which in prehyb steps were performed in 96 well plates.
For prehybridization, gametophytes were moved through a 3-step ethanol series into PBS
pH 7.5, 20 minutes for each wash, permeabilized in 0.2M HCl for 20 minutes, neutralized
with 2x washes in PBS, and treated with 0.1 mM Proteinase K for 10 minutes at room
temperature. Proteinase K digestion was stopped with three 2 minute washes in PBS, 1%
glycine, and PBS. Gametophytes were then postfixed in 4% paraformaldehyde for 20
minutes at room temperature. After a 90 min. prehybridization at 55°C in hybridization
solution, hybridization was performed for 15-18 hours in screw cap tubes set in a 55°
water bath with 100 µL hybridization solution (6x SSC, 3% SDS, 50% formamide, 0.1
mg/mL tRNA) and 1 ng/µL denatured probe. Posthybridization washes were performed
in screw cap tubes. 2-10 minute low stringency washes (0.2x SSC, 0.1% SDS) at 55°C,
and 1-2 minute high stringency wash (2x SSC) at room temperature with gentle agitation.
RNase digestion was performed with 10µg/mL RNaseA in 2x SSC for 30 minutes at
37°C, and then gametophytes were rinsed for 2 minutes in 2x SSC at room temperature.
Three final washes were performed: low stringency for 10 minutes at 55°C, high
52
stringency for 2 minutes at room temperature, and Tris-buffered saline (TBS pH 7.5) for
2 minutes at room temperature. Gametophytes were then incubated for 2 hours in 1%
blocking solution (Roche) for 2 hours, then 2 hours in a 1/1000 dilution of anti-DIG AP
fab fragments (Roche) in 1% blocking solution, washed 4 times for 10 minutes each with
agitation at RT (100 mM Tris-base pH 7.5, 150 mM NaCl, 0.3% (v/v) Tween 20).
Detection was performed with 1x color substrate solution (NBT/BCIP, Roche) in
detection buffer (100 mM Tris-base pH 9.5,150 mM NaCl), with overnight incubations.
Gametophytes were mounted in 50% glycerol and photos for each experiment were taken
under the same light and exposure conditions on a Leica DM LB microscope with
imaging system, and exported to Adobe Photoshop CS4.
Results
Strategy to Identify Genes Enriched During
Apogamy Commitment
Using the optimized conditions for apogamy induction in C. richardii, and the
timing of apogamy commitment under the experimental conditions (Chapter 2; Cordle, et
al. 2007), I designed a strategy to identify genes that may play important roles in
apogamy commitment. I employed the SSH method to normalize over-represented
mRNA sequences from, and amplify only those sequences that are enriched in, the 12day-old gametophyte transcriptome (the experimental cDNA population, after apogamy
commitment) as compared to the 9-day-old transcriptome (the driver cDNA population,
before apogamy commitment). I call this normalized, subtracted cDNA library the
apogamy library.
53
The C. richardii genome is not sequenced, and sequence information is limited to
approximately 3000 ESTs in public databases. Therefore, to learn more about the
functions of the genes in the apogamy library, I chose to identify their closest
Arabidopsis homologues using BLASTx of the apogamy sequences against the NCBI
non-redundant Arabidopsis protein database, and examine the expression patterns of
these Arabidopsis genes in silico by compiling vast publicly available microarray data
using Genevestigator (Zimmerman et al., 2004; Zimmerman et al., 2005).
Validation of the Apogamy Library
The successful normalization and subtraction of the library was validated by
colony hybridization using a probe which is specific to the C. richardii RuBisCo small
subunit, which without normalization, would be expected to constitute approximately
50% of the cloned cDNA sequences (Ellis, 1981). I found that this sequence comprised
0.2% of the apogamy library clones (2 out of 902 total clones), representing a more than
200-fold normalization and/or subtraction.
To confirm that the apogamy library represents transcripts enriched in day 12
gametophytes compared with day 9 gametophytes, 5 library clones whose homologues in
Arabidopsis show flower or embryo-specific or embryo-enhanced expression (Fig. 17)
and one clone that has no apparent homologue in other organisms (P3F12) were chosen
for relative RT-PCR analysis (Fig 12). All of the sequences chosen showed higher
expression in day 12 gametophytes compared to day 9 gametophytes in this experiment
(Fig. 12). However, not all of these sequences increased steadily over that time period,
for instance the sequences encoding a glycine-rich protein and an RNA recognition
motif-containing protein appear to peak in expression in day 11 gametophytes, and have
54
Figure 12 Relative RT-PCR analyses of select sequences from the apogamy library.
Expression levels are relative to a UBQ10 internal standard. Experiment
was performed once.
55
Figure 13 RT-qPCR analyses of select sequences from the apogamy library.
Results are the combination of 2 technical and 2 biological
replicates. Asterisk indicates a statistically significant increase in
gene expression (Student’s t-test; p < 0.05)
56
decreased in expression by day 12 (Fig. 12). Nevertheless, the expression levels of these
genes, and all others, were higher at day 12 compared to day 9, which suggests that the
apogamy library contains genes genuinely upregulated during this time period. The
expression patterns of these same genes in gametophytes cultured on non-inductive basal
fern medium are very different than the glucose-grown gametophytes (Fig.12). Indeed, on
basal medium, they all appear to be expressed at a lower level in day 12 gametophytes
compared to day 9 gametophytes (Fig. 12). Differing expression patterns between basal
and rich medium would be expected of genes that are necessary for the induction of
apogamy, or for those genes that are directly or indirectly regulated by glucose.
Three of these genes, plus 7 other library clones, were chosen for RT-qPCR
analysis (Fig. 13). These 10 genes represent diverse molecular function activities, based
on their slimmed plant Gene Ontology mappings (Fig. 15). Specifically, the molecular
functions mapped to these sequences are transcription factor activity, hydrolase,
transporter, RNA binding, protein binding, and enzyme regulator activity. Also, the
molecular function of the sequences that encode the senescence associated protein,
UNC93-like protein, and nucleolar essential protein are unknown. The RT-qPCR
experiments are the combination of 2 technical and 2 biological replicates, and all
sequences examined show a significant increase in expression from day 9 gametophytes
to day 12 gametophytes (Fig. 13). These experiments provide further evidence that the
clones in the apogamy library represent genes that increase in expression during the time
period when C. richardii gametophytes are undergoing apogamy commitment.
57
Composition of the Apogamy Library
From 441 sequenced clones, 306 unique sequences (unigenes) were identified
(Table 2). Among these unigenes, 156 had significant BLASTx hits (defined in this study
as an E-value at or below 1E-3) against the NCBI non-redundant protein database using
the Blast2GO 3000 program (Conesa et al., 2005; Conesa and Gotz, 2008). Among the
remaining 150 unigenes, 14 could be identified with significant BLASTn hits against
NCBI land plant reference RNA sequences or with BLASTx hits against JGI genome
databases (Physcomitrella patens and Selaginella moellendorffii). The remaining 136
unigenes had no significant hits from any database. All but 4 of the sequences without
significant hits were smaller than 200 nucleotides and/or contained long poly(A)+ strings.
The 4 longer unigenes were between 374-595 nucleotides and, not having significant hits
from any database, including the P. aquilinum gametophyte transcriptome (Der et al.,
2011), are presumably C. richardii specific. Alternatively, they may be present in P.
aquilinum but expressed only in the sporophyte, or expressed in the P. aquilinum
gametophyte but not detected in the transcriptome analysis by Der et al. (2001).
The 170 unigenes identified with confidence were chosen for further analysis. In
the process of identifying these 170 genes, the top BLASTx hits for each sequence were
collected and analyzed (Fig 14). To prevent bias, the top hits included in this table are
restricted to those whose genomes are completely sequenced, in assembly, or that have
large-scale EST projects completed (at least 25,000 ESTs in GenBank). 156 genes from
the apogamy library had top blast hits among these organisms and 3 species dominate
these top hits, the moss P. patens, the conifer Picea sitchensis (Sitka spruce), and the
lycophyte S. moellendorffii. These three organisms comprise over 50% of the top BLAST
58
Table 2 The 170 apogamy library clones identified with confidence
Clone
name
Clone Identity
Clone
length
(nt)
Best Hit
E-value
Query
Coverage
(%)
P1A10
lipid transfer protein
398
8.10E-06
42
P1A11
casein kinase 2 subunit beta
129
1.50E-15
100
P1A12
heat shock 70
266
1.90E-15
82
P1A2
glycine-rich protein
803
8.30E-17
69
P1A7
transketolase
485
1.70E-24
41
P1A9
pyrophosphate-dependent
phosphofructokinase beta subunit
383
2.40E-50
98
P1B11
60S ribosomal subunit
164
1.10E-07
98
P1B3
calcium-dependent phosphotriesterase-like
636
9.40E-13
48
P1B7
glutathione S-transeferase
388
5.30E-26
98
P1B8
nitrite reductase
306
5.30E-26
67
P1B9
uncharacterized protein
186
2.20E-11
100
P1C10
fructose bisophosphate class I
168
7.70E-17
98
P1C12
L-ascorbate peroxidase
236
5.50E-31
99
P1C2
thylakoid lumenal protein
112
2.00E-06
77
P1C4
catalase
117
1.50E-12
97
P1C6
60S L7A ribosomal protein
1103
3.30E114
70
P1C7
translation initiation factor
200
1.00E-08
99
P1C9
flavoprotein monooxygenase
247
1.20E-09
53
P1D1
chlorophyll a/b binding protein
570
3.60E-55
59
P1D11
calreticulin precursor
263
1.00E-37
99
P1D12
light rearvesting complex II protein LHCB6
320
1.2E-6
39
P1D4
UDP-glucoronosyl transeferase family protein 264
1.9-07
79
P1D5
glutathione S transferase
1039
60E-64
66
P1D9
delta tonoplast integral protein
516
3.00E-35
66
P1E10
serine esterase domain-containing protein
465
7.10E-10
43
P1E11
phosphoglycerate kinase
517
9.40E-53
66
P1E12
immunophilin
144
3.80E-11
79
P1E5
cytoplasmic hydroxyacylglutathione
hydrolase
349
7.80E-25
91
P1E6
plastid transcriptionally active (PTAC17)
431
1.00E-06
20
59
Table 2 Continued
P1E7
carbonic anhydrase
425
3.00E-29
84
P1F2
10 kd photosystem II polypeptide
295
3.90E-25
85
P1F4
cytochrome C
821
6.40E-44
48
P1F8
FTSH10 protease
279
7.90E-14
90
P1G10
multidrug resistance protein
153
1.70E-11
98
P1G4
augmenter of liver regeneration
327
1.00E-13
35
P1G7
hydroxycinnamoyl shikimate/quinate
hydroxycinnamoyltransferase-like
486
2.30E-42
99
P1G8
uncharacterized protein
685
5.10E-72
84
P1H1
alpha-expansin 15
808
9.20E-64
69
P1H10
transducin family protein
249
2.60E-17
86
P1H11
eukaryotic translation initiation factor SUI1
family protein
314
2.30E-13
45
P1H9
rhodanese domain-containing protein
559
6.80E-64
92
P2A1
ribosomal protein L1
285
1.00E-05
31
P2A11
HMGB1 transcription factor
628
4.70E-15
22
P2A12
DEAD box RNA helicase
114
5.00E-06
60
P2A2
acyl-synthetase
946
4.80E-44
40
P2A4
cysteine protease
493
1.30E-19
43
P2A7
BTB domain-containing protein
405
1.90E-07
45
P2A9
cathespin B-like cysteine protease
217
1.6E-14
66
P2B1
60S ribosomal protein L17
334
1.6E-27
82
P2B12
24-sterol-C-methyltransferase
448
10E-16
50
P2B3
elongation factor TU
237
1.50E-28
98
P2B6
chloroplast light-harvesting complex II
protein
125
1.50E-12
98
P2C2
small nuclear riboprotein F
630
1.80E-35
36
P2C5
LURP1-related protein
906
3.30E-39
68
P2C7
26S proteasome non-ATPase regulatory
subunit 13 (RPN9)
447
3.20E-23
40
P2C8
26S proteasome regulatory subunit
814
8.10E100
97
P2D1
citrate synthase
230
7.80E-33
99
P2D10
glutaredoxin-related protein
538
8.20E-29
55
P2D12
elongation factor 1A
1040
1.90E137
72
P2D2
zeanthin epoxidase
98
2.00E-07
97
60
Table 2 Continued
P2E12
flavanoid 3'-hydroxylase (transparent testa 7) 562
1.00E-35
98
P2E11
70 kD DNA-binding replication protein A70
415
2.80E-27
99
P2E3
chloroplastic NADPH-protochlorophyllide
oxidoreductase
297
5.70E-44
94
P2E4
ribulose bisphosphate carboxylase oxygenase 1022
activase
2.70E104
65
P2E5
40S ribosomal protein
374
4.70E-06
69
P2E6
flavonoid 3-hydroxylase
562
7.70E-39
98
P2F1
GDSL esterase/lipase protein
227
7.10E-10
99
P2F4
boron transporter
191
3.60E-06
98
P2F5
ankyrin repeat domain-containing protein
854
2.70E-56
58
P2F9
peptidylprolyl isomerase
144
2.00E-14
79
P2F8
geranylgeranyl diphosphate reductase
203
1.00E-08
48
P2G10
HTPG chaperone-family protein
189
6.00E-25
100
P2G11
uncharacterized protein
488
5.90E-65
99
P2G12
snakin-1 (gibberellin-regulated protein)
375
3.20E-18
69
P2G4
cyclin-dependent kinase regulatory subunit
313
4.60E-22
77
P2G5
uncharacterized protein
189
3.60E-06
66
P2G9
sodium-bile acid cotransporter-like protein
327
2.00E-25
80
P2H6
23S ribosomal RNA
113
1.60E-06
90
P2H7
endo-beta-D-glucanase precursor
231
2.60E-12
97
P3A1
alpha-beta-hydrolase domain-containing
protein
734
2.30E-93
91
P3A10
lanatoside 15-O-acetylesterase
223
1.90E-10
73
P3A2
chlorophyll a oxygenase
332
4.80E-11
47
P3A4
rubisco subunit binding protein beta
673
3.80E-24
32
P3A6
peroxisomal-2-hydroxy-acid oxidase 2
481
4.30E-20
47
P3A8
SCF ubiquitin ligase
233
3.50E-09
48
P3B1
alpha-glucan phosphorylase
317
7.30E-47
99
P3B10
LisH/CRA/RING-U-box domain-containing
protein
159
4.90E-19
100
P3B12
60S ribosomal protein
398
1.10E-10
48
P3B4
senescence-associated protein
215
2.70E-14
97
P3B5
ubiquitin-conjugating enzyme E2 36
215
5.30E-18
62
P3B8
eIF2 beta subunit protein
605
2.40E-26
31
P3C1
early nodule-specific protein
392
8.40E-24
99
61
Table 2 Continued
P3C10
long chain fatty acid CoA ligase
467
4.00E-42
40
P3C3
protochlorophyllide reductase
392
5.00E-06
19
P3D1
actin
405
9.30E-63
99
P3D12
protein phosphatase
236
4.40E-20
82
P3D5
sec61 transport protein
495
2.10E-70
82
P3E10
hypersensitive response protein
117
7.00E-13
97
P3E1
60S acidic ribosomal protein P0
216
1.30E-24
98
P3E11
fructose-bisphosphate aldolase
408
1.10E-44
70
P3E12
rRNA 2'-O-methyltransferase (fibrillarin 2)
418
3.60E-70
99
P3F3
dienelactone hydrolase family protein
231
4.0E-11
97
P3F1
actin depolymerizing factor
134
4.0E-51
21
P3F4
map 4 kinase alpha 1
563
90E-06
93
P3E3
non-yellowing protein 1
834
7.20E-06
21
P3F11
methionine synthase
112
9.30E-10
91
P3G2
phytochrome-associated serine/threonine
protein phosphatase
193
7.80E-25
93
P3G3
uncharacterized protein
230
3.90E-16
97
P3G5
pyrophosphate-energized vacuolar membrane 455
protein
1.10E-63
99
P3H11
14-3-3 protein
197
1.80E-26
98
P3H3
DNAJ heatshock protein
249
1.00E-10
24
P3H4
photosystem I protein
159
2.30E-21
98
P4A6
hydroxymethylbutenyl 4-diphosphate
synthase
649
2.70E-69
71
P4A8
macrophage migration inhibitory factor
94
2.00E-07
98
P4B2
superoxide dismutase
374
5.20E-53
95
P4B3
RNA recognition motif protein
322
8.80E-37
98
P4B12
copper transporter
215
2.00E-06
39
P4C2
thylakoid lumenal 29.8 kDa protein
1036
2.90E-56
53
P4C3
zinc-binding hydrolase
329
2.00E-09
17
P4C5
beta-glucosidase G3
315
1.90E-10
49
P4D1
plastidic glucose transporter
540
1.20E-22
37
P4D11
calcium-dependent phosphotriesterase-like
635
4.90E-12
48
P4D9
nucleolar essential protein
317
1.90E-42
98
P4E11
40S ribosomal protein
118
2.00E-08
71
P4E2
S-adenosyl methionine synthetase 2
487
5.90E-33
48
62
Table 2 Continued
P4E7
ran-binding zinc finger domain-containing
protein
800
2.30E-06
19
P4E9
adenosine kinase
330
2.60E-33
80
P4E10
aminocyclopropanecarboxylate oxidase
362
2.00E-04
9
P4F12
DNAJ heatshock N-terminal-domaincontaining protein
397
5.60E-23
92
P4F4
pentacopeptide repeat-containing protein
378
6.50E-24
71
P4F8
DNAJ heatshock protein
534
2.10E-13
50
P4G11
soluble starch synthase I
477
4.50E-33
54
P4G12
chlorophyll a/b binding protein
269
3.90E-37
84
P4G2
3,5-epimerase-4-reductase
207
1.20E-30
100
P4G3
magnesium cheletase subunit D
327
3.10E-42
99
P4G7
uncharacterized protein
97
2.00E-09
86
P4G8
histone H1 linker
818
3.00E-09
15
P4H3
transcription factore Hecate2
320
4.00E-04
43
P4H8
translation elongation factor I alpha
783
1.30E115
83
P5A10
peroxin 22
307
1.30E-13
44
P5A2
ferredoxin-NADP+ reductase
432
4.20E-15
30
P5A4
60S ribosomal protein
115
3.40E-12
96
P5A6
S-adenosyl methionine synthetase
664
7.00E-76
75
P5B12
BP5A protein
237
3.50E-09
50
P5B2
pentacopeptide repeat-containing protein
422
3.20E-26
88
P5B8
glycine dehydrogenase
264
7.00E-11
34
P5B9
photosystem I reaction center subunit IV-A
375
1.90E-10
28
P5C1
ATP-citrate lyase A-1
480
5.40E-47
74
P5C12
biotin carboxyl carrier protein
742
3.50E-40
70
P5C4
eukaryotic translation initiation factor 2gamma
494
4.50E-57
78
P5C5
30S ribosomal protein S13
428
5.20E-13
41
P5D12
glycine cleavage system H protein
222
2.40E-13
91
P5D5
monodehydroascorbate reductase
601
1.20E-65
93
P5D9
DNA photolyase protein
241
3.60E-14
75
P5E2
UNC93-like
237
6.80E-13
100
P5E4
RNA binding protein 47C
233
2.80E-19
97
P5E6
light harvesting complex
408
1.70E-24
74
63
Table 2 Continued
P5E7
sugar transporter
280
5.00E-07
45
P5F10
chlorophyll a/b binding protein 1
243
6.50E-32
91
P5F11
delta-aminolevulinic acid dehydratase
107
9.30E-10
98
P5F12
ketol-acid reductoisomerase
471
2.70E-54
93
P5F2
plastid transcriptionally active 4 (PTAC4)
206
8.00E118
53
P5F7
malate dehydrogenase 2
348
2.20E-48
99
P5F9
transducin/WD40 domain-containing protein
258
3.60E-30
97
P5G11
chalcone synthase
142
1.70E-16
99
P5G9
senescence-associated protein
294
1.60E-46
98
P5H12
translation elongation factor TS
187
2.80E-14
83
P5H2
chlorophyll a/b binding protein
235
1.10E-18
63
P5H9
30S ribosomal protein S31
323
3.30E-07
62
hits for the 156 genes (Fig. 14a). The remaining top BLAST hits were a variety of
angiosperms and, surprisingly, one gene in Carboxydothermus hydrogenoformans, a
thermophilic bacterium (Fig. 14a and b). This sequence is predicted to encode a glycine
cleavage system H protein, and, although this particular region of the sequence is most
highly similar to an extremophile, it is unlikely to represent contamination. The sequence
could be amplified from two separate biological samples of gametophytes in RT-qPCR
analysis, and shows increasing expression over the 9-12 day apogamy commitment
period (Fig. 13). Overall, though, the majority of the sequences identified in the apogamy
library are related to dicotyledonous angiosperms (Fig. 14b). Monocots and dicots
together comprise 38% of the top hit species from the sequence identifications (Fig. 14b).
GO-slim Mapping and Comparative Analysis with the
P. aquilinum Gametophyte Transcriptome
The 163 unigenes were annotated with GO terms, enzyme codes, and InterPRO
domain/motif information. Annotations were then mapped to their parent plant GO-slim
64
Figure 14 Top hit species and classifications. (A) The number of genes from the
apogamy library with best E-value BLASTx best hits from the given
organisms. Only organisms with genome sequencing completed, in
assembly, or with large-scale EST projects were included. (B) The
classifications of the species from (A) into their respective land plant
phylogeny clades.
65
terms (Fig. 15). 58% of the evidence codes were inferred from electronic annotation,
16.5% from direct experimental evidence (direct assay), and 6.6% and 5.8% from
sequence or structure similarity and reviewed computation analysis, respectively (data
not shown), accounting for over 85% of the annotations. Within the biological process
domain, the term mapped to approximately 13.5% of the apogamy library unigenes is
response to stress (22 unigenes), followed by 12.3% involved in catabolic processes (20
unigenes), and 10.4% (17 unigenes) each involved in generation of precursor metabolites
and energy, translation, and photosynthesis (Fig. 15a). 40.5% of the apogamy library
unigenes are mapped to the plastid (66 unigenes), the term that dominates the cellular
component domain, followed by approximately 12.3% (20 unigenes) each mapped to the
mitochondrion and thylakoid (Fig. 15b). Over 50% of the terms under the molecular
function domain are mapped to nucleotide binding (20.2%, 33 unigenes), protein binding
(17.2%, 28 unigenes), and hydrolase activity (15.3%, 25 unigenes) (Fig. 15c).
It would be interesting to compare the apogamy library plant GO-slim terms with
those mapped to the whole C. richardii genome to learn which terms are overrepresented
in the apogamy library. However, the C. richardii genome is not sequenced and the EST
coverage is limited to 5125 clones representing approximately 4000 genes from
sporophyte (sporophyll) tissue and germinating spores (Salmi et al., 2005). Therefore, I
performed a statistical assessment of the differences in GO-slim terms between the
apogamy library and the gametophyte transcriptome of the homosporous fern P.
aquilinum (Der et al., 2011), the only large sequence data set for any fern species. I found
a number of plant GO-slim categories that are over-represented (Fisher’s Exact Test, p <
0.05) in the apogamy library compared to the P. aqulinum gametophyte
66
Figure 15 Distribution of plant GO-slim categories mapped to the 170 unigenes in
the apogamy cDNA library.
67
transcriptome. These categories are shown in Figure 16. The biological process
categories over-represented are mainly involved in metabolism (secondary metabolic
process, catabolic process, and photosynthesis) and stress response (response to stress,
abiotic stimulus, biotic stimulus, and external stimulus) (Fig. 16a). This is consistent with
previous reports that suggest an increase in metabolic processes and stress response
mechanisms underlie the induction of haploid sporophytes from gametophyte cells
(Raghavan, 1989; Segui-Simarro, 2010). Also over-represented in the apogamy library,
compared to the P. aquilinum gametophyte transcriptome, are the embryonic
development and flower development plant GO-slim terms (Fig. 16a). This is interesting
because the source tissue for the apogamy library is, of course, fern gametophytes, and
these two GO terms are mapped to genes that function in early sporophyte development
and the development of angiosperm sporophytic sexual structures. Obviously, flowers are
structures that are not present in ferns, but structures that are homologous to flowers may
be present in the gametophytes of ferns. For instance, the antheridia may be analogous to
the anthers of flowers because the end product of both structures is sperm. The plastid
and thylakoid are among most highly over-represented GO-slim cellular compartment
terms mapped to the apogamy library compared to the P. aquilinum transcriptome (Fig.
16b). These proteins are likely involved in photosynthesis, which is also an overrepresented biological process term. Nucleolus and ribosome components and
translation/nucleic acid binding functions are also over-represented, consistent with an
increase in the production of ribosome and translation factor biological activity (Fig16a).
Components of the vacuole are also over-represented. The vacuole is an organelle that
enables plants to respond to changing environmental conditions (including stressors),
68
Figure 16 Apogamy library enriched GO-slim terms. (A) Biological process plant GO
Slim categories over-represented in the 12-day cDNA library (grey bars)
compared to the Pteridium aquilinum gametophyte transcriptome (black
bars). (B) Cellular compartment and molecular function plant GO-slim
categories over-represented in the 12-day cDNA library compared to the
Pteridium aquilinum gametophyte transcriptome. Statistical assessment of
annotation differences was performed using the Fisher’s Exact Test (p <
0.05) corrected for multiple testing.
69
store excess nutrients, and maintain optimal metabolic conditions in the cytosol
(Martinoia et al., 2006).
in silico Expression Patterns of Apogamy Library
Closest Arabidopsis Homologues
Using BLASTx against the NCBI Arabidopsis non-redundant protein database,
the closest Arabidopsis homologues of the 170 unigenes of the apogamy library were
identified. Only those sequences that returned BLASTx hit scores of 1E-5 or lower were
considered for this analysis; 8 sequences did not make this cutoff, so 162 Arabidopsis
genes were considered. The locus tags for these 162 genes were entered into the
Genevestigator program (Zimmerman et al., 2004; Zimmerman et al., 2005), and a
metaprofile analysis was performed. Genevestigator mines high quality microarray data
from public repositories and integrates that data into biologically meaningful gene
expression profiles (Zimmerman et al., 2004). All gene profiles were hierarchically
clustered, and from those clustered genes I isolated the profiles of those that are
expressed mainly, or upregulated in, the floral organs and seed structures (Fig. 17). These
genes could be separated into 4 general categories: those that have increased expression
in the seed and organs of the flower but are absent from the stamen and anthers (Fig
17A), those that have increased expression in the stamen and anthers and may or may not
show elevated expression levels in the seed and other flower organs (Fig 17B), those
genes that show elevated or specific expression in the embryo (Figure 17C), and genes
that show specific or highly elevated expression in other flower organs or seed structures
such as the seed coat, suspensor, ovule, petal, or sepal (Fig 17D). Among those genes
70
Figure 17 in silico expression patterns of Arabidopsis homologues of selected unigenes
from the apogamy library. Anatomical meta-profile heat maps were generated
with data mined from over 6000 high quality microarray experiments and
clustered using the Genevestigator program.
71
expressed mainly in the seed and flower organs and the genes that are specific or
upregulated in the embryo, are interspersed various ribosomal proteins and translation
initiation and elongation factors (Fig 17A and C). This is indicative of the importance of
de novo protein synthesis for these tissues and organs. Additionally, specific
transcriptional profiles for different ribosomal proteins are in line with previous reports of
developmental and tissue-specific expression for many proteins associated with
ribosomes (McIntosh and Bonham-Smith, 2006). In addition to ribosomal proteins, there
are also proteins that are associated with the nucleolus, such as the snoRNA binding
protein fibrillarin, and the nucleolar protein, both upregulated in the Arabidopsis embryo
(Fig. 17C). There may be novel developmental functions of the nucleolus that are not
fully understood (Pendle et al., 2005). Suggestive of this is the TORMOZ gene of
Arabidopsis, which is involved in the specification of the first planes of mitosis in the
developing embryo, whose homologues in S. pombe is predicted to function in 18S rRNA
biogenesis (Griffith et al, 2007).
Genes in the apogamy library whose homologues showed elevated or specific
expression in Arabidopsis flower and seed organs and tissues were chosen for whole
mount in situ analysis in C. richardii gametophytes. The reason for this approach was
two-fold: to investigate whether, as in Arabidopsis, these genes also showed increased
expression in homologous structures in the fern gametophytes, and to find those genes
that show a specific or increased expression in the mature C. richardii egg and/or
archegonia. I have made 3 observations which suggest that perceiving the inductive
signal for the development of apogamy in C. richardii gametophytes requires a functional
archegonia or egg. First, under my culture conditions, the first functional archegonia
72
begin to develop on C. richardii gametophytes between 8 and 10 days of culture. The
timing of apogamy commitment (10 days on inductive medium) approximately coincides
with this, occurring after 10 days of culture on inductive medium, suggesting the need for
mature eggs and/or archegonia, or perhaps “post-functional” eggs in the process of
degenerating after not being fertilized. Second, I have never successfully induced
apogamy from wild-type male gametophytes of C. richardii. Finally, and perhaps the
most convincingly, I have never successfully induced apogamy from the fem1-1tra2
mutant C. richardii gametophytes which posses an “intersex” phenotype, producing
normal antheridia with functional sperm, but abnormal archegonia and non-functional
eggs (Strain et al., 2001). Thus, it appears that the induction of apogamy depends upon a
functional egg and/or archegonium, suggesting that some genes that are expressed in
these structures are necessary for the inductive process.
Whole Mount in situ Analyses in C. richardii
Gametophytes
Whole mount in situ analyses were performed in C. richardii gametophytes for 13
sequences from the apogamy library whose homologues in Arabidopsis were
preferentially expressed in the seed or flower structures: glycine rich protein, heat shock
70, and RNA recognition motif-containing protein (Fig 17A); FTSH protease, WD40
repeat, 14-3-3- protein, protein kinase CK2 regulator (Fig 17B), UNC93-like protein,
nucleolar essential protein, zinc finger (Fig 17C). Also included in the in situ analyses
were two apogamy library sequences with interesting identity but were not identified with
high confidence: a sequence weakly similar to LEUNIG, a co-repressor of the floral
homeotic gene AGAMOUS also involved in lateral organ patterning in Arabidopsis (Liu
73
Figure 18 Whole mount in situs of nucleolar essential protein and RNA recognition motif
clones. Nucleolar essential protein antisense probes against 9-day (A) and 12day (B) gametophytes, in which a slight increase in staining intensity is
evident. Nucleolar essential protein sense probe control against a 12-day
gametophyte is depicted in (C). (D) Antisense RNA recognition motif
antisense probe against 9-day (D) and 12-day (E) gametophytes, and sense
probe control against a 12-day gametophyte (F). Very little difference in
staining intensity is detectable in these samples probes with the RNA
recognition motif probe.
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Figure 19 The BTB and FTSH protease whole mount in situs show preferential
expression in early embryo and gametophyte structures. An antisense BTB
probe (A) shows staining of an early sporophyte embryo (white arrow) 72
hours post-fertilization compared to sense control (D). Antisense FTSH
protease probe against 12-day gametophytes shows intense staining of
developing antheridia (B, white arrows) compared to mature antheridium
(B, black arrow). Antisense FTSH protease probes also show intense
staining of the archegonial cells (E, thick white arrows), but not the egg
cells at the base of the archegonia (E, thin white arrow). Rubisco small
subunit antisense (C) and sense (F) against 12-day gametophyte prothalli
are included here for signal to background comparisons.
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Figure 20 UNC93-like whole mount in situs show staining in egg cells of 12-day
gametophytes. UNC93-like antisense probes against 9-day (A)
gametophyte grown on rich medium has little signal, while antisense
probes against 12-day gametophyte (B) grown on rich medium shows
intense signal in the archegonial cells and the egg cells (white arrows), as
well as throughout the prothallus. UNC93-like sense probe against 12-day
gametophyte (C) shows no staining in prothallus, archegonia or eggs
(white arrows).
76
and Meyerowitz, 1995; Connor and Liu, 2000; Stahle et al., 2009), and a SNF2-like
sequence, a family of genes involved in chromatin remodeling and other functions in
plants (Knizewski et al., 2008). Additionally, a sequence very similar to a BTB domaincontaining gene in the moss P. patens, with no obvious homologue in Arabidopsis was
examined. 10 of these sequences showed no specific expression in gametophyte
structures, instead showing signal over the entire prothallus of gametophytes grown on
rich medium, and when detectable, an increase in expression from gametophytes cultured
for 9 days to gametophytes cultured for 12 days (Fig 18 A-F). The whole mount in situ
results for 6 representative sequences, including the 3 that show enhanced expression in
specific gametophytic structures, are shown in Figures 18-20.
The whole mount in situ results for most of the genes tested had very low levels
of staining, presumably due to the low expression levels of the genes, thus were difficult
to distinguish from background staining levels. A slight increase in staining could be
detected for most genes between 9 day and 12 day gametophytes, as can be seen in the in
situ expressions for the nucleolar essential protein clone (Fig. 18A-C) and the RNA
recognition motif clone (Fig. 18D-F). However, 3 clones showed specific expression
patterns in the structures of the gametophytes, or in early sporophyte embryos developing
from gametophytes grown on basal fern medium (Fig. 19 and 20).
It is conceivable that genes involved in triggering the process of apogamy may
also play a role in embryogenesis derived from syngamy. One sequence examined,
encoding a BTB-domain containing protein, was upregulated in the early embryo, 72
hours after fertilization (Fig. 19A). Antisense (Fig. 19C) and sense (Fig. 19F) staining for
the Rubisco small subunit transcript are included in this figure to aid the reader in
77
comparisons of positive staining from background staining. There are many BTB
domain-containing proteins in Arabidopsis, and it is unclear whether this C, richardii
sequence is homologous to any of the genes that encode BTB domain-containing proteins
in Arabidopsis. However, this sequence shows 25% amino acid identity and 40%
similarity to proteins in both the moss P. patens and the lycophyte S. moellendorfii. This
information, together with the evidence of its upregulation in the early embryo suggests a
role for this gene in early embryo development, perhaps specifically for lower plants.
Another sequence, encoding an FTSH protease, was found to be upregulated in
the young antheridia (Fig. 19B) and the archegonia cells (Fig. 19E, thick arrows), but
absent or expressed at a lower level in egg cells (Fig. 19E, thin arrows) of 12 day
gametophytes grown on 2.5% glucose. Interestingly, the Arabidopsis homologue of this
sequence is upregulated in the anthers, pollen and endosperm of Arabidopsis flowers and
seeds (Fig. 17B). The possibility that antheridia of ferns and anthers of flowers are
homologous structures is not surprising because both are structures in which sperm (or
pollen) develop. Perhaps more interestingly, FTSH protease expression in the endosperm
of Arabidopsis may suggest an evolutionary relationship between fern archegonial cells
and endosperm in angiosperm seeds.
Staining for the UNC93-like clone was observed throughout the prothallus of 12
day gametophytes grown on rich medium, compared to 9 day gametophytes (Fig. 20A
and B) Also in 12 day gametophytes, intense staining for the UNC93-like probe was
detected in the archegonia and the egg cells that sit at the base of the archegonia (Fig
20B, arrows point to eggs) compared to the sense probe (Fig. 20C). This is the only probe
that showed whole mount in situ staining of egg cells.
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Discussion
In this chapter I described the creation and analysis of a normalized, subtracted
cDNA library that represents genes that are upregulated during apogamy commitment in
the gametophytes of the fern C. richardii. Among the clones in this library, 170
sequenced clones could be identified with confidence through BLAST searches of NCBI
and JGI databases. I found that the 3 highest represented species in the compiled BLAST
top hit data were a moss, P. patens, a gymnosperm, P. sitchensis, and a lycophyte, S.
moellendorffii. It is perhaps not surprising that so many genes identified from the
apogamy library from C. richardii are most closely related to genes in a moss, lycophyte
and gymnosperm, considering the fact that within the land plant phylogeny, the position
of the ferns is nested between the branches of these three clades (Chapter 1, Fig. 2).
Angiosperms were the clade that represented the most top BLAST hits, with over
38% of the apogamy library genes showing the highest homology. Among the land plant
phylogeny, angiosperms are the most divergent from the basal lineages, and the most
distant relatives of the ferns (Chapter 1, Fig. 2). The fact that angiosperms comprise the
highest percentage of top BLASTx hits may be explained by the overrepresentation of
angiosperms, especially dicots, among those land plants that have sequenced genomes,
which can bias evolutionary comparisons (Jackson et al., 2006). In fact, 11 out of the 13
land plant families represented among plants with sequenced genomes are angiosperms,
which is likely a side effect of, historically, the almost exclusive emphasis on sequencing
of plants with small genomes and of economically important plant species (Jackson et al.,
2006). There are currently no ferns with sequenced genomes, and only recently P.
aquilinum with transcriptome data (Der et al., 2011). It is likely that, if this analysis were
79
repeated in the future when the genome sequences of more lower plants are completed,
the results would be dramatically different, showing a greater percentage of sequences
most highly related to other ferns and “lower” plants.
The GO-slim terms mapped to the apogamy library genes showed a
preponderance of terms involved in stress response, metabolism, translation and
photosynthesis. A comparison to the GO-slim terms mapped to the entire gametophyte
transcriptome of the homosporous fern P. aquilinum, proved these processes significantly
enriched in the apogamy library. In addition to these biological processes, the cellular
compartments and molecular functions that may be associated with these processes, such
as the plastid (metabolism), thylakoid (photosynthesis), vacuole (stress and metabolism),
and ribosome (translation), are also over-represented. An over-representation of genes
associated with the nucleolus may be interpreted as necessary for rRNA transcription and
processing for the ribosomes, but the nucleolus is also implicated in a multitude of RNA
processing functions and also the sensing of stress in plants (Brown and Shaw, 2008).
It would be ideal to compare the apogamy library to the transcriptome of the C.
richardi gametophyte, but the latter is not available for comparison. The only fern
gametophyte transcriptome available for comparison is that of P. aquilinum (Der et al,
2009). P. aquilinum is an homosporous fern, and its gametophytes are amenable to
apogamy induction and the process of apogamy has been thoroughly studied in this fern
using tissue culture techniques (Steeves et al., 1955; Whittier and Steeves, 1960, 1962;
Whittier, 1964; Whittier and Pratt, 1971; Whittier, 1975). Additionally, P. aquilinum is
relatively closely related to C. richardii, within the very large order of Polypodiales. P.
aquilinum is a member of the Dennstaedtiaceae family within the Polypodiales (Smith et
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al., 2006; Der et al., 2009), which is the sister family to Pteridaceae (Smith et al., 2006).
C. richardii is a member of the Pteridaceae family (Hasebe et al., 1995; Pryer et al., 1995,
Schneider et al., 2004; Smith et al., 2006; Schuettpelz et al., 2007) and its sister taxa
among these ferns are the Acrostichum, the only other aquatic species in this family
(Smith et al., 2006; Schuettpelz et al., 2007).
The in silico expression analysis of the apogamy library Arabidopsis homologues
uncovered a number of genes that were induced or specifically expressed in the flower
organs and/or seed structures. It is interesting to consider what those genes are doing in
the fern gametophytes. The common ancestor of land plants is likely to have had a
multicellular and dominant gametophyte generation (Niklas and Kutschera, 2010), and
the current hypothesis is that the evolution of the sporophyte body of land plants occurred
through the recruitment of genes involved in gametophyte function of their ancestors. In
line with this is the finding that nearly 70% of the genes in the genome of Arabidopsis
thaliana have homologues that are expressed in the gametophyte tissues of the moss P.
patens (Nishiyama et al., 2003). Of course, the genes in the apogamy library were
isolated from a gametophytic system that had been induced to produce sporophytes, so it
is not surprising to see genes from this library whose homologues are specific for
sporophyte structures in Arabidopsis. The interesting aspect of these observations is the
fact that these genes are being expressed in Arabidopsis structures that do not exist in
ferns. This may be evidence supporting the scenario that these genes have been recruited
from their ancestors to perform functions in the unique sexual structures of flowers in
angiosperms. It would be interesting to examine the expression patterns of the P. patens
and Arabidopsis homologues of genes showing preferential expression in the C. richardii
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early embryo (BTB), developing antheridia and archegonial cells (FTSH protease) and
egg cells (UNC93-like).
This does not preclude the possibility that the flower organs and seed structures of
angiosperms have homologous structures in ferns. When examining the expression
patterns of the FTSH protease clone, I found that this sequence is induced in the
developing antheridia and archegonial cells of 12-day gametophytes grown on 2.5%
glucose. The homologue of this gene in Arabidopsis shows upregulation in the anther and
pollen as well as in the endosperm of the seed. Pollen is the male gametophyte of an
angiosperm. Each pollen grain contains 2 non-flagellated sperms and one generative cell.
The pollen initial is formed via meiosis in the anther of the flower, followed by two
rounds of mitosis to form the three-celled pollen grains. The antheridia of fern
gametophytes is where the sperm develop via mitosis, and in C. richardii each
antheridium produces 32 coiled and flagellated sperms after 4 rounds of cell division and
subsequent development. These similarities in function suggest that the pollen (and
perhaps the anthers) of angiosperm flowers, and the antheridia of fern gametophytes, are
homologous structures, and the similarity in genes expression of FTSH protease between
the two organisms supports this view. However, while the final products for both are
sperm, antheridia and anthers may not go through homologous developmental pathways
to reach that end. The FTSH protease may be a gene that is generally involved in mitosis,
or some other process that is not specific for the production of sperm.
One could also consider the possibility that the archegonia, which form a canal
through which the sperm swim to fertilize the egg, which lies at the base of the canal, and
the endosperm of seeds, are homologous structures. The archegonium of ferns cradles the
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developing embryo as it develops, providing nutrients and protecting it until it is
independent. Angiosperms lack a morphologically distinct archegonium, but their
embryo sacs contain the binucleate central cell, which, as a product of double
fertilization, forms the triploid endosperm that nourishes the developing embryo upon
seed germination. Upon first comparison it may seem like the nucellar tissue or the
integuments surrounding the embryo sac are the analogous structures to the archegnium
in ferns (Chapter 1, Fig. 3). But these structures are sporophytic in origin, and as such
seem unlikely be analogous to the gametophytic archegonia of lower plants. The origins
of double fertilization and the triploid embryo of modern angiosperms has long been an
unresolved question in plant biology (Friedman and Williams, 2004). The major
questions are: what was the ancestral state of the female gametophyte? Did the earliest
flowering plants have double fertilization, and if so, did it create a triploid or a diploid
endosperm? The answer to these questions has changed as the resolution of the basal
lineages of the angiosperms has become clearer (Williams and Friedman, 2002; Friedman
and Williams, 2004). The current view is that the ancestral state of the embryo sac was a
4 celled structure that produced a diploid endosperm upon double fertilization, and that
the 7-celled, 8 nucleate embryo sac of modern angiosperms is a reiteration of the 4-celled
ancestral structure (Friedman et al., 2003; Friedman and Williams, 2003; Friedman and
Williams, 2004). In this scenario it is possible that the embryo sac itself may have
evolved from egg cell and a three-tiered archegonium, typical of most archegonial
structures in extant ferns (Bell, 1960; Raghavan, 1989). Indeed, the cell that comprises
the neck canal of the archegonium, which eventually breaks down to allow sperm to
enter, is generally binucleate in extant ferns, but there are some primitive ferns which
83
have mononucleate neck canal cells (Nishida and Sakuma, 1961; Raghavan, 1989). Thus,
one can imagine the canal cell as the progenitor of the central cell of the modern day
angiosperm embryo sac.
The whole mount in situ assays revealed the increased expression of the UNC93like gene in the eggs of the gametophyte prothallus undergoing apogamy commitment.
The expression profile of this gene fit the expectation of a gene that is likely involved in
apogamy induction in C. richardii, i.e., upregulated during apogamy commitment,
specifically expressed or upregulated in the egg cell, and having an embryo-specific
expression pattern in Arabidopsis. Furthermore, despite its ubiquitous distribution, the
function of this gene is unknown in plants. As such, I chose the UNC93-like gene to
investigate further in Arabidopsis. The details of these investigations are in Chapter 4.
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CHAPTER IV
THE UNC93-LIKE GENE IN ARABIDOPSIS
AND C. RICHARDII
Introduction
In the previous chapter I presented evidence of a gene, UNC93-like that has
increased expression during apogamy commitment in C. richardii gametophytes. This
gene also shows expression in the mature antheridia and eggs of gametophytes grown on
2.5% glucose. My hypothesis is that mature and functional eggs are necessary for C.
richardii gametophytes to become capable of apogamy (see discussion in Chapter 3). The
temporal and spatial expression pattern of the UNC93-like gene in C. richardii fits the
profile of a gene that may be fundamentally involved in the process of apogamy in this
fern. Additionally, in silico expression pattern analysis of the UNC93-like homologue in
Arabidopsis indicates that this gene is most highly expressed in the embryo, which is the
young sporophyte. This suggests a role for AtUNC93-like in early sporophyte growth
and/or development. While this does not directly implicate UNC93-like as a gene
involved in triggering asexual sporophytes in C. richardii, many genes involved in
zygotic embryo development, when mutated or ectopically expressed, induce asexual
embryo development (Mayer et al., 1998; Lotan et al., 1998; Hecht et al., 2001; Boutiliier
et al., 2002; Okano et al., 2009), so a gene induced during apogamy induction may also
play a role in zygotic embryo development. Another possibility is that the UNC93-like
gene product is essential for a mature egg and the latter indirectly controls apogamy. It is
equally possible that the UNC93-like gene product is essential for the early development
of both sexual and apogamous sporophytes. In either case, a better understanding of the
85
function of the UNC93-like gene in Arabidopsis will provide crucial information for how
it may function in apogamy in C. richardii. In this chapter I present the characterization
of the UNC93-like gene in Arabidopsis thaliana. Its function in plants is unknown.
The UNC93 gene was first discovered in Ceaenorhabditis elegans as a gene that
encodes a novel transmembrane protein involved in the regulation of muscle contraction
(Greenwald and Horvitz, 1980; Levin and Horvitz, 1992). A rare semi-dominant allele of
unc93 in C. elegans gives the characteristic sterile, sluggish, and uncoordinated
phenotype for which the gene is named, while unc93 null alleles have no phenotype
(Greenwald and Horvitz, 1980; Levin and Horvitz, 1992). Suppressors of the unc93
phenotype in C. elegans, sup9 and sup18, encode a two-pore potassium channel and a
novel transmembrane protein, respectively, suggesting that unc93 is a component of a
multiprotein complex involved in potassium transport (Greenwald and Horvitz, 1980,
1986; Levin and Horvitz, 1993; De Stacio et al., 1997, de la Cruz et al., 2003). Two other
suppressors of the unc93 dominant mutation encode a chromatin remodeling factor and
an inorganic phosphatase (Long and Horvitz, 2006). An UNC93::GFP fusion protein is
found in the membranes of the C. elegans body wall, vulval, anal, and intestinal muscles,
as well as in dense bodies, which are structures that perform structural and signaling
functions in muscle cells (de la Cruz et al., 2003; Lecroisey et al., 2007). The
UNC93::GFP construct is also found in 4 muscle neurons in the head (de la Cruz et al.,
2003).
In humans, there are two UNC93 paralogs. UNC93A is expressed in the testis,
ovary, small intestine and spleen, but the function of this gene has not yet been elucidated
(Liu et al., 2002). The UNC93B1 gene is upregulated in the heart and antigen-presenting
86
cells (Isnardi et al., 2008) This gene is involved in immunity by regulating nucleotidesensing Toll-like receptors. UNC93B1 transports a subset of these receptors from the ER
to endolysosomes where they activate signaling cascades and engage pathogens. (Isnardi
et al., 2008).
Potassium is the most abundant cation in plants, and unlike other soil cations, it is
not incorporated into the organic matter of the plant, instead serving as a major
osmoticum (Ho and Tsay, 2010). The physiological responses to potassium starvation
include growth arrest, impaired phloem transport of sucrose, and reduced water content
(Ho and Tsay, 2010). The reduced water content decreases turgor pressure, which is
required for cell expansion in the elongation zone of roots (Dolan and Davies, 2004).
Plants, therefore, have a number of root morphology responses to potassium starvation
including reduced primary root growth, decreased lateral root length and number, and
increased root hair elongation (Jung et al., 2009, Shin and Schachtman, 2004). How
plants detect and respond to changing potassium levels is not fully understood, but some
components of the potassium deficiency response have been elucidated. Under low
potassium conditions, an influx potassium channel, AKT1, and a high affinity potassium
transporter, HAK5, are responsible for potassium uptake (Rubio et al., 2008; Gierth et al.,
2005). ROS production by NADPH oxidase and a type III peroxidase mediates the
transcription of the HAK5 gene (Shin and Schachtman, 2004; Kim et al., 2010). ROS
production, the activation of HAK5, as well as the promotion of root morphological
responses, are eliminated in the absence of ethylene and in ethylene insensitive mutants,
indicating an important role for this gaseous plant hormone in potassium sensing (Jung et
al., 2009).
87
The internal transport of potassium relies on other potassium channel proteins that
are incorporated into the membranes and envelopes of organelles. The Arabidopsis
genome contains 15 genes whose proteins are capable of forming potassium channels
(Lebaudy et al., Szczerba et al., 2009). Based on their structure, there are 3 classes of
proteins: 9 relatively well characterized Shaker-like K+ channels, 5 tandem pore K+
(TPK) channels, and 1 K ir -like (KCO3) channel (Maser et al., 2001; Very and Sentenac,
2003; Ward et al., 2009; Voelker et al., 2010). The latter 6 proteins are targeted to the
vacuolar membrane (Czempinski et al., 2002; Schonknecht et al., 2002), with the
exception of TPK4, which is targeted to the plasma membrane and the ER (Voelker et al.,
2006; Latz et al., 2007). The proteins likely form homodimers (Voelker et al., 2006) and
most of them interact with 14-3-3 proteins in a phosphorylation-dependent manner (Latz
et al., 2007), which is also true for their animal orthologues (Plant et al., 2005). Many
details of potassium sensing, signaling, and transport in plant cells have yet to be
elucidated.
In this chapter I describe two transcripts that originate from the sole AtUNC93like gene in the Arabidopsis genome, and examine their spatial distribution in various
Arabidopsis tissues. I also describe the phenotypes of an AtUNC93-like T-DNA
insertional mutant line, detailing a number of vegetative, root, and reproductive defects.
Based on the prediction that the C. elegans UNC93 protein regulates a two-pore
potassium channel, I performed a number of experiments to investigate the possibility
that AtUNC93-like is involved in K+ uptake or sensing, and whether K+ plays a role in
the induction of apogamy in C. richardii gametophytes. My experiments and
observations reveal little evidence that the AtUNC93-like gene is involved in K+ uptake
88
in Arabidopsis, or that K+ plays a role in apogamy beyond supplying an essential nutrient
for growth. I do find, however, that the AtUNC93-like gene is involved in embryo
development, as a proportion of seeds undergo abortion before maturity, and that the
homozygous T-DNA plants are unable to maintain the viability of a proportion of their
gametophytes, whether male or female, but AtUNC93-like does not appear to play a
critical role in gametophyte function.
Materials and Methods
Plant Materials and Growth Conditions
Arabidopsis wild-type plants utilized in this study were Columbia ecotype. Seeds
for the AtUNC93-like homozygous T-DNA line, SALK_010430 were acquired from the
Arabidopsis Biological Resource Center (ABRC; Alonso et al., 2003). Homozygous TDNA lines for the 6 tandem-pore potassium channel genes in the Arabidopsis genome,
two of which are putative orthologs to sup9 and sup10 in C. elegans, were obtained from
ABRC. These lines are: SALK_146903 (TPK1), SALK_007975 (TPK2), SALK_049137
(TPK3), SALK_053858 (TPK4), SALK_123690 (TPK5), SALK_048607 (KCO3). The
C. richardii her mutant gametophytes were used for 3’ RACE and degenerate PCR, and
the self-sterile fem mutant was used for the apogamy induction experiments (Banks,
1994).
When grown in soil, Arabidopsis seeds were cold-treated at 4°C for 2-3 days on
filter paper soaked with 10 mM KNO3, and then sown on plant mix # 1 (Beautiful Land
Products, West Branch, IA). When cultured on agar medium, unless [K+] was varied,
both Arabidopsis plants and C. richardii gametophytes were grown on ½ strength MS
medium (pH 5.7 for Arabidopsis and pH 6 for C. richardii), supplemented with 0.5%
89
sucrose and 50 µg/mL ampicillin for the prevention of contamination. All Arabidopsis
growth conditions were under a 16-hour light, 8-hour dark schedule at 21-25°C. C.
richardii gametophytes were cultured under continuous light (100 µM m2 s-1) in a 28°C
incubator.
For Arabidopsis seed sterilization, seeds were agitated in 0.1% Tween 20 for 30
minutes, soaked in 100% ethanol for 2 minutes, 1.5% Na hypochlorite for 5 minutes,
rinsed 5 times in sterile H 2 O, and plated on the appropriate medium. Plates were stored in
the dark at 4°C for 2 days to stratify seeds before placing under the light conditions
described. C. richardii spores were sterilized as previously described (Cordle et al.,
2007), and plates dark treated for 3 days at room temperature before moving plates to the
incubator conditions described above.
Bioinformatics
The Arabidopsis UNC93-like protein sequences were acquired from the NCBI
protein database, and the AtUNC93-like predicted alternate transcript sequences are from
The Arabidopsis Information Resource (TAIR). Amino acid alignments were generated
with ClustalW2 (Larkin et al., 2007) with default parameters, and the UNC93-like
domain boundaries were defined with information from the Conserved Domain Database
(Marchler-Bauer et al., 2011). Transmembrane domain predictions and N- and C-terminal
domain conformations were generated with the PredictProtein program (Rost et al.,
2003). Potential protein interactions with Arabidopsis UNC93-like protein were
generated with the GeneMania program (Warde-Farley et al., 2010).
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Root growth assays, Fresh Tissue Weights
and K+ in Apogamy
For the root growth assay, Arabidopsis seedlings were grown on agar plates as
described above, with plates held steady at a 90° angle from horizontal. Photographs
were taken on the given days with the DinoXcope 1.4 (AnMo Electronics Corporation).
For obtaining fresh weights of shoots and roots, Arabidopsis seeds were sterilized,
plated on appropriate medium, and cold-treated for 2 days. After moving to light, plates
were positioned vertically and plants were grown for 15 days before removing tissue for
weighing. Aerial tissue was excised from roots; all tissues were blotted gently to remove
any moisture, and immediately weighed in batches.
The experiment examining the role of K+ in apogamy induction from C. richardii
gametophytes was performed on 0.8% agar and ½ strength MS medium, either with or
without the addition of 2.5% glucose. Gametophytes were transferred to fresh plates
every 14 days to prevent drying conditions, and total apogamy counts were made after 45
days of culture. Modifications were made to the [K+] by replacement of KNO 3 and
KH 2 PO 4 in the MS medium with NH 4 NO 3 and NH 4 H 2 PO 4 , respectively. KCl was added
to the medium to raise [K+] above that found in ½ MS medium (above 10 mM). There is
a trace amount of K+ in the agar used to solidify the medium (Xu et al., 2006), so the 0
mM K+ plates refer to the lack of K+ in the MS medium, but not a complete lack of K+
altogether. K+ - depleted medium for Arabidopsis plants was made in the same fashion
except 0.5% sucrose was added and the pH was adjusted to 5.7.
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Arabidopsis Seed Area and Seed Weight
For data on seed size, photographs of mature WT and unc93-like seeds were taken
with the DinoXcope camera (AnMo Electronics). Images were processed with ImageJ.
Briefly, images were converted to binary, threshold adjusted, and areas of outlined seeds
were measured. Seed area measurements are an average of 25 seeds for each WT and
unc93-like. Seed weights were obtained by measuring 10-20 mg of mature seeds,
counting the number of seeds, and dividing the weight by the number of seeds. Over 400
WT seeds and 800 unc93-like seeds are represented in this experiment.
RT-PCR, 3’ RACE, PCR, and Degenerate PCR
For RT-PCR, degenerate RT-PCR, and 3’ RACE, RNA extraction was performed
by grinding frozen tissues in liquid nitrogen to a fine powder and extracting RNA using
the guanidinium thiocyanate and acid phenol method (Chomczynski and Sacchi, 1987)
Before precipitation with isopropanol, 0.4x volume of a high salt solution (0.8M sodium
citrate, 1.2M sodium chloride) was added to minimize the precipitation of
polysaccharides. The RNA pellet was dissolved in freshly deionized formamide, heated
to 70ºC for 10 minutes in the presence of 0.7M NaCl and 1% CTAB, and extracted twice
with chloroform to remove precipitated polysaccharides. After adding 1x volume of
DEPC-treated water, total RNA was recovered with standard ethanol precipitation. RNA
quality and quantity was analyzed with the NanoDrop 1000 (Thermo Scientific) and
native gel electrophoresis. 300 ng of total RNA was used for cDNA synthesis with the
Superscript III enzyme (NEB) using 9-nucleotide random primers, for RT-PCR reactions,
and with the modified oligo (dT) primer for 3’ RACE (Table 3). 1 µL of the resulting
cDNA was used undiluted in subsequent 25 µL PCR reactions. For 3’ RACE, a
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subsequent PCR was performed with nested primers and a 1/500 dilution of the original
PCR products.
The degenerate RT-PCR product and 3' RACE products were cloned into the
pCRII vector (Invitrogen) and transformed into TOP10 (Invitrogen) E. coli cells. Plasmid
DNA was isolated and purified with the Wizard Plus Minipreps DNA Purification
System (Promega), and submitted for sequencing on the ABI 3730 DNA analyzer
(Applied Biosystems) at the Center for Comparative Genomics (University of Iowa).
For the determination of the genotypes of the reciprocal cross progeny, genomic
DNA was extracted from progeny. A single leaf was ground in a centrifuge tube with a
pestle and 600 µL of extraction buffer added (200 mM Tris-Hcl pH 7.5, 250 mM NaCl,
25 mM EDTA, 0.5% SDS) and briefly vortexed. Plant debris was pelleted in a tabletop
centrifuge for 1 minute and supernatant transferred to a fresh tube. 500 µ of isopropanol
was added and the mixture incubated at room temperature for 2 minutes. DNA was spun
down in a table-top centrifuge for 5 minutes. The pellet was dried gently and resuspended
in 100 µL TE. 1µl of the resulting DNA was used in 2 separate PCR reactions for each
plant: one using the AtUNC93-like gene specific RP and LP primers (amplicon indicates
a WT allele present in plant) and one using the AtUNC93-like RP and a primer specific
for the T-DNA border (amplicon indicates a T-DNA allele). PCR products were
separated on 1% agarose gels and visualized with ethidium bromide staining and UV
imaging.
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Table 3 Primers used for RT-PCR, 3’ RACE, PCR and degenerate PCR
Sequence Name
Primer Sequence
3’RACE gene specific
5’-CAGTCTGGGAAGGTCGATTC-3’
3’RACE nested gene
specific
3’RACE modified
oligo (dT)
3’RACE adapter
specific 1
3’ RACE adapter
specific 2
5’ gene specific
5’-CCTGTTACAACTTGGCCTCTT-3’
5’ degenerate
5’-TGGCATATCATGCAGCAcaraayytnsa-3’
AtUNC93-long LP
5’-CAGTTGAAGAAACACATTTCACG-3’
AtUNC93-long RP
5’-TCGAATCTGAATGACATGTTGAG-3’
AtUNC93-short LP
5’-GAATGTATTTACCCACGACGAA-3’
AtUNC93-short RP
5’-AGTTGCATACTTCCCAAGTCTC-3’
AtUNC93-both LP
5’-GCTGTTTGGGAATCTCATCAC-3’
AtUNC93-both RP
5’-AACTCAGCCCAAACAAATGC-3’
5’-CAGTGATAACGCAGCATACAGGTGAGATGAG(T)5-3’
5’-CAGTGATAACGCAGCATACAG-3’
5’-GCAGCATACAGGTGAGATGAG-3’
5’-TTCCAATTATCTGGTTCGATGC-3’
Alexander staining, Tissue clearing, Optics and
Photography
Alexander staining was utilized for detecting pollen abnormalities. The anthers of
freshly opened flowers were removed from plants and placed on microscope slides. 3
drops of Alexander stain (20% ethanol, 0.05% malachite green, 0.25% Fuchsin acid,
0.025% orange G, 10% (w/v) phenol, 10% (w/v) chloral hydrate, 0.7 M glacial acetic
acid, 50% glycerol) were placed on top of the anthers and gentle pressure was applied to
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push the pollen out of the anthers. Slides were imaged after a 15-minute incubation at
room temperature. For Hoechst-dye staining of pollen grains was performed as described
in Park et al., 1998. Briefly, freshly opened flowers were placed into an eppendorf tube
with 300 µL of extraction buffer (0.1% Triton X-100, 0.1 M sodium phosphate, 1 mM
EDTA, 0.8 µg/mL Hoechst 33342), vortexed briefly, and then spun in a tabletop
centrifuge for 1 minute. The pollen pellet was transferred to a slide and a coverslip added.
After a 30-minute incubation in the dark, pollen was imaged via brightfield and UV epiillumination with a Leica DMRB microscope. Images were imported into and processed
in Adobe Photoshop CS4 (Adobe Systems Inc.).
For imaging, siliques of various ages were removed from plants, fixed under
vacuum for 15 minutes in ice-cold FAA. Fresh FAA was added, and siliques were kept
overnight at 4°C in this solution. Siliques were then moved through an ethanol series at
room temperature and finally immersed in chloral hydrate solution (2.5 g chloral hydrate
in 1 mL of 30% glycerol). Siliques were allowed to clear in this solution for 2 days, then
pictures were taken with a DinoXcope camera (AnMo Electronics).
Results
CrUNC93-like and AtUNC93-like Proteins are
Conserved
The original CrUNC93-like clone from the apogamy library was 220 nucleotides
long. After performing 5’ RACE and amplifying the 3’ region of the transcript using a
sequence-specific primer along with a degenerate primer (see Materials and Methods), a
CrUNC93- like transcript sequence of 1149 nucleotides was obtained that spanned the
UNC93 domain through the 3’ portion of the transcript (Fig. 21A). From the Arabidopsis
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Figure 21 UNC93-like protein alignments and Arabidopsis UNC93-like gene structure.
The C. richardii UNC93-like predicted protein sequence aligned with the
Arabidopsis “Short” and “Long” UNC93-like proteins from alternative
splicing (A). The black bar in alignments indicates the UNC93 domain,
amino acids in red are predicted transmembrane domains, and “I” and “O”
designate predicted inside and outside plasma membrane domains for the
Arabidopsis (top) and C. richardii (bottom) proteins. The blue dashes at the
N-terminal region of the C. richardii protein sequence indicate the region for
which no amino acid sequence may be inferred because the transcript
sequence is unknown. (B) is a diagram of the AtUNC93-like gene and
transcripts. Blue boxes are exons, light blue boxes are 5’- and 3’-
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UNC93-like gene, two alternate transcripts are produced, of which the longer transcript is
predicted to code for a protein with 11 transmembrane domains, and a shorter transcript
that is predicted to code for a protein with 12 transmembrane domains (Fig. 21A and B).
Based on the conformation of the proteins in C. elegans and humans (Levin and Horvitz,
1992; Isnardi et al., 2008), and the PredictProtein algorithm (Rost et al., 2003) the larger
AtUNC93-like protein likely is inserted into the membrane with its N-terminal domain
inside the membrane, and the C-terminal domain on the outside (Fig. 21A). In contrast,
the smaller AtUNC93-like protein likely has both its N-terminal and C-terminal domains
inside the cell or organelle (Fig. 21A). The predicted amino acid sequence of the
CrUNC93-like protein aligns with the large and the small Arabidopsis proteins with
37.9% identity, 52.1% similarity, and 39.1% identity, 54.3% similarity, respectively (Fig.
21A). Within the UNC93 domain, the C. richardii proteins are 56.9% identical and
83.7% similar, indicating a high degree of conservation for this domain. The CrUNC93like protein is predicted to encode a protein with 10 transmembrane domains, with both
N-terminal and C-terminal domains outside of the cell (Fig. 21A). Note that the entire 5’
sequence of the CrUNC93-like transcript has not been elucidated (Fig. 21A). It is likely
that this 5’ sequence would encode another transmembrane domain, as is present in both
the small and large Arabidopsis UNC93-like proteins. It can be surmised, then, that the
CrUNC93-like protein encodes a protein with 11 transmembrane domains, with the Nterminal domain inside the membrane and the C-terminal domain outside the membrane,
which is a topology more similar to the large UNC93-like protein in Arabidopsis (Fig.
21A).
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Figure 22 Phenotypes of a homozygous T-DNA insertion mutant of AtUNC93-like. (A)
Primers specific for the long and short AtUNC93-like transcript amplify both
(black arrows) from the flower (F), leaf (L), and root (R) tissues of WT plants,
but none in those same tissues from unc93-like (T-DNA). Primers that
amplify both transcripts, that hybridize upstream of the T-DNA insertion,
detect UNC93-like transcript in both flower and root of WT and unc93-like
(black arrows) (B) unc93-like plants are smaller, darker green, and have
shorter petioles (arrows) than WT. (C) unc93-like roots produce more lateral
roots than WT plants. (D) wavy root phenotype, compared to WT, of unc93like roots grown on 90° vertical plates. Bar = 1 cm in all pictures except (C)
where white bar = 1mm
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Figure 23 Number of lateral roots from plants grown on medium containing varying
concentrations of K+. Bars are standard error, and asterisks indicate
significance (Student’s t-test, p < 0.05)
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Vegetative Phenotypes of the Arabidopsis
unc93-like Mutant
The Arabidopsis T-DNA insertional mutant line SALK_010430 is homozygous
for a T-DNA insertion into the AtUNC93-like gene near the junction of the 8th exon and
8th intron (Figure 21B). This T-DNA insertion disrupts the coding region of both the
large and small transcript for this gene. Accordingly, using two sets of primers specific
for the large and small transcripts, respectively, reverse transcriptase PCR failed to detect
either transcript in this mutant. Notably, both sets of primers are downstream of the TDNA insertion. However, using a set of primers upstream of the T-DNA insertion, in a
region that is shared by both transcripts, an amplicon was detected with RT-PCR (Figure
22A). This may indicate that this T-DNA line is not a knockout mutation, instead
generating a truncated transcript and possible truncated protein that may provide partial
function or dominant negative effects.
This T-DNA mutant line displays a number of vegetative and reproductive
phenotypes (Figure 22). The mutant plants have smaller leaves and shorter petioles thus
have a smaller circumference than wild-type (WT) plants (Figure 22B). Additionally, the
T-DNA plants are dark green compared to WT plants (Figure 22B), and are 1-2 weeks
delayed in development compared to WT plants, having on average the same number of
leaves at flowering time as WT plants (unc93 11.5 ± 0.63, WT 11.8 ± 0.85, n = 10; t =
0.896, p > 0.05).
When grown on sterile agar plates, unc93-like plants produce more lateral roots
than WT plants (Figure 22C). Therefore, the mutant plants, while having less aerial tissue
than WT plants, were proliferating more root tissue than WT plants. This was verified in
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Figure 24 Fresh root and shoot weights of unc93-like and WT plants. Bars indicate
standard error and asterisks indicate statistical significance (Student’s ttest, p < 0.05).
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two ways, by directly counting number of lateral roots emerged from the main root of
plants grown vertically for 12 days on agar medium with varying concentrations of K+
(Fig. 23) and by measuring the fresh tissue weight of WT and unc93-like shoots and roots
after 16 days of growth on agar medium with 10 mM K+ (Fig. 24). It was found that
unc93-like plants develop significantly more lateral roots than WT plants when grown on
agar medium for 12 days (Fig. 23). Furthermore, varying amounts of K+ in the medium
does not appear to affect this phenotype, with unc93-like plants consistently developing
more lateral roots than WT plants in all conditions except with no added K+, on which
neither unc93-like nor WT plants developed roots (Fig. 23). The fresh root and shoot
weight of unc93-like and WT plants were determined after 16 days of growth on agar
medium (Fig. 24). WT plants have significantly more aerial tissue at this time period than
unc93-like plants, but the root tissue weights did not differ significantly (Fig. 24). Since it
is visually clear that unc93-like plants have more extensive lateral roots than WT plants
after 10 or more days of growth (Fig. 22C), it is surprising that the difference is not
significant with regard to weight. Roots are very thin and delicate structures, and this type
of analysis may not be sensitive enough to detect the putative difference in weight
between the approximately 40 plants of each measured. Alternatively, while WT roots do
not grow longer than unc93-like roots (data not shown), they may be thicker, which
would explain the discrepancy between these two experiments.
In addition to having less aerial tissues and more lateral roots, unc93-like roots
consistently grew in a wavy pattern on vertical agar medium (Figure 22D). Root waving,
as well as other unusual root growth patterns, can be induced from WT roots by tilting
agar plates 30° from vertical (Migliaccio et al., 2009). This is a well-characterized root
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Figure 25 Phenotypes of kco3 and tpk1 potassium channel mutants on 0 mM K+ differs
from unc93-like and WT plants. (A) kco3 and (D) tpk1 mutant plants grow
roots in the presence of extreme K+ starvation. This phenotype differs from
that of unc93-like plants (B and E) or WT plants (C and F).
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response to the mechanical stimulation (Migliaccio and Piconese, 2001) of penetrating
the agar medium. However, my growth conditions had the agar plates completely
vertical, such that roots did not penetrate the agar medium, and WT plants grown in
parallel or on the same plates as unc93-like plants did not show wavy root growth (Figure
22D). Moreover, this was a phenotype observed consistently when growing the plants on
agar medium, even when WT and unc93-like were cultured on the same plates (data not
shown). This suggests that this is a phenotype caused by the unc93-like mutation rather
than a normal thigmomorphogenic response to growth conditions.
Comparison of unc93-like Phenotypes with K+
Channel Mutants
I also obtained homozygous T-DNA lines for the 5 TPK and KCO3 potassium
channel-encoding genes in the Arabidopsis genome, to investigate whether any of these
putative knockout lines phenocopy the unc93-like mutant. I grew plants in soil as well as
on agar medium with optimal and suboptimal potassium concentrations. I found that the
plants of all 6 potassium channel mutant lines have phenotypes indistinguishable from
WT plants (data not shown). Except in the case of growth on 0 mM potassium agar plates,
where tpk1 and kco3 produced more roots than the unc93-like mutant or WT plants (Fig.
25). The significance of this is unknown, but it may indicate that these 2 potassium
channel mutants have a defect in potassium signaling, and produce roots even in the
absence of K+. Potassium signaling is normally required for root growth (Dolan and
Davies, 2004). Other than this phenotype in tpk1 and kco3, all of the potassium channel
mutant lines had phenotypes that resembled WT plants, thus did not phenocopy unc93like.
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Reproductive Phenotypes of Arabidopsis unc93-like
Mutant
The unc93-like plants also exhibit abnormal reproductive phenotypes. Seeds from
unc93-like plants are significantly smaller in size than WT seeds (Fig. 26). Accordingly,
the unc93-like seeds, at 61.4 seeds/mg are also almost 27% lighter in weight than WT
seeds, which contain 45 seeds/mg. This could be indicative of the retardation of embryo
or endosperm development. Additionally, siliques from unc93-like plants have 27.6%
fewer seeds than WT plants (Fig. 27A). While some of those missing seeds are caused by
failed fertilizations, suggesting a possible gametophytic effect for this gene, there is also
evidence of seed abortion in unc93-like mutant siliques (Fig. 27B), also implicating a role
for the AtUNC93-like gene in early sporophyte development.
To examine whether there is a male gametophyte defect in the unc93-like mutant,
I examined pollen viability with Alexander staining (Alexander, 1969). This method
stains the pollen coats green and the cytoplasm of viable pollen dark red. Aborted pollen
appears as empty green spore coats. Alexander staining of pollen from unc93-like plants
showed a large fraction of pollen in this line that has aborted, as indicated by empty green
pollen coats lacking functional (red) cytoplasm (Fig. 28B), while WT pollen did not
display any obvious defects (Fig. 28A). Arabidopsis pollen normally contains 2 sperm
cells and a generative cell. To examine whether there was any nuclear defect in the pollen
of the unc93-like mutant, I stained WT and unc93-like pollen with Hoechst 33334 dye and
imaged the pollen under epi-fluorescence (Fig. 28C and D). I found no obvious nuclear
abnormalities in the viable pollen grains of WT or unc93-like mutants, as indicated by the
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Figure 26 Seed sizes of unc93-like and WT plants. Bars indicate standard error and
asterisk indicates significance (Student’s t-test, p < 0.05).
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Figure 27 unc93-like plants have fewer seeds in their mature siliques than WT plants. (A)
The seeds from 16 siliques for both WT and unc93-like plants were examined,
representing 5 separate plants for each. Bars indicate standard error and
asterisk indicates significance (Student’s t-test, p < 0.05) (B) Siliques of
unc93-like plants have empty regions (white arrow) indicative of failed
fertilizations, and shriveled seeds (black arrow) indicative of embryo abortion.
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Figure 28 Examination of pollen viability in the unc93-like mutant. (A) WT pollen stained
with Alexander stain showing no defective pollen grains. (B) Pollen abortion
in the unc93-like mutant plant revealed by the presence of empty spore coats.
(C) Staining of WT pollen nuclei with Hoechst dye showing two, brightlystaining sperm cells and one diffuse vegetative nucleus in each. (D) Mature
unc93-like pollen does not have any obvious nuclear defects.
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presence of 2 densely staining sperm nuclei and one diffusely staining vegetative cell
nucleus in all pollen imaged (Fig. 28C and D).
Based on the Alexander stain evidence that there is pollen abnormality and failed
fertilization events in unc93-like siliques, I investigated the possibility that the mutant
unc93-like allele affects female and/or male gametophyte function. I crossed WT plants
to homozygous T-DNA plants to generate plants heterozygous for the T-DNA allele (TDNA +). Then, I performed reciprocal crosses with flowers from these heterozygous
plants and those of WT plants. If gametophyte function is not affected, then I would
expect to see a 1:1 ratio of T-DNA +: ++ plants in the next generation for both crosses. If
female gametophyte function is negatively affected by the T-DNA allele, I would expect
to see fewer heterozygous plants from the cross using heterozygotes as female. If male
gametophyte function is affected, then I would expect to see a reduction in the 1:1 ratio
of T-DNA+: ++ plants from the cross that uses heterozygotes as the male donor. The
genotypes of these F 1 plants from these crosses were determined with PCR. It is
important to note that siliques generated from the cross using heterozygotes as females
resulted in approximately 2 times fewer seeds than those crosses using heterozygotes as
males (Fig. 29). There is no obvious difference in flower architecture between
heterozygous plants and WT plants to explain this difference (data not shown), so it
suggested a female gametophyte deficiency. However, this is not the case. The T-DNA
allele is passed with equal efficiency through both the Arabidopsis female and male
gametophytes, with a ratio of 46:50 (T-DNA+: ++) F1 plants from the heterozygous
female cross, and a 51:55 (T-DNA+: ++) F1 plants from the heterozygous male cross.
Neither of these is significantly different than a 1:1 ratio (X2 test; p > 0.05). These results
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Figure 29 Significantly fewer seeds produced from crosses performed with females
heterozygous for the unc93-like mutant allele. Bars indicate standard error,
asterisk indicates significance (Students t-test, p < 0.0001)
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Figure 30 The effect of K+ concentration on apogamy induction in C. richardii.
Gametophytes were grown on medium with varying concentrations of
potassium in the presence or absence of 2.5% glucose. Asterisk = statistical
significance (Student’s t-test, p < 0.05). Glc refers to rich medium, BFM
refers to basal fern medium. The agar naturally contains a small amount of
K+, and 0 mM indicates no added K+ to the medium. Empty set symbol
indicates that all gametophytes died.
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seem to be in conflict with the following two observations. First, there are significantly
fewer seeds in the siliques from the crosses of heterozgous females to the WT males and
second, that I have already detected a pollen defect in unc93-like plants, suggesting that
the unc93-like allele affects pollen function. Based on the reciprocal cross results, the
AtUNC93-like gene does not appear to be necessary for gametophyte function. Instead,
just the presence of a mutant allele in the parent plant (of either pollen or embryo sac)
renders equal numbers of the gametophytes of both sexes inviable, regardless of which
allele the gametes carry. This suggests that the unc93-like allele creates a maternal
environment that negatively affects the viability of both male and female gametophytes.
The Role of K+ in the Induction of Apogamy
from C. richardii Gametophytes
The UNC93 protein in C. elegans is predicted to associate with components of a
two-pore potassium channel, perhaps performing a regulatory role for potassium uptake
(de la Cruz et al., 2003). Based on this information, I performed an experiment with C.
richardii gametophytes to determine if potassium plays a role in apogamy induction in
this fern. I cultured fem mutant gametophytes, from which there is no risk of selffertilization because they do not form sperm, on 2.5% glucose or no glucose plates,
varying in the concentration of potassium. I then tallied the percentage of gametophytes
that formed apogamous structures after 45 days of growth. I found that, grown on
medium containing 2.5% glucose (previously shown to be inductive for apogamy), in the
absence of potassium, an average of 8% of gametophytes produce apogamy after 45 days
(Fig. 30). Note that there is a trace amount of potassium in the agar used for solidifying
the media, enough for the gametophytes to survive and proliferate, albeit to a much lesser
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extent than at higher potassium levels. This percentage increases significantly to 55% and
above 70% for 5mM potassium and 10mM potassium, respectively (Fig. 30). 10mM
potassium is the optimum concentration of potassium under normal growth conditions of
1/2x MS medium. Potassium concentrations 2 times higher than 10 mM produce a
slightly lower percentage of apogamy in the presence of 2.5% glucose, and gametophytes
grown on glucose with potassium concentrations higher than 20 mM do not survive,
presumably due to osmotic effects (Fig. 30). On basal fern medium, with no glucose,
approximately 5% of gametophytes produce apogamy after 45 days of culture. This
percentage increases to between 15% and 30% for gametophytes grown on medium
containing potassium concentrations between 5 mM and 20 mM, and reduces on 50 mM
potassium, and gametophytes do not survive on higher concentrations of potassium (Fig
30). Therefore, while there is a significant increase in the incidence of apogamy with
increasing potassium concentration on rich medium, there is no corresponding difference
in the incidence of apogamy on basal medium. This suggests that potassium does not play
a role in the induction of apogamy, or that potassium plays a role in apogamy when
combined with glucose. An alternative view would be that potassium is necessary for the
induction of apogamy by glucose, although this effect would be difficult to distinguish
from the role of potassium in general plant cell growth.
Discussion
I have described in this chapter experiments investigating the role of the UNC93like gene in Arabidopsis. This gene was isolated from C. richardii gametophytes
committed to apogamy, and identified as being induced in the embryos of Arabidopsis
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based on in silico analysis. In C. richardii gametophytes, UNC93-like is induced in the
female reproductive structure, the archegonia, and in egg cells.
The AtUNC93-like gene produces two transcripts, and the expression level of the
large transcript is increased in Arabidopsis embryos according to in silico analysis using
Genevestigator (Zimmerman et al., 2004; Chpt. 3, Fig. 17C). Conflicting results were
obtained using another probe on the ATH genome microarray chip. Both transcripts of
the AtUNC93-like gene are detected with this second probe set which indicates
expression in nearly all tissues of the plant with upregulation in the roots (data not
shown). The CrUNC93-like protein more closely resembles the protein encoded by the
large AtUNC93-like transcript based on transmembrane domain predictions. It is
unknown whether the CrUNC93-like gene has multiple mRNA splice forms. If it does,
the alternate forms are unlikely to be expressed in the gametophytes undergoing apogamy
commitment because 3’RACE and degenerate PCR experiments amplified only one
sequence from this fern tissue. It is also unknown whether CrUNC93-like is represented
by only one gene in the fern genome. The Arabidopsis genome contains only one gene,
At3g09470, which encodes a protein with an UNC93 domain.
Because the UNC93 protein namesake in C. elegans is predicted to regulate a
multi-component two-pore potassium channel, I examined how the unc93-like mutant is
affected in responses to K+ starvation. I found that the unc93-like vegetative phenotypes
are unchanged when the plants are K+ challenged, including the production of excess
lateral roots. Furthermore, the unc93-like plants do not phenocopy tpk1-5 or kco3 mutant
plants, which resemble WT plants in all respects except the ability to form roots on
medium lacking potassium. The fact that these two mutants, tpk1 and kco3, have a
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phenotype that differs from both WT and unc93-like plants suggest that the UNC93-like
protein is not involved in the regulation of these two potassium channels. However, all
five of the TPK proteins are likely to form heterodimers in vivo (Voelker et al., 2006),
and the expression patterns of most of these genes significantly overlaps (Deeken et al.
2003; Philippar et al. 2003). The WT phenotypes of these mutants may suggest that they
can compensate for each other when one of the genes is mutated. The creation of double
or more mutant lines for these genes may reveal phenotypes that are similar to the unc93like mutant.
Lateral root formation and length is inhibited in WT plants when exogenous K+
levels are limited (Jung et al., 2009, Shin and Schachtman, 2004). The fact that the
unc93-like mutant continues to make excessive lateral roots, compared to WT plants, even
in K+ starvation conditions is consistent with UNC93-like playing a role in K+ uptake,
sensing, or signaling. Additionally, the small aerial tissues of unc93-like plants may be an
indication that the mutant plants are not receiving enough K+ to proliferate.
The unc93-like mutants have approximately 27% fewer seeds in their mature
siliques than WT plants. Analysis of developing seeds in young siliques revealed that this
reduced seed set is due to fertilization failures and seed abortion combined. In contrast,
reciprocal crosses between plants heterozygous for the T-DNA insertion showed equal
transmission efficiency through both male and female gametophytes. Combining these
two seemingly contradictory results, the most plausible explanation is that, either one or
both, fertilization and seed development was impaired when the female donor was the
heterozygote. In agreement with this explanation, a fraction of the pollen grains of unc93like homozygous mutants aborted. These data may be explained by UNC93-like protein
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being involved in the ability of a parent plant to maintain viable gametophytes, while not
being essential for gametophyte function, per se. It is conceivable, but unlikely, that a
reciprocal cross of this type would provide evidence of a low overall functional pollen
count for a male donor. The procedure for manually crossing Arabidopsis flowers
involves the copious application of donor pollen to a stigma—more than enough to
fertilize the ~50 embryo sacs within a given gynoecium. Therefore, fewer embryo sacs
will result in fewer seeds, regardless of how much pollen is applied to a stigma.
An experiment was performed in which the role of potassium concentration on the
induction of apogamy in C. richardii gametophytes was examined. There was a slight
increase in the percentage of apogamy induced from gametophytes on basal medium as
the potassium concentration increased to 20 mM. However, this increase is not
statistically significant. A much larger increase in the percentage of gametophytes
producing apogamy was obtained with increasing potassium concentration in the
presence of 2.5% glucose. The peak percentage of apogamy was reached, around 70%,
at10 mM K+ in medium supplemented with glucose, which is the optimal concentration
of K+ in standard plant media. C. richardii fem1 gametophytes have displayed a similar
peak level of apogamy in previous experiments when cultured on glucose (Chpt. 2, Fig.
11). In those experiments, basal levels of apogamy (induced from medium that does not
contain sugars) never exceeded 5%, so it is notable that over 20% of gametophytes
exhibited apogamous structures in this experiment on basal medium with potassium
concentrations between 5 and 20 mM. Previous experiments were conducted on 10 mM
potassium-containing medium. The high level of apogamy on basal medium may be
attributed to some other cultural conditions between the experiments. Possible sources of
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variation include different light bulbs in the incubation chamber, or the freshly acquired
MS medium that was used for the experiments in this chapter. Varying light conditions
and nutrient levels are known to affect apogamy induction in some ferns (Raghavan,
1989). Alternatively, it may be that potassium is necessary for the induction of apogamy
by glucose. Because potassium is necessary for plant cell growth, this effect would be
difficult to distinguish from the role that potassium plays in the normal growth and
development of plant cells. If potassium is necessary for apogamy induction by glucose,
and the UNC93-like protein is in fact involved in potassium uptake or transport, it would
be important to induce it during apogamy commitment. To my knowledge, there is no
published evidence of potassium playing a role in the induction of apogamy in other
plants. Overall, this experiment may suggest that potassium is necessary for the induction
of apogamy by glucose or that potassium concentration does not play a role in apogamy
beyond providing the necessary nutrients for growth.
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CHAPTER V
SUMMARY AND FUTURE DIRECTIONS
Land plants partition the two major events of a sexual life cycle, meiosis and
fertilization, to two different multicellular generations, the diploid sporophyte and the
haploid gametophyte. Despite the obvious evolutionary benefits of meiotic recombination
and fertilization, many plants have evolved modes of asexual reproduction that avoid the
processes of meiosis and fertilization altogether. These plants will produce asexual
sporophytes directly from the cells of or around the gametophyte. The genes that drive
these asexual processes are unknown, but are likely to at least partially overlap with those
involved in zygotic embryogenesis. Defining and characterizing genes involved in these
asexual processes is of interest not only agriculturally, but also for the insight they will
provide on the evolution of asexuality, the alternation of generations, and the sporophyte
body of land plants. In this thesis, the genes that are induced during the process of
apogamy induction in the fern C. richardii are elucidated and examined.
The process of apogamy occurs in approximately 10% of ferns in nature and may
also be induced in the laboratory from many fern gametophytes that normally reproduce
sexually (Sheffield and Bell, 1987; Raghavan, 1989). I discovered that 2.5% glucose
medium is optimal for the induction of apogamy in the fern C. richardii (Ch. I). In
Chapter II, I described the haploid sporophyte structures that these gametophytes produce
in response to culture on this medium. While obligate apogamy had been previously
described from hybrid and aneuploid strains of this fern (Hickock, 1977, 1979), the
induction of apogamy from this fern had not previously been described in the literature.
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As is the case with induced apogamy in other ferns, the apogamous sporophytes induced
from C. richardii do not have the normal morphology of zygote-derived sporophytes but
do share some or all of the features of zygotic sporophytes including vascular tissue and
stomata.
I found that fern gametophytes begin committing to the production of induced
apogamous sporophytes after 10 days growth on 2.5% glucose medium. This
commitment timing provided an opportunity to isolate the genes induced during the
process of apogamy induction in this fern. I created a subtracted, normalized “apogamy”
cDNA library that contains approximately 900 clones representing genes induced in C.
richardii gametophytes at the time of apogamy commitment. Among the 306 unigenes
identified from the sequenced clones in this library, 170 could be identified with
sufficient confidence to examine as a representation of the library as a whole (Chapter
III). 306 unique genes gives 3-fold coverage of the 902 clones in the apogamy library,
which is equivalent to 95% confidence that each apogamy library gene is represented at
least once in these sequence data.
The apogamy library is enriched in genes that are involved in stress response and
metabolism compared to the entire transcriptome of the fern P. aquilinum. This suggests
a role for nutrient flux, and perhaps sugar sensing or signaling, in apogamy induction in
C. richardii. Additionally, inductive microspore embryogenesis in angiosperms
universally requires a stress treatment to induce an embryogenetic program from the
haploid spores (Shariatpanahi et al., 2006; Segui-Simarro, 2010). Induction of stress
response genes may be a requirement for induced apogamy from both angiosperm and C.
richardii gametophytes.
119
The apogamy library contains many sequences whose homologues in Arabidopsis
show enhanced or specific expression in flowers and seeds, two structures that do not
exist in ferns. It is tempting to speculate on the function of these genes in ferns. It may be
that these are sporophyte-specific genes in C. richardii that were induced to express in
the gameotphyte through apogamy induction, or they could be involved in gametophyte
function and/or development, or both. The prevailing hypothesis for the evolution of
alternation of generations is that the modern sporophyte of the land plants evolved from
an ancestor that had a dominant, multicellular gametophyte with meiosis proceeding
directly from the zygote (Chapter I). As such, achieving development of the sporophyte
body plan would likely involve the recruitment of gametophyte genes for related or novel
functions in the sporophyte. The Arabidopsis homologues of the apogamy library
sequences that have sporophyte-specific expression patterns are interesting for
investigations into asexual processes and the evolution of the alternation of generations,
both subjects of particular interest in plant biology. The apogamy library will be a rich
resource for future investigations in this laboratory.
One of the apogamy library genes with enhanced expression in Arabidopsis
embryos was the UNC93-like gene. Whole mount in situ analyses in C. richardii
gametophytes undergoing apogamy commitment suggested that this sequence was highly
expressed in the egg cells of gametophytes. Based on my observations of induction of
apogamy in C. richardii, I had hypothesized that mature, functional eggs or archegonia
were required for the ability of C. richardii gametophytes to undergo apogamy (Chapter
III). Therefore, the UNC93-like gene was chosen for further functional analysis in
Arabidopsis. UNC93-like is a homologue of the unc93 gene in C. elegans, a membrane
120
bound protein that is predicted to regulate a two-pore potassium channel during the
coordination of muscle contraction (Levin and Horvitz, 1992; de la Cruz et al., 2003).
This gene is uncharacterized in plants.
A homozygous T-DNA insertional line in the AtUNC93-like gene shows a
number of vegetative and reproductive phenotypes, including small plant stature, retarded
growth and delayed flowering. Siliques have a reduced seed set compared to WT plants
and this is probably due to a combination of embryo abortion and gametophyte lethality
(Chapter IV). The mutant allele does not appear to play a critical role in gametophyte
viability, per se, because the allele is passed with equal efficiency as the WT allele
through both male and female gametophytes to the next generation. RT-PCR experiments
indicated that plants of this T-DNA line produce a partial UNC93-like transcript from
upstream of the T-DNA insertion site, which may be translated into protein. If this is the
case, and the protein has partial function, this mutation may be recessive or may cause
dominant negative effects. Evidence for one or the other may be obtained by examining
the phenotypes of plants heterozygous for the mutation. A dominant or semi-dominant
negative effect will produce heterozygous plants with mutant phenotypes similar or
identical to those homozygous for the allele. I have preliminary evidence that
heterozygous plants have retarded growth resulting in a later flowering time than WT,
suggesting that the T-DNA mutant allele is a semi-dominant mutation. These
observations will have to be quantified before a conclusion can be made regarding the
nature of this allele. Additionally, it would be informative to examine the phenotypes of
plants belonging to a true knockout line for the AtUNC93-like gene. There are several
insertional mutant lines available for this gene. If a knockout of the AtUNC93-like gene is
121
not lethal, and the phenotype resembles the phenotype of the current T-DNA line, this
would be evidence that the allele of the current T-DNA line is recessive.
While I found in silico expression evidence that the two transcripts of the
AtUNC93-like gene are differentially expressed, my RT-PCR analysis found the presence
of both transcripts in all tissues examined (Chapter IV). Accordingly, the first
experiments that should be completed regarding the further characterization of the
AtUNC93 gene are in situ hybridizations to examine at the tissue level and where the two
transcripts of this gene are being expressed in the WT tissues of Arabidopsis.
Additionally, it would be very interesting and informative to use this methodology to
examine the locations of the AtUNC93-like transcripts in Arabidopsis tissues that are
undergoing somatic embryogenesis. Multiple gain- and loss- of function Arabidopsis
mutations which produce somatic embryos in developmental processes that resemble
apomixis (Chapter I). If the expression of AtUNC93-like gene is found to be significantly
altered in these mutant backgrounds compared to WT, or specifically expressed in or
around the somatic embryos themselves, this would be strong evidence of a role for the
UNC93-like gene in asexual reproduction. I have acquired a fis2 mutant line, from which
60% of embryo sacs produce endosperm and embryogenic growth in the absence of
fertilization (Chaudhury et al., 1997). These tissues undergoing somatic embryogenesis
will be used for in situ analyses with the AtUNC93-like probes. These in situ experiments
are ongoing in our lab.
Of course, the transcriptional profile does not always match the location of the
encoded protein. The UNC93-like protein is predicted to be a transmembrane protein.
There are a number of ways to identify the tissue and subcellular location of the
122
AtUNC93-like gene products. For the tissue specific location of the AtUNC93-like
protein a gene trap line is preferable to transformation with a reporter construct, as it
retains the native promoter elements and genetic milieu. I have already acquired a gene
trap line from the TRAPPER collection (Sundaresan et al., 1995) that is annotated as
being inserted into the AtUNC93-like gene immediately after the start codon.
Unfortunately, this line does not have any appreciable GUS expression, and both the long
and short AtUNC93-like transcripts are detected in the seedlings of plants homozygous
for this construct, so its usefulness for mutant-phenotype analysis is limited as well.
Alternatively, one could create GFP and YFP fusion constructs with the coding
sequences of the two transcripts, driven by a native promoter region, transform plants
with this construct and then examine the tissue-specific and subcellular location of the
two proteins with confocal microscopy. Antibodies specific to each of the two proteins
could be used in immunolocalization analyses.
There is evidence of embryo abortion occurring in the unc93-like homozygous TDNA mutant. It will be important to follow up on this observation by observing embryo
development in the seeds of this mutant to find the exact cause of embryo death. The seed
abortion in this line could be evidence of a developmental defect affecting the embryo or
the endosperm, or it could simply be the result of the failure of the parent plant to transfer
sufficient nutrients to the developing embryos so that a portion of the embryos abort. This
later scenario is supported by evidence of the failure of parent plants to maintain the
viability of a proportion of their respective male and female gametophytes (Chapter IV).
The ultimate test of the role CrUNC93-like role in apogamy is to create C.
richardii lines that under-, over-, and ectopically express the protein. In order to achieve
123
this, an efficient and reliable transformation protocol needs to be developed for C.
richarii. The only reported transformation method for C. richardii is direct bombardment
of DNA into gametophyte cells (Rutherford et al., 2004). We have found this method to
be expensive and inefficient (data not shown). Our lab is currently developing an
Agrobacterium-mediated, stable transformation protocol. Once available, C. richardii
gametophytes will be transformed with the CrUNC93-like full-length cDNA under the
control of the strong, nearly ubiquitously expressed 35S promoter. Our previous
experiments indicate that the 35S promoter works efficiently in this fern. The phenotype
of the transformants, especially their ability to apogamously produce sporophytes, will be
examined.
Other interesting genes in the apogamy library, such as FTSH protease, BTB
domain, and LEUNIG-like can also be tested using this transformation protocol. In
addition, it can be used to test ectopic expression of those genes with gain-of –function,
somatic embryogenesis phenotypes in Arabidopsis, such as WUSCHEL, BABYBOOM,
and SERK1.
124
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