A Simple Metabolic Switch May Activate Apomixis in <I>Arabidopsis

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12-2018

A Simple Metabolic Switch May Activate Apomixis in Arabidopsis thaliana

David Alan Sherwood Utah State University

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A SIMPLE METABOLIC SWITCH MAY ACTIVATE APOMIXIS IN

ARABIDOPSIS THALIANA

David Alan Sherwood

A dissertation submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

Plant Science – Molecular Biology

Approved:

______John G. Carman Ph.D. Bruce Bugbee Ph.D. Major Professor Committee Member

______Lee Rickords Ph.D. Steve Larson Ph.D. Committee Member Committee Member

______Zhongde Wang Ph.D. Laurens H. Smith Ph.D. Committee Member Interim Vice President for Research and Dean of the School of Graduate Studies

UTAH STATE UNIVERSITY Logan, Utah

2018 ii

iii

ABSTRACT

A Simple Metabolic Switch May Activate Apomixis in Arabidopsis thaliana

David Alan Sherwood, Doctor of Philosophy

Utah State University, 2018

Major Professor: Dr. John G. Carman Department: Plants, Soils and Climate

Apomixis, asexual or clonal seed production in plants, can decrease the cost of producing hybrid seed and enable currently open pollinated crops to be converted to more vigorous and higher yielding hybrids. Apomixis can also accelerate plant breeding programs by enabling the selection and perpetuation of individuals with unique genetic combinations rather than using uniform inbreds. Facultative apomictic plants can switch between sexual and apomictic reproduction based on stress signaling. RNAseq studies identified differentially expressed genes and enriched GO categories suggesting that a stress-induced down regulation of growth phase genes may be responsible for the shift from apomictic to sexual reproduction. The initiation of stress signaling during female meiosis (megasporogenesis) was analyzed in the wildtype obligately sexual Arabidopsis thaliana by the co-analysis of meiocyte callose accumulation and chromosome condensation. The onset of callose accumulation generally occurred within 3 hours after the lights came on in the morning and coincided with pachytene, the 3rd step of meiotic prophase I that generally fails in apomictic plants. Topical application of glucose or brassinosteroids, within the correct developmental window, was used to halt stress iv signaling and shift metabolism to a pro-growth status. This triggered high rates of 1st division meiotic restitution, reminiscent of Taraxicum type diplospory that lacked callose. Dyads of unreduced megaspores then formed, and the chalazal most cell of the dyad in these ovules produced an unreduced eight nucleate polygonum type embryo sac.

A similar topical application of glucose and brassinosteroids near the end of embryosac maturation (8 nucleate stage prior to the fusion of the two central cells) was necessary to trigger parthenogenesis in either haploid or unreduced (diploid) ovules. These results suggest that fully functional apomixis might be achieved in wild-type obligately sexual

Arabidopsis thaliana by shifting metabolic signals within appropriate developmental windows. This discovery also suggests that apomixis is not a mutationally derived condition caused by ‘broken genes’, but rather an alternative mode of reproduction anciently imbedded in the genomes of angiosperms that can be triggered by the appropriate metabolic signals.

PUBLIC ABSTRACT

A Simple Metabolic Switch May Activate Apomixis in Arabidopsis thaliana

David Alan Sherwood

Apomixis, asexual or clonal seed production in plants, can decrease the cost of producing hybrid seed and enable currently open pollinated crops to be converted to more vigorous and higher yielding hybrids that can reproduce themselves through their own seed. Sexual reproduction may be triggered by a programmed stress signaling event that occurs in both the meiocyte, just prior to meiosis, and later in the egg just prior to embryo sac maturation. The prevention of stress signaling and the activation of a pro- growth signal prior to meiosis triggered apomeiosis, the first half of apomixis. The same approach was used prior to embryo sac maturation to trigger parthenogenesis, the second half of apomixis. This discovery suggests that apomixis exists as a program that can be activated by the appropriate metabolic signal at the appropriate developmental stages.

Therefore, apomixis may be alternative mode of reproduction rather a ‘broken’ form of sexual reproduction.

ACKNOWLEDGMENTS

I would like to pour my heart out in gratitude to my dedicated wife who has sacrificed much of her time with me in raising 3 active boys while school and research consumed most of my daily thoughts, time and energy. We won’t get those years back but we do cherish our days together as they will continue to come one by one for the rest of eternity.

A very special thanks to my adviser, Dr. John Carman, who has guided me through my graduate learning process and has engaged me in endless discussions that have bettered my critical thinking. I would like to thank my committee members for their support and contribution, Dr. Bruce Bugbee, Dr. Lee Rickords, Dr. Steve Larson and Dr. Zhongde Wang.

David Alan Sherwood

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CONTENTS

Page

ABSTRACT ...... iii PUBLIC ASTRACT ...... v ACKNOWLEDGEMENTS ...... vi CONTENTS ...... vii LIST OF TABLES ...... ix LIST OF FIGURES ...... x CHAPTER I. INTRODUCTION ...... 1

LITERATURE CITED ...... 16

II. STRESS INDUCED SEXUAL REPRODUCTION IN APOMICTIC BOECHERA WAS CORRELATED WITH DOWN-REGULATION OF GROWTH PHASE GENES ...... 25

ABSTRACT ...... 25 INTRODUCTION ...... 27 MATERIALS AND METHODS ...... 28 RESULTS AND DISCUSSION ...... 30 LITERATURE CITED ...... 43

III. METABOLICALLY SWITCHING MEIOSIS INTO APOMEIOSIS ...... 45

ABSTRACT ...... 45 INTRODUCTION ...... 46 MATERIALS AND METHODS ...... 49 RESULTS AND DISCUSSION ...... 53 LITERATURE CITED ...... 68

IV. PROGRAMMING THE EGG FOR PARTHENOGENESIS AND TRIGGERING AUTONMOUS ENDOSPERM FORMATION ...... 77

ABSTRACT ...... 77 INTRODUCTION ...... 78 MATERIALS AND METHODS ...... 79 viii

RESULTS AND DISCUSSION ...... 81 LITERATURE CITED ...... 87

V. SUMMARY ...... 89

LITERATURE CITED ...... 91

VI. CURRICULUM VITAE ...... 92

LIST OF TABLES

Table Page

2.1 Differentially expressed genes across species (control growth conditions) ...... 31

2.2 Differentially expressed genes within species across control vs. treatment ...... 31

2.3 Enriched biological pathway GO’s involved in meiosis ...... 33

2.4 Enriched biological pathway GO’s involved in stress response ...... 34

2.5 Enriched molecular function GO’s of interest involved in stress response ...... 35

2.6 Significant Differentially Expressed NAC genes (LOG2 Fold Change) within species comparison across t (treatment/stress) vs. c (control) conditions ...... 36

2.7 NAC genes identified by Shah et al., 2016, as differentially expressed (LOG2 Fold Change) between B. cf. gunnisoniana vs. B. stricta ...... 37

2.8 NAC genes (LOG2 Fold Change) across species/reproductive mode grown under control conditions ...... 38

2.9 Enriched Biological Pathway GO’s for plant hormones ...... 39

2.10 Enriched Biological Pathway GO’s common to all apomicts ...... 41

2.11 Enriched GO’s of interest for cellular components de-regulated within species but across t (treatment/stress) vs. c (control) conditions ...... 41

LIST OF FIGURES

Figure Page

1.1 Stress switch hypothesis ...... 5

3.1 REDOX Signaling ...... 47

3.2 TOR Signaling ...... 49

3.3 Arabidopsis meiotic timing experimental outline ...... 50

3.4 Meiotic Prophase - simultaneous staining of chromosomes and callose ...... 54

3.5 Timing of meiotic initiation in Arabidopsis thaliana ...... 56

3.6 Timing of treatments leading to apomeiotic conversion ...... 57

3.7 Treated Arabidopsis ovules undergoing Taraxacum type or Antennaria type diplospory ...... 58

3.8 Early tetrad staged Arabidopsis pistil stained for callose ...... 59

3.9 Late tetrad Arabidopsis pistils treated pre-meiotically with 50 mM glucose stained for callose ...... 60

3.10 Late tetrad Arabidopsis pistils treated pre-meiotically with 50 mM glucose stained with dapi and for callose ...... 62

4.1 The involvement of stress signaling during the evolution of the meiocyte and the egg ...... 79

4.2 Haploid parthenogenesis with endosperm in Arabidopsis ...... 82

4.3 Parthenogenesis (possibly diploid) with endosperm in Arabidopsis ...... 83 xi

4.4 Parthenogenesis without endosperm in Arabidopsis ...... 84

CHAPTER 1

INTRODUCTION

Apomixis is considered the holy grail of agriculture because it is a form of reproduction

that enables clonal or asexual seed formation in plants. It provides a platform to stabilize

specific favorable genetic combinations in all crops and to fix heterozygosity in currently self-pollinated crops such as soy, wheat and rice. Apomictic reproduction differs from sexual reproduction by i) apomeiosis, the formation of a genetically unreduced clonal egg and ii) parthenogenesis, the development of an embryo from the unreduced egg

without fertilization. The potential value of apomixis has led teams of scientists over

many generations to spend their lives in the pursuit of recreating apomixis in crop plants.

Why then is apomixis still a mystery?

The history of apomixis research

Over the decades the search for finding simple apomixis genes that can be transferred

into crop plants has been met with increasing complexity. The island of knowledge has

grown, but so have the shores of uncertainty. Yet this island of knowledge may not be

well rounded, as most apomixis research has been driven by a single over-riding

assumption, i.e., the simple mutation hypothesis. However, the origin of apomixis is an

evolutionary conundrum because the two processes needed for apomixis, i.e. i)

apomeiosis and ii) parthenogenesis, are independently inviable suggesting that multiple

simultaneous mutations would be required.

Even so, for the last couple of decades research labs world-wide have focused on

finding “apomixis genes,” a pursuit that is based on the “simple mutation” hypothesis for 2 the repeated de novo evolution of apomixis. This approach has yielded limited success wherein the unreduced gametes formed through gene knockouts generally fail to develop parthenogenetically (Ravi et al., 2008; d’Erfurth et al., 2009; Olmedo-Monfil et al.,

2010; Singh et al., 2011).

Some of the gene knockouts that lead to the first of two components of apomixis, i.e. apomeiosis, do so by altering epigenetic processes. For example, Olmedo-Monfil et al (2010) discovered that ARGONAUTE9 is involved in a small RNA pathway that restricts female gamete formation to the megaspore mother cell. Knocking out components of the pathway led to the formation of unreduced embryo sacs that were not parthenogenetically competent. Also, the loss of function in MULTICOPY

SUPPRESSOR OF IRA 1, an evolutionarily conserved Polycomb group (PcG) chromatin-remodeling complex, initiates non-viable parthenogenetic embryo development from meiotically derived haploid egg cells (Guitton and Berger, 2005).

Additional examples regarding the involvement of epigenetic processes that differentiate apomictic from sexual reproduction are reviewed by Rodrigues and Koltunow (2005) and Hand and Koltunow (2014).

These studies and the advent of expression profiling techniques have advanced the simple mutation hypothesis into a new and popular hybridization-induced epigenetic

“deregulation of sex” hypothesis that is shifting the focus of research onto elucidating the epigenetic differences between sexual and apomictic reproduction (Koltunow and

Grossniklaus, 2003; Tucker and Koltunow, 2009; Sharbel et al., 2010; Rodrigues et al.,

2010; Armenta-Medina et al., 2011). These new ideas are still limited to identifying simple mutations or gene knockouts that have a large-scale effect. 3

Limitations to the “simple mutation” and “deregulation of sex” hypotheses

These hypotheses are limited by the observation that gene knockouts typically only

disrupt the machinery used to regulate components of the sexual pathway. They also fail

to account for the fact that apomixis often occurs in tandem with sex within an

individual. Furthermore, in such cases the expression or initiation of either the sexual or

the apomictic developmental program is influenced by environmental factors (stress) in

facultative apomictic plants (Asker and Jerling, 1992; Carman et al., 2011) and cyclically apomictic animals (Suomalainen et al., 1987).

If apomixis is an intact and entirely different program regulated by environmental

signals, then current mounting research results may be building a skewed island of

knowledge. These gene knockouts that appear to trigger components of the pathway,

such as unreduced gamete formation (labeled as apomeiosis) or triggering egg activation

(labeled as parthenogenesis), likely fail because they are simply disrupting a component

of the sexual program, i.e., they are still sexual.

Alternatively, we hypothesize that apomeiosis and parthenogenesis are two steps

of a complete program that can only be initiated when signaled by the correct metabolic

pathway. This hypothesis assumes that apomixis co-evolved with the sexual pathway

during early eukaryogenesis. Hence, the last common ancestor of extant eukaryotes

would have been a cyclical apomict able to rapidly reproduce under favorable conditions

and resort to the sexual pathway under stress conditions (Carman et al., 2011; Hojsgaard

et al., 2014). Understanding how higher plants built on this platform may provide

valuable insights into understanding apomixis and rounding out our island of knowledge. 4

If apomixis is evolutionarily ancient, but also conserved in many eukaryote genomes, then its expression may simply be induced by silent metabolic signaling pathways which can be activated by the right trigger. This concept could readily explain how the complex multi-step process of apomixis could arise independently in many different eukaryotic species, including angiosperms (Hojsgaard et al., 2014) and animals

(Suomalainen et al., 1987).

The complexity of apomixis

The absence of parthenogenesis in the experimentally derived unreduced gametes mentioned above may be due to the complexity of the developmental programs. For example, upon fertilization, eggs of sexual plants and animals produce embryos.

Preclusion of embryogenesis prior to and its activation after fertilization are in part regulated by complex pre and post-transcriptional gene silencing processes, i.e. imprinting. These involve small RNAs and Polycomb group (PcG) repressive protein complexes that remodel chromatin through histone modifications (Rodrigues et al., 2010;

Armenta-Medina et al., 2011; Singh et al., 2011). For example, mutations in the

FERTILIZATION INDEPENDENT SEED (FIS) class of plant genes, which regulate epigenetic gene imprinting and seed development (Guitton et al., 2004), by-pass the requirement for fertilization to initiate endosperm development but produce inviable seeds (Chaudhury et al., 1997). Stress signaling may activate these and other processes in a cascade of events coupling meiosis to syngamy because stress signaling is central to sexual gametogenesis in both plants (McInnis et al., 2006) and animals (Ramalho-Santos et al., 2009). Interestingly, the shift from apomictic to sexual reproduction in plants and animals is also initiated by stress (Fig. 1.1; and as reviewed in Suomalainen et al., 1987; 5

Asker and Jerling, 1992; Carman et al., 2011). How this functional sex-apomixis switch

works is the central question that may solve the current evolutionary conundrum explaining the origin of apomixis. Environmentally regulated shifts that trigger fully

functional pre-existing program s (i.e. either apomixis or sex) is more parsimonious.

Apomeiosis & Parthenogenesis Meiosis & Syngamy Switch Stress-induced Based on perception shift towards sex of environmental conditions

Fig. 1.1 Stress Switch Hypothesis. Possible pathway for observed shifts toward sexually produced progeny among facultative and cyclical apomicts. In obligate sexuals or apomicts, reproductive tissues incorrectly perceive and transmit the presence or absence of stress, respectively.

Physiology of stress signaling

The formation of reactive oxygen species (ROS), especially H2O2, is central to both

biotic and abiotic stress signaling (Shlomai, 2010). A stress-signal or oxidative burst, programmed or otherwise, can re-structure the entire function of the cell by 6 simultaneously modifying the proteome, the metabolome, the epigenome and the transcriptome (detailed in Chapter 3). Therefore, an oxidative burst might operate as the sex-apomixis switch. Apomixis may be a default reproductive pathway that occurs unless stress signaling triggers the sexual pathway. Obligately sexual plants and animals may rely on a programed oxidative burst to activate the sexual pathway.

A specific example of an important role for stress signaling in meiosis is the

Mei2 gene. It encodes an RNA binding protein considered to be a master regulator of meiosis and is required for its initiation (Harigaya and Yamamoto, 2007; Pawlowski et al., 2007). In yeast, during mitosis, Mei2 transcripts accumulate but the Mie2 protein moieties remain inactive in the cytoplasm. When under stress (nutrient starvation) the

Mei2 proteins bind to small RNA (meiRNA) and are moved to the nucleus forming a visible dot (Shimada et al., 2003; Yamashita et al., 1998). This coincides with the onset of meiosis. As Mei2 is conserved between yeast and plants (Kaur et al., 2006), this process may be another part of the complex cascade of events activated by a stress signal resulting in the coupling of meiosis and syngamy in the sexual program.

Cavalier-Smith (2010) and Gross and Bhattacharya (2010) argue that stress signaling, particularly ROS signaling, played a central role in the evolution of meiosis during eukaryogenesis and its timing-of-onset during the life cycle. As such, stress signaling may be the foundation upon which both the meiocyte and the egg evolved.

Therefore, a massively restructured stress-induced sexual program likely prohibits simple gene knockouts from thwarting the ensuing sexual reproduction pathway.

Callose, evidence of the stress-induced meiotic switch

The timing and location of the oxidative burst(s) that may initiate meiosis can be traced to the formation of callose, a polysaccharide that is deposited in cell walls as a response to both biotic and abiotic stress. It is found in a thick layer in the cell wall around and between the developing tetrad of spores of most angiosperms. It also surrounds the meiocyte at the very end of MMC maturation just prior to meiosis at stage 2-III of ovule development (Schneitz et al., 1995). This event is a defining moment which will be discussed in more detail below.

In Arabidopsis callose formation in ovules increases under stress conditions in a

dosage dependent manner until it leads to apoptosis and ovule abortion (Sun et al.,

2004), a mechanism to balance fecundity with resources. Callose deposition in

Arabidopsis has also been shown to correlate with levels of H2O2 production (Luna et

al., 2011) and is activated by calcium (Karin and Christer, 1989).

This stress-induced polysaccharide also forms, albeit transiently and to a much

lesser extent, around the cell plate of active mitotically dividing cells (Stone, 2005).

Although an oxidative burst precedes mitosis by stimulating cell expansion (Foreman et

al., 2003) and division (Livanos et al., 2012), the strength of an oxidative burst and other

signals may, in a dosage-dependent manner, initiate a downstream reduction/oxidation

(redox) signal used to distinguish somatic mitosis like divisions from germ-cell meiosis.

The apomeiotic germ-cells of apomictic angiosperms do not produce thick layers

of callose like their related sexual taxa unless stress induces meiosis (Carman et al.,

1991; Tucker et al., 2001; see Chapter 3). This pattern substantiates the hypothesis that a

stress-induced switch from apomictic to sexual reproduction occurs during late stage 2- 8

III of MMC maturation when callose forms immediately prior to meiosis (Schneitz et al.,

1995).

The beginning of stage 2-III of MMC maturation marks the beginning of

leptotene, the first step in meiotic prophase I, during which the chromosomes begin to

condense (She et al., 2013; She and Baroux, 2014). The second and third steps of

meiotic prophase I, zygotene (sister chromatids begin to synapse prior to crossing over)

and pachytene (crossing over occurs), may be preceded by a stress signal since

chromosome pairing and homologous recombination is triggered due to oxidative

damage and stress signaling (Rahavi and Kovalchuk, 2013). Apomictic plants

undergoing Taraxacum-type diplosporous apomeiosis (1st division restitution followed

by mitotic like division) are thought to proceed normally until the zygotene stage of

prophase with little or no synapsis occurring and meiotic crossing over completely

failing (Crane and Carman, 1987). Does this happen simply due to a lack of a stress

signal at this defining moment? Is this why apomictic plants produce little to no callose

in their apomeiotic ovules?

Metabolic stress signaling

ROS is a normal part of metabolism in both sexual and apomictic plants. The redox

buffer system is designed to quench it (Chapter 3). Under stress conditions the metabolic

machinery is recruited to amplify the stress signal thereby shutting down growth and

activating autophagy (Chapter 3) via the inactivation of a kinase named TARGET OF

RAPAMYCIN (TOR). TOR is a central control point. When TOR is active it stimulates

growth through the activation of many down-stream targets (Chapter 3). Although some

hormonal pathways are associated with active TOR, and glucose leads to its activation in 9

plants, many stress pathways can act as a ‘kill switch’ to shut down TOR to stop growth

and even trigger autophagy (Chapter 3).

It is important to note that apomicts are still capable of meiosis if stressed

enough. They may simply be prevented from activating meiosis because they exhibit a

higher level of stress tolerance throughout their life cycle via the upregulation of the

redox buffer system (Chapter 3). Therefore, in an obligately sexual plant, would adding

glucose or hormonal signals associated with active TOR signaling send the MMC into a

growth phase triggering true apomeiosis rather than a pre-programmed stress-signal-

induced survival/sporulation (meiotic) phase?

Stress signaling during egg maturation

Stress signaling is also involved in programming the sexual egg as it may be evolutionarily derived from the same ancestral cell as the stress-induced meiocyte

(Chapter 4). Complex signaling pathways regulate the formation of the sexual female megagametophyte which contains the egg. Localized auxin biosynthesis is responsible for patterning and specifying antipodal, egg, and synergid cell types while the central cell (CC) is more resistant to under/over expression of auxin biosynthesis during development (Pagnussat et al., 2009; Panoli et al., 2015). Near the end of female megagametophyte development the CC becomes a source of metabolic stress signaling

(stages FG6 to FG7; Martin et al., 2013) that programs the sexual egg (Chapter 4). Could this stress signaling in the CC be responsible for imprinting the sexual pathway into the maturing egg cell?

Defining the sexual egg

As a consequence of reprogramming to a pluripotent state, the egg epigenome becomes

very open and transcriptionally permissive. This poses potential regulatory hazards, and

it may be why RNA polymerase II levels are reduced in the mature egg (Pillot et al.,

2010). Consequently, after fertilization early development is primarily driven by

maternally provided transcripts until the maternal to zygotic transition (MTZ), identified

by inhibiting Polymerase II and new RNA transcription (Baroux et al., 2008). MTZ occurs prior to the globular stage in Arabidopsis (Baroux et al., 2008). Interestingly the

MTZ is the point at which most gene knockouts that trigger parthenogenesis tend to fail

(Guitton and Berger, 2005). Take note that the MTZ is not a highly-defined period since the earliest low-level expression of paternal or even zygotic genes occurs much earlier, is variable according to species, and differs according to the direction of hybrid crosses

(Vielle-Calzada et al., 2001; Toro-De León et al., 2016).

The artificial activation of the sexual egg (without fertilization) in mammals by

an enzymatically-active sperm-specific phospholipase C triggers appropriate calcium

signaling patterns and activates the maternally directed developmental program that

proceeds normally until the MTZ (blastocyst stage in mammals) at which point

development fails (Saunders et al., 2002).

Research is revealing that the same pathway exists in angiosperms where calcium

signaling is triggered upon fertilization of both the egg cell and the CC (Denninger et al.,

2014). Fertilization independent calcium signaling via ionophore incubation initiates cell

wall establishment, a major egg activation event (Antoine et al., 2000). However, proper

development through the MTZ may also be due to additional factors normally provided 11 by pollen sperm cells, such as transcripts aimed at further reprogramming the epigenome, transcriptome, and proteome (Russell et al., 2012). This suggests that the programming of the sexually formed egg is not complete.

Interestingly, in plants, viable haploid zygotes can be generated after fertilization despite eliminating the paternal genome. This is done by pollinating with irradiated pollen to remove the paternal genome (Froelicher et al., 2007; De Witte and Keulemans,

1994; Bouvier et al., 1993), or it can also be done during early mitotic divisions, possibly at the first division, due to improper attachment of the spindle microtubules to a mutated form of the centromere specific histone CENH3 (Ravi and Chan, 2010). The zygote develops normally as a haploid. As a mature plant spontaneous doubling of the genome creates diploid (doubled haploid) branches that are fertile while remaining haploid branches are not fertile.

This mechanism of haploid induction is interesting because it likely occurs prior to the MTZ and yet these haploid embryos continue to develop normally through this developmental transition without any genetic contribution from the paternal genome, which was lost much earlier. What factors are delivered by the sperm cell, other than the genome, that make possible the development of a sexual egg to proceed normally through the MTZ?

Another mechanism of haploid induction was recently elucidated in maize. Here, pollen sperm cells were found to contain protein-based components (including a sperm- specific phospholipase) that are important for egg activation and proper development of haploid plants (Kelliher et al., 2017). 12

These examples illustrate that factors in the pollen sperm cell other than the

paternal genome might be required for the sexual egg to develop past the MTZ.

Apomictically formed eggs may be parthenogenetically competent because an entire and

active embryony program is provided maternally, as appears to be the case in

autonomous apomicts where pollination is not required for embryo or endosperm

formation. However, it cannot be ruled out that sperm cells provide non-genome signals

to either the egg or early developing embryo that are important in pseudogamously

apomictic plants (those that require CC fertilization for endosperm formation that

supports continued embryo development).

In sexual plants, ROS production by the CC during the late stages of embryo sac

formation may program the sexual egg for syngamy dependence, thereby setting the

requirement for syngamy to restore proper development. Absence of a ROS signal in an

apomictic embryo sac may program the egg differently, and possibly the CC, enabling

fully functional autonomous or pseudogamous parthenogenesis. If glucose and/or

hormone signaling can switch meiosis to apomeiosis could a similar signaling pathway

also program the egg for parthenogenesis?

Endosperm is required to support the growing embryo

Properly timed endosperm formation is necessary to support the growth of the

developing embryo. Fertilization of the CC immediately stimulates endosperm

development, which initially forms syncytially. The timing of cellularization influences the total amount of endosperm produced and the eventual size of the embryo. Interploidy crosses reveal that early cellularization leads to small seed size while late cellularization leads to large seed size (Scott et.al., 1998). However, if cellularization occurs too late it 13 cannot support the developing embryo, and this leads to abortion as is the case in FIS mutants (Chaudhury et al., 1997).

FIS and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) genes are part of the multi-protein complex termed the Polycomb group repressive complex 2 (PRC2).

Although PRC2 forms other complexes (Chanvivattana, 2004), FIS-PRC2 is involved in imprinting by suppressing the initiation of endosperm development prior to fertilization

(Luo et al., 2000). Mutations in this complex permit the initiation of endosperm in the absence of fertilization, but the endosperm fails to cellularize (Hehenberger, 2012). The timing of cellularization is negatively regulated by maternally expressed AGL62, which inhibits endosperm cellularization (Hehenberger, 2012). AGL62 is silenced by FIS-

PRC2 until after fertilization. Maternally-supplied FIS-PRC2 silences auxin biosynthesis genes of paternal origin (Hsieh et al., 2011).

These processes illustrate that endosperm development is suppressed by the sexual program until after fertilization. In the absence of stress-signal induced programming of the CC, would autonomous endosperm (AE) development and proper cellularization occur? For example, the ectopic production of auxin triggers AE formation, and in vitro culturing experiments exhibit high frequency AE in wild type

Arabidopsis thaliana (Figueiredo et al., 2015) but it probably still requires expression of

AGL62 for cellularization since the authors reported only checking at 3d after treatment which is not enough time for cellularization (Kradolfer et al., 2013). A parthenogenetic egg may also have the capacity to trigger AE formation by causing a synergid cell to fuse with the CC thus inducing endosperm formation, as is found in the PRC2 mutant

MULTICOPY SUPPRESSOR OF IRA1 (Motomura et al., 2016). 14

Pollination in sexual plants involves fertilization of both the egg and the CC. This process is termed double fertilization which occurs via two sperm nuclei that originate from a single pollen grain (Russell et al., 1992). Most apomictic plants are pseudogamous apomicts (Nogler et al., 1984; Asker and Jerling, 1992) that require fertilization for endosperm formation, but no genetic contribution from the sperm nuclei is made to the clonally formed parthenogenetic egg.

CONCLUSIONS

The study of naturally occurring cyclical or facultative apomicts can pave the way for understanding how the natural program works, which may provide a better platform for engineering into crops. RNAseq analysis from pistils of sexual and apomictic species of

Boechera grown under control and stress conditions led to the identification of differentially expressed genes (DE) and enriched gene ontology (GO) categories that indicate apomicts are metabolically more active than their sexual counterparts until stressed (Chapter II). To further investigate the role of stress signaling in triggering meiosis, I analyzed the timing of the zygotene & pachytene stages of meiosis I and correlated them to the development of callose, a stress induced polysaccharide. The timing of these events identifies a defining moment where stress signaling triggers the committal step of meiosis (Chapter III). The initiation of meiotic stress signaling was thwarted by a pro-growth signal induced by a topical application of glucose or the hormone brassinosteroid. This resulted in high-frequency apomeiosis and the lack of callose (Chapter III). Parthenogenesis did not occur automatically in un-reduced embryo sacs. Rather it required a second application of the same pro-growth signals that were 15 used pre-meiotically (Chapter IV). Presumably these signals stopped the CC stress signaling from programming developing eggs prior to them becoming fully mature.

Furthermore, parthenogenesis could be triggered in both haploid reduced eggs as well as experimentally unreduced diploid eggs.

The simplicity of this approach is remarkable, and the evidence presented herein strongly suggests that apomixis capacities are be embedded in the core developmental pathways of angiospermous genomes as alternative modes of reproduction that can be accessed without disruptive gene knockouts. Furthermore, this discovery could enable the engineering of a capacity to switch apomixis on and off as needed for plant breeding or other purposes. 16

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CHAPTER 2

STRESS INDUCED SEXUAL REPRODUCTION IN APOMICTIC BOECHERA

IS CORRELATED WITH DOWN-REGULATION OF GROWTH PHASE

GENES

ABSTRACT

Apomixis, or asexual plant reproduction through clonal seed, can greatly accelerate plant breeding programs and enables stable hybrids to be perpetually used in agriculture. The quest for understanding the genetic control of apomixis has been wrought with complexity. Modern molecular tools such as RNA sequencing-based expression profiling (RNASeq) can quickly uncover a wealth of data that is often analyzed at a high level by using gene ontology (GO) categories. Enriched GO analysis was performed on

RNASeq data from whole pistils of three apomictic and one sexual Boechera

(Brassicaceae) species grown under control and stress conditions. The three apomictic species differed from each other in important cytological ways. Boechera cf. gunnisoniana is a triploid diplosporous apomict. Boechera exilis x retrofracta is a diploid apomict that forms meiotic tetrads that often fail in favor of adventitious embryo sac formation (apospory). Boechera lignifera is a diploid apomict that forms diplosporous dyads that are surprisingly encased in a callosic cell wall similar to sexual tetrads. The two diploid apomicts reduced the regulation of metabolic stress when exposed to stress conditions and became sexual. In contrast the sexual diploid and apomictic triploid increased the regulation of stress through increased expression of stress response genes when exposed to the same stress conditions. Interestingly, all three 26 apomictic species down regulated genes associated with pro-growth states such as amino acid, protein and ribosome synthesis when exposed to stress conditions. 27

INTRODUCTION

In 1951, stress-induced shifts from apomeiosis to meiosis in pollen mother cells (PMC)

of Boechera were reported (Bocher, 1951). Four reduced (1n) microspores tended to form by meiosis (tetrads) instead of two unreduced (2n) microspores by apomeiosis

(dyads). Bocher speculated that these stress-induced shifts from apomixis to sex in male anthers might also occur in ovules of apomictic Boechera. To investigate Bocher’s suspicion, sexual and apomictic Boechera were grown in well-watered, droughted, and droughted plus high heat conditions. Heat and drought stress induced high frequency shifts in two Boechera apomicts from Taraxacum type diplospory (1st division meiotic

restitution resulting in two unreduced megaspores) to normal meiosis with four reduced

megaspores (Mateo de Arias, 2015). These results were consistent with previous reports

of apparent stress induced shifts from apomixis to sex in other angiosperms (Knox &

Heslop-Harrison, 1963; Knox, 1967; Evans & Knox, 1969; Saran & Dewet, 1976).

Sufficient numbers of pistils to produce quantitative RT-PCR (qPCR) and

RNASeq expression profiles of pistils (MMC to early embryo sac stages) of well-

watered and droughted plants were also collected from these plants (Mateo de Arias,

2015). Strong divergences between species were observed in gene expression among

species regardless of treatment. Only minor differences were observed between control

plants and stress-treated plants within species (Shilling, 2016). The objective of the

present study was to probe these minor differences in gene expression between treated

and control plants to determine if they provide clues concerning how and why stress

induces a shift from apomixis to sex. By understanding genetic and molecular 28

mechanisms of the apomixis to sex switch, it should be possible to formulate hypotheses

concerning how a switch from sex to apomixis might be engineered.

MATERIALS AND METHODS

Plant materials

Plant growth and stress treatment procedures, cytological analyses, qPCR and RNASeq

analyses, and gene expression profiling analyses including the identification of

differentially expressed genes were previously reported (Mateo de Arias, 2015;

Schilling, 2016). A more in-depth analyses of differential gene expression is reported here. Enriched gene ontology analyses are also reported.

Gene Analysis

For differential gene expression and gene ontology analyses, expression counts for

Boechera genes that mapped to a single TAIR10 locus were summed

(https://www.arabidopsis.org/). A low-count filter was applied such that genes were retained if their replicate mean transcript count was ≥ 5 for each taxa by treatment category and if at least one replicate mean transcript count among the categories was ≥

10. Seven gene expression comparisons were then conducted: four that compared control versus treatment samples within taxa, three that compared control samples of the three apomicts to control samples of the sexual. The data were then transformed and normalized using the DESeq2 R package, and differentially expressed (DE) genes were identified using edgeR (FDR, P ≤ 0.05). Both programs were accessed through BRB-

ArrayTools 4.6.0 (developed by Dr. Richard Simon and the BRB-ArrayTools

Development Team, https://brb.nci.nih.gov/BRB-ArrayTools/). Raw RNASeq data will 29 be deposited with the NCBI Gene Expression Omnibus (GEO) upon submittal of publications.

Boechera loci that mapped to TAIR10 genes were used for GO analyses.

Normalized expression values of all retained genes (both DE genes and non-DE genes) were used to identify enriched GO categories within the three divisions, molecular function, biological process and cellular component. This was accomplished by averaging the normalized replicate values of each taxa by treatment combinations and then obtaining the Log base 2 values of the ratio of the treatment means being compared for each gene. These ratios (+ and – value) were then used to identify significantly enriched GO categories by Panther Enrichment Tests (4/13/2017 release, 8/8/2018 GO database release, PantherTM Classification System 76, Ver. 13.1). Bonferroni corrections are conservative in GO enrichment analyses

(http://go.pantherdb.org/tools/compareToRefList.jsp) and were not used. My enrichment objectives were to i) identify molecular pathways that putatively cause sex apomixis switching based on less conservative GO analyses, and ii) conducted thereafter specific experiments (Chapter 3 & 4) to test the postulated pathways. While, Bonferroni corrections were not performed, GO analyses were conducted at the P ≤ 0.05 level of confidence. Instead of listing all of the GO data from these analyses the DE genes and enriched GO categories can be regenerated with updated GO annotations by downloading the raw RNASeq file deposited with NCBI GEO. 30

RESULTS AND DISCUSSION

For RNASeq, three replicate samples of immature pistils of sexual B. stricta,

diplosporous B. lignifera, diplosporous B. cf. gunnisoniana and diplosporous and

aposporous B. exilis x retrofracta were obtained from well-watered and droughted plants.

Mateyo et al., (2015) cytologically analyzed these and documented statistically

significant stress-induced shifts from apomeiosis to meiosis among the three apomicts

from which this RNAseq data set was derived. These produced 306 M reads (12.76 ±

1.94 M SD per library), which represented 26,072 B. stricta genes (a mapping efficiency of 0.90 ± 0.03 SD). Blastn to TAIR10 failed for 837 Boechera genes (Shilling, 2016). Of the B. stricta genes, 18,911 corresponded to separate Arabidopsis TAIR10 genes. An additional 6324 B. stricta genes corresponded to 2413 TAIR10 genes (21,324 TAIR10 matches total). Of the 6324 genes, two Boechera genes corresponded to each of 1765

TAIR10 genes, three corresponded to 594, four to 226, and 5-56 to 577.

A strength of this study was the ability to analyze across treatment (well-watered

vs. droughted) effects within species. This ruled out confounding effects of species

differences observed in other studies discussed below where apomicts were compared to

sexuals of a different species. As conducted herein, commonalities among the three

different apomicts can easily be identified, which helps to further narrow down potential

pathways responsible for apomixis to sex switching. This is in contrast to other studies

where differences in reproductive mode (sex vs. apomixis) are confounded by species or

genotype differences. For example, in a recent publication (Tang et al., 2017), flower

transcriptomes at four stages of floral development of a sexual diploid, Boehmeria

tricuspis, and of an apomictic triploid (Antennaria type diplospory), Boehmeria

tricuspis, were compared. A large number of differentially expressed genes was 31

observed (18,899 unigenes). Enriched GO categories identified that plant hormone signal

transduction, cell cycle regulation, and transcription factor regulation differentiated the

two genotypes. These differences were reported to be due to reproductive mode

(apomixis vs. sex) or ploidy levels (diploid vs triploid). However, this comparison was

confounded by possible genotypic or ecotypic differences as well which may have

masked the ability to identify differences due to apomixis or ploidy.

A large number of differentially expressed genes were identified in the current

study when comparing apomictic and sexual Boechera across species (Schilling, 2016;

and Table 2.1). However, in the current study, stress treatments that cytologically shifted

apomictic reproduction to sexual reproduction within species produced few differentially

expressed genes (Table 2.2). This suggests that most differentially expressed genes

identified in the across-species comparisons are likely due to species differences rather

than differences in mode of reproduction. However, if apomictic plants are ‘wired’

differently than sexual plants, then it is possible to miss important information unless

cross-species comparisons are also carefully analyzed.

TableTable 1. 2.1 Differentially Differentially Expressed Expressed genes acrossgenes speciesacross (controlspecies growth (control conditions). growth conditions).

B. exilis x retrofracta B. cf. gunnisoniana B. lignifera vs. B. stricta vs. B. stricta vs. B. stricta # of up-regulated genes 3184 1751 3255 # of down-regulated genes 3600 1868 3234

TableTable 2. 2Differentially.2 Differentially Expressed genes expressed within species genes across within control species vs. treatment across (stress) control vs. treatment (stress)

B. stricta B. exilis x retrofracta B. lignifera B. cf. gunnisoniana control vs. treatment control vs. treatment control vs. treatment control vs. treatment # of up-regulated genes 79 309 43 15 # of down-regulated genes 46 133 103 9 32

Despite a low number of differentially expressed (DE) genes within species but across treatments, many enriched GO categories were observed (based on expression levels of all genes). GO category enrichment was used to uncover biological processes that may be responsible for this shift in reproductive mode based on being under or over represented. As such, enriched Biological Pathway GO categories (1491 unique GO categories) were predominately analyzed. Where noted, enriched Molecular Function

(1063 unique GO categories) and Cellular Component (550 unique GO categories) GO’s provide supporting information.

The three apomictic species analyzed differed from each other in their apomictic modes and behavior. Hence, comparisons of the sexual taxon against each of the three apomictic taxa were considered separately. In control conditions, ovules of apomict B. exilis x retrofracta often undergo female meiosis, but the surviving megaspores often degenerate while the embryo sac forms adventitiously (aposporously) from an unreduced somatic cell of the ovule wall. In stress conditions many Biological Pathway GO categories involving the activation of meiosis were underrepresented in this species

(Table 2.3), but similar changes were not observed in the strictly sexual B. stricta under stress conditions. Many of the same meiosis specific categories were overrepresented in the other two apomicts (B. lignifera and B. cf. gunnisoniana) when exposed to stress, and these shifts toward meiotic gene expression were correlated with the stress-induced activation of meiosis as observed cytologically (Mateo de Arias, 2015). Boechera c.f. gunnisoniana is a great example of a diplosporous apomict. However, because it is a triploid it is difficult to compare gene expression levels with the sexual diploid or the other highly aposporous apomictic diploid. Lastly, an odd discovery was made in B. lignifera, a diplosporous apomict. In control conditions, staining for callose revealed that 33

Table 2.3 Enriched Biological Pathway GO’s of interest involved in meiosis. De-regulated Table 3. Enriched Biological Pathway GO's of interest involved in meiotic processes that are de-regulated within species but across t within(treatment/stress) species vs. but c (control) across conditions t (treatment/stress) vs. c (control) conditions

GO ID GO term B. lignifera B. exilis x retrofracta B. cf. gunnisoniana t/c t/c t/c # of # of # of P value +/- P value +/- P value +/- genes genes genes GO:0007127 meiosis I 54 0.00013 + 54 1.18E-05 - 54 0.00717 + GO:0007135 meiosis II 14 0.00225 - GO:0051321 meiotic cell cycle 123 0.00162 + 124 8.56E-14 - 124 3.7E-07 + GO:1903046 meiotic cell cycle process 102 0.0008 + 103 4.11E-12 - 103 0.00027 + GO:0045132 meiotic chromosome segregation 44 0.00013 + 44 9.65E-07 - 45 0.00937 + GO:0051307 meiotic chromosome separation 12 0.0042 + 12 0.000848 - 12 0.0338 + GO:0033206 meiotic cytokinesis 7 0.000629 - GO:0140013 meiotic nuclear division 76 5.1E-05 + 76 6.52E-11 - 77 0.00043 + chromosome organization involved in GO:0070192 37 0.00059 37 3.06E-05 meiotic cell cycle + - GO:0110029 negative regulation of meiosis I 3 0.00983 - 3 0.038 + GO:0051447 negative regulation of meiotic cell cycle 3 0.00983 - 3 0.038 + negative regulation of meiotic nuclear GO:0045835 3 0.00983 3 0.038 division - + GO:0051446 positive regulation of meiotic cell cycle 7 0.0131 + GO:0007131 reciprocal meiotic recombination 42 1.8E-05 + 42 0.000083 - 42 0.0185 + GO:0060631 regulation of meiosis I 4 0.00476 - 4 0.0154 + GO:0051445 regulation of meiotic cell cycle 15 0.0274 - 15 0.00046 + GO:0040020 regulation of meiotic nuclear division 12 0.00401 + regulation of reciprocal meiotic GO:0010520 3 0.00983 3 0.038 recombination - +

callose forms around the diplosporous dyads in a pattern similar to that observed in

sexual tetrads (unpublished data). Generally, callose deposits in the walls of

diplosporous MMC and meiocytes has been greatly reduced (Carman et al., 1992; Peel et

al., 1997). However, it has been observed to form in high concentrations, similar to that

observed herein, in diplosporous dyad walls of Paspalum minus (Bonilla and Quarin,

1997), triploid Taraxacum atricapillum (Musiał et al., 2015), and Chondrilla juncea

(Musiał and Kościńska-Pająk, 2017).

The use of these unique apomicts for comparison purposes provides both

weaknesses to be aware of and strengths that can be capitalized on. One weakness is the

difficulty of comparing different apomicts due to ploidy differences or their partially 34

sexual nature. However, this is also a strength that can be used as a restrictive filter to

identify patterns common to the three apomicts. With this in mind, of the enriched

Biological Pathway GO categories observed between control and stressed sexual B.

stricta, 70% were over-represented because of stress (309 out of 439). The two diploid

apomicts responded to stress in an opposite manner: 10 – 20% of the enriched Biological

Pathway GO categories were over-represented when stressed (149 out of 801 and 62 out

of 625 in B. exilis x retrofracta and B. lignifera respectively). In the triploid apomict,

93% of the enriched Biological Pathway GO categories were over-represented because

of stress (586 out of 633). This coincides with the genetic responses to stress, where

Biological Pathway GO categories were under-represented because of stress conditions

in diploid apomictic B. lignifera and B. exilis x retrofracta and over-represented when

the triploid apomictic B. cf. gunnisoniana and the diploid sexual B. stricta were stressed

(Table 2.4). A different pattern is seen in the Molecular Function GO categories wherein

the triploid apomict follows a pattern similar to the other diploid apomicts (Table 2.5).

Table 2.4 Enriched Biological Pathway GO’s of interest involved in stress response. Table 4. Enriched Biological Pathway GO's of interest involved in stress response that are de-regulated within species but across t (treatment/stress) vs. Dec (control)-regulated conditions within species but across t (treatment/stress) vs. c (control) conditions.

GO ID GO term B. lignifera B. exilis x retrofracta B. stricta B. cf. gunnisoniana t/c t/c t/c t/c # of # of # of # of P value +/- P value +/- P value +/- P value +/- genes genes genes genes GO:0006950 response to stress 2293 0 - 2257 3.34E-02 - 2289 3.93E-03 + 2298 3.99E-11 + GO:0006970 response to osmotic stress 498 0 - 500 1.49E-06 + GO:0006979 response to oxidative stress 310 2.14E-03 - 319 1.05E-04 + GO:0033554 cellular response to stress 660 2.84E-03 - 661 4.48E-08 - 659 4.20E-10 + GO:0047484 regulation of response to osmotic stress 23 2.46E-03 + GO:0034599 cellular response to oxidative stress 72 1.71E-03 - GO:0080134 regulation of response to stress 259 2.30E-06 + GO:1902882 regulation of response to oxidative stress 8 2.91E-03 + GO:0009651 response to salt stress 443 0 - GO:1990170 stress response to cadmium ion 2 1.89E-02 - 2 2.75E-02 - 35

Table 2.5 Enriched Molecular Function GO’s of interest involved in stress response. TableDe- 5.regulated Enriched Molecular within Function GO's species of interest involvedbut across in stress response t (treatment/stress) that are de-regulated within species vs. butc across(control) t (treatment/stress) conditions. vs. c (control) conditions

GO ID GO term B. lignifera B. exilis x retrofracta B. stricta B. cf. gunnisoniana t/c t/c t/c t/c # of # of # of # of P value +/- P value +/- P value +/- P value +/- genes genes genes genes GO:0015036 disulfide oxidoreductase activity 56 1.68E-01 - 56 3.81E-01 - 55 2.09E-04 - GO:0003913 DNA photolyase activity 5 9.87E-02 - 5 2.09E-02 - 5 6.97E-01 - GO:0015038 glutathione disulfide oxidoreductase activity 15 6.90E-03 - 15 2.23E-01 - 15 4.72E-02 - GO:0031072 heat shock protein binding 30 9.26E-03 - 30 2.45E-05 - 30 4.65E-02 - GO:0000702 oxidized base lesion DNA N-glycosylase activity 7 6.96E-01 - 7 1.66E-01 - 7 5.15E-01 - GO:0008534 oxidized purine nucleobase lesion DNA N-glycosyl5 7.49E-01 - 5 4.09E-01 - 5 2.43E-01 - GO:0016667 oxidoreductase activity, acting on a sulfur group o92 1.47E-02 - 92 7.05E-01 - 92 1.05E-06 - oxidoreductase activity, acting on a sulfur group GO:0016673 2 3.28E-01 2 7.09E-01 2 9.61E-01 of donors, iron-sulfur protein as acceptor - - -

oxidoreductase activity, acting on a sulfur group GO:0016672 of donors, quinone or similar compound as 3 5.95E-02 - 3 4.23E-01 - 3 1.70E-01 - acceptor oxidoreductase activity, acting on hydrogen as GO:0046995 1 6.35E-01 1 2.50E-01 1 6.28E-01 donor, with other known acceptors + + + GO:0016651 oxidoreductase activity, acting on NAD(P)H 91 2.93E-06 - 91 8.33E-01 - 91 8.70E-02 - GO:0016652 oxidoreductase activity, acting on NAD(P)H, NAD(8 3.29E-01 - 7 9.73E-01 - 8 8.93E-01 - oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular GO:0016713 4 1.15E-01 4 4.53E-02 8 4.11E-02 4 4.99E-01 oxygen, reduced iron-sulfur protein as one + + + + donor, and incorporation of one atom of oxygen oxidoreductase activity, acting on superoxide GO:0016721 8 7.42E-02 8 1.01E-01 8 7.79E-01 radicals as acceptor - - - oxidoreductase activity, acting on the CH-CH GO:0016635 group of donors, quinone or related compound 9 1.59E-01 - 9 4.04E-01 - 9 6.57E-01 - as acceptor oxidoreductase activity, acting on the CH-OH GO:0016899 14 9.30E-01 15 4.06E-03 15 7.75E-01 group of donors, oxygen as acceptor + + + GO:0016724 oxidoreductase activity, oxidizing metal ions, oxyg5 1.51E-02 + 5 7.46E-02 + 5 1.26E-02 + GO:0031559 oxidosqualene cyclase activity 6 8.21E-02 + 4 6.44E-01 + 6 2.70E-01 + GO:0015037 peptide disulfide oxidoreductase activity 15 6.90E-03 - 15 2.23E-01 - 15 4.72E-02 - GO:0019789 SUMO transferase activity 5 4.32E-01 - 5 3.08E-01 - 5 8.11E-01 - GO:0061630 ubiquitin protein ligase activity 134 8.91E-01 + 133 1.21E-03 + 133 3.01E-05 + GO:0044390 ubiquitin-like protein conjugating enzyme binding 23 2.96E-01 + 22 6.46E-03 + 22 7.84E-01 + GO:0061659 ubiquitin-like protein ligase activity 135 8.41E-01 + 134 1.01E-03 + 134 2.68E-05 + GO:0019787 ubiquitin-like protein transferase activity 353 8.92E-01 + 349 4.26E-03 + 349 3.11E-05 + GO:0004842 ubiquitin-protein transferase activity 345 7.91E-01 + 341 2.43E-03 + 341 1.48E-05 +

NAC transcription factors typically undergo abiotic-stress induced transcriptional reprogramming (Nakashima et al., 2012). However, within each of the species tested, stress-induced changes in NAC gene expression levels greater than 2-fold within pistil tissue were almost non-existent except for five genes in B. exilis x retrofracta, one in B. lignifera, and one in B. stricta out of 102 unique NAC genes listed in the TAIR database 36

(Table 2.6). This result is surprising especially because evidence of stress response exists

(Tables 2.4, 2.5).

Shah et al. (2016) reported a partial list of NAC transcription factors that were

deregulated in a comparison between apomictic B. gunnisoniana and sexual B. stricta in

seedling tissue grown in control conditions. However, the same NAC genes in these

same two species showed almost no difference in gene expression levels in pistil tissue

also grown in control conditions in our lab since the fold differences were less than 2

(Log2 based fold differences between -1 and 1; Table 2.7). This observation may be due

to sampling of different tissues (whole seedlings and, in some cases, premeiotic floral

buds in the Shah study vs. pistils ranging from premeiotic to early embryo sac formation

in the present study). Additionally, differences in NAC gene expression may be mostly

due to species differences rather than reproductive differences. For example, when

comparing each apomict to sexual B. stricta grown under control conditions, I identified

that across all species a combined total of about one third of NAC genes (38 out of 102)

showed expression differences greater than 2-fold, and these were clearly species

differences since they did not show expression fold differences within species across

treatments (compare Tables 2.6 and 2.8). Furthermore, there was very little overlap of

differentially expressed NAC genes when comparing each of the three apomicts to the

sexual (Table 2.8).

Table 2.6 Significant Differentially Expressed NAC genes (LOG2 Fold Change) within speciesTable 6. Significant comparison Differentially across Expressed t (treatment/stress) NAC genes (LOG2 Fold Change) vs. cwithin (control) species comparision conditions of stress vs control conditions

Arabidopsis locus ID Locus name B. exilis x retrofracta B. cf. gunnisoniana B. lignifera B. stricta AT3G04070 NAC047 5.34 #N/A -2.38 #N/A AT1G69490 NAC-LIKE, ACTIVATED BY AP3/PI(NAP) 3.08 #N/A #N/A #N/A AT3G15500 NAC055 3.07 #N/A #N/A #N/A AT5G07680 NAC080 1.47 #N/A #N/A #N/A AT3G49530 NAC062 -0.60 #N/A #N/A -0.45 37

TableTable 7. 2.7 NAC NAC genes genes identified identified by Shah et by al. Shahas differentially et al., 2016, expressed as differentially (LOG2 Fold Change) expressed between (LOGB. cf. 2 Foldgunnisoniana Change) vs. between B. stricta. B.These cf. same gunnisoniana genes are analyzed vs. B. instricta. the same These species same grown genes under arecontrol analyzed inconditions the same in thespecies curent grown study. under control conditions in the current study.

Arabidopsis locus ID Locus name Shah et. al. 2016 Current Study Up-regulated in Apo-1 AT4G28530 NAC074 5.63 #N/A AT1G69490 NAC-LIKE,ACTIVATEDBYAP3/PI(NAP) 3.10 #N/A AT3G15500 NAC055 2.87 -1.06 AT1G76420 CUPSHAPEDCOTYLEDON3(CUC3) 2.69 #N/A AT2G46770 NAC043 2.43 #N/A AT4G27410 RESPONSIVETODESICCATION26(RD26) 2.43 0.7853 AT5G17260 NAC086 2.28 #N/A AT3G44290 NAC060 2.22 2.37 AT5G53950 CUC2 2.11 #N/A AT5G64060 NAC103 1.88 #N/A AT1G52890 NAC019 1.86 #N/A AT1G01720 NAC002 1.81 0.846 AT5G66300 NAC105 1.69 #N/A AT1G77450 NAC032 1.60 #N/A AT2G27300 NTM1-LIKE8(NTL8) 1.41 #N/A AT5G07680 NAC080 1.41 #N/A AT3G18400 NAC058 1.33 #N/A AT5G56620 NAC099 1.28 #N/A AT2G24430 NAC038 1.27 #N/A AT5G61430 NAC100 1.26 #N/A AT1G32510 NAC011 1.11 #N/A AT1G02250 NAC005 1.00 #N/A Down-regulated in Apo-1 AT1G01010 NAC001 -2.38 #N/A AT1G26870 NAC009 -2.70 #N/A AT1G32770 NAC012 -1.48 #N/A AT1G33280 NAC015 -1.16 #N/A AT1G52880 NOAPICALMERISTEM(NAM) -1.11 #N/A AT2G17040 NAC036 -2.97 2.43 AT2G33480 NAC041 -1.23 #N/A AT2G43000 NAC042 -3.77 #N/A AT3G12977 NAC-like -1.24 #N/A AT3G15510 NAC056 -1.38 #N/A

Gene expression profiling using custom built microarrays was also performed on

sexual and aposporous Hypericum perforatum. Despite not discovering very many genes

they reported that plant hormones probably play a key role in gene regulation and the

expression of apospory in this species (Galla et al., 2017). The enriched Biological 38

Pathway GO categories in the present study showed varied responses to plant hormones

(Table 2.9). Plant hormone responses were also reported to be differentially expressed in the aforementioned Boehmeria study (Tang et al., 2017) and in a comparison of laser assisted microdissection of sexual diploid Arabidopsis thaliana and triploid apomictic

Boechera gunnisoniana MMC’s (Schmidt et al., 2014).

Table 2.8 NAC genes (LOG2 Fold Change) across species/reproductive mode grown underTable 8. controlNAC genes conditions (LOG2 Fold Change) across species/reproductive mode grown under control conditions

Arabidopsis locus ID B. cf. gunnisoniana B. lignifera vs. B. B. exilis x retrofracta vs. B. stricta stricta vs. B. stricta AT1G60280 -5.98 #N/A -5.85 AT3G15500 -1.06 #N/A -3.89 AT4G27410 0.79 #N/A -0.94 AT1G01720 0.85 #N/A -1.28 AT1G32870 0.92 -0.59 #N/A AT5G13180 1.18 #N/A 0.75 AT3G01600 1.79 -2.33 -0.59 AT5G22290 1.83 2.22 1.78 AT3G44290 2.37 2.10 3.19 AT2G17040 2.43 #N/A #N/A AT1G69490 #N/A -3.33 -2.26 AT1G61110 #N/A -2.12 #N/A AT5G39820 #N/A -1.94 #N/A AT1G02250 #N/A -1.56 -1.55 AT5G50820 #N/A -1.2345 #N/A AT3G15170 #N/A -1.12 1.51 AT1G01010 #N/A -1.0924 #N/A AT4G01540 #N/A -0.89 -1.14 AT1G52890 #N/A -0.58 -2.93 AT5G64060 #N/A -0.58 1.32 AT1G25580 #N/A -0.3642 #N/A AT5G09330 #N/A -0.3427 #N/A AT3G10500 #N/A 0.54 0.38 AT2G02450 #N/A 1.1071 #N/A AT5G07680 #N/A 1.29 -2.06 AT5G61430 #N/A 1.6379 #N/A AT3G49530 #N/A #N/A -2.14 AT5G14000 #N/A #N/A -1.68 AT1G77450 #N/A #N/A -1.54 AT4G01550 #N/A #N/A -1.27 AT5G08790 #N/A #N/A -1.26 AT5G63790 #N/A #N/A -1.15 AT1G34180 #N/A #N/A -0.58 AT5G24590 #N/A #N/A -0.57 AT3G10490 #N/A #N/A 0.39 AT3G10480 #N/A #N/A 0.78 AT5G53950 #N/A #N/A 1.36 AT5G41090 #N/A #N/A 1.98 39

Table 2.9 Enriched Biological Pathway GO’s for plant hormones. De-regulated within speciesTable 9. Enriched but Biologicalacross Pathway t (treatment/stress) GO's for plant hormones vs. de-regulated c (control) within species conditions but across t (treatment/stress) vs. c (control) conditions GO ID GO term B. lignifera B. exilis x retrofracta B. stricta B. cf. gunnisoniana t vs. c t vs. c t vs. c t vs. c # of # of # of # of P value +/- P value +/- P value +/- P value +/- genes genes genes genes GO:0071215 cellular response to abscisic 176 1.67E-03 176 1.97E-02 acid stimulus - + GO:0009737 response to abscisic acid 443 4.99E-08 - GO:0009687 abscisic acid metabolic process 18 4.09E-03 + GO:0060918 auxin transport 66 3.66E-02 + GO:0009733 response to auxin 280 1.78E-02 - GO:0071367 cellular response to 62 3.59E-03 brassinosteroid stimulus - GO:0009741 response to brassinosteroid 80 2.01E-02 - GO:0071368 cellular response to cytokinin 45 2.77E-02 stimulus - GO:0009735 response to cytokinin 192 1.04E-02 - GO:0009692 ethylene metabolic process 18 4.34E-03 - GO:0010476 gibberellin mediated signaling 49 3.87E-02 pathway + GO:0009739 response to gibberellin 109 1.94E-02 - GO:0071395 cellular response to jasmonic 58 1.78E-03 acid stimulus + GO:0009867 jasmonic acid mediated 57 2.08E-03 signaling pathway + GO:0009694 jasmonic acid metabolic 24 3.10E-04 24 5.08E-03 process - + GO:0080140 regulation of jasmonic acid 3 4.46E-02 metabolic process + GO:0071446 cellular response to salicylic 33 3.79E-02 36 2.75E-03 acid stimulus - + GO:0010337 regulation of salicylic acid 13 2.75E-03 metabolic process + GO:0009751 response to salicylic acid 138 3.65E-02 + GO:0009863 salicylic acid mediated 30 1.39E-02 signaling pathway -

Another study employed a comparison of transcript profiles from micro-dissected ovules of apomictic and sexual Boechera across four different stages in ovule development. They reported a heterochronic shift in gene expression patterns and concluded that apomixis may be related to a downregulation of genes, which occurred in the apomict during megaspore mother cell formation. This phenomenon was suggested to be due to the ineffectiveness of transcription factor binding in the hybrid genome 40

(Sharbel et al., 2010). However, this early study was limited by older technologies that may have introduced a sampling bias in the mRNA’s that were sequenced through

SuperSAGE tags (restriction enzymes were used to ligate adapters to a subset of total mRNA’s for sequencing). This may be why their results contradict the current study where there is a relatively even split between genes that were up or down regulated

(Table 2.1 and 2.2).

Of great interest are the few Biological Pathway GO categories that were deregulated in all three apomicts under the stress conditions that shifted apomeiosis to meiosis. These categories, which were not downregulated in the sexual but were downregulated under stress in all three apomicts, involved amino acid, protein and ribosome synthesis (Table 2.10).

The analysis of Cellular Component GO categories that were deregulated in all 3 apomicts outlines a similar pattern wherein stress shuts down growth phase gene expression, i.e. ribosome and mitochondrial components (Table 2.11). This suggests that under stress conditions, the apomicts down regulate pro-growth signals. Activating a stress signal, which shuts down a pro-growth state and triggers autophagy, appears to be central to activating meiosis (Chapter 3). Therefore, a signal that counteracts stress signaling and stimulates a pro-growth state during megagametophyte formation may be the type of metabolic signal that could switch sex to apomixis in sexual plants. 41

TableTable 10. 2.10Enriched Enriched Biological Pathway Biological GO's common Pathway to all apomicts GO’s that common are de-regulated to all within apomicts. species but acrossDe-regulated t (treatment/stress) vs. c (control) conditions within species but across t (treatment/stress) vs. c (control) conditions.

GO ID GO term B. lignifera B. exilis x retrofracta B. cf. gunnisoniana t vs. c t vs. c t vs. c # of # of # of P value +/- P value +/- P value +/- genes genes genes GO:0043604 amide biosynthetic process 469 0 - 471 0 - 470 1.90E-06 - GO:0043043 peptide biosynthetic process 428 0 - 429 0 - 428 1.20E-06 - GO:0006457 protein folding 209 2.07E-03 - 209 1.59E-03 - 208 7.01E-03 - GO:0022613 ribonucleoprotein complex biogenesis 271 9.30E-09 - 270 0 - 270 3.11E-02 - GO:0042254 ribosome biogenesis 229 0.2.2E-07 - 229 3.90E-15 - 229 3.02E-03 - GO:0006412 translation 423 0 - 424 0 - 423 3.60E-07 -

Table 2.11 Enriched GO’s of interest for cellular components de-regulated within species butTable 11.acr Enrichedoss t GO's (treatment/stress) of interest for cellular components vs. c de-regulated(control) within conditions species but across t (treatment/stress) vs. c (control) conditions

GO ID GO term B. lignifera B. exilis x retrofracta B. stricta B. cf. gunnisoniana t vs. c t vs. c t vs. c t vs. c # of # of # of # of P value +/- P value +/- P value +/- P value +/- genes genes genes genes GO:0022626 cytosolic ribosome 231 0.00E+00 - 230 0.00E+00 - 231 1.43E-11 - GO:0005730 nucleolus 390 1.11E-16 - 388 0.00E+00 - 388 1.92E-02 - 388 1.07E-01 - GO:0015934 large ribosomal subunit 134 9.05E-14 - 134 7.67E-14 - 134 1.71E-10 - GO:1990904 ribonucleoprotein complex 688 0.00E+00 - 685 0.00E+00 - 689 4.24E-08 - GO:0044391 ribosomal subunit 216 0.00E+00 - 216 0.00E+00 - 216 2.00E-14 - GO:0005840 ribosome 82 0.00E+00 - 345 0.00E+00 - 346 0.00E+00 - GO:0015935 small ribosomal subunit 2.65E-13 - 82 9.54E-12 - 82 1.78E-05 - GO:0022626 cytosolic ribosome 231 0.00E+00 - 230 0.00E+00 - 231 1.43E-11 -

GO:0030964 NADH dehydrogenase complex 58 8.04E-04 - 58 7.58E-04 - 57 1.47E-05 -

GO:0005740 mitochondrial envelope 317 8.47E-03 - 318 5.99E-05 - 316 1.48E-05 -

GO:0005743 mitochondrial inner membrane 253 1.69E-02 - 253 3.71E-04 - 252 3.19E-06 -

GO:0031966 mitochondrial membrane 306 1.02E-02 - 307 1.22E-04 - 305 2.22E-05 -

GO:0044455 mitochondrial membrane part 185 1.36E-03 - 185 4.85E-04 - 184 9.42E-08 -

GO:0044429 mitochondrial part 434 1.03E-03 - 435 1.33E-05 - 433 1.65E-05 -

GO:0098798 mitochondrial protein complex 185 1.37E-03 - 185 9.49E-05 - 184 1.02E-09 -

GO:0005746 mitochondrial respiratory chain 91 1.54E-03 - 91 6.74E-04 - 90 1.19E-05 - inner mitochondrial membrane GO:0098800 protein complex 136 6.12E-04 - 136 4.20E-04 - 135 1.76E-08 -

GO:0070469 respiratory chain 99 8.02E-04 - 99 4.65E-04 - 98 5.98E-06 -

GO:0098803 respiratory chain complex 90 1.03E-03 - 90 7.68E-04 - 89 7.25E-06 -

GO:0045271 respiratory chain complex I 58 8.04E-04 - 58 7.58E-04 - 57 1.47E-05 - 42

CONCLUSIONS

RNASeq data can be a powerful tool that unearths a wealth of data that can be challenging to analyze and can leave the investigation in a hole that is difficult to climb out of. The risk is building an intellectual ladder that leads nowhere. The design of the present study allowed for sex apomixis comparisons to be made within the same genotype. This allowed for the extraction of valuable information for use in building testable hypotheses. In the current study it was important to separate out species differences that may otherwise confound the valuable information I was seeking. My results suggest that perhaps many hypotheses generated from previously published studies might be seriously confounded by species differences and not representative of reproductive differences. Based on the information gathered from the present study, I hypothesize that apomixis occurs under growth phase metabolic states that can be shut off through stress thus triggering the meiotic pathway. 43

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46043

CHAPTER 3

METABOLICALLY SWITCHING MEIOSIS TO APOMEIOSIS

ABSTRACT

The formation of callosic cell walls in meiocytes just prior to meiosis does not occur in many plants that undergo Taraxacum-type diplospory (1st division restitution). However, such apomicts are facultative and many will undergo meiosis and form callose when stressed. In wild-type obligately sexual Arabidopsis thaliana, the timing of stress signaling, in relation to specific stages within meiotic prophase I of female meiocytes, was analyzed by simultaneous callose and chromosome staining. Stress-induced callose formation in the walls of female meiocytes coincided with mid prophase I, possibly the zygotene to pachytene stage. Stress signaling responsible for callose formation was effectively thwarted by topical in situ applications of glucose and/or brassinosteroid to the exterior wall of the pistil hours before meiosis. Here, < 10 % of meiocytes of treated pistils produced callose and a tetrad of megaspores. Most meiocytes lacked callose and underwent 1st division restitution to produce dyads of unreduced megaspores, which is typical of Taraxacum type diplosporous apomicts in nature. Many of the resulting functional megaspores were observed to undergo presumed unreduced gametophyte formation. These findings indicate that apomeiosis competencies exist in A. thaliana and can be triggered by a pro-growth metabolic signal that inactivates sex and induces apomixis.

INTRODUCTION

Stress signaling is central to sexual gametogenesis in both plants (McInnis et al., 2006)

and animals (Ramalho-Santos et al., 2009). Interestingly, a shift from apomictic to

sexual reproduction in many plants and animals is also initiated by stress (Fig. 1.1;

Suomalainen et al., 1987; Asker and Jerling, 1992; Carman et al., 2011; Sarhanova et al.,

2012; Rodrigo et al., 2017; Mateo de Arias, 2015; Gao, 2018). Cavalier-Smith (2010),

Gross and Bhattacharya (2010) and Horandl and Hadacek (2013) argue that stress

signaling played a central role in the evolution of meiosis during eukaryogenesis and its

timing-of-onset during the life cycle. As such, stress signaling may be the foundation

upon which the meiocyte evolved, and it may be the functional sex-apomixis switch.

At the hub of both biotic and abiotic stress signaling is the formation of reactive oxygen species (ROS) such as H2O2 (Shlomai, 2010). As a signaling molecule, H2O2

and other ROS post-translationally modify target proteins by oxidizing thiol groups thus

forming disulfide bonds that reversibly alter protein structure and function or inactivate

catalytic sites. Specificity is achieved by localized production, concatenate hormone or

Ca+2 signaling, with targeted secondary oxidation occurring via glutaredoxins or

thioredoxins (Winterbourn and Hampton, 2008). Target proteins containing reduction-

oxidation (redox) sensitive thiol groups include i) signal transduction pathway proteins,

such as phosphatases (Tanner et al., 2011; Fig. 3.1), ii) cell cycle regulating mitogen

activated protein kinases (Kovtun et al., 2000), iii) embryogenesis regulating proteins

(Ufer et al., 2010), iv) many transcription factors, v) RNA binding proteins that direct

DNA methylation, and vi) proteins involved in histone acetylation, deacetylation or methylation (Sundar et al., 2010; Shlomai, 2010). Thus oxidative stress, or a 47

Fig. 3.1 REDOX Signaling. As an example, phosphatases can be temporarily or permanently inactivated via H2O2 stress signal.

programmed oxidative burst, can re-structure the proteome, the metabolome, the epigenome and the transcriptome. It is thus feasible that an oxidative burst is responsible for the sex-apomixis switch. In obligately sexual eukaryotes this stress signal may have

evolved to be a fixed part of the reproductive program. As an example, evidence exists

that increased ROS caused by increased day length triggers meiosis in an apomictic

Ranunculus auricomus (Klatt et al., 2016).

The formation of callose, a polysaccharide composed of glucose residues linked

by β-1,3-linkages, provides clues into the timing and location of the oxidative burst(s)

that may initiate meiosis. Callose is found in a thick layer in the cell wall around

meiocytes of most angiosperms. It surrounds the meiocyte at the very end of megaspore

mother cell (MMC) maturation just prior to meiosis at stage 2-III of ovule development

(Schneitz et al., 1995). In microsporocytes callose walls thicken at pachytene, the third

step of meiotic prophase I (Stronghill et al., 2014).

In Arabidopsis callose formation in ovules increases under stress conditions in a dosage dependent manner until it leads to apoptosis and ovule abortion (Sun et al., 48

2004), a mechanism that balances fecundity with resources. Callose deposition in

Arabidopsis has also been shown to correlate with levels of H2O2 production (Luna et al.,

2011) and is activated by calcium (Karin and Christer, 1989). This is significant because

diplosporously-apomictic angiosperms stained for callose deposition in the ovule, across

monocots and dicots, have revealed that most either lack callose or it infrequently occurs

in various but indistinct places throughout the ovule but not immediately around the

meiocyte in a thick layer as found in related sexual taxa (Carman et al., 1991; Tucker et

al., 2001). A lack of callose-inducing (meiotic-like) redox stress signaling in

diplosporous apomicts supports the hypothesis of a redox-based signal transduction

pathway regulating the switch between apomeiosis and meiosis.

The balance between stress and growth pivots on the signal transduction enzyme

known as TARGET OF RAPAMYCIN (TOR). Many stress pathways act as a kill switch

to shut down TOR, stop growth and trigger autophagy (Fig. 3.2). Glucose, the primary fuel of plant cells, stimulates (TOR) to activate growth by turning on many downstream pathways including protein synthesis and brassinosteroid (BR) signaling (Gupta et al.,

2015; Roustan et al., 2016; Zhang et al., 2016). While high levels of H2O2 can shutdown

TOR signaling, many processes are dependant on some H2O2 signaling, which is

common to actively respiring mitochondria. For example, the activation of

brassinosteroid (BR) signaling, which stimulates growth, requires H2O2 to (reversibly)

oxidize the transcription factor BRASSINAZOLE-RESISTANT1 (BZR1) (Tian et al.,

2018).

Fig. 3.2 TOR signaling. This simplified diagram outlines that the balance between growth and autophagy which is regulated by TOR.

Here, I evaluated the possiblity of thwarting the stress signal that triggers meiosis and activates TOR signaling and growth by the topical application of glucose and/or brassinolide (BR). The object of this paper was to identify the timing of the stress signal that triggers meiosis in A. thaliana and then test if glucose or BR can induce apomeiosis presumably by activating TOR signaling.

MATERIALS AND METHODS

Plant materials

Arabidopsis thaliana (ecotype Col 0) seed were hand sown in square pots (85 mm wide x 95 mm tall, 350 mL volume), which were filled with Sunshine Mix #1 potting soil

(Sun Gro Horticulture Canada Ltd, Vancouver, BC). Seedlings were grown in a controlled temperature room (20°C) with 14 h photoperiod (14/10 day/night) from soft- white fluorescent bulbs until the four-leaf stage before being thinned down to 3-4 plants per pot. Plants were then transferred to a 16 h photoperiod (16/8 day/night) at warmer temperature (22°C) under a mix of soft-white fluorescent bulbs and 1000 W high- 50

pressure sodium-vapor lamps. Shelves were positioned to provide a photosynthetic

photon flux of about 250 µmol m-2 sec-1 at the soil surface. Plants were watered with tap

water or periodically with a 15:20:20 (250 mg L-1) nutrient solution.

Meiotic Timing

Pistils to be collected for microscopy were measured with an ocular micrometer after

opening the sepal buds. One hour before the start of the photoperiod, floral buds containing pistils that were 0.6 mm to 0.7 mm long, corresponding to stage 2-III of ovule development where the pre-meiotic MMC is almost mature and both the inner and outer integument walls are beginning to bud (Schneitz et al., 1995), were marked on the sepal

with a small point permanent marker. After floral buds were marked, the pots were

randomly assigned to two harvest times (Fig. 3.3). Meiotic stages were then determined

by microscopy for pistils harvested at the start of the photoperiod and 3 h thereafter.

Fig. 3.3 Arabidopsis meiotic timing experimental outline. Chart outlining the harvest time points followed for the Arabidopsis meiotic staging experiment.

Pre-Meiotic Treatments

Arabidopsis pistils containing ovules predominately staged at 2-III, just as the two integument walls started to bud out, were treated within 45 min prior to the start of the photoperiod (lights on). Floral buds were opened by separating sepals, and a bead of solution containing 1) 2 µM BR (MilliporeSigma, St. Louis, MO), 2) 50 mM glucose, or

3) deionized water was wrapped around the pistil inside the flower bud. After soaking

for about 1 min, the entire tip of the infloresence was sumberged into a PCR tube containing the appropriate solution to maintain concentration and minimize evaporation.

Soaking times ranged from 3-8 h prior to rinsing and air drying. Pistils were then harvested 12 and 24 h later for analysis at the tetrad stage and the functional megaspore

(FMS) to early 2-nucleate embryo sac (ES2) stages, respectively. Differential interference contrast microscopy (DIC) and confocal microscopy were used to analyze ovule development and analyze callose and DAPI staining. Arabidopsis pistils containing ovules predominately staged at 2-I prior to the budding of integument walls were treated similarly, but harvest periods were delayed an additional 24 h.

Parthenogenesis and autonomous endosperm tests

Arabidopsis pistils containing ovules predominately staged at 2-III, just as the two

integument walls started to bud out, were treated within 45 min prior to the start of the

photoperiod (lights on). Floral buds were opened by separating sepals and the tip of the

stigma was clipped off using the tip of a insulin needle. Then a bead of solution

containing 1) 2 µM BR and 50 mM glucose, or 2) deionized water was wrapped around

the pistil inside the flower bud. After soaking for about 1 min (and marking the stem of

the floral bud with a small permanent marker) the entire tip of the infloresence was 52

submerged into a PCR tube containing the appropriate solution to maintain concentration

and minimize evaporation. Soaking times ranged from 3-8 h prior to rinsing and air

drying. Pistils that did not soak in solution dried out and shriveled on the wound end and

were not used. Pistils were then harvested 7 and 10 d later and cleared for DIC analysis

at the early embryo stage to search for signs of early embryonic or endosperm growth.

DIC microscopy

Pistils were fixed in formalin acetic acid alcohol (FAA) overnight, cleared in 2:1 benzyl

benzoate : dibutyl phthalate (Crane & Carman, 1987), and mounted horizontally on

slides. Ovaries were studied using a BX53 microscope (Olympus, Center Valley, PA,

USA) equipped with DIC optics. Details of ovule development were photographed using

a MicroFire 599809 camera (Olympus).

Confocal microscopy

Pistils were fixed overnight in formalin acetic acid alcohol (FAA). Chromosomes were then stained by soaking pistils in 95% ethanol containing 0.1 µg DAPI per mL. Callosic walls were then stained by soaking the DAPI stained pistils in an aqueous solution containing 136 µM aniline blue (Millipore Sigma, Darmstadt, Germany) and 100 mM

NaOH. These were then progressively changed to a sucrose saturated 100 mM NaOH aqueous solution by using mixtures of the callose stain and the sucrose solution to maintain a high pH (modified from Peel et al., 1997). Imaging was achieved using a

×63/1.3 NA oil immersion objective. Simultaneous DAPI and callose fluorescence was detected with a Zeiss LSM 510 meta laser scanning confocal microscope equipped with an argon-ion laser, using single wavelength excitation at 408 nm and a dichroic reflector 53 at HFT488 nm with detection at 450 nm – 650 nm. Callose was distinguished from DAPI staining with detection at 550 nm – 650 nm.

RESULTS AND DISCUSSION

Female meiocytes developed to stage 2-III of MMC differentiation, which coincides

with the end of the premeiotic S-phase and the beginning of meiotic prophase I, without

any detectable callose formation (Fig. 3.4a). During the first step of prophase I,

leptotene, the chromosomes begin to condense (Fig. 3.4 a,b). The earliest detection of

callose, coincided with the second and third steps of meiotic prophase I, zygotene

(chromosome pairing) and pachytene (crossing over) (Fig. 3.4b-d).

This defining moment of callose formation may be preceded by a stress signal,

since homologous recombination is triggered due to oxidative damage (Rahavi and

Kovalchuk, 2013). This is also the stage where meiosis ‘fails’ in diplosporously

apomictic Elymus (Crane and Carman, 1987). It may be at this stage when a

programmed pre-meiotic oxidative burst initiates the zygotene and pachytene stages of

meiotic prophase I and subsequently programs the epigenome committing it to the

sexual pathway. In microsporocytes callose walls also thicken at the pachytene stage

(Stronghill et al., 2014). Callose deposition was present during metaphase I (Fig. 3.4e)

and during meiosis II (Fig. 3.4f).

The premeiotic S phase is about twice as long as premitotic S phase (Watanabe et

al., 2001; Munoz-Velasco et al., 2013). Essential differences exist between S-phase for

meiosis and S-phase for mitosis. This suggests that the MMC is already programmed

and capable of meiosis before this oxidative burst occurs. Experiments topically 54

a b c

d e f

Fig. 3.4 Meiotic Prophase - simultaneous staining of chromosomes and callose. DAPI (red) stained Arabidopsis ovules also showing callose fluorescence (green overlay) from early meiotic prophase I leptotene (a), through zygotene/pachytene (b-d), metaphase (e), and the first meiotic division (f).

applying H2O2 to apomictic Boechera pistils triggered meiosis within hours (Gao, 2018).

This is strong evidence that the long “premeiotic” S-phase does not irreversibly determine meiosis or apomeiosis.

Variation in pistil length occurs at the time of meiosis initiation (Armstrong and

Jones, 2003) even though developmental staging is generally correlated with pistil length

(Smyth et al., 1990). In this respect, cytologists often collect plant tissue samples within the first few hours of daylight when the meiotic metaphase plate is more likely to be found (http://www.ars-grin.gov/npgs/images/w6/grasseval/root-tip_technique.htm). 55

These observations suggest that pistils arriving at the meiotic developmental checkpoint too late in the day or at night are delayed from continuing into meiosis until the following morning.

In wild type sexual Arabidopsis, the formation of callose and the mid meiotic prophase I stages, zygotene/pachytene, tended to occur within 3 h after the start of the photoperiod in pistils 0.6 mm – 0.7 mm in size (Fig. 3.5). At this stage in meiocyte development, stress signaling may be a switch that must be activated to continue meiosis in meiocytes. If this is the case then it is reasonable to wonder if the switch may work in reverse, that is, could apomeiosis be activated as late as this stage by replacing stress signaling with a pro-growth state?

Evidence suggests that apomictic plants buffer against stress better than sexual plants due to the upregulation of stress-defense pathways, i.e. a more robust redox buffer system (Chapter 2). Osmotic and salt challenges that stunted root growth of sexual

Arabidopsis and sexual Boechera stricta seedlings had no effect on root growth of apomictic diploid Boechera divaricarpa or apomictic triploid Boechera gunnisoniana

(Shah et al., 2016). This may be due to polyploidy, which can induce non-linear effects on gene expression, increased cell size, an altered metabolism, and increased stress tolerance (Schoenfelder et al., 2015). Since polyploidy is a common feature of apomictic plants (Galdeano et al., 2016; Delgado et al., 2016; Burges et al., 2014) and can even induce apomixis (Quarin et al., 2001), polyploidy, increased-cell-size, other changes in metabolism, or redox buffering may be responsible for improving stress tolerance and preventing germline stress signaling.

Fig. 3.5 Timing of meiotic initiation in Arabidopsis thaliana. Pistils were 0.6 mm to 0.7 mm in size. 773 ovules and 675 ovules (15 pistils each time sample) were analyzed at the first and second time samples, respectively.

Topical treatments of BR or glucose were used to test if a pre-meiotic pro-growth change in metabolic state can reverse stress signaling and activate apomeiosis. The chemicals BR at 2 µM (53 pistils) or glucose at 50 mM (48 pistils), were applied in the mornings prior to lights on (Fig. 3.6). Both chemicals induced apomeiotic dyad formation in ovules of appropriately staged pistils. DIC analyses were difficult and subjective. Nevertheless, water treated controls produced distinct tetrads.

Treatments, BR at 2 µM (32 pistils) or glucose at 50 mM (31 pistils), applied prior to lights on at early 2-III stage of MMC maturation, after both the inner and outer integument walls start to bud hours before meiosis occurs, were effective at activating apomeiosis. These were harvested 12 and 24 h after treatment, at the developmental stages encompassing late tetrad through early functional megaspore (FMS). Treatments,

BR at 2 µM (21 pistils) or glucose at 50 mM (17 pistils), were also effective at inducing apomeiotic dyad formation when applied 24 h earlier at stage 1-II, prior to the budding 57 of integument walls while the MMC was still distinguishable but small. In these cases, harvests were postponed for an additionl 24 h harvesting at 36 and 48 h after treatment

(12 and 24 h after expected meiosis).

Fig. 3.6 Timing of treatments leading to apomeiotic conversion. Pistils were treated with 2 µM BR or 50mM glucose at either stage 1-II or 2-III of MMC maturation. They were then harvested at either the expected Tetrad or FMS/ES2 stages. Dark grey represents time pistils were treated. Light grey represents time allowed for continued development while still attached to the plant prior to harvesting. 58

Unreduced FMS were generally produced by Taraxacum type diplospory (Fig.

3.6a,b). Parietal cells were also frequently observed. Absence of meiosis with possible onset of Antennaria type diplospory (early vacuolate MMC enlargement) was also observed (Fig. 3.7c,d).

a b

c d

Fig. 3.7 Treated Arabidopsis ovules undergoing Taraxacum type or Antennaria type diplospory. Ovules treated pre-meiotically with 50 mM glucose. Taraxacum type diplospory (a,b), or early vacuolate enlargement, possible Antennaria type diplospory (c,d). 59

The above experiment was repeated and combined with callose staining to better

evaluate the frequency of apomeiosis in treated pistils. Untreated controls (10 pistils)

showed callose staining in 100% of their ovules, which started as early as the late 2-III

ovule stage, where both the inner and outer integument walls were budding just prior to

meiosis, and persisted into the early 2 nucleate embryo sac stage. (Fig. 3.9).

a b

c d

Fig. 3.8 Early tetrad staged Arabidopsis pistil stained for callose. a,b) Early tetrad stage ovules a) DIC and b) callose fluorescence. c,d) an FMS staged ovule, c) DIC and d) callose fluorescence. 100% of the ovules within the pistils staged between early meiosis and late FMS contained fluorescently stained callose that was readily visible within the appropriate focal planes. 60

Pre-meiotically treated pistils, 12 with BR and 14 with glucose, lacked callose staining except for a few tetrads, which fluoresced brightly (Fig. 3.9 a,b). The observation that a few ovules fluoresced brightly was evidence that the staining procedure worked equally well with treated and untreated controls. Arabidopsis Col 0 pistils contain an average of 57 ovules (Nahar et al., 2012). Combining DIC analyses with callose staining reduced the subjectivity of analyses and identified a 79% - 95%

a b

c d e

Fig. 3.9 Late tetrad Arabidopsis pistils treated pre-meiotically with 50 mM glucose stained for callose. Most ovules in the treated pistils contained dyads without callose, while in this pistil a,b) two tetrads formed, both of which produced bright callosic walls, a) DIC, b) callose fluorescence within the ovules of the pistil. c,d,e) apomeiotic ovule with light callose staining on micropylar cap, c) DIC of the ovule, d) callose fluorescence, e) DIC with callose fluorescence overlay. 61 conversion to apomeiosis (3 to 12 callose stained meiotic ovules per pistil) when treated within the appropriate stage (Fig. 3.9a,b). Some FMS-staged dyads (3 out of 100’s) showed light callose presence on the micropylar cap (Fig. 3.9c,d,e), as was also observed in apomictic Elymus (Carman et al., 1991; Peel et al., 1997). Absence of callose florescence in most ovules of treated pistils is strong evidence of Taraxacum-type diplospory (Fig. 3.9-3.10).

The experiment was repeated again but the pistils treated pre-meiotically (8 with

BR, 11 with glucose) were allowed to continue to grow on the plant for 2 to 3 d. These were then DIC analyzed at later stages in development. Ovules appeared to develop normally from the FMS stage through to the mature embryo sac stage.

Three additional pistils treated premeiotically with 50 mM glucose were allowed to self-fertilize and seeds were allowed to ripen. The seeds were then sprouted and grown without apparent abnormalities.

Pistils treated pre-meiotically (34 pistils treated with a combination of 2µM BR and 50mM glucose) were also tested for signs of parthenogenesis or autonomous endosperm formation. The tips (stigmas) of these pistils were clipped when they were treated premeiotically to prevent future self-fertilization. Control pistils (24) were also clipped premeiotically and treated with deionized water. While soaking scar tissue quickly developed at the cut end of the pistil lacking the stigma, which may have prevented shriveling of the pistil tip after rinsing and air-drying. These were allowed to grow for 10 d after their pre-meiotic treatments. None of these pistils contained ovules with embryos or endosperm. 62

b c

Fig. 3.10 Late tetrad Arabidopsis pistils treated pre-meiotically with 50 mM glucose stained with dapi and for callose. a) the left ovule contains a sexually reduced FMS- staged tetrad with degenerating callosic megaspores (DAPI stained with callose fluorescence overlay). The right ovule is developing into an FMS-staged Taraxacum type dyad. b-d) An FMS staged Taraxacum type dyad located between two tetrads with fluorescently stained callose b) DIC image, c) callose fluorescence, d) DAPI stained with callose fluorescence overlay.

Pre-meiotic treatment with glucose or brassinosteroid apparently prevented the stress signaling that normally induces meiosis and callose formation and instead activated a pro-growth metabolic state. As a result, we observed high frequency apomeiosis and unreduced embryo sac formation. This suggests that 1) apomeiosis is an intact program in otherwise obligately sexual Arabidopsis, 2) meiosis requires a stress signal for activation leaving apomeiosis as a possible default reproductive program, and

3) ROS may originate due to metabolic stress (which was altered by application of BR or glucose) rather than being produced from other sources such as chloroplasts or peroxisomes (Demidchik, 2015).

The balance between metabolic stress and favorable growth conditions can be tipped by the extent of calcium signaling from the endoplasmic reticulum (ER). In favorable conditions small amounts of calcium are released in localized bursts from the

ER and quickly absorbed by the closely associated mitochondria (Rizzuto et al., 1998) located at the mitochondrial associated endoplasmic reticulum membrane (MAM). The calicum then simultaneously activates energy demanding cellular processes while increasing the proton motive force and ATP production (McCormack and Dentonc,

1993; Poburko et al., 2011). This system depends on the signaling molecule inositol

1,4,5 trisphosphate (IP3), which is formed by PHOSPHOLIPASE C (Williams and

Katan, 2004). IP3 works in concert with glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) and locally produced NADH to bind to inositol tris-phosphate receptor protein (IP3R) channels in the MAM to release calcium from the ER (Rizzuto et al.,

1998; Patterson et al., 2004). The system functions as a feedback loop that tightly regulates cellular levels of free-ATP (Mak et al., 1999). 64

TOR is a key signal transduction enzyme located within the MAM that is the central control point relating the balance between a pro-growth signal and stress signaling. Under favorable conditions, an abundance of glucose activates TOR signaling, which can reprogram the epigenome and change the transcriptome in under 2 h to stimulate growth (Xiong et al., 2013). Active TOR is a kinase that has many down stream targets. It directly phosphorylates S6K (Xiong and Sheen, 2012) increasing ribosome biogenesis and translational capacity. In Arabidopsis S6k2 phosphorylates

BIN2 activating brassinosteroid (BR) signaling and growth (Xiong et al., 2017).

More recently discovered downstream targets of TOR include novel regulators of the S-phase genes of the cell cycle pathway (Xiong and Sheen, 2013). A relationship between TOR and auxin signaling is also evident (Schepetilnikov etal., 2013; Deng et al., 2016), though case specific pathways need to be determined.

The molecular components used for stress signaling are coopted from those that regulate normal metabolism. Current research is unraveling incredible amounts of detail involved in the pathway. A major player is stress-associated calcium signaling. It is initiated by the oxidation of the previously mentioned IP3R channel from the luminal side of the ER by ERO1α in the MAM (Kang et al., 2008; Li et al., 2009; Anelli et al.,

2011). This stress-associated calcium release over stimulates mitochondria causing an over-reduced mitochondrial electron transport chain (ETC) (Camacho-Pereira, 2009;

Møller et al., 2001) thereby activating an alternative NADH dehydrogenase (Rasmusson et al., 2004) to regenerate NAD+ to alleviate it. It also activates respiratory burst oxidase homologue proteins that amplify the ROS signal and fine tune the spatial control of ROS production (Torres et al., 2002; Grant et al., 2000; Fluhr, 2009). Superoxide dismutase 65

and the ascorbate-glutathione redox buffer system reduce ROS to water by indirectly

shuttling electrons from NADPH (Noctor and Foyer, 1998). In the process H2O2 likely

regulates TOR by oxidizing two completely conserved cysteine residues to make a

disulfide bridge in the FATC domain reducing its functional lifespan (Dames et al.,

2005). Shutting down TOR shuts down growth and activates autophagy (Pu and

Bassham, 2013). It is interesting to note that a temporary down-regulation of TOR is necessary to trigger stress signaling and to activate the meiotic pathway in yeast and

Drosophila (Wei et al., 2014).

What would trigger a stress-induced calcium release from the ER? The

Endoplasmic Reticulum Quality Control (ERQC) system refers to the molecular machinery involved in sensing the proper folding of new proteins. The presence of improperly folded proteins triggers the ER-associated protein degredation (ERAD) pathway to retrotranslocate them into the cytosol where they are tagged with ubiquinone for degradation (Vembar and Brodsky, 2008). Consequently, metabolic changes that overly stress the ER trigger the unfolded protein response (UPR, Ron and Walter, 2007) and a stress-associated calcium release into the MAM. This triggers the amplification of the stress signal as outlined above via ROS production and deactivates TOR (located in the MAM) thereby shutting down metabolism and growth and activating autophagy (Pu and Bassham, 2013).

Evidence that these pathways are active in differentiating modes of reproduction

was obtained in a comparision of MMC transcripts produced by sexual Arabidopsis and

the closely related apomictic Boechera gunnisoniana. The apomict MMC transcripts

were enriched in genes for auxin signaling and auxin response transcription factors 66

whereas the sexual MMC transcripts were enriched in ubiquitin-dependent degradation

pathways (Schmidt et al., 2014). Therefore, the simple addition of glucose (or BR) to

pre-meiotic MMC in the present study may have activated apomixis in obligately sexual

plants presumably by activating TOR signaling and sending the germline into a growth

phase rather than a pre-programmed stress-signal induced survival/sporulation phase.

CONCLUSIONS

Meiosis may be activated by a metabolically programmed stress response. Glucose-TOR

signaling prevents stress-signaling-induced meiosis and enables apomeiosis as a default

program. Metabolic stress during gametogenesis may have evolved as a programmed

step establishing meiosis and the sexual pathway as the dominant form of reproduction

among plants with a similar pattern occurring in yeast and Drosophila (Wei et al., 2014).

Interestingly the unreduced eggs in the unreduced embryo sacs of the present study did

not produce parthenogenetic embryos. Neither parthenogenesis or autonomous

endosperm formation occurred in unreduced embryo sacs in the absence of fertilization

(prevented by clipping the stigma; n = 34 pistils). Since the meiocyte and the egg share

a similar evolutionary origin (Cavalier-Smith, 2010; Gross and Bhattacharya, 2010),

which later, through the evolution of multicellularity and alternation of generations in

plants, became developmentally separated during the life cycle, stress signaling may also

be a part of programming eggs for syngamy through imprinting. In this respect, the

central cell of the megagametophyte produces ROS during late megagametophyte

maturation (Martin et al., 2013), which may be responsible for programming the egg for

syngamy. Similar treatments may be required at this stage in development to enable 67 programming the egg for parthenogenesis. If this is the case, apomixis may be a relic of eukaryogenesis that repeatedly emerges when conditions permit, such as hybrid or polyploid-induced upregulation of stress defense pathways that affect all life cycle phases (Shah et al., 2016). 68

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CHAPTER 4

PROGRAMMING THE EGG FOR PARTHENOGENESIS AND

TRIGGERING AUTONOMOUS ENDOSPERM FORMATION

ABSTRACT

Parthenogenesis is the development of an embryo from the unreduced egg without

fertilization and is the second half of apomixis or clonal/asexual seed formation. The

analysis of genetic knockouts has produced little progress towards unraveling the genetic

control of parthenogenesis. This may be due to programmed stress signaling events that

occur prior to the maturation of the egg that may be programming it for the requirement of syngamy. To test this, stigmas of Arabidopsis pistils were clipped to prevent self- fertilization and the pistils were treated in situ with the topical application of glucose and brassinosteroid during mid to late embryo sac formation to change stress signaling into a pro-growth signal prior to the end of embryo sac maturation. This led to the low frequency activation of haploid parthenogenesis from sexually reduced eggs and possibly diploid parthenogenesis from experimentally generated apomeiotically unreduced eggs.

No signs of embryos or endosperm occurred in water treated controls. The data suggest that in most cases autonomous endosperm formation, with full development beyond the stage of cellularization, accompany both haploid and diploid parthenogenesis when the central cell and egg cell are appropriately programmed.

INTRODUCTION

Apomixis, asexual reproduction in plants through seed, often occurs in tandem with sex even within the same individual. In such facultative apomicts the expression or initiation of either the sexual or the apomictic developmental program is often influenced by environmental stress (Asker and Jerling, 1992; Carman et al., 2011; Mateo de Arias,

2015). The same is true for cyclically apomictic animals (Suomalainen et al., 1987). It has been hypothesized that apomixis is an intact polyphenic alternative to sex that is regulated by metabolic signals (Carmen et al., 2011; Hojsgaard et al., 2014). If this is correct, then gene knockouts that appear to trigger components of the pathway, such as unreduced gamete formation (apomeiosis) or egg activation (parthenogenesis), should fail due to disrupting a component of a stress-induced sexual program rather than triggering metabolic pathways that activate an alternative apomixis program.

Angiosperms have a much-reduced gametophyte generation relative to their lower-plant ancestors, e.g., ferns and gymnosperms. However, it is still a complex multi- cellular stage that, through deep evolution, was originally derived from the ancient single-celled eukaryotic common ancestor. Here, the sexual egg (gamete) and the meiotic zygote shared their stress induced developmental origins (Cavalier-Smith, 2010;

Gross and Bhattacharya, 2010; Fig. 4.1). Therefore, stress signaling may be responsible for programing both the sexual egg and the meiocyte (Chapter 3). For example, in

Arabidopsis thaliana the central cell (CC) becomes a major source of reactive oxygen species (ROS) during late stages of female gametophyte maturation just prior to pollination (stages FG6 to FG7; Martin et al., 2013). The CC is in close proximity to the maturing egg cell and this may be the time when the egg is programmed and committed to a sexual pathway. In this Chapter, the experimental approach that previously enabled 79

Fig. 4.1 The involvement of stress signaling during the evolution of the meiocyte and the egg. These derived cells may have involved stress signal induced programming in their shared ancestry.

the first half of apomixis (apomeiosis) in a sexual plant was applied to test if it would also enable the second half of apomixis (parthenogenesis). If the topical application of glucose and brassinosteroid (BR) could prevent stress signaling in the CC and egg by triggering a pro-growth signal then it may activate parthenogenesis in the otherwise obligately sexual Arabidopsis thaliana.

MATERIALS AND METHODS

Plant materials

Arabidopsis thaliana (ecotype Col 0) seed were hand sown in square pots (85 mm wide

x 95 mm tall, 350 mL volume), which were filled with Sunshine Mix #1 potting soil

(Sun Gro Horticulture Canada Ltd, Vancouver, BC). Seedlings were grown in a

controlled temperature room (20°C) with 14 h photoperiod (14/10 day/night) from soft-

white fluorescent bulbs until the four-leaf stage before being thinned down to 3-4 plants

per pot. Plants were then transferred to a 16 h photoperiod (16/8 day/night) at warmer

temperature (22°C) under a mix of soft-white fluorescent bulbs and 1000 W high-

pressure sodium-vapor lamps. Shelves were positioned to provide a photosynthetic 80 photon flux of about 250 µmol m-2 sec-1 at the soil surface. Plants were watered with tap water or periodically with a 15:20:20 (250 mg L-1) nutrient solution.

Parthenogenesis test

Pistils to be treated were marked prior to meiosis, emasculated, and tracked until 1) fully mature embryo sacs formed (FG7), or 2) FG4 to FG6 staged embryo sacs formed, which occur just prior to ROS signaling events originating from the CC (Martin et al., 2013).

The stigmas of pistils containing 1) sexually reduced eggs, or 2) unreduced eggs produced as described previously (Chapter 3) were clipped. The pistils were then immediately treated with a bead of solution containing 1) deionized water or 2) 10 µM

BR in a 50 mM glucose solution, which was wrapped around the pistil inside the attached flower bud. The entire tip of the infloresence was then submerged into a PCR tube containing the appropriate solution to maintain the concentration and to minimize evaporation. Soaking times ranged from 3 to 6 h prior to rising and air drying. Pistils were then harvested 5 - 10 d after treatment and fixed in formalin acetic acid alcohol

(FAA) overnight, cleared in 2:1 benzyl benzoate : dibutyl phthalate (Crane & Carman,

1987), and mounted horizontally on slides. Ovaries were studied using a BX53 microscope (Olympus, Center Valley, PA, USA) equipped with DIC optics. Details of ovule development were photographed using a MicroFire 599809 camera (Olympus).

RESULTS AND DISCUSSION

Induction of parthenogenesis in FG7 staged embryo sacs (fully mature) through topical application of glucose and BR was unsuccessful. Treatment of both the wild type reduced and the experimentally unreduced embryo sacs (Chapter 3) that were fully mature did not produced parthenogenetic events (n=22, and 18 pistils respectively).

Induction of parthenogenesis by topical applications of glucose and BR when FG4-FG6 staged embryo sacs were treated was successful in a few cases. Treatment at this stage, prior to CC ROS production in Arabidopsis, activated parthenogenesis in a small percentage of sexually reduced egg sacs (7 total events occurred in 6 out of 23 pistils;

Fig. 4.2) and in presumed apomeiotically unreduced egg sacs that were also treated premeiotically as described in chapter 3 (3 events occurred in 2 of 7 pistils, Fig. 4.3, 4).

The low frequency may be due to the low penetrance of the chemicals through the many cell layers of the more mature pistil and ovule wall.

Some of the parthenogenetic tests from sexually reduced embryo sacs were allowed to mature beyond 10 d before harvesting and clearing. One out of 6 pistils, the pistil wall of which was with damaged by the dissecting needle contained two parthenogenetic events, but the seed coat for each of these events was too opaque to obtain quality pictures.

The experiment was then repeated, and some of the sexually reduced embryo sacs that were treated at the FG4 - FG6 stage were then allowed to fully mature and set dry seed (one ripe seed in one pistil out of 12). This one seed sprouted but the seedling grew very slowly (took about 3x longer than normal control plants), but it eventually became a full-sized plant that set ripe seed. This may be due to chromosome doubling 82

Fig. 4.2 Haploid parthenogenesis with endosperm in Arabidopsis. FG4 to FG6 staged embryo sacs were treated with 10µM BR and 50mM glucose in emasculated Arabidopsis pistils. Stigmas were clipped prior to embryo sac maturation to prevent fertilization. Note the early development of the embryo relative the stage of endosperm development.

and the production of dihaploid seed. Flow cytometry of leaf material was too variable to be diagnostic of ploidy level.

There is a possibility that these parthenogenetic events were actually pollinated

and fertilized developing embryos despite the measures taken to prevent this occurrence.

However, this is unlikely to be the case because no events occurred in the non-treated

control pistils (n=43 pistils).

In Arabidopsis thaliana, the central cell (CC) becomes a major source of ROS

during the late stages of embryo sac maturation just prior to pollination (stages FG6 to 83

Fig. 4.3 Parthenogenesis (possibly diploid) with endosperm in Arabidopsis. Induced by treating FG4 to FG6 staged embryo sacs with 10 µM BR and 50 mM glucose in emasculated Arabidopsis pistils. Stigmas were clipped prior to embryo sac maturation to prevent fertilization.

FG7; Martin et al., 2013). This also coincides with the demethylation of the CC genome, the mobilization of transposable elements (TE), and the production of siRNA’s involved

in transgenerationally-silencing TE’s and imprinted genes in the egg (Gehring et al.,

2009). These events may be programing the egg (Erdmann et al., 2017) so that it is 84

Fig. 4.4 Parthenogenesis without endosperm in Arabidopsis. Parthenogenetic embryo is failing without endosperm (in possible diploid embryo sac). Induced in an FG4 to FG6 staged embryo sacs by treatment with 10µM BR and 50mM glucose in an emasculated Arabidopsis pistil. Stigma was clipped prior to embryo sac maturation to prevent fertilization.

sexually competent and set for syngamy. For example, after fertilization early development of the egg is primarily driven by maternally provided transcripts until the maternal to zygotic transition (MTZ), which was identified by inhibiting Polymerase II

and new RNA transcription (Baroux et al., 2008). MTZ occurs prior to the globular stage

in Arabidopsis (Baroux et al., 2008). Interestingly the MTZ is the point at which most gene knockouts that trigger parthenogenesis tend to fail (Guitton and Berger, 2005),

indicating that the maternally provided program is incomplete and does not activate

zygotic transcription.

Proper development through the MTZ may also be due to additional factors

normally provided by pollen sperm cells, such as transcripts aimed at further

reprogramming the epigenome, transcriptome, and proteome (Russell et al., 2012). It 85 may be that the paternally provided program completes the maternally provided program. This possibility is corroborated by the fact that, in plants, viable haploid zygotes can be generated after fertilization despite eliminating the paternal genome via a variety of ways (Bouvier et al., 1993; De Witte and Keulemans, 1994; Froelicher et al.,

2007; Ravi and Chan, 2010; Kelliher et al., 2017). If the stress-induced sexually programmed egg requires both maternal and paternal inputs to complete the developmental program and activate zygotic transcription, then parthenogenetic eggs must provide the entire program via a pro-growth signal during the maturation stage of the egg. If apomixis is a complete functioning program, rather than a derived and broken version of the sexual program, then it may be possible to activate fully functioning apomixis in currently obligately sexual plants simply by providing the appropriate metabolic signal within the right developmental windows.

Whether the apomictic egg and CC are programmed for full autonomous parthenogenesis or whether there is a fertilization requirement (pseudogamous apomixis) is irrelevant to the existence of the program. Both processes are variations built on the same platform that may have originated from an ancestral single celled cyclically apomictic eukaryote. The difference between the two may simply be the level to which programming occurs in the CC and the egg as the embryo sac matures. For example, autonomous apomixis is most common among apomicts that exhibit Antennaria type diplospory (Asker and Jerling, 1992). This may be due to reduced levels of stress signaling and a stronger pro-growth metabolic state at both developmental checkpoints, i.e. during apomeiosis and parthenogenesis.

CONCLUSIONS

Since stress signaling is responsible for triggering meiosis and the formation of reduced gametes (Chapter 3), it appears from the present study that it might weave together these

two steps to form sexual reproduction. These steps have become developmentally

separated through the evolution of alternating generations. I show herein that the

presence of stress signaling in sexual Arabidopsis can be switched to a pro-growth signal,

at these two developmental stages, enabling both apomeiosis and parthenogenesis. These

results provide an evolutionarily parsimonious explanation for the repeated occurrence of

apomixis in plants and animals, i.e. simple polyploidization or hybridization may

occasionally induce changes in stress tolerance (Shah et al., 2016). My research provides

compelling evidence that apomixis may have been embedded early into eukaryotic

evolution as a default reproductive program in growth favoring environments with stress

signaling triggering the alternative, i.e. sexual reproduction.

The low frequency of inducing parthenogenesis in the present study may be due

to the difficulty of the glucose BR solution diffusing through many cell layers (thicker

pistil wall and ovule walls) to reach the CC and egg. As a next step, genetic engineering may enable cell or tissue specific activation of the glucose-TOR and BR signaling

pathways to induce high frequency parthenogenesis in otherwise obligately sexual crops. 87

LITERATURE CITED

Asker SE, Jerling L. 1992. Apomixis in Plants. Boca Raton: CRC Press.

Baroux C, Autran D, Gillmor CS, Grimanelli D, Grossniklaus U. 2008. The maternal to zygotic transition in animals and plants. Cold Spring Harb Symp Quant Biol 73:89–

100

Carman JG, Jamison M, Elliott E, Dwivedi KK, Naumova TN. 2011. Apospory appears to accelerate onset of meiosis and sexual embryo sac formation in sorghum ovules. BMC Plant Biology 11:9, http://www.biomedcentral.com/1471-2229/11/9

Erdmann RM, Hoffmann A, Walter HK, Wagenknecht HA, Grob-Hardt R,

Gehring M. 2017. Molecular movement in the Arabidopsis thaliana female gametophyte. Plant Reproduction 30:141-146

Gehring M, Bubb KL, Henikoff S. 2009. Extensive Demethylation of Repetitive

Elements During Seed Development Underlies Gene Imprinting Science 324:1447–1451

Guitton AE, Berger F. 2005. Loss of function of MULTICOPY SUPPRESSOR of IRA

1 produces nonviable parthenogenetic embryos in Arabidopsis. Curr Biol 15:750–754

Kelliher T, Starr D, Richbourg L, Chintamanani S, Delzer B, Nuccio ML Green J,

Chen Z, McCuiston J, Wang W, Liebler T, Bullock P, Martin B. 2017.

MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction.

Nature 542:105

Martin MV, Fiol DF, Sundaresan V, Zabaleta EJ, Pagnussat GC. 2013. oiwa, a

Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide

Dismutase, Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac

Development and Fertilization in Arabidopsis. The Plant Cell 25:1573–1591 88

Ravi M, Chan SWL. 2010. Haploid plants produced by centromere-mediated genome

elimination. Nature 464:615-618

Russell SC, Gou X, Wong CE, Wang X, Yuan T, Wei X, Bhalla PL, Singh M. 2012.

Genomic profiling of rice sperm cell transcripts reveals conserved and distinct elements

in the flowering plant male germ lineage. New Phytologist 195:560–573

Suomalainen E, Saura A, Lokki J. 1987. Cytology and evolution in parthenogenesis.

CRC Press, Baca Raton, FL

Bouvier L, Zhang YX, Lespinasse Y. 1993. Two methods of haploidization in pear,

Pyrus communis L., greenhouse seedling selection, in situ parthenogenesis induced by

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Ollitrault P. 2007. Induced parthenogenesis in mandarin for haploid production:

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Depletion of Key Meiotic Genes and Transcriptome-Wide Abiotic Stress

Reprogramming Mark Early Preparatory Events Ahead of Apomeiotic Transition.

Frontiers in Plant Science 7:1539 89

CHAPTER 5

SUMMARY

In the quest to understand the molecular mechanisms regulating apomictic reproduction

in plants, I first took a ‘30,000 foot’ perspective to identify a regulatory pattern (stress)

that differentiates sexual from apomictic reproduction in nature (Chapter 2). Using this

perspective as a guide I boiled it down to a testable hypothesis. This required analyzing

the timing of early callose formation and the initiation of meiotic prophase I, in

particular, pachytene in Arabidopsis thaliana, to correlate stress signaling with the

defining step of meiotic commitment. I also tested the influence of H2O2 stress signaling

on initiating meiotic onset in facultatively apomictic Boechera as demonstrated by Gao

(2018).

Recent publications report that sexual Arabidopsis thaliana exhibits stress signaling

during embryo sac maturation. These two stress signaling events, during early meiosis

and during embryo sac maturation, may be responsible for programming the entire

sexual pathway.

The possible effects of TARGET OF RAPAMYCIN (TOR) signaling on activating

apomeiosis or parthenogenesis in sexual wild-type Arabidopsis thaliana was analyzed

via the timely topical application of glucose or brassinosteroid (BR). DAPI or callose

fluorescence staining techniques and confocal or differential interference contrast (DIC)

microscopy were used to analyze the results of these experiments.

My null hypothesis was that either stress signaling or TOR activation (via

glucose or BR signaling) would have no effect on meiosis/apomeiosis or

parthenogenesis. 90

Based on the literature reviewed herein, I expected to find a response in both the sexual and apomictic plants, switching their mode of reproduction according to the appropriate metabolic stimuli induced by the applied chemicals. I observed that meiosis can be activated in apomictic plants at the appropriate developmental stage by applying H2O2

and that sexual plants were competent to undergo apomeiosis when metabolically

stimulated within a specific developmental window with glucose or BR. Furthermore, my

studies provide strong evidence that parthenogenesis, of either 1) the sexually reduced

egg or 2) the apomeiotically unreduced egg (created experimentally), can also be

metabolically triggered by similar signaling patterns within the right developmental

window.

This remarkably simple discovery represents a paradigm shift for apomixis

research. It suggests that apomixis is a default reproductive program regulated by a

simple metabolic switch. Facultative (or cyclical) apomixis may be of ancient

evolutionary origin, deeply embedded in the genomes of even highly derived plant

species. If this is correct, the goal of engineering apomixis into important crop species

will be greatly simplified.

LITERATURE CITED

Gao L. 2018. PhD Dissertation. Pharmacologically induced meiosis apomeiosis interconversions in Boechera, Arabidopsis and Vigna. https://digitalcommons.usu.edu/etd/5192

CURRICULUM VITAE

David Sherwood (435)5356176 [email protected] CAREER OBJECTIVE To learn the scientific language and develop the thought processes that will aid me in my life-long commitment to continuing research in biological processes related to health, quality of life and sustainability.

EDUCATION 2018 Ph.D. in Plant Science – Molecular Biology, Plant, Soils and Climate Department, Utah State University. 2008 B.S. in Biology, Eastern Washington University.

RESEARCH INTERESTS The molecular biology and biochemistry of health, nutrition and sustainability.

PROFESSIONAL EXPERIENCE Sherwood Pet Health – Global Pet Food Company developing solutions to common health problems through better nutrition. http://sherwoodpethealth.com/ NiTOR – Protein Nutrition and Sports Supplement Company helping pro-athletes achieve new world records through better nutrition and education. https://nitorperformance.com/

REFEREED JOURNAL ARTICLES Windham MD, Beck JB, Li F-W, Allphin L, Carman JG, Sherwood DA, Rushworth CA, Sigel E, Alexander PJ Bailey CD, Al-Shehbaz IA. 2015. Searching for diamonds in the apomictic rough. I: A case study involving Boechera lignifera (Brassicaceae). Systematic Botany 40: 1031-1044 http://dx.doi.org/10.1600/036364415X690076

A Simple Metabolic Switch May Activate Apomixis in &lt;I&gt;Arabidopsis

A Simple Metabolic Switch May Activate Apomixis in <I>Arabidopsis