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Transcript Profiling during male meiosis in Petunia hybrida proefschrift

Transcript Profiling to analyse gene expression during male meiosis in Petunia hybrida

Op woensdag 6 oktober 2004 om 15.30 u precies in de Aula van de Radboud Universiteit Nijmegen, Comeniuslaan 2

Transcript profiling to analyse gene expression during male meiosis in Receptie na afloop van Petunia hybrida de promotie in de aula

Filip Cnudde

Filip Cnudde [email protected]

Paranimfen:

Janny Peters ([email protected])

Filip Cnudde Stefan Royaert ([email protected]) bw-cnudde 23-08-2004 14:49 Pagina 2 bw-cnudde 23-08-2004 14:49 Pagina 1

Transcript profiling to analyse gene expression during male meiosis in

Petunia hybrida bw-cnudde 23-08-2004 14:49 Pagina 2 bw-cnudde 23-08-2004 14:49 Pagina 3

Transcript profiling to analyse gene expression during male meiosis in Petunia hybrida

Een wetenschappelijke proeve op het gebied van de Natuurwetenschappen, Wiskunde en Informatica

Proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de Rector Magnificus prof. dr. C.W.P.M. Blom volgens besluit van het College van Decanen in het openbaar te verdedigen op woensdag 6 oktober 2004 des namiddags om 3.30 uur precies

door

Filip Cnudde

geboren op 10 maart 1973 te Gent bw-cnudde 23-08-2004 14:49 Pagina 4

Promotor: Prof. Dr. T. Gerats

Manuscriptcommissie: Prof. Dr. U. Grossniklaus, Universität Zürich, Switzerland Prof. Dr. H. de Jong, Wageningen Universiteit Prof. Dr. K. Goethals, Universiteit Gent, België

Design: Martien Frijns ISBN 90-90184414 Printed by: PrintParners Ipskamp, Enschede bw-cnudde 23-08-2004 14:49 Pagina 5

Contents

Chapter I 7 General introduction

Chapter II 55 Transcript profiling on developing Petunia hybrida floral organs

Chapter III 73 Expression analysis during meiosis in Petunia hybrida anthers using cDNA-AFLP-based transcript profiling

Chapter IV 89 cDNA-AFLP-based transcript profiling enables the assembly of a comprehensive catalogue of genes modulated during male meiosis

Chapter V 123 Clustering genes expressed during male meiosis

Chapter VI 147 Cellular localisation of a selected subset of modulated Petunia hybrida transcripts by in situ hybridisation

Summary and perspectives 159

Samenvatting en perspectieven 163

Dankwoord 166

Curriculum vitae 168

Appendix 169 bw-cnudde 23-08-2004 14:49 Pagina 6

Wanneer ik niet weet waar ik mee bezig ben – ben ik onderzoek aan het doen.

Uit: Duizend wegen naar wijsheid, David Baird bw-cnudde 23-08-2004 14:49 Pagina 7

Chapter I

General introduction

submitted as a review to Plant Biology bw-cnudde 23-08-2004 14:49 Pagina 8

8 CHAPTER I

History

In 1883 the Belgian cytologist Edouard van Beneden made an amazing observation: fertilised eggs of the parasitic nematode Ascaris megalocephala contain four chromosomes, whereas the sperm and the egg nuclei each contain only two. Building on this observation, which was published without any comments, August Weismann (1887) speculated on the existence of a reductive cell division during the sexual life cycle to compensate for the fusion of gametes at fertilisation. Farmer and Moore (1905) introduced the term meiosis to describe this specialised form of cell division (from the Greek meioun: to diminish). Since these early observations, meiosis has been the subject of intense scientific scrutiny for more than a century. Cytogeneticists have documented numerous cytological analyses of meiosis for over 100 years. Most often these were carried out in plants and gave detailed descriptions of the behaviour of meiotic chromosomes. The molecular details underlying some of these unique structural and behavioural aspects of meiotic chromosomes are only now beginning to be unraveled. Data from different model systems suggest that the overall mechanisms of meiosis are conserved from yeast to mammals and often use homologous genes for comparable functions. However, intriguing differences exist between species, providing a challenge for geneticists and molecular biologists to comprehend the cellular processes surrounding meiosis and the rules governing genetic inheritance.

Introduction

The life cycle of sexually reproducing eukaryotes is characterised by an alternation of diploid and haploid generations (Fig. 1). A diploid cell contains two versions of each chromosome, one of maternal and one of paternal origin, which are called homologous chromosomes, whereas haploid cells have only one copy of each chromosome. The transition from diploid to haploid takes place at meiosis, a specialised set of two cell divisions by which four haploid gametes are produced from one diploid parental cell. In the case of pollen tetrads in plants, all four haploid nuclei give rise to four different cells, while in female meiosis only one haploid nucleus ends up in the oocyte. Chromosome numbers are reduced during meiosis because a single round of DNA replication is followed by two successive rounds of chromosome segregation. This contrasts with mitosis, during which a single round of chromosome segregation follows each round of DNA replication, and the chromosome number remains constant at the end of each cycle (Fig. 1). Fusion of two gametes during sexual reproduction restores the diploid chromosome complement in the zygote. bw-cnudde 23-08-2004 14:49 Pagina 9

Introduction 9

S

Figure 1 Alternation of diploid and haploid generations in the life cycle of sexually reproducing eukaryotes (after Petronczki et al., 2003)

Meiosis is a key feature of eukaryotic sexual reproduction and fulfils two primary functions. First, it serves to reduce the chromosome number of gametes twofold in anticipation of fertilisation and the reconstitution of the diploid state. Second, the high- frequency genetic exchange events not only ensure proper chromosome disjunction but also contribute to genetic diversity upon which natural selection can act. The structure and behaviour of meiotic prophase I chromosomes are very different from those observed for prophase chromosomes in mitotically dividing cells (Fig. 2). In meiosis, chromosomes replicate and compact; homologous chromosomes pair to identify partners and then these partners segregate away from each other at the first (reductional) division, while sister chromatids separate during the second (equational) division at meiosis II. The second division of meiosis resembles mitosis: sister chromatids separate and segregate. The first division, however, is unique. There are at least four crucial differences between chromosome behaviour during meiosis I and mitosis (Moore and Orr-Weaver, 1998; Zickler and Kleckner, 1998; Petronczki et al., 2003). The first is pairing and recombination between maternal and paternal chromosomes. The resulting crossovers collaborate with cohesion between sister chromatids to form temporary connections (chiasmata) between homologues that allow them to orient themselves towards opposite poles of the meiosis I spindle. Correct bw-cnudde 23-08-2004 14:49 Pagina 10

10 CHAPTER I

Figure 2 Schematic representation of the mitotic (left) and meiotic (right) cell cycle (see also appendix)

segregation at the meiosis I division depends on these crossover events between the DNA molecules of homologous chromosomes (Moore and Orr-Weaver, 1998). As a result of crossing-over, each offspring will carry genes from all four grandparents. If no crossing-over would occur, the offspring would only inherit genes from two grandparents. Second, sister kinetochores always attach to microtubules from the same pole, which is known as co-orientation or monopolar attachment. This ensures that sister chromatids are now pulled to the same pole at the end of the first meiotic division. Monopolar attachment is fundamentally different from the mode of attachment in mitosis and meiosis II, where sister kinetochores are attached to opposite poles (bipolar). The third major difference between meiosis I and mitosis is that cohesion between sister chromatids in the vicinity of centromeres is maintained until the onset of the second meiotic division (Moore and Orr-Weaver, 1998; Moore et al., 1998). Finally, DNA replication is inhibited in between the two meiotic divisions. Proliferating mitotic cells rely on alternating rounds of DNA replication during S-phase and segregation of the duplicated chromatids during M-phase or mitosis. This rule has to be violated between the two meiotic divisions to allow the formation of haploid nuclei. Meiotic cells, therefore, suppress S-phase after meiosis I (Petronczki et al., 2003). bw-cnudde 23-08-2004 14:49 Pagina 11

Introduction 11

Whence meiosis?

Sexual reproduction among living creatures seems so common to everyone that the asexual cases (e.g. budding in yeasts, cutting and grafting of plants) are sometimes perceived as a curiosity of nature. In fact, in the evolution of living organisms, sexual reproduction has proven to be a successful strategy (Bell, 1982). This is due to its ability to recombine the genomes of individuals, resulting in increased genetic diversity that makes coping with selection more efficient (Rice and Chippindale, 2001). Although the rate of reproduction is slower, sexually reproducing organisms evolve more rapidly because the fast removal of deleterious mutations and the quick adoption of advantageous mutations allow a better adaptation to changes in the environment. Sexual reproduction is overwhelmingly popular among eukaryotes, despite the higher costs – sexual reproduction demands more organic resources than asexual reproduction (Siller, 2001). Remarkably, it is fully absent in Archea and Bacteria, although some of the components of sexual reproduction are present among the latter. Mechanisms for gene recombination, genome packaging, separation of parental and replicated genomes, and mechanisms for the association of genomes with membranes are some of the tools needed in sexual reproduction that are already present among Archea and Bacteria. On the other hand, both kingdoms lack microtubules, a nuclear envelope and chromosomes with telomeres. These differences are related to the absence of a true mitotic process among prokaryotes (Lewis, 2001). The vast majority of prokaryotes have main circular genomes (Drlica and Bendich, 2000) that exclude the possibility of a regular meiosis with recombination. The exceptional prokaryotes with linear genomes do not have true telomeres, while all studied eukaryotes have conserved telomeric structures. It is then reasonable to conclude that the development of linear chromosomes with differentiated telomeres is an important step prior to the evolution of meiosis (Solari, 2002). Another prerequisite for meiosis is the doubling of the chromosome content. Diploidy could have arisen in response to the need for repair of damage via recombination off a homologue (Maynard Smith and Szathmary, 1995). Some models have hypothesised that meiosis evolved as a way to rescue a cell from the diploidy resulting from fusion (Cleveland, 1947; Margulis and Sagan, 1986). The constant pressure for a further amelioration of mechanistic problems that arose in diploid cells exposed to high levels of DNA damage could have provided short-term advantages. In turn, these could have provided time for long-term advantages to come into play, required for the survival of meiosis over evolutionary time (Kleckner, 1996). According to Dawkins (1982), meiosis reveals diploids as elaborate chemostats that have evolved entirely to maximise the fitness of the haploid organisms they carry: their gametes. bw-cnudde 23-08-2004 14:49 Pagina 12

12 CHAPTER I

Yeast is very useful to study the evolution of meiosis, since it has two life cycles: a) an asexual (clonal) one, and b) an alternation of haploid/diploid phases with sexual reproduction (meiosis and conjugation). Thus, yeast can be kept in culture either in the haploid or diploid form, both dividing by mitosis. The budding yeast, Saccharomyces cerevisiae, is mostly diploid and has a sophisticated meiosis, similar in many ways to that of higher eukaryotes. On the other hand, the fission yeast, Schizosaccharomyces pombe, is a more primitive unicellular fungus that is phylogenetically distant from S. cerevisiae; both species are estimated to have diverged over 330 million years ago (Cliften et al., 2003). S. pombe is usually in a haploid condition and has a significantly simpler meiosis, which may show some of the most primitive features of meiotic mechanisms. Apart from a different mechanism of chromosome pairing through oscillatory movements, it also lacks one of the most conserved structural features of meiosis: a protein apparatus specific for the meiotic prophase, called the ‘synaptonemal complex’ (SC) (Solari, 2002).

Meiosis evolved from a mitosis-like process

In spite of the differences shown in Fig. 2, numerous features are shared between mitosis and meiosis. The relationship between mitosis and meiosis is obvious from microscopical observations, as meiosis recruits the existing mitotic machinery for genome separation. A growing pool of molecular data is adding more evidence for this relationship. It is therefore assumed that meiosis has evolved from a mitosis-like process. Kleckner (1996) suggested that during evolution of meiosis, crossovers appeared first, before the occurrence of two divisions without an intervening round of DNA replication. Sequential reductional and equational divisions in the absence of crossing-over should be disastrous: homologues would segregate randomly at the first division. But the presence of crossovers during a mitotic cell cycle would be relatively harmless: crossovers would create interhomologue connections that would readily disappear when sister chromatids separated. The use of the homologous chromosome as a recombination partner during meiosis is in contrast to the situation in vegetative cells, where the sister chromatid is the preferred partner for the mitotic recombinational repair of double strand breaks (DSBs) (Paques and Haber, 1999). This switch in recombination partner preference was a crucial event in the development of sex, since crossover events must occur between homologues, rather than between sisters, if they are to afford the connection between homologues that will allow them to orient toward opposite spindle poles (Villeneuve and Hillers, 2001). Recombination occurs at a 100-to 1,000-fold higher frequency during meiosis than during vegetative growth (Paques and Haber, 1999). This elevation is essential to ensure that each pair of homologues will enjoy at least one crossover in every meiosis. This, in bw-cnudde 23-08-2004 14:49 Pagina 13

Introduction 13

turn, suggests that an important step in the development of meiosis has been the generation of a means to greatly stimulate the frequency of recombination. This is done by deliberate introduction of double strand breaks in the DNA by the Spo11 endonuclease enzyme (Keeney, 2001). Double Strand Break Repair (DSBR) by homologous recombination can result in either crossover or noncrossover products, but only crossovers between homologues ensure correct segregation at meiosis I. Thus, it is not sufficient to promote the use of the homologue as the recombination partner: a meiotic cell must also ensure that recombination between homologues results in crossing-over. In Saccharomyces cerevisiae 30%-50% of meiotic recombination events are associated with a crossing-over, compared to 5%-20% in somatic cells (Paques and Haber, 1999). Possibly a modified single-division cell cycle functioned as an intermediate stage before full two-division meiosis could evolve (Kleckner, 1996). Such a cell cycle would always yield rational segregation of genetic material if connected homologues would always undergo reductional segregation while unconnected homologues would always give equational segregation (Simchen and Hugerat, 1993). In fact, several yeast mutations confer single-division meiosis in which chromosomes undergo a mixture of reductional and equational segregation. Such a ‘mixed meiosis’ condition may have been an intermediate stage in the evolution of a full two-division meiosis. Since there are no extant lineages that appear to have split off the eukaryotic tree before the evolution of meiosis (Cavalier-Smith, 2002), it is virtually certain that sex and meiosis already had evolved in our last common ancestor with Arabidopsis. However, many questions remain. There is no clear understanding of how and when meiosis evolved and most theories remain solely hypothetical.

An overview of meiosis

Although meiosis is a continous process, it can be subdivided into a number of cytologically distinct stages, each of which is characterised by a unique chromosome morphology (Fig. 3). A typical feature of meiosis is the complex and prolonged prophase I that follows replication and meiotic interphase. The diffuse chromosomes begin to condense at the leptotene stage of prophase I to produce a long, thin thread with a proteinaceous central axis. Although each chromosome consists of two sister chromatids, these chromatids are closely apposed and cannot be distinguished from each other. In the zygotene stage, synapsis between two homologues begins and their rope-like proteinaceous axes are brought together to form a synaptonemal complex (SC). At pachytene, homologous chromosomes have synapsed along their entire bw-cnudde 23-08-2004 14:49 Pagina 14

14 CHAPTER I

Figure 3 Schematic overview of meiotic stages (after Bhatt et al., 2001), (see also appendix)

length. The chiasmata, which are the visible manifestations of crossover recombination, become visible in the diplotene stage. This stage is characterised by the progressive shortening and condensation of the bivalents, separation of the homologues except at chiasmata and continued presence of the nuclear membrane and nucleolus. As meiosis proceeds from prophase I into metaphase I, the highly condensed bivalents become attached to the spindle and align at the equator of the cell with the centromeres directed to opposite poles. At anaphase I, homologous chromosomes - each consisting of two chromatids - migrate to the opposite poles of the cell. Telophase I leads to the dyad stage in which the two polar groups of chromosomes partially decondense before embarking on the second meiotic division. The chromosomes recondense at prophase II and at metaphase II become aligned prior to the second division. At anaphase II individual chromatids are separated and migrate to the spindle poles. As a result, late anaphase II cells contain four groups of chromatids, which gradually reform into tetrads of haploid interphase nuclei. Cytokinesis then occurs producing four haploid cells (Ross et al., 1996; Armstrong and Jones, 2001). bw-cnudde 23-08-2004 14:49 Pagina 15

Introduction 15

Pairing of homologous chromosomes: many aspects remain obscure

For homologues to properly disjoin at meiosis I, they must pair, recombine, and synapse. Early in meiosis I prophase, pairing of replicated maternal and paternal chromosomes occurs (reviewed in Walker and Hawley, 2000). The underlying mechanisms by which initial pairing is achieved, remain largely obscure, making this fundamental process one of the least understood steps in the study of meiosis. In many organisms, homologue pairing is clearly distinct from chromosome synapsis. Pairing refers to the side-by-side alignment of homologues, perhaps at a distance, whereas synapsis refers to the intimate association of chromosomes specifically in the context of the synaptonemal complex or SC (Roeder, 1997). Furthermore, several observations indicate that pairing not only precedes, but also is separable from synapsis. First, in certain meiotic mutants, chromosomes pair but fail to synapse (Moreau et al., 1985; Loidl et al., 1994; Weiner and Kleckner, 1994; Nag et al., 1995). Second, in triploids, all three homologues line up side by side, but only two chromosomes engage in synapsis in any given region (Loidl, 1990). Third, the SC can form between non- homologous chromosomes (von Wettstein et al., 1984; Sherman et al., 1989) whereas pairing (by definition) involves homologues (Roeder, 1997). Unexpectedly, a premeiotic association of centromeres occurs in both tapetal cells and meiocytes of polyploids, whereas in diploid wheat, homologous centromeres associate only during the meiotic prophase (Bhatt et al., 2001). Also in some other organisms, including S. cerevisiae, premeiotic pairing has been observed, but fluorescent in situ hybridisation (FISH) analyses on premeiotic cells of maize, mice and humans demonstrate that this is not a general feature of meiosis in diploid nuclei (Scherthan et al., 1996; Bass et al., 1997). It has been proposed that the mechanism of early centromeric association facilitates premeiotic sorting in polyploids, a mechanism that remains inactivated in diploids (Martinez-Perez et al., 2000). Studies in hexaploid wheat have shown that between 70 and 80% of the premeiotic centromere associations start out as non-homologous interactions (Aragon-Alcaide et al., 1997; Maestra et al., 2002) and that the Ph1 locus is needed to resolve these incorrectly paired centromeres (Martinez-Perez et al., 2001). It is generally thought that if homology is identified by a trial-and-error process, there must be a mechanism in the early prophase cell that increases the number or efficiency of random contacts. One such mechanism could be a widespread phenomenon known as bouquet formation: the clustering of telomeres, which are randomly distributed in the premeiotic interphase and early leptotene, to a small region of the nuclear envelope during zygotene. It seems to be a consistent motif of the pairing process of nearly all eukaryotic species of different kingdoms studied so far (Dernburg bw-cnudde 23-08-2004 14:49 Pagina 16

16 CHAPTER I

et al., 1995; Scherthan, 2001). The formation of the meiotic bouquet is a dramatic example of chromosome movement that occurs in the absence of a spindle. This telomere-mediated reorganisation of the early meiotic prophase nucleus has the effect of reducing the search space required for chromosomes to find their partners; its mechanism is currently unknown. Using three-dimensional FISH imaging on maize meiocytes, Bass et al. (2000) provided cytological evidence that the bouquet coincides with homologous chromosome pairing and synapsis, suggestive of a role for telomeres in the homology search process. However, the meiotic bouquet is not essential for homologue association because maize homologues in an oat addition line associate even though they are not part of the oat bouquet. This is analogous to the situation in a yeast mutant, in which bouquet formation is abolished while homologues still associate (Trelles-Sticken et al., 2000). In Arabidopsis thaliana, telomeres appear to cluster during the meiotic prophase, but instead of clustering on the nuclear envelope, they associate on the nucleolus prior to homologous synapsis (Armstrong et al., 2001). Recently, Carlton et al. (2003) carried out a time course analysis of bouquet formation in cultured rye anthers and developed a computer simulation of chromosome movement during bouquet formation. Examining mutants defective in telomere function could help to further clarify the mechanism and role of bouquet formation in meiosis. Obviously, after the chromosomes are brought into close proximity, some regulated process is required to accomplish correct discrimination of homologues. In general, chromosome pairing and alignment mechanisms can be divided into mechanisms that require DNA double strand breaks (DSBs) for formation and those that are DSB- independent (Page and Hawley, 2003). The DSB-dependent class of mechanisms likely involves a homology search on the basis of the DNA sequence itself. In contrast, DSB- independent processes might include mechanisms such as the maintenance of premeiotic pairings or the pairing of homologues on the basis of the aggregation of sequence-specific DNA-binding proteins. The degree to which each of these pairing mechanisms is used in meiosis appears to vary among organisms. In the complete absence of DSBs, homologue pairing is abolished in S. cerevisiae (Lichten, 2001), but occurs normally in Caenorhabditis elegans (Dernburg et al., 1998) and in both sexes of Drosophila (McKim et al., 1998). S. cerevisiae uses a genome-wide DNA-based homology search, which allows even DNA sequences as short as 1kb to find their homologous counterparts. However, this solution cannot apply exhaustively to organisms with large genomes and extensive repetitive sequence families. In maize, for instance, 100-200 retroelement families account for over 50% of the genome (Sanmiguel et al., 1996), making each part of the genome resemble every other part on the megabase scale. This would make identification of DNA homology insufficient to establish the presence of a homologous bw-cnudde 23-08-2004 14:49 Pagina 17

Introduction 17

locus. Therefore, other recognition mechanisms at the chromosomal and chromatin level are likely to be at play as well.

Figure 4 Diagram of the synaptonemal complex (after Roeder, 1997)

Synapsis

Pairing is followed by the formation of the SC (Albini and Jones, 1987; Heyting, 1996). Synapsis is defined as the intimate association of homologues in the context of the mature SC, a proteinaceous zipper-like structure that has a width of approximately 100 nm (Sym et al., 1993). At full synapsis, the entire structure (paired homologues plus SC) is called a meiotic bivalent. SC formation initiates during leptotene with the assembly of axial elements along the pairs of sister chromatids. In the following zygotene stage, a central element forms between the two homologous axial elements, which are now referred to as lateral elements. At pachytene, the tripartite SC is completed with structures called transverse filaments extending from the central element to the lateral elements. The chromatin of each pair of sister chromatids is organised into loops attached at the base to the lateral elements (Fig. 4) (Heyting, 1996). Finally, during diplotene, the SC is disassembled. bw-cnudde 23-08-2004 14:49 Pagina 18

18 CHAPTER I

Though characteristic of most meiotic cells, the SC remains one of the mysteries of meiosis and its function is still poorly understood. In at least some organisms, the SC serves to hold homologues together during the processes of chromosome compaction and condensation that occur during the transition from leptotene to zygotene (Zickler and Kleckner, 1999). In most organisms, SC assembly seems to be a prerequisite for proper chiasma distribution (Sym and Roeder, 1994). However, meiosis is still possible even in the absence of synapsis. A notable example is S. pombe, which produces plenty of exchanges and performs reductional segregation of homologous chromosomes without producing an SC (Bahler et al., 1993). S. pombe also lacks interference, raising the possibility that the intimate synapsis of maternal and paternal chromosomes during pachytene causes interference (see further).

The relationship between synapsis and the initiation of meiotic recombination

Over the past decade, genetic analyses in yeast have revolutionised our understanding of meiosis. Traditionally, it has been assumed that synapsis is required for meiotic recombination and that chromosomes synapse before recombination is initiated. However, recent studies in yeast indicated that this assumption is incorrect. Time- course analyses in S. cerevisiae showed that the initiation of both recombination and chromosome pairing occurs in early leptotene, well before synapsis (Padmore et al., 1991). Furthermore, recombination intermediates were observed well in advance of SC formation (Schwacha and Kleckner, 1994, 1995). The initial resistance among many working in the field of meiosis to accept these surprising observations was silenced upon further support from yeast mutants. In S. cerevisiae, mutants that completely suppress recombination also block the formation of the synaptonemal complex. In contrast, other mutants can block synapsis while allowing near normal levels of meiotic recombination. This led to a reordering of the meiotic events, which promptly outdated generations of textbook material. As noted by Roeder (1997): “Synapsis is not required for recombination in yeast; instead steps in the recombination pathway appear to be required for synapsis. Mutants that do not sustain DSBs fail to make SC.” Attempts to extend “the fall of the classical view of meiosis”, to quote Hawley and Arbel (1993), to other organisms were frustrated by the observation that recombination is not essential for synapsis in Drosophila oocytes (McKim et al., 1998) or in the nematode Caenorhabditis elegans (Dernburg et al., 1998). In contrast with yeast, the formation of fully synapsed bivalents and SC takes place in the absence of bw-cnudde 23-08-2004 14:49 Pagina 19

Introduction 19

recombination in these organisms. Moreover, time-course studies have shown that DSB formation occurs after synapsis in Drosophila (Jang et al., 2003). Walker and Hawley (2000) suggested conveniently that this different relationship between meiotic events is caused by the greater genome complexity in higher eukaryotes. However, recent studies in mice (Mahadevaiah et al., 2001) and Arabidopsis (Grelon et al., 2001) demonstrated that, as in yeast, recombinational DNA double strand break formation precedes and is required for synapsis. Taken together, these results indicate that recombination and synapsis can occur quite independently, but in most meiotic systems both processes are tightly coupled by one or more regulatory links of varying strength. Compared with other eukaryotes, Drosophila and C. elegans have developed different control mechanisms for SC formation.

Meiotic recombination

The Double Strand Break Repair (DSBR) model of meiotic recombination

In 1983, Szostak et al. proposed that meiotic recombination was initiated by the breakage of both DNA strands in a duplex, followed by strand invasion of a homologous partner. Subsequent experimental support has established the Double Strand Break Repair (DSBR) pathway. Until today, it is used as a guideline for meiotic recombination in yeast and most probably in other eukaryotes including plants (Fig. 5) (Xu et al., 1995). Lesions initiating meiotic recombination have been unambiguously identified as DSBs formed during leptotene in S. cerevisiae. First, meiosis-specific DSBs have been physically detected at a number of recombination hot spots (Sun et al., 1989; Cao et al., 1990; Goldway et al., 1993; Bullard et al., 1996). Second, these breaks appear and disappear with the kinetics expected for an early intermediate in the exchange process (Padmore et al., 1991). And third, the overall frequency of DSBs and their distribution throughout the genome are consistent with the observed frequency and distribution of meiotic recombination events in S. cerevisiae (Baudat and Nicolas, 1997; Klein et al., 1996; Wu and Lichten, 1994). A meiosis-specific Spo11 endonuclease has been identified as the key enzyme for the generation of DSBs in many organisms, including Arabidopsis (Grelon et al., 2001). In S. cerevisiae at least 11 proteins in addition to Spo11 are required for DSBs (Keeney, 2001; Pecina et al., 2002). These findings, along with genetic interaction studies (Kee and Keeney, 2002), are consistent with the suggestion that Spo11 acts as part of a complex with other proteins, and that efficient DSB formation may require the assembly of a specialised meiotic chromosome structure (Blat et al., 2002). bw-cnudde 23-08-2004 14:49 Pagina 20

20 CHAPTER I

Figure 5 Double Strand Break Repair Model of meiotic recombination (Szostak et al., 1983); (after Roeder, 1997), (see also appendix)

According to the model, double strand cleavage is followed by exonucleolytic digestion to expose single-stranded tails with 3’ termini. These single-stranded tails invade an uncut homologous duplex where they promote repair synthesis followed by branch migration to produce two Holliday junctions. Thus, in contrast to mitotic cells, meiosis- initiated DSBs are repaired using the homologous chromosome, not the sister chromatid, as a template for repair (Paques and Haber, 1999). Each Holliday junction will then be cut in one of two orientations. Cleaving the two junctions in the same orientation results in a noncrossover product or gene conversion, whereas resolution of these junctions in opposite directions results in a reciprocal recombination product (crossover), creating a cytologically visible joint between the homologues (chiasma). Only crossovers provide the required physical connection between homologues and contribute to a correct segregation at meiosis I. The identification of yeast mutants blocked at different steps in the repair process and the demonstration of double Holliday junctions seem to vindicate the DSBR model. However, studies of heteroduplex DNA make it necessary to question certain aspects of the model. Unexpectedly, physical assays failed to detect heteroduplex DNA until relatively late in meiotic prophase, around the same time as mature crossover products (Nag and Petes, 1993; Goyon and Lichten, 1993). According to the model, however, bw-cnudde 23-08-2004 14:49 Pagina 21

Introduction 21

heteroduplex DNA should be produced early in the recombination process and certainly should be present in joint molecules. Fig. 5 illustrates how the DSBR model predicts that hybrid DNA (containing one strand from each of the two recombining duplexes) is formed on both sides of the initiating DSB. Furthermore, hybrid DNA to the left of the DSB should be on a different chromatid than hybrid DNA to the right of the DSB. In genetic studies, however, these expectations were not met. Instead, the data suggest frequent asymmetry in individual recombination reactions. Most events were found to be one-sided; when two-sided events were detected, both regions of hybrid DNA were in the same chromatid. Attempts have been made to reconcile these observations with the basic tenets of the DSBR model, but the explanations proposed remain to be tested (Porter et al., 1993; Schwacha and Kleckner, 1995; Gilbertson and Stahl, 1996). Finally, in a recent model proposed by Allers and Lichten (2001) crossover and noncrossover pathways diverge soon after single-strand invasion and initial repair synthesis. Their experiments examining the kinetics of formation of crossover and noncrossover products indicated that the canonical double Holliday junction intermediate gives rise mainly to crossover products, whereas most noncrossover recombinants arise earlier via a different pathway. The big questions that remain to be answered are when, and how, the decision to create a crossover or noncrossover is made. Recent work in S. cerevisiae suggests that the Sgs1 helicase plays a specific role in this process by favouring the production of noncrossovers at the expense of crossovers (Rockmill et al., 2003). In conclusion, the initation of recombination by DSBs created by the Spo11 protein is a universal ‘truth’ in all eukaryotes. However, it is clear from the above that the basic mechanisms of meiotic recombination, lying at the heart of meiosis, are still not fully understood. Undoubtedly, their further unravelment poses a serious challenge for future generations of scientists and will require a coordinated effort with the integration of different techniques and approaches.

Somatic recombination

The mismatch repair system (MMR) plays an important role during meiotic recombination in systems ranging from yeast to humans (Roeder, 1995). The mismatch correction machinery responsible for correction of mismatches during meiotic recombination in yeast is closely related to the E. coli machinery involved in the repair of replication errors. Homologous recombination during meiosis is in many respects quite different from somatic or mitotic recombination. Meiotic recombination is initiated by the creation of DSBs by a meiosis-specific topoisomerase II-like protein, Spo11. Another special feature of meiosis is the high level of crossing-over, a desired outcome generating bw-cnudde 23-08-2004 14:49 Pagina 22

22 CHAPTER I

more genetic diversity in the gametes. DSBs are critical lesions in eukaryotic genomes. If left unrepaired, they result in cell death. If imprecisely repaired, the damage leads to mutations and chromosomal rearrangements. In principle, breaks can be repaired by two modes, via homologous recombination (HR) and via illegitimate recombination or non- homologous end joining (NHEJ). NHEJ connects the broken DNA ends irrespective of their sequence information. This process is therefore error-prone and can lead to the creation of various deletions or small insertions. In contrast, HR is the most conservative type of repair because it uses the genetic information stored in the sister chromatid - the preferred partner in somatic recombination - to repair the DSB with high fidelity. DSBs are repaired by both HR and NHEJ in all cell types examined; however, the preferred mechanism of recombinational DNA repair differs significantly in a given organism. In particular, yeast cells rely almost entirely on homology-based recombinational DNA repair, whereas in mammalian and plant cells non-homologous recombination is much more frequent than homologous recombination (Puchta and Hohn, 1996). This was concluded after encountering repeated failures in developing accurate gene targeting strategies and because of the high frequency of end-fusions of transfected DNA (Haber, 1999). The basis of the strong preference for illegitimate recombination in mammalian and plant cells is not fully understood. Unlike animals, plants have no fixed germline. For this reason, cells destined to form plant gametes are not derived from conserved populations of cells set aside early in embryogenesis, but are instead formed from meristems that - prior to floral induction - generated vegetative cells. As a consequence, somatic changes in the genome can thus become heritable. It has been shown that mitotic recombination events play an important role in plant evolution (Das et al., 1990). Theoretically they should create a formidable problem for maintaining genome integrity. Though somatic crossovers have been reported in plants, their spontaneous frequency of occurrence is very low (Vig, 1973; Carlson, 1974; Harrison and Carpenter, 1977). Factors that introduce unspecific DSBs in DNA, such as X-rays (Tovar and Lichtenstein, 1992; Lebel et al., 1993), or DNA-damaging chemicals such as methyl methanesulfonate (Puchta et al., 1995), were shown to enhance homologous recombination in somatic plant cells. To ensure that the meiotic DSBs are repaired by homologous recombination, the mechanism of NHEJ is switched off in meiosis.

The frequency of meiotic recombination is not constant

Analysis using classical markers forms the basis for the present understanding of meiotic recombination. These indicated that meiotic recombination events are distributed bw-cnudde 23-08-2004 14:49 Pagina 23

Introduction 23

Figure 6 Chiasmata hold homologous chromosomes together by virtue of cohesion between sister chromatids (after van Veen and Hawley, 2003). (A) Normal bivalent. The sister chromatids in the vicinity of the centromere do not separate until meiosis II. Sister chromatid cohesion distal to the exchange is removed at anaphase I. (B) A bivalent held together only by a distal exchange. Such bivalents often fail to segregate properly because insufficient cohesion may be available to hold homologous chromosomes together during meiosis I. (C) A bivalent with a ‘two-strand double’ crossover event. Like the example presented in (B), the two homologues are connected only by sister chromatid cohesion in a short region and might separate precociously.

unevenly throughout eukaryotic genomes. As a result, there is no simple linear relationship between genetic and physical map distances. Instead, the frequency of crossovers per unit physical distance can vary substantially, even within a single chromosome. This has been demonstrated in yeast (for reviews, see Lichten and Goldman, 1995; Petes, 2001; Koren et al., 2002), but also in cereals (Heslop-Harrison, 1991), in tomato (Tanksley, 1992), maize (Xu et al., 1995) and, more extensively, in chromosome 4 of Arabidopsis (Schmidt et al., 1995). The regions in which many crossovers occur are termed recombination hot spots; regions with relatively few crossovers are called cold spots. In Arabidopsis chromosome 4, the average distance of 1cM is 150kb, but the frequency of recombination varies along the whole chromosome, ranging from 30kb/cM (hot spot) to more than 550kb/cM (cold spot). No clear correlation can be found with specific chromosomal regions, except for some suppression being detected around the centromere. This contrasts with the picture emerging from tomato and wheat chromosomes, in which recombination is strongly suppressed over large intervals around the centromere, and maximal recombination occurs in the proximal regions. bw-cnudde 23-08-2004 14:49 Pagina 24

24 CHAPTER I

The position of chiasmata on the bivalent affects their efficiency in chromosome segregation. In general, exchanges don’t occur close to kinetochores since otherwise, insufficient cohesion near the centromeres would remain for meiosis II (Petronczki et al., 2003). Already half a century ago, Sears (1952) associated the apparent cases of meiosis II missegregation in wheat with aberrant, far too proximal exchanges. In order to permit homologous chromosome segregation during meiosis I, cohesion between sister chromatids is destroyed distal to, but not proximal to, chiasmata (Fig. 6A). Cohesion in the vicinity of centromeres is preserved until the second meiotic division and is used to orient and segregate chromatids on meiosis II spindles. Centromeres therefore suppress the formation of exchanges in their vicinity (Lambie and Roeder, 1988), which permits the accumulation of repetitive sequences and ‘junk’ DNA. Also distal exchanges are not favourable; Hassold et al. (1995) showed that human trisomies of chromosome 16, which originate in oocytes, appear to be associated with recombination events occurring close to the ends of a bivalent. The weakness of a single exchange close to the telomere can be explained by a reduced sister chromatid cohesion distal to the exchange (Fig. 6B). In this case insufficient cohesion may be available to hold homologous chromosomes together during meiosis I (Petronczki et al., 2003). In plants and other organisms, recombination is concentrated in gene-rich regions (Civardi et al., 1994; Gill et al., 1996). In contrast, in C. elegans, recombination occurs preferentially in gene-poor regions (Barnes et al., 1995); mutation of its rec-1 gene abolishes this bias (Zetka and Rose, 1995). Still, little is known about the molecular basis of hot spot activity in systems other than fungi. Another approach to estimate genetic recombination values is the direct scoring of chiasmata through microscopical analysis on preparations of diplotene chromosomes (e.g. Havekes et al., 1997). Moreover, since in Arabidopsis thaliana each bivalent can be marked by FISH, chiasma analysis can be further refined to determine individual bivalent chiasma frequencies (Sanchez-Moran et al., 2002). For Arabidopsis, a wild-type mean chiasma frequency of 9.24 per cell was determined, with individual bivalent chiasma frequencies varying according to chromosome size; chromosome 1 had the highest mean chiasma frequency (2.14) while the acrocentric chromosomes had the lowest frequencies, 1.54 and 1.56 for chromosomes 2 and 4, respectively (Armstrong and Jones, 2003). Caution is required when comparing the results of a chiasma screening with the number of crossovers as determined by a marker-based approach. Using microscopy each crossover is readily detectable, while (preferentially) a backcross population must be screened to measure recombination between genetic markers. Since a crossover in a bivalent consists of a reciprocal exchange between two nonsister chromatids, the end result will be two reciprocally recombined and two original, non-affected chromatids bw-cnudde 23-08-2004 14:49 Pagina 25

Introduction 25

(Fig. 5). Ultimately, for every crossover taking place in each bivalent, the resulting haploid gametes – the four microspores in male meiosis or the one surviving megaspore in female meiosis – have an equal chance to acquire either the recombined or the non- recombined allele. Consequently, the estimated recombination values (based on a dense grid of markers) must be multiplied by a factor two in order to compare them with chiasma frequencies. Knowledge about the densities of meiotic crossover events on chromosomes is important for the construction of high-density molecular linkage maps, which are indispensable for map based cloning, marker-assisted breeding and quantitative trait mapping experiments (Peters et al., 2003).

Factors influencing the frequency of meiotic recombination

Relative to the small size of the Saccharomyces cerevisiae genome, meiotic recombination occurs at extremely high levels, on the order of 260 events per nucleus per meiosis (Bishop et al., 1992; Rockmill et al., 1995). Recombination in a given organism can vary considerably due to many factors. Plant breeders know by experience that the amount of recombination between marker loci can be increased or decreased by environmental factors such as temperature during gametogenesis (Baker et al., 1976). Recently, Abdullah and Borts (2001) showed that meiotic recombination frequencies are affected by nutritional states in Saccharomyces cerevisiae. However, most frequently, such differences have a genetic basis. In many organisms, recombination during male and female gametogenesis occurs at different rates (Burt et al., 1991), but there is no consistency in the direction of the difference. In humans, for example, females have significantly more meiotic recombination than males, with total map lengths of 44 and 28 Morgans, respectively (Broman et al., 1998). The sex difference in recombination rate is most extreme in the exceptional case of Drosophila, where no recombination at all is observed in male gametes. In plants, up to more than 10-fold higher recombination frequencies during female meiosis have been reported in several tomato crosses (Ganal and Tanksley, 1996). However, data from other plants such as potato and pearl millet suggest that this is not a general phenomenon (Busso et al., 1995; Gebhardt et al., 1991). In S. cerevisiae, it has been shown that hot spots correspond to the sites of the meiosis-specific DSBs that initiate recombination. DSBs do not occur at a specific DNA sequence but rather at regions that undergo meiosis-specific modifications resulting in an open chromatin configuration. This chromatin transition at active DSB sites, detected by Ohta et al. (1994) as a quantitative increase in micrococcal nuclease (MNase) sensitivity prior to DSB formation, is coupled with premeiotic DNA bw-cnudde 23-08-2004 14:49 Pagina 26

26 CHAPTER I

replication (Murakami et al., 2003). Borde et al. (2000) showed earlier that delaying meiotic DNA replication also delayed the initiation of recombination. This delay in DSB formation could represent the time required to remodel chromatin to a meiosis- specific recombination-competent form, for example through histone modifications. Since an open chromatin structure is required, hot spots can be found in transcriptionally active chromatin regions. A dense chromatin packaging reduces the accessibility of DNA to the recombination machinery. For example, human DNA is compacted in meiosis 25-fold more than yeast DNA (Loidl et al., 1995), resulting in a recombination rate that is 300 times lower compared to yeast: 1cM per 3kb in S. cerevisiae (Petes et al., 1991) versus 1cM per 1000kb in humans (Broman et al., 1998). When comparing different organisms, Anderson and Stack (2002) have shown that recombination frequencies correlate better with total pachytene SC lengths and gene number than with total genome size. Recently, Kleckner et al. (2003) discussed several models on how chromosome axis length would determine recombination frequencies. Current evidence favours the explanation that the total number of DSBs is the factor that determines the number of crossovers. This number is proportional to total SC length. In an attempt to build a high-density AFLP map in Petunia hybrida, Strommer et al. (2002) reported low levels of recombination in clusters of tightly linked markers. Linkage analysis in Petunia using transgenic lines had revealed earlier that recombination is suppressed in a wide cross relative to an inbred recombination assay (Robbins et al., 1995). It has been confirmed in other plant species that sequence diversity in interspecific crosses (Ganal and Tanksley, 1996) and heterozygosity (Barth et al., 2001) influence meiotic recombination and cannot be ignored in genetic mapping of genomes and backcross breeding. As a general rule, the level of recombination is inversely proportional to the number of available polymorphisms, confronting us with the following strange paradox: the higher our capacity to detect recombination, the rarer it occurs. Information on meiotic genetic recombination in Arabidopsis derives from a number of sources, including conventional genetic mapping (Alonso-Blanco et al., 1998; Peters et al., 2001), tetrad analysis exploiting the quartet mutation (Copenhaver et al., 1998), and selective meiotic marker-based systems (Barth et al., 2000). Sex differences in recombination frequency have also been observed in Arabidopsis (Vizir and Korol, 1990; Barth et al., 2000; Armstrong and Jones, 2001). Further, Sanchez-Moran et al. (2002) observed a significant natural variation in meiotic recombination frequency among 8 accessions of Arabidopsis thaliana by analysing chiasma frequency variation. Because chiasmata can be scored rapidly in large samples of meiocytes, this represents an efficient approach to assaying genome-wide levels of recombination as well as the distribution of recombination events among and within chromosomes. These findings indicate the existence of recombination regulatory elements in Arabidopsis, for which there is obvious bw-cnudde 23-08-2004 14:49 Pagina 27

Introduction 27

interest to experimentally modify recombination frequencies. The potential practical implications of such developments for plant breeding are considerable. Finally, several genes that are essential for normal meiotic levels of recombination have been identified and characterised. These are merely genes that encode proteins required for the pairing and synapsis of chromosomes or that are involved in catalysing key steps in DNA breakage, repair, and recombination (Roeder, 1997; Zickler and Kleckner, 1999). Disrupting these genes usually results in extreme phenotypes that have depressed levels of recombination (e.g. Alani et al., 1989; Baker et al., 1996; Grelon et al., 2001). An exceptional plant mutant locus known to modulate the level of meiotic recombination is Rm1 in Petunia, which is thought to alter the spatial distribution of meiotic crossing-overs (Cornu et al., 1989). In Arabidopsis the X-ray hypersensitive xrs4- mutant has a significant deficiency in somatic recombination, accompanied by meiotic hyper-recombination (Masson and Paszkowski, 1997). The Ph1 locus in hexaploid wheat drastically affects recombination by controlling the correct pairing of homologous chromosomes, which is more difficult to achieve in polyploid species. Deletion of the Ph1 locus leads to the synapsis of non-homologous chromosomes and induces recombination between the different genomes (Martinez-Perez et al., 2001). In Saccharomyces cerevisiae the frequency of homologous recombination during meiosis can be substantially augmented at a targeted site by a simple modification of the Spo11 protein (Pecina et al., 2002).

Crossover interference

Meiotic crossing-over is a highly regulated process that takes place at least once between each pair of homologues during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division. When two exchanges occur on the same chromosome arm, they are almost always widely spaced. This phenomenon, known as crossover interference, was first observed in Drosophila nearly a century ago (Muller, 1916) and is believed to involve the transmission of an inhibitory signal from one crossover site to nearby potential sites of crossing-over. Interference is thought not to arise due to the inhibition of DSB formation but, instead, from a reduction in the probability of a break being converted into a double Holliday junction and crossover. It is thought that the purpose of interference is to ensure that each bivalent produces at least one exchange, irrespective of chromosome size. Though most species form on average about two crossovers per bivalent, only one chiasma is actually needed to hold homologous chromosomes together. Some organisms, such as S. pombe, show no interference and have high rates of reciprocal recombination (Bahler et al., 1993), bw-cnudde 23-08-2004 14:49 Pagina 28

28 CHAPTER I

whereas others such as C. elegans restrict the number of exchanges per chromosome to one (Barnes et al., 1995). Interference in this case is so extreme that a single exchange inhibits the formation of a second exchange on the same chromosome (Petronczki et al., 2003). In a recent study, Hillers and Villeneuve (2003) demonstrated that during C. elegans meiosis, fusion of two or three whole chromosomes removes the requirement for placing a chiasma in each of the constituent chromosomes, indicating that these fusions are now perceived by the organism as a single chromosome ‘unit’ that typically receives one crossover. Why has crossover interference evolved when it clearly is not necessary? Recent work suggests that not only too few crossovers are detrimental, but that also too many can be harmful (Rockmill et al., 2003). A possible explanation may be the requirement for sufficient sister chromatid cohesion to hold the homologues together. As shown in Fig. 6C, closely linked double crossover bivalents in which both crossovers involve the same two chromatids, so-called ‘two-strand doubles’, will be held together only by cohesion of sister chromatids in the short interval between the two exchanges. This may not be enough to ensure homologue-homologue conjunction. Although this problem would not be shared by those types of double exchange that involve more than two chromatids, such as three-strand and four-strand doubles, it may simply have proved easier to space doubles far apart than to try and restrict double exchange events in a fashion that precludes the occurrence of two-strand doubles (van Veen and Hawley, 2003). In most species, meiotic recombination occurs in the context of a prominent proteinaceous structure, the synaptonemal complex (SC), which is assembled between homologous chromosomes during meiotic prophase. Several lines of evidence suggest that the SC is responsible for crossover interference. Yeast mutants have been identified that eliminate interference by preventing SC formation (Sym and Roeder, 1994). Furthermore, Schizosaccharomyces pombe and Aspergillus nidulans fail to make SC’s and do not exhibit crossover interference (Egel-Mitani et al., 1982; Bahler et al., 1993; Kohli and Bahler, 1994). Finally, studies in grasshoppers, mice and humans have demonstrated a positive correlation between the length of the synaptonemal complex and the number of crossovers (Quevedo et al., 1997; Lynn et al., 2002). In addition to crossovers that are subject to interference, a noninterference pathway for meiotic crossing-over has been proposed, based on mutant analysis (Zalevsky et al., 1999). In yeast, a large proportion of meiotic recombination events are resolved as crossovers. In contrast, Copenhaver et al. (2002) found >10 times more double strand breaks than crossovers during meiosis in Arabidopsis, suggesting a large excess of gene conversions over crossovers in plants. Since only crossovers contribute to a correct segregation of homologous chromosomes at the first meiotic division, the basis for this phenomenon is not yet understood. bw-cnudde 23-08-2004 14:49 Pagina 29

Introduction 29

The quartet (qrt) mutation in Arabidopsis (Preuss et al., 1994; Copenhaver et al., 1998) causes the four products of male meiosis to remain attached after cytokinesis, allowing tetrad analysis in a higher plant to study meiotic recombination. The crossover distribution in meiotic tetrads can be analysed by scoring the segregation of PCR-based molecular markers. However, despite this handsome method, gene conversion events have not yet been observed in Arabidopsis (Copenhaver et al., 1998).

Sister chromatid cohesion

Sister chromatid cohesion is essential for the proper distribution of sister chromatids to daughter cells. Premature loss of sister chromatid cohesion can result in aneuploid meiotic products, which in turn may contribute to the production of unviable or abnormal progeny. Cohesion between sister chromatids is provided by a multisubunit protein complex called cohesin, of which most of the components are well conserved across the kingdoms and between the mitotic and meiotic cell cycles. In mitosis, cohesion is released at once all along the sister chromatids, at the metaphase-to- anaphase transition (Nasmyth, 2001). Therefore, the two sister chromatids can move towards opposite poles at anaphase. A cell-cycle-regulated ubiquitin-protein ligase, known as the anaphase-promoting complex or cyclosome, targets one of the cohesins subunits for proteolytic degradation, causing dissociation of the sister chromatids (Irniger et al., 1995). In meiosis, sister chromatid cohesion is established concomitantly with DNA replication, like in mitosis (Toth et al., 1999; Skibbens et al., 1999). However, meiotic sister chromatid cohesion and recombination are modified in such a way that reciprocal exchanges occur preferentially between homologous chromosomes, resulting in chiasmata that ensure a correct segregation at the first meiotic division (van Heemst and Heyting, 2000). Chiasmata only hold homologues together by virtue of cohesion between sister chromatids, which is maintained throughout those regions of the chromosome that have not undergone exchanges. The loss of sister chromatid cohesion in meiosis occurs in a highly regulated two- step process (Miyazaki and Orr-Weaver, 1994). Cohesion along chromosome arms is released at anaphase I, resolving chiasmata and allowing homologues to separate to opposite spindle poles. Cohesion near the centromeres is maintained until anaphase II, when sister chromatids separate during the second meiotic division that resembles mitosis. This stepwise separation of sister chromatids implies the operation of two types of chromatid cohesion that differ in mechanisms and/or regulation (Roeder, 1997). A meiosis-specific cohesin complex has first been identified in S. cerevisiae and S. pombe, providing the foundation for defining the regulation of meiotic cohesion at a molecular bw-cnudde 23-08-2004 14:49 Pagina 30

30 CHAPTER I

Figure 7 Schematic representation of the regulation of sister chromatid cohesion in mitosis (A) and meiosis (B); (after Dej and Orr-Weaver, 2000), (see also appendix)

level (Klein et al., 1999; Watanabe and Nurse, 1999). In mammals, all cohesin proteins so far examined are parts of components of synaptonemal complexes (Eijpe et al., 2000; Prieto et al., 2001; Revenkova et al., 2001; Lee et al., 2003). Their role is not clear, but it has been proposed that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes. The changes in sister chromatid cohesion between meiosis I and meiosis II are accompanied by a change in kinetochore-spindle association: in meiosis I, sister kinetochores associate with the same spindle pole and act as a single unit, an arrangement known as co-orientation, whereas during meiosis II, sister kinetochores must associate with opposite spindle poles, known as bi-orientation (Suja et al., 1999) (Fig. 7). Recent functional genomics research in S. cerevisiae resulted in the identification of a group of proteins, termed monopolins, that are required for monopolar attachment at meiosis I (Toth et al., 2000; Rabitsch et al., 2003). In meiotic cells depleted for monopolins, homologues fail to segregate, despite normal recombination and regulation of sister chromatid cohesion, because sister kinetochores erroneously form bipolar microtubule attachments that are resisted by sister chromatid cohesion. bw-cnudde 23-08-2004 14:49 Pagina 31

Introduction 31

Meiotic chromosome segregation

The faithful transmission of chromosomes to daughter cells during cell division is fundamental to the survival of all living organisms. Errors in meiotic chromosome segregation are the leading cause of miscarriage and are also responsible for genetic disorders such as Down’s syndrome, which is caused by an extra copy of chromosome 21 (Hassold and Hunt, 2001). To solve the unusual constraints of homologue and sister chromatid sorting in mitosis and meiosis, eukaryotic cells have evolved three structures: first, the spindle, a complex microtubule machine; second, the centromere, a specialised locus of the chromatid that organises the assembly of a microtubule-binding complex called the kinetochore; and third, sister chromatid cohesion, cross-links between sister chromatids that form during DNA replication. The meiosis I division is distinct from mitosis and meiosis II in that sister chromatids remain associated with each other, whereas homologous chromosomes move to opposite poles of the spindle apparatus. Proper chromosome segregation at meiosis I depends on crossing-over to establish chiasmata, which are stable connections between homologues that persist after the SC has disassembled and recombination intermediates have been resolved (Roeder, 1997). The consequences of a failure to undergo exchange are dependent on the meiotic system under examination. In plants, achiasmate pairs of homologues simply fall apart and the resulting univalents are free to segregate randomly and may arrive at the same pole. This missegregation is referred to as ‘non-disjunction’ and results in aneuploid gametes. In many animal male meiosis systems, apoptotic arrest is triggered, while in other species, such as Drosophila, a backup process, called distributive segregation, exists that can ensure the segregation of those chromosomes that fail to undergo exchange (Hawley and Theurkauf, 1993). Missegregation during meiosis I also occurs when the sister kinetochores fail to orient together, causing the sister chromatids to disjoin. Disjoined sister chromatids are not only subject to random segregation in meiosis I but are also incapable of regular disjunction in meiosis II (Fig. 7). Homologous chromosomes become attached to microtubules from the same or opposite spindle poles. Only attachment to microtubules from opposite poles results in a stable configuration that is maintained until anaphase. If homologues attach to microtubules from the same pole they dissociate and try again, while anaphase is delayed. The recognition that chromosomes are properly oriented depends on the mechanical tension that results when homologues are pulled towards opposite spindle poles, and this pulling is resisted by chiasmata (for review, see McKim and Hawley, 1995). Tension on all pairs of sister chromatids is thought to be a signal for their bw-cnudde 23-08-2004 14:49 Pagina 32

32 CHAPTER I

successful bipolar attachment and for the cell to inactivate cohesion and initiate anaphase (Ault and Nicklas, 1989). The importance of tension has been demonstrated by artificially applying tension to homologues that are attached to the same spindle pole. If a micromanipulation needle is used to apply an opposing force then the otherwise unstable monopolar attachment is stimulated (Ault and Nicklas, 1989). The mechanical tension is converted to a chemical signal at kinetochores by tension-sensitive protein phosphorylation (Nicklas et al., 1995): after chromosomes attach properly, tension results in kinetochore dephosphorylation and gives a ‘go ahead’ signal to progress further through the meiotic cell cycle.

Progression through the meiotic cell cycle

Putting everything together: As cells proceed through meiosis, their chromosomes undergo a series of specialised and closely linked events, including meiotic cohesion, assembly and activation of recombination complexes, pairing and synapsis, and reductional and equational divisions. To carry out these events, cells muster an impressive array of proteins ranging from mitotic proteins that are recruited to new roles to meiosis-specific proteins with specialised functions. The multiple appearances of these proteins emphasise that all aspects of meiotic chromosome behaviour are closely intertwined (Forsburg, 2002). An example for this strong interrelation of meiotic functions can be found in the Arabidopsis swi1 mutant; it seems that SWI1 has a pivotal role, as it appears to be required for sister chromatid cohesion, axial element formation and recombination (Mercier et al., 2003). We have already discussed the close relationship between the initiation of meiotic recombination and synapsis. Furthermore, several lines of evidence indicate a mechanistic linking between meiotic events and the meiotic S-phase, when DNA replication takes place. Recently, Borde et al. (2000) showed compelling evidence that DSB formation requires passage through the meiotic S-phase. It has been proposed that passage of the replication fork is required to establish a structure permissive for DSB formation, or/and that factors involved in DSB formation, such as Spo11, might assemble onto DNA during replication (Forsburg, 2002). Earlier it had been shown by Watanabe et al. (2001) that like in mitosis, the cohesion system is built as early as S- phase, when the two sister chromatids originate (Nasmyth, 2001). Significant differences have been noticed between the mitotic and meiotic cell cycle. In most organisms, premeiotic S-phase is substantially longer than premitotic S-phase and requires other initation factors for DNA replication. For example, in Saccharomyces cerevisiae, the B-type cyclins Clb5 and Clb6 are not essential for DNA replication or for bw-cnudde 23-08-2004 14:49 Pagina 33

Introduction 33

viability in mitosis but are absolutely required for normal meiotic S-phase (Stuart and Wittenberg, 1998). A key question in the cell-cycle field is how processes that seem independent become properly coordinated. As in the mitotic cell cycle, checkpoint machinery operates in meiosis monitoring meiotic progression and ensuring the proper order of cell-cycle events by arresting or delaying the cycle in response to defects and cellular processes. The downstream targets of such surveillance mechanisms are likely to be master regulators, such as Ndt80, a meiosis-specific transcription factor in yeast (Tung et al., 2000), and p53, a tumour suppressor protein promoting apoptosis upon cell-cycle arrest in mammals (Vousden, 2000). The role of the checkpoint machinery in monitoring and ordering the phases of the mitotic and meiotic cell cycle is well documented in yeast and animals (Roeder and Bailis, 2000; Handel et al., 1999). However, there is limited information on the control of meiotic progression in plants. Two important checkpoints have been shown to operate in meiosis. The pachytene checkpoint inhibits cell-cycle progression until recombination intermediates have been resolved or when defects in recombination or synapsis occur (Roeder and Bailis, 2000). This damage checkpoint only monitors ongoing recombination events; there is, unexpectedly, no checkpoint to ensure that recombination is initiated (Roeder, 1997). Yeast mutants that fail to make any DSBs (e.g. spo11) still undergo both meiotic divisions, resulting in massive chromosome missegregation and inviable meiotic products (Klapholz et al., 1985). Mutations further downstream the recombination pathway (e.g. dmc1) cause cells to arrest at the pachytene stage with unrepaired DSBs, preventing meiotic divisions (Bishop et al., 1992). The pachytene checkpoint has also been found in animals (Edel- mann et al., 1996), but remains to be identified in plants (Garcia et al., 2003). Surprisingly, a mutation in AtDMC1, the Arabidopsis homologue of DMC1, does not cause arrest but leads to random chromosome segregation in meiosis and the production of defective spores (Couteau et al., 1999). Additional evidence of the lack of the pachytene checkpoint in plants comes from the large collection of maize meiotic mutants (e.g. Chan and Cande, 1998). The different mutants are defective in various stages of meiosis but none exhibit a checkpoint response. Hence the control of meiotic progression in plants may differ from that in yeast (Reddy et al., 2003). Recently, Preuss and Britt (2003) suggested that Arabidopsis may have a less stringent response than other organisms to double strand breaks. Their analysis of gamma-radiation-hypersensitive mutants supported the argument for a DNA damage-dependent but not DSB-dependent cell cycle checkpoint in Arabidopsis. The spindle checkpoint or metaphase checkpoint is also operative during mitosis (Amon, 1999) and monitors the proper orientation of chromosomes on the spindle apparatus. This checkpoint prevents cells from exiting metaphase I in response to bw-cnudde 23-08-2004 14:49 Pagina 34

34 CHAPTER I

signals generated by spindle damage or by incomplete or defective kinetochore attachment. It is a biological curiosity that the spindle checkpoint in mammals operates in males but not in females. This could provide an explanation for the high incidence of meiotic non-disjunction observed in human females, leading to a variety of birth defects and a relatively high frequency of miscarriages (Lemaire-Adkins et al., 1997; Hassold and Hunt, 2001).

Why is female meiosis more error-prone? First of all, the system for error detection is more extensive in male meiosis than in female meiosis. For example, the tension- sensitive spindle checkpoint is completely absent in oocytes in mammals (Lemaire- Adkins et al., 1997; Hassold and Hunt, 2001) and in Drosophila (McKim and Hawley, 1995). Second, there is a higher incidence of errors in chiasma placement, a process that occurs before birth in human females. Improperly positioned exchanges make bivalents more susceptible to non-disjunction. And third, there is an age-dependent impairment of the segregation machinery. Especially in older women, an important weakness lies in the ability to build and operate meiotic spindles (Champion and Hawley, 2002). What are the effects of such a high error rate? For the most part, meiotic failures are blunted by the strong requirement for euploidy in human foetuses. There are no viable monosomies for the human autosomes. However, a few trisomic zygotes are capable of survival. These are trisomies for the sex chromosomes (XXX, XXY, XYY), trisomy 21 (Down’s syndrome), trisomy 18 and trisomy 13. Aneuploidy is the most common recognised cause of mental retardation. The effects of these defects on the life of these people, and those of their families, are substantial. Genetic screens in budding yeast have identified several components of the meiotic checkpoints. Subsequently, homologues have been identified in higher eukaryotes showing that these pathways are highly conserved. Furthermore, these screenings indicated that the mechanisms to monitor meiotic recombination and mitotic DNA damage are related but not interchangeable (Lydall et al., 1996). In plants, the Arabidopsis AtATM gene, characterised by Garcia et al. (2003), was the first gene that was shown to be involved in the mediation of meiotic checkpoint responses. The Atm protein is a protein kinase whose activity is induced by DNA damage, particularly DNA double strand breaks, in somatic cells as well as during meiosis. Compared to the extensively studied mitotic cell cycle control systems, where complete checkpoint signaling pathways have been described (for review, see Straight, 1997) it becomes clear that still a lot of research is required to unravel the regulatory mechanisms that guide a plant cell through meiosis. bw-cnudde 23-08-2004 14:49 Pagina 35

Introduction 35

Box 1: “We humans are simply not very good at meiosis”

When you observe meiotically dividing cells through a microscope, it is easy to become impressed by the mysterious pairing of the homologous chromosomes, by the amazingly con- densed state of a metaphase I bivalent, and most of all by the sight of segregating chromosomes or chromatids, moving confidently towards their preset destination. The succession of different mei- otic figures, melting smoothly into one another, and the buildup towards the dramatic segregation scenes, seem like a perfectly produced play, an impressive ballet under the expert choreography of our master-director: the cell. Filled with awe for the regulatory mechanisms that guide the cell through the complex process of meiosis, it is easy to forget that sometimes, things go wrong. In meiosis, however, the consequences of such errors can be devastating. Unfortunately, we humans are simply not very good at meiosis (Champion and Hawley, 2002). The frequency of meiotic failure in humans is difficult to estimate because most aneuploid zygotes spontaneously abort early during a pregnancy that often passes unnoticed. According to Hassold and Hunt (2001) at least 10-30% of human conceptions are chromosomally abnormal and the frequency of errors increases markedly with advancing maternal, but not paternal age. Interestingly, the vast majority of human aneuploidies is caused by non-disjunction in the oocyte.

Model systems in the study of meiosis

Because meiosis is embedded in a cellular developmental pathway (gametogenesis, sporogenesis), it is to be expected that many species-specific variations of the regulation of the meiotic cell cycle exist (Van Heemst and Heyting, 2000). Therefore, it is important to study meiosis in a range of model organisms. An overview of the features of meiosis in different species is presented in Table 1. The budding yeast Saccharomyces cerevisiae is an ideal organism for studies of meiosis for a multiplicity of reasons. When diploid yeast cells are starved for carbon and nitrogen, almost all cells of the population enter meiosis, and they proceed through the meiotic divisions in a fairly synchronous manner. This efficiency and synchrony permits temporal analyses of the meiotic process on a nearly pure population without the need for separating meiocytes from surrounding cell types. Another significant strength of the Saccharomyces cerevisiae model is tetrad analysis. The ability to recover and analyse all four products of the individual meioses allows detailed investigation of the mechanisms of meiotic recombination (Roeder, 1995). Furthermore, recombination hot spots are well characterised and have been used as target sites in numerous experiments. Added up to the sophisticated genetics, the powerful transformation system and the availability of its full genomic sequence, it is not surprising that bw-cnudde 23-08-2004 14:49 Pagina 36

36 CHAPTER I

Table 1 Summary of features of meiosis in different species (after Shaw and Moore, 1998)

Saccharomyces cerevisiae is the most commonly used model organism in the study of meiosis. A second yeast strain that is frequently used is Schizosaccharomyces pombe. This more primitive fungus shows the features of a simpler meiosis, making it an interesting reference organism. Given the overwhelming strength of fungal model systems, research on meiosis in other eukaryotes follows at a respectable distance. A genome project also exists for Drosophila melanogaster. Furthermore, the investigation of meiosis in this model organism has the powerful advantage of extensive cytological data and a valuable collection of mutations affecting meiosis, an inheritance accumulated over seven decades (Orr-Weaver, 1995). A particular area of research has focused on the proper segregation of achiasmate chromosomes. In Drosophila, which possesses chromosomes that never recombine, these mechanisms are nearly 100% effective (Hawley and Theurkauf, 1993). The hermaphrodite nematode Caenorhabditis elegans is a particularly useful experimental system for investigating meiosis. C. elegans is essentially a ‘meiosis machine’: The germline accounts for more than half of the cell nuclei in the adult organism, providing an abundant and easily accessible source of meiotic cells (Schedl, 1997). Premeiotic nuclei and nuclei at all stages of meiotic prophase are present simultaneously in a clear temporal/spatial gradient along the distal-proximal axis of the gonad, so that each germline represents a continuum of progression into and through meiotic prophase. The accessibility of the germline material and the availability of the full genomic sequence make C. elegans particularly suitable for gene expression profiling experiments using DNA microarrays (e.g. Reinke et al., 2000). Until recently, the study of mammalian meiosis was hindered by the relative paucity of analytical techniques with which to study chromosomal events and outcomes in the less accessible mammalian germline (Cohen and Pollard, 2001). The mouse is a frequently used model system because its spermatogenesis is well characterised (e.g. de bw-cnudde 23-08-2004 14:49 Pagina 37

Introduction 37

Rooij, 2001) and commences in the postnatal mouse fairly synchronously (Vergowen et al., 1991). In mammalian females the entire population of oocytes enters meioses I in a near-synchronous fashion and progresses into early diplonema. Shortly thereafter, they undergo meiotic arrest and retain their pachytene/diplotene morphology until hormones stimulate the resumption of meiosis I during the estrous cycle in a small cohort of oocytes. Apart from the molecular analysis and mutant studies, a strong emphasis on immunocytological analysis is characteristic of the field of mammalian meiosis. Mouse and rat spermatocytes are often used for protein localisation studies using immunofluorescence labeling (e.g. Goedecke et al., 1999; Eijpe et al., 2000). Research on human meiosis is closely linked with reproductive medicine studying infertility problems. Recently, there have been considerable advances in our understanding of the genetic causes underlying male infertility (Krausz et al., 2003). Unfortunately, much less is known about the genetics of female infertility. Furthermore, there is an important connection with cancer research, since many types of cancer are caused by defective DNA repair (Rajewsky et al., 1998).

Rechearch on meiosis in plants

A good understanding of the meiotic process is crucial for research on reproduction, fertility, genetics and breeding, and in the plant field, has important implications for crop production. One of the aims of current meiosis research is to understand whether and to what extent meiotic processes and controls are conserved across all eukaryotes and, in this context, the inclusion of plant models is very important (Armstrong and Jones, 2003). Meiosis marks the transition from the sporophytic to the gametophytic generation in the life cycle of all plants (Dickinson, 1994). In seed plants male meiosis takes place in the developing anthers. In Arabidopsis each anther locule contains around 30 microsporocytes or pollen mother cells (PMCs), which enter and proceed through meiosis with a high degree of synchrony (Armstrong et al., 2001). Meiosis in the PMC produces a tetrad of haploid microspores, which are then released from a common wall and go on to develop into the male gametophytes (pollen grains). In the female reproductive tissues or ovules, meiosis takes place in the megaspore mother cell (MMC) and results in the formation of four haploid megaspores. The three spores closest to the micropyle of the ovule undergo programmed cell death leaving the chalazal megaspore to develop into the female gametophyte (the embryo sac) (Yang and Sundaresan, 2000). Interestingly, in most angiosperms male and female meiosis do not occur simultaneously: by the time male meiosis is complete, female meiosis is still at prophase I (Armstrong and Jones, 2001). bw-cnudde 23-08-2004 14:49 Pagina 38

38 CHAPTER I

Cytogenetic analysis

Because of the accessibility and the abundance of male meiocytes in the anthers, combined with the relatively prolonged prophase, plants have been an important source of knowledge about meiosis at the cytological level (Dawe, 1998; Zickler and Kleckner, 1999). Many of the cytogenetic advances of the 20th century, including detailed descriptions of chromosome behaviour during meiosis, were based on Liliaceous plants, with their large chromosomes and good chromosome morphology. Especially the correlation between the length of the floral bud with the cytological stage of meiosis made the anthers of Lilium longiflorum a superb model system for cytological and biochemical studies (Riggs, 1997). Analysis of meiosis in Arabidopsis thaliana is a growing area of research. Arabidopsis has a number of positive attributes for meiotic studies, including a large pool of tagged meiotic mutants and the molecular tools available to characterise them (Caryl et al., 2003). In contrast to the progress of molecular and genetic studies in Arabidopsis thaliana, cytological research on this plant has been regarded as difficult because of its small chromosomes. The development of molecular cytogenetic techniques, together with improved cytology, have enabled Ross et al. (1996) to conduct a first detailed analysis of normal meiotic chromosome behaviour in Arabidopsis, paving the way for the characterisation of a number of meiotic mutants (Ross et al., 1997; Caryl et al., 2003). Fransz et al. (1998) used the extended bivalents of meiotic pachytene cells, which are 20-25 times longer than mitotic metaphase chromosomes, to present a detailed karyotype of Arabidopsis and to demonstrate the power of the FISH technique for high resolution physical mapping at the cytological level. A detailed staging of floral development in Arabidopsis thaliana (Smyth et al., 1990) and anther development in tobacco (Koltunow et al., 1990) based on developmental landmarks linked with morphometric data and meiotic stages is particularly helpful for research on meiosis, since it facilitates the rapid and efficient preparation of meiocytes for cytogenetic analysis (Armstrong and Jones, 2003). A similar developmental staging is also available for Petunia hybrida (Porceddu et al., 1999). Recently, Armstrong et al. (2003) presented a meiotic time-course for Arabidopsis thaliana, based on in vivo radioactive labeling of cells in meiotic S-phase, followed by immunocytological detection at successive sampling times. This method allowed a precise measuring of the overall duration of meiosis: from the end of meiotic S-phase to the tetrad stage, meiosis in Arabidopsis thaliana takes 33h, which is about three times longer than in a mitotic cell cycle. Given the greater complexity of meiosis, this is not surprising. In most other eukaryotes, meiosis takes significantly longer than in plants, ranging from 4 days in Drosophila (Chandley and Bateman, 1962), to 11.5 days in the mouse (Oakberg, 1956) bw-cnudde 23-08-2004 14:49 Pagina 39

Introduction 39

and 24.3 days in humans (Heller and Clermont, 1963). On the other hand, meiosis in yeast has been found to occur very rapidly, meiosis is initiated and completed within 9h (Padmore et al., 1991). Furthermore, in their time-course study, Armstrong et al. (2003) measured the duration of individual meiotic stages in Arabidopsis, which varied considerably (Fig. 8). The establishment of such a temporal framework of the meiotic pathway provides an essential tool for determining the relative timing and durations of key molecular events in relation to cytologically defined landmarks of meiotic progression. Much of the recent progress in the detailed analysis of meiosis in plants has come from 3-D optical imaging methods applied to well-preserved cells and tissues. Traditional cytological methods such as squashing and spreading, performed on fixed, dead material, often cause considerable disruption and can mask important features. Therefore, new methods come into use, such as green fluorescent protein fusions and imaging techniques, which are more suitable to reveal all the subtleties of the dynamic process of meiosis in living organisms (Shaw and Moore, 1998). The detailed characterisation of the meiosis-specific Switch1 protein in Arabidopsis thaliana (Mercier et al., 2003) using a combination of genetic analysis and (immuno)cytology gives a good example of the strength of these new methods.

Dissecting plants meiosis using mutant analysis

Research on meiosis in plants has yielded large numbers of meiotic mutants (Gottschalk and Klein, 1976; Golubovskaya, 1979; Koduro and Rao, 1981; Kaul and Murthy, 1985; Singh, 1993) especially from species such as maize, tomato, pea, wheat and rye, which have been extensively studied cytogenetically. Unfortunately, since the large genome size of the majority of these plant species made the isolation of the corresponding genes difficult, it has not been possible to identify and characterise the genes affected in these mutants at a molecular level. It was not until the identification of a number of meiotic mutants from Arabidopsis thaliana that this problem could be overcome and molecular studies on meiosis in plants became possible. At present, almost all plant meiotic genes cloned are Arabidopsis genes (see Table 2 for a list that is growing rapidly) (Bhatt et al., 2001; Mercier et al., 2001a; Caryl et al., 2003). Only recently, the first maize meiotic gene, poor homologous synapsis1 (phs1), of a large mutant collection has been cloned (Pawlowski et al., 2004). So far, almost all the cloned genes were isolated via insertion tagging (transposon or Agrobacterium T-DNA). Two different strategies have been used: 1) forward genetics, which is based on the screening for mutated lines affected in meiosis, and subsequent cloning of the tagged gene; and 2) reverse genetics, which implies the selection of an bw-cnudde 23-08-2004 14:49 Pagina 40

40 CHAPTER I

Figure 8 Duration of individual meiotic stages in Arabidopsis (after Armstrong et al., 2003) S, G2 premeiotic DNA replication; L leptotene; Z/P zygotene/pachytene; D diplotene; T tetrad

insertion in a candidate gene and then analysing the phenotype (Mercier et al., 2001a, Peters et al., 2003). For forward genetics in Arabidopsis large mutant collections (e.g. the Versailles T-DNA mutant collection: http://weedsworld.arabidopsis.org.uk/Vol2ii/pelletier.html, TAIR: http://www.arabidopsis.org/) are available. However, screening for meiotic mutants is a laborious task: numerous plants exhibiting reduced fertility or infertility are selected for further investigation at the cytological level. In a screening for Arabidopsis mutants affected in somatic and meiotic recombination, Masson and Pazkowski (1997) selected several lines according to the hypersensitivity of these plants to X-ray irradiation. Reverse genetics is a second approach to study meiotic functions in plants by using data collected in other organisms, especially Saccharomyces cerevisiae (Roeder, 1997; Zickler and Kleckner, 1999; Dresser, 2000) and Schizosaccharomyces pombe (Davis and Smith, 2001). The identification of homologues in Arabidopsis was particularly successful for genes involved in meiotic recombination such as, for example, DMC1 (Klimyuk and Jones, 1997), MRE11 (Hartung and Puchta, 1999), and SPO11 (Hartung and Puchta, 2000). Systematic sequencing of the DNA flanking T-DNA insertions is currently underway, which greatly facilitates the screening process and makes mutants readily available through existing services from, for example, the SALK Institute (http://www.arabidopsis.org/abrc/tdna_ecker.html) and the Arabidopsis TILLING service (http://tilling.fhcrc.org:9366/home.html). However, meiotic genes often share surprisingly little sequence similarity. Therefore, the number of target genes for reverse genetics is relatively limited. Recently, the first double meiotic mutants have been reported in Arabidopsis (Grelon et al., 2003; Mercier et al., 2003; Reddy et al., 2003), contributing to a further characterisation of the mutated genes. However, double mutants are difficult to obtain in the field of meiosis because many mutants are sterile (Table 2). The increasing rhythm of discovery and characterisation of meiotic genes will undoubtedly lead to a better view of the highly regulated molecular control of plant meiosis, in comparison with organisms from other kingdoms. bw-cnudde 23-08-2004 14:49 Pagina 41

Arabidopsis Mutagen SterilityCloned Sequence References mutant used gene? Similarity

asy1 T-DNA M/F Yes (f) ScHOP1 Ross et al., 1997 Caryl et al., 2000

syn1 or T-DNA M/F Yes (f) ScREC8 Bhatt et al., 1999 dif1 transposon Bai et al., 1999

swi1 or T-DNA M or M/F Yes (f) - Motamayor et al., 2000 dyad EMS Siddiqi et al., 2000 Mercier et al., 2001b Agashe et al., 2002 Mercier et al., 2003

ms5 or T-DNA F Yes (f) - Ross et al., 1997 tdm1 or T-DNA Glover et al., 1998 pollenless3 T-DNA Sanders et al., 1999

atSpo11-1 T-DNA M/F Yes (f) ScSPO11 Grelon et al., 2001

atDmc1 T-DNA M/F Yes (r) ScDMC1 Couteau et al., 1999 EcRECA

atRad50 T-DNA M/F Yes (r) ScRAD50 Gallego et al., 2001

ask1 transposon F Yes (f) ScSKP1 Yang et al., 1999 Yang and Ma, 2001

mei1 or T-DNA M/F Yes (f) HsTOPBP1 Ross et al., 1997 mcd1 T-DNA He and Mascarenhas, 1998 Yang and McCormick, 2002 Grelon et al., 2003

dsy1 T-DNA M/F no - Ross et al., 1997

ms4 T-DNA F no - Chaudhury et al., 1994

tam EMS M* no - Magnard et al., 2001

dsy10 T-DNA M/F no - Cai and Makaroff, 2001

solo dancers transposon M/F Yes (f) AtCDC2a Azumi et al., 2002 (sds) AtCDC2b

atMre11 T-DNA M/F Yes (r) ScMRE11 Bundock and Hooykaas, 2002

atk1** transposon M Yes (f) ScKAR3 Chen et al., 2002

atATM T-DNA M/F Yes (r) ScMEC1 Garcia et al., 2003

tes or fast neutron/ M Yes (f) NtNACK1 Spielman et al., 1997 std EMS Hülskamp et al., 1997 Yang et al., 2003a

mmd1 or transposon M Yes (f) AtMS1 Yang et al., 2003b duet Reddy et al., 2003

ahp2 T-DNA M/F Yes (f) SpMEU13 Schommer et al., 2003

At= Arabidopsis thaliana, Ec= Escherichia coli, Nt= Nicotiana tabacum, Sc= Saccharomyces cerevisiae , Sp= Table 2 Arabidopsis Schizosaccharomyces pombe, Hs= Homo sapiens M, F: male sterile, female sterile thaliana mutants (f), (r): isolated by forward or reverse genetics, respectively affected in meiosis * tam plants are fertile, but male meiosis is delayed ** atk1 mutants show reduced male fertility bw-cnudde 23-08-2004 14:49 Pagina 42

42 CHAPTER I

Meiosis: a conserved process with intriguing differences

The application of molecular and modern cell biological techniques in a range of different model species is beginning to show that the classical picture of meiosis is an oversimplification (Shaw and Moore, 1998). Although many meiotic processes are highly conserved among organisms, an increasing number of differences become apparent in features thought to be an integral part of meiosis. For instance, it is clear that the mechanisms of DSB formation, recombination and chiasmata formation are conserved in systems ranging from yeast to humans; SC formation on the other hand is not obligatory, and none of these mechanisms is absolutely required for meiosis. Differences in the meiotic pathway have, for example, been observed in polyploid plants, which include up to 70% of flowering plants (Masterton, 1994). To produce viable gametes, polyploids must behave effectively as diploids during meiosis, allowing only true homologous chromosomes to pair. Martinez-Perez et al. (2000) demonstrated that centromeres associate early during anther development in polyploid species. In contrast, centromeres in diploids only associate at the onset of meiotic prophase. Surprisingly, meiosis appears to be shorter in polyploid plants than in their diploid progenitors (Bennett and Smith, 1972). Apparently, the more chromosome sets are present, the shorter the time required to sort them. One aspect of meiosis that may vary significantly between organisms concerns how the cell controls the progression through the meiotic cell cycle. Regulation of the meiotic cell cycle is best understood in yeast (reviewed by Lee and Amon, 2001), in which numerous mutations have been identified that block chromosome synapsis and/or recombination and induce pachytene arrest (Roeder, 1997). This pachytene checkpoint also has been shown to operate in several other organisms, such as Drosophila, Caenorhabditis elegans, and mouse. However, mutations in genes that trigger pachytene arrest in these organisms do not appear to activate a typical meiotic checkpoint in plants, suggesting that plants do not use the same meiotic checkpoint control mechanisms (Yang et al., 2003b). These differences suggest that currently no single species will provide a correct model for the rest. A detailed understanding of the meiotic process is necessary in each species of interest, and it remains to be demonstrated how far information from other kingdoms is directly comparable and interchangeable. Such cross-kingdom comparisons, based on homology studies complemented with mutant analysis data, can lead to the specification of the ‘core’ meiotic machinery, conserved during evolution. It is to be expected that functional genomics strategies, supported by bioinformatics, will play an essential role in this field. bw-cnudde 23-08-2004 14:49 Pagina 43

Introduction 43

Aim and outline of this thesis

My work as described in this thesis is the result of a solid technological foundation, a challenging biological process in a non-everyday model system, and two different working environments as sources of inspiration: the labs of Plant Genetics in Gent and Nijmegen. The application of the cDNA-AFLP technology to various biological questions plays a central role throughout this thesis. The combination of this functional genomics strategy with a subsequent gene- specific approach was used to contribute to the unraveling of the complex process of meiosis at the molecular level. A general introduction on meiosis is found in Chapter I. A first aim of this thesis was the analysis of flower development in Petunia hybrida and was done in collaboration with Andrea Porceddu and Mario Pezzotti from the universities of Perugia and Verona, respectively. They provided flower organ-specific mRNA samples for a gene expression analysis study. At that time the in house developed cDNA-AFLP Transcript Profiling technology was being optimised at the Department of Plant Genetics in Gent, which proved to be an adequate tool for such an analysis. The setup and results from these experiments are described in Chapter II. In Chapter III the use of cDNA-AFLP as a tool to unravel meiosis is described. Much attention is given to the setup of these experiments. Since our study of flower development in Petunia hybrida strongly focused on male and female gametogenesis, it came as a surprise that almost no cDNA fragments with homology to any known meiotic genes could be retrieved. After considering several plausible explanations, we embarked on a risky enterprise: the elaboration of a functional genomics strategy aiming at the molecular dissection of male meiosis in Petunia hybrida. Using cDNA-AFLP-based transcript profiling, we were able to build a comprehensive inventory of genes with a modulated expression pattern during male meiosis. The characterisation of these fragments and the functional classification based on literature datasearches is described in Chapter IV. Chapter V describes the cluster analysis to group genes based on their expression pattern. Finally, in Chapter VI, the results of in situ hybridisation experiments on 5 selected genes are presented as a demonstration of the possible follow-up and future prospects of the research on meiosis and the functional analysis of meiotic genes. bw-cnudde 23-08-2004 14:49 Pagina 44

44 CHAPTER I

References

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Chapter II

Transcript profiling on developing Petunia hybrida floral organs

Filip Cnudde, Chiaraluce Moretti1, Andrea Porceddu2, Mario Pezzotti3, Tom Gerats

1Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy 2Institute of Plant Genetics-CNR sez Perugia. Via Madonna Alta 130 01626 Perugia. 3Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 7, 37134 Verona, Italy

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56 CHAPTER II

Abstract

The cDNA-AFLP transcript profiling technique was used to analyse gene expression during flower development in Petunia hybrida. Reproductive and vegetative floral organs were sampled at five developmental stages and gene expression profiles were compared. This allowed us to assemble an inventory of genes expressed mainly in anthers during microspore development and in ovaries during macrosporogenesis. About 6,000 transcript tags were generated, 354 of which showed a modulated and/or organ-specific expression pattern. Stamen-specific transcripts exhibiting an upregulation in gene expression were well represented in our screening. Ovary-specific transcripts were less frequently observed and often displayed a constant level of gene expression. Of 194 fragments characterised further by sequencing, 35% showed homology with known genes in a database search. They belong to a wide range of gene classes, such as proteases, transcription factors and genes involved in metabolism, cell cycle and disease resistance. The usefulness of cDNA-AFLP transcript profiling as a tool to unravel complex developmental processes at the molecular level is discussed.

Introduction

Genetic studies have identified two classes of consecutively acting genes involved in the early steps of flower development. First, meristem identity genes are required for the regulation of the floral meristem formation. Second, homeotic genes are expressed in discrete domains of the flower meristem to specify the identity of floral organ types (Coen and Meyerowitz, 1991). Little information is available on the downstream genes involved in subsequent processes of floral organ morphogenesis and tissue differentiation. These genes should include those that: (1) regulate floral organ compartment differentiation, (2) control floral shape and size, (3) initiate cell- and tissue-specification programs, and (4) activate organ-specific gene subsets (Meyerowitz, 1997). The elucidation of genes belonging to the above categories is both an intriguing and a challenging task. Ovule development is particularly well described at the morphological level (Schneitz et al., 1995), but the underlying genetic and molecular mechanisms are just beginning to be unraveled (for reviews, see Drews et al., 1998; Gasser et al., 1998; Grossniklaus and Schneitz, 1998; Schneitz et al., 1998). A number of interesting ovule identity mutants have been identified in Petunia hybrida and Arabidopsis thaliana, leading to the cloning of the first regulatory genes involved in ovule development (Angenent and Colombo, 1996). Differential screening of cDNA libraries has also been used for the isolation of bw-cnudde 23-08-2004 14:49 Pagina 57

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genes associated with ovule development (Gasser et al., 1989; Nadeau et al., 1996), although the inaccessibility of the ovule within the ovaries and the difficulty in harvesting adequate amounts of tissue have been, and still are, major obstacles. In contrast, much attention has been devoted to the genetic control of anther development (for reviews, see Koltunow et al., 1990; Scott et al., 1991b; Goldberg et al., 1993). This exceptional interest can be partly attributed to 1) the synchronicity of anther development, and 2) the strong allometric relationship between anther development and bud length (Koltunow et al., 1990; Scott et al., 1991a). In early DNA/RNA hybridisation experiments it was estimated that approximately 25,000 genes are expressed in the tobacco anther, of which 10,000 are thought to be anther- specific (Kamalay and Goldberg, 1980, 1984). Anther-specific genes have been cloned from a number of plant species such as tobacco (Koltunow et al., 1990), Brassica napus (Scott et al., 1991a), Arabidopsis (Rubinelli et al., 1998) and rice (Tsuchiya et al., 1992). Less genomic information is available on the morphogenesis of vegetative floral organs. Petunia hybrida is considered as a model system for studying morphogenesis of floral organs because, among other advantages, it has a large flower and a strong allometric relation between bud size and developmental processes in reproductive organs. The identification of expressed sequence tags (ESTs) has proven to be a powerful and rapid approach to identify genes that are preferentially expressed in certain tissues or cell types (Adams et al., 1995). The expression profile of these sequences can then be studied using microarray-based technologies (Brown and Botstein, 1999). Though powerful, this approach is expensive and can be readily applied only to model species for which significant genomic information is available (Baldwin et al., 1999). Present additional limitations include the sensitivity of microarrays and the reduced specificity because of potential cross-hybridisation problems with genes belonging to the same family (Breyne and Zabeau, 2001). Since relatively little genomic sequence information is available for Petunia hybrida, we employed cDNA-AFLP transcript profiling technology to identify genes that exhibit a modulated expression during male and female sporogenesis as compared to vegetative tissues. This sensitive and reproducible method does not require prior sequence information and allows the identification of as yet unidentified genes. Moreover, small amounts of RNA are sufficient as starting material, allowing the analysis of small samples (Breyne et al., 2002). Direct comparison of 6,000 amplicons yielded 354 candidate cDNA fragments displaying a modulated and/or organ-specific gene expression pattern. 35 Ovary-fragments and 159 anther-fragments were further analysed by direct sequencing. The majority of the cDNA fragments yielded no significant homology in a database search, although homologies with diverse classes of known sporogenesis genes were retrieved, indicating that our screening was basically effective. bw-cnudde 23-08-2004 14:49 Pagina 58

58 CHAPTER II

Experimental procedures

Plant material

Petunia hybrida (line W115) plants were grown under standard greenhouse conditions (21°C; 16h photoperiod). Flowers were collected at five different developmental stages recognised on the basis of petal length and corresponding with stages 4-8 as published in Porceddu et al. (1999). Samples were taken separately from sepals, petals, anthers, style and stigma, and ovaries. Root and leaf samples were also collected and frozen in liquid N2.

mRNA isolation and cDNA synthesis

Total RNA was isolated from about 2g each of floral or vegetative organs using the TRIZOL method (Gibco-BRL, Paisley, UK). Total RNA concentration was determined spectrophotometrically and adjusted to a final concentration of 1µg/µl. A poly(A)+ RNA fraction was isolated from 1mg total RNA using an mRNA purification kit from Amersham Pharmacia (Uppsala, Sweden) according to the manufacturer’s instructions. First and second cDNA strands were synthesised according to standard protocols (Sambrook et al., 1996). The resulting double stranded cDNA was subsequently purified by phenol extraction and ethanol precipitation.

cDNA-AFLP analysis

The cDNA-AFLP-based transcript profiling procedure was performed according to Breyne et al. (2002). Double stranded cDNA (500ng) was used for cDNA-AFLP analysis. The restriction enzymes used were BstYI and MseI (New England Biolabs, Beverly, Mass.). For preamplifactions, an MseI-primer without selective nucleotides was combined with a BstYI-primer containing a T at the 3’-end. The amplification mixtures obtained were diluted 600-fold and 5µl was used for final selective amplifications following a described procedure (Breyne et al., 2002). BstT-primers and MseI-primers with two and one selective nucleotides, respectively, were used for the cDNA-AFLP analysis, and all 64 possible primer combinations were performed. The 33P-ATP-labeled fragments were separated on a 5% polyacrylamide gel. Dried gels were exposed to Kodak Biomax films in addition to scanning in a Phospho-imager (Molecular Dynamics). bw-cnudde 23-08-2004 14:49 Pagina 59

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Characterisation of AFLP fragments

Selected fragments were excised from the polyacrylamide gel, suspended in H2O and eluted DNA was reamplified using the same PCR conditions and the same primer combination as for selective amplification. Reamplified products were checked on a 1% agarose gel. Sequence information was obtained by direct sequencing using the selective BstYI-primer as a sequencing primer. The sequences obtained were compared to those in the GenBank database using BLAST sequence alignments (Altschul et al., 1997).

Results

Developmental staging of Petunia flower buds

Flowers of Petunia hybrida line W115 were harvested and classified into five developmental stages according to petal length. These stages correspond to stages 4-8 as defined by Porceddu et al. (1999). At each stage, we harvested samples from sepals, petals, stamens, ovaries and style/stigma. During these developmental stages, Petunia sepals increase in length (from 9 to 14mm), almost exclusively through cell expansion. Petals show a 5-fold increase in length (5.75-24.75mm) through cell division and expansion (Martin and Gerats, 1993; Reale et al., 2002). It has already been demonstrated that bud length is tightly linked to the stages of postmeiotic microspore development and to megasporogenesis in Petunia (Porceddu et al., 1999); at stage 4, most of the ovules harbour a megaspore mother cell, while at developmental stages 5, 6 and 7, dyads, triads and tetrads, respectively, are predominant. At stage 8 only tetrads and megaspores are detected (data not shown). At the beginning of stage 4, the first phase of anther development has already been completed (Porceddu et al., 1999): the anther has acquired its characteristic four-lobed shape; cell differentiation has occurred and all major anther tissues have been specified; the microspore mother cells have undergone meiosis and tetrads of haploid microspores are present in the pollen sac. During the developmental range monitored in our work (stages 4-8), microspores differentiate into pollen grains, the anther enlarges and is pushed upwards in the flower by filament extension, tissue degeneration occurs and the anther enters a dehiscence program ending with the release of pollen grains. bw-cnudde 23-08-2004 14:49 Pagina 60

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cDNA-AFLP transcript profiling

cDNAs were synthesised from 1µg poly(A+) RNA of different floral organs sampled at the five developmental stages described above. We focused on the analysis of gene expression in anthers and carpels, while samples from sepals, petals and style/stigma served as a reference. cDNA was also prepared from roots and leaves as a second control for vegetative tissues.

Figure 1 A-E Sensitivity and selectivity of cDNA-AFLP. B, C Transcript profiles from anthers and ovaries, respectively, sampled at five developmental stages (stages 4-8 as defined by Porceddu et al., 1999). Two selective nucleotides were used: one for the BstT-primer and one for the Mse- primer (+1/+1). A, D Profiles of the ten pooled preamplification products from anthers and ovaries and using a third selective nucleotide; this third nucleotide was added to the BstT-primer in panel A (+2/+1) and to the Mse-primer in panel D (+1/+2). An extra nucleotide increases the sensitivity and the selectivity of cDNA- AFLP transcript profiling: each transcript tag from a +1/+1 combination reappears more intensely in one of the four lanes of the +2/+1 and +1/+2 primer combinations. Panel E represents the +1/+1 profiles for some of the reference samples: St/St style-stigma, R roots, L leaves

The transcript profiling technology used in this experiment is based on the original cDNA-AFLP method described by Bachem et al. (1996). The method was adapted such that theoretically only one unique restriction fragment is obtained from each cDNA (Breyne and Zabeau, 2001). The radiolabeled cDNA fragments were separated on high- resolution polyacrylamide gels. We primarily analysed fragments that ranged in length from 50-400 basepairs. For the majority of the gels, the preamplification products of separate developmental stages were pooled for the reference samples (sepals, petals and style/stigma) to reduce the number of lanes per primer combination. When BstYI/MseI templates were amplified using primers with two selective nucleotides, a high number of fragments per lane was generated, mostly corresponding to abundant mRNAs. The addition of a third selective nucleotide decreased the number of fragments to an average of 90 for each primer combination and enhanced the sensitivity, allowing detection of less highly expressed genes. In a check for the reliability of the transcript profiling method (Fig. 1), we could bw-cnudde 23-08-2004 14:49 Pagina 61

Transcript profiling on floral organs 61

confirm that each transcript tag amplified by primers with two selective nucleotides (+1/+1) reappeared more intensely in one of the four lanes of the +2/+1 or the +1/+2 primer combinations. Using BstYI and MseI primers carrying two or one selective nucleotides, respectively, gives a total of 128 possible primer combinations. With half of these combinations performed, around 6,000 transcripts could be detected. In silico analysis of full-length cDNAs from Arabidopsis thaliana has shown that 80% of all transcripts are cut by both BstYI and MseI restriction enzymes and that 68.8% are represented as a BstYI-MseI 5’- 3’ fragment (Breyne et al., 2003). The collection of available full-length cDNA sequences in Petunia hybrida is too limited for such an analysis, but if we assume that a comparable number of messenger RNAs is tagged by this enzyme combination and that 10% of the restriction fragments generated are either too small or too large to be analysed on polyacrylamide gels (Breyne et al., 2002), we may estimate that approximately 30% of the Petunia transcriptome has been analysed by applying half of the BstYI/MseI primer combinations. This would lead to an estimate of around 20,000 transcripts for the whole transcriptome. A typical polyacrylamide gel is shown in Fig. 2.

Figure 2 cDNA-AFLP during floral development For the screening for gametogenesis-related transcript fragments, all 64 possible BstT/Mse +2/+1 primer combinations were carried out. Expression levels are compared during 5 developmental stages (stages 4-8 as defined by Porceddu et al., 1999) for anthers and ovaries. The preamplification products of petals, sepals and style- stigma are pooled prior to amplification. These samples serve as a reference, together with roots and leaves. Arrows indicate candidate gametogenesis-genes with a modulated or reproductive organ- specific expression pattern. S sepals; P petals, St/St style-stigma, R roots, L leaves bw-cnudde 23-08-2004 14:49 Pagina 62

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Analysis of cDNA-AFLP expression patterns and characterisation by sequencing

Of the 6,000 AFLP tags analysed, 354 gene fragments (6%) were found to be organ- specific or modulated during the developmental range tested. As our study focuses on changes in gene expression in developing reproductive organs, we report a classification of fragments based on their expression profile in these organs (Table 1). Fragments expressed exclusively in anther (or ovary) are denoted as anther (or ovary)-specific. Fragments found also in other organs but with a clearly different expression pattern in anthers or ovaries were denoted as anther (or ovary)-non-specific. Fragments classified as non-modulated (and specific or non-specific) typically exhibited a constant and high expression level in the stages tested. Most of the ovary-modulated fragments were also present in the style and stigma; moreover, most of the ovary fragments overall were non-modulated. Also, 72 of the anther-specific and 74 of the anther-non-specific fragments were highly induced. Finally, 18 fragments were (non)-modulated in both anthers and ovaries. Several examples of selected cDNA-AFLP expression profiles are shown in Fig. 3.

Figure 3 Examples of selected cDNA-AFLP expression patterns. +2/+1 Transcript profiles from anthers and ovaries, sampled at five developmental stages (stages 4-8 as defined by Porceddu et al., 1999), compared to control tissues S sepals, P petals, St/St style-stigma, R roots, L leaves bw-cnudde 23-08-2004 14:49 Pagina 63

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Table 1 Classification of differentially expressed cDNA fragments

In total, 354 transcript fragments were isolated from gels, reamplified by PCR and sequenced. Direct sequencing of the PCR products gave high quality sequence for 194 fragments (55 %). For the remaining transcript tags no clear sequence information was obtained, presumably because these cDNAs were a mixture of co-migrating fragments. These were not further characterised. The success rate of direct sequencing can be increased by using cDNA-AFLP primers containing an additional selective nucleotide. This reduces the complexity of the amplification mixture 4-fold and will lead to less co- migrating fragments. As an alternative, PCR products can be cloned and individual colonies can be sequenced (Breyne et al., 2002). The sequences obtained were compared to those present in the GenBank database using the BLAST program (Altschul et al., 1997); 71 fragments (36%) show significant homology (maximum E-value 0.001) to genes with known function, while another 10 (5%) match genes without allocated function (ESTs or putative proteins; Table 2). The remaining 59% either represent sequences of previously uncharacterised genes, or share no homology, presumably because the AFLP tags are too short and/or originate from the 3’-end region of the transcripts, which is often assumed to be less conserved. Of the fragments with ovary-specific or (non)-modulated expression, 42 were successfully sequenced. Only 11 (25%) displayed significant homology with known genes in a database search (Table 2). This could reflect the backlog in the molecular analysis of megasporogenesis compared to the more extensively studied anther development. Homologies were found with proline-rich proteins, a peroxidase, a tobacco tumour-related protein eliciting the hypersensitive response and a PR-protein. They can be added to a growing list of abundantly expressed pistil genes that encode defence-related proteins. Pistils are particularly vulnerable to pathogen infection because they are rich in precursors of macromolecules required for supporting pollen tube growth (Harris et al., 1984). A fragment was found that exhibited homology with the Capsicum KNOLLE gene, which encodes a cytokinesis-specific protein related to syntaxins, a protein family involved in vesicular trafficking. KNOLLE mRNA has previously been shown to accumulate preferentially in organs with actively dividing tissues, such as inflorescence and floral meristems (Lukowitz et al., 1996). A total of 142 bw-cnudde 23-08-2004 14:49 Pagina 64

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Table 2 Relevant homologies of sequences of AFLP fragments to sequences in the database bw-cnudde 23-08-2004 14:49 Pagina 65

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anther-fragments were successfully sequenced; for 62 (49%) of these a significant homology was found. Based on sequence homology we could relate some of the genes identified to physiological processes occurring during anther development. A first group of genes that could be discerned is involved in dessiccation tolerance and was upregulated during pollen maturation. Petunia hybrida has pollen with a water content of 10% of its fresh weight at dispersal. Reserves consist of cytoplasmic mono-, oligo- and polysaccharides, whereas starch is absent (Baker and Baker, 1979). bw-cnudde 23-08-2004 14:49 Pagina 66

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A cDNA fragment with an induced expression profile in anthers showed homology to a delta 1-pyrroline-5-carboxylate synthetase, the rate-limiting enzyme in the biosynthesis of proline (Yoshiba et al., 1999). Proline represents about 50% of the amino acid pool of Petunia pollen and is utilised for protein synthesis during pollen germination (Zhang et al., 1982) and as a protectant against dessication (Zhang and Croes, 1983). Fragments homologous to sucrose phosphate synthase, Na+/H+ antiporter and betaine aldehyde dehydrogenase also indicate the occurrence of drought tolerance processes in developing anthers. Sucrose has been shown to play a role in the acquired tolerance to severe dehydration in the resurrection plant Craterostigma plantagineum (Ingram et al., 1997). Na+/H+ antiporter anther-specific induction has already been described during dessication in Oryza sativa (Fukuda et al., 1999) and the involvement of betaine aldehyde dehydrogenase in dessication has been demonstrated in sorghum (Wood et al., 1996). Another group of genes could be related with tapetum degradation. Inorganic pyrophosphatase (V-PPase) might supply a driving force for loading sugars from the cytosol into the vacuole (Mitsuda et al., 2001). Yet another group of fragments identified genes expressed in the pollen grain that may play a role during pollen germination. An ‘early’ anther-specific cDNA fragment expressed up to the time of microspore mitosis showed homology to the Arabidopsis FERTILIZATION INDEPENDENT ENDOSPERM (FIE) gene. FIE encodes a WD polycomb group protein that represses endosperm development in the absence of pollination and fertilisation (Ohad et al., 1999). Luo et al. (2000) demonstrated that FIE::GUS fusion proteins were expressed in the female gametophyte and transiently in developing microspores. A ‘late’ anther-specific cDNA was homologous to the MALE STERILITY 2 (MS2) gene, encoding a fatty acyl reductase involved in the formation of the pollen wall (Aarts et al., 1997). Its importance in pollen development was demonstrated by the isolation of a transposon-tagged male-sterile mutant in Arabidopsis (Aarts et al.; 1993). Flavonoids and other phenylpropanoids are present in large amounts in the exine layer of pollen grains (Mascarenhas, 1990). A key enzyme in phenylpropanoid metabolism, phenylalanine ammonia lyase, was induced upon pollen maturation. Anther-specific cDNA fragments were also isolated that show homology to genes functioning during pollen tube growth. Their mRNA accumulates during pollen maturation and is translated upon germination. Calmodulin, a Ca2+-interacting protein and profilin, an actin-binding protein, both play a role in signaling pathways involving protein kinase and phosphatase cascades that may regulate pollen tube growth by acting on the actin-based cytoskeleton organisation and assembly (Franklin-Tong et al., 1999). Homologues of LAT59, a tomato pectate lyase (Wing et al., 1990) and a pectin esterase- like protein (Mu et al., 1994) may be involved in pectin degradation during germination and pollen tube growth. bw-cnudde 23-08-2004 14:49 Pagina 67

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Maybe not unexpectedly, anther-specific induction was observed for a cDNA fragment homologous to an S-locus F-box protein involved in self-incompatibility. Finally, the isolation of cDNA fragments exhibiting homology to transcription factors such as a zinc finger transcription factor and a bHLH transcription factor are an indication of the high sensitivity of our transcript profiling experiment. An interesting expression pattern was observed for a protein kinase homologue present in all floral organs but not in roots and leaves.

Discussion

The advantages of cDNA-AFLP transcript profiling

The combination of developmentally staged floral organ samples with the cDNA-AFLP transcript profiling technique allowed us to perform a large screening for genes exhibiting a modulated and/or organ-specific expression pattern during gametogenesis in Petunia hybrida flower buds. The identification of hundreds of transcript tags together with the generation of reliable expression data prove that cDNA-AFLP transcript profiling is a valuable tool for high-throughput expression analysis. This is certainly true for non-model species, where it can be used as a valid alternative to microarrays. Compared to subtractive hybridisation library screening, cDNA-AFLP transcript profiling allows a more thorough analysis because of the direct comparison of gene expression levels in different organs during a range of developmental stages. Further characterisation of the cDNA fragments showed that they corresponded to diverse classes of known gametogenesis genes, indicating that our screening was effective. Genes active in the same pathways are often co-expressed, allowing the identification of ongoing biological processes and their mutual coordination. In agreement with recent studies (Durrant et al., 2000; Breyne et al., 2002) a large number of as yet unknown genes was identified, proving that cDNA-AFLP transcript profiling is an efficient technique to identify previously unknown transcripts.

Number of anther-specific genes identified lower than expected

Previous estimations of the number of genes specifically expressed during gametogenesis have focused on the male gametophyte. Early DNA/RNA hybridisation experiments indicated that about 25,000 genes are expressed in the tobacco anther at stage 6 (Kamalay and Goldberg, 1980). Approximately 10,000 of these genes were estimated to bw-cnudde 23-08-2004 14:49 Pagina 68

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be anther-specific because their mRNA was not detectably expressed in other floral or vegetative organs (Kamalay and Goldberg; 1980, 1984). Based on these figures, and taking the smaller genome content into account, Sanders et al. (1999) predicted 8,000 genes to be expressed during the equivalent stage of anther development in Arabidopsis, of which 3500 exhibited an anther-specific expression pattern. Stage 6 in tobacco anther development (Koltunow et al., 1990) corresponds to stage 3 in Petunia (Porceddu et al., 1999); most specialised cell types are still present within the anther and the microspore nuclei divide in the locules. It is likely that many additional genes might be active immediately after meiosis and during the early stages of microspore development. Similar experiments on mature pollen of Tradescantia and maize indicate that 20,000 and 24,000 genes, respectively, are transcribed, about 10% of which are considered to be pollen-specific (Willing and Mascarenhas, 1984; Willing et al., 1988). Looking at our trancript profiling experiment with the choice of restriction enzymes and half of all possible primer combinations carried out, we estimate that 30% of the transcriptome has been visualised on gels, resulting in 6,000 AFLP tags. Since each expressed gene is theoretically represented by one fragment, approximately 20,000 genes are transcribed in anthers of Petunia hybrida during postmeiotic microspore development and pollen maturation. The number of generated fragments did not differ significantly between all organs tested, suggesting that an overall equal number of genes is expressed. In our screening, 138 anther-specific cDNA fragments were identified. This suggests that no more than 500 genes with an anther-specific expression pattern can be expected in a genome-wide screening. How can this difference from the 10,000 genes proposed by Kamalay and Goldberg (1980, 1984) be explained? First, the sensitivity of the cDNA-AFLP transcript profiling method is higher than that of a hybridisation-based technique (Breyne and Zabeau, 2001); up to one mRNA copy per cell can be visualised on a gel (Bachem et al., 1998). This leads to a more stringent definition of anther- specificity because it allows the detection of low levels of expression in other floral and vegetative organs. Second, 10,000 genes corresponds to the number of anther mRNAs not shared with leaf. Although it was shown that this mRNA set is distinct between anthers and ovaries, the selection of anther-specific genes was more precise in our experiment, which allows a direct comparison of expression levels for each individual anther-expressed gene with ovary, style-stigma, sepal, petal, root and leaf. Third, with cDNA-AFLP transcript profiling, gene numbers are determined unambiguously by counting fragments on a polyacrylamide gel, whereas in the RNA/DNA hybridisation experiments of Kamalay and Goldberg, gene numbers were estimated in an indirect way based on mRNA complexities. bw-cnudde 23-08-2004 14:49 Pagina 69

Transcript profiling on floral organs 69

Gene expression patterns during male gametophyte development

With our approach, it is possible to group genes based on their expression pattern and to search for a possible function based on their co-expression with already defined genes. Mascarenhas (1990) distinghuished two patterns of pollen gene expression. ‘Early’ genes are transcribed soon after meiosis and are reduced or undetectable in mature pollen. Transcripts of the ‘late’ genes are first detected around the time of microspore mitosis and continue to accumulate as pollen matures. In our transcript profiling experiment we screened for genes expressed in anthers displaying a modulated or anther-specific expression pattern. Of these, 60% belong to the class of ‘late’ genes, showing an increase in gene expression starting from stage 6 and 7 up to the end of pollen maturation. These accumulating mRNAs belong to genes involved in pollen maturation. A set of mRNAs is stored in mature pollen for mobilisation upon pollen germination and pollen tube growth. Around 25% of anther genes are repressed; in most cases the gene expression level decreases from stage 4 to stage 6 (microspore mitosis). They belong to the class of ‘early’ genes that encode cytoskeletal proteins and proteins needed for pollen wall biosynthesis and energy production; most of these genes are expressed in the tapetum. We propose a third class of gene expression, comprising the remaining 15% of anther-specific genes showing a non-modulated gene expression pattern, but one that displayed anther-specificity or a high level of expression compared to other plant organs. Genes involved in signaling and cell-cell interactions such as subtilisin-like serine protease and calmodulin belong to this class.

Conclusion

We have demonstrated that cDNA-AFLP transcript profiling is a suitable method for the identification of genes involved in male and female gametogenesis in Petunia hybrida. By comparing gene expression profiles of floral organ-specific samples during several stages of flower development, modulated and anther- or ovary-specific cDNA fragments were encountered, including many as yet unidentified genes. The generated cDNA tags can readily be used as probes for further in situ hybridisation experiments or for mutational analysis. Further characterisation of these sets of genes will help to unravel the control mechanisms and genetic networks underlying male and female gametogenesis in plants. bw-cnudde 23-08-2004 14:49 Pagina 70

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Acknowledgements

Filip Cnudde was payed partly by the Flanders Fund for Scientific Research (FWO), and partly by the Flemish Institute for Biotechnology (VIB) and partly by a grant from the subfaculty of Biology, University of Nijmegen.

References

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Franklin-Tong, V.E. (1999) Signaling in pollination. Curr. Opin. Plant Biol. 2, 490-495. Fukuda, A., Nakamura, A., and Tanaka, Y. (1999) Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. Biochym. Biophys. Acta 1446, 149-155. Gasser, C.S., Budelier, K.A., Smith, A.G., Shah, D.M., and Fraley, R.T. (1989) Isolation of tissue-specific cDNAs from tomato pistils. Plant Cell 1, 15-24. Gasser, C.S., Broadhvest, J., and Hauser, B.A. (1998) Genetic analysis of ovule development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 1-24. Goldberg, R.B., Beals, T.P., and Sanders, P.M. (1993) Anther development: Basic principles and practical applications. Plant Cell 5, 1217-1229. Grossniklaus, U., and Schneitz, K. (1998) The molecular and genetic basis of ovule and megagametophyte development. Semin. Cell. Dev. Biol. 9, 227-238. Harris, P.J., Anderson, M.A., Bacic, A., and Clarke, A.E. (1984) Cell-cell recognition in plants with special reference to the pollen-stigma interaction. Oxford Surv. Plant Mol. Cell Biol. 1, 161-203. Ingram, J., Chandler, J.W., Gallagher, L., Salamini, F., and Bartels, D. (1997) Analysis of cDNA clones encoding sucrose-phosphate synthase in relation to sugar interconversions associated with dehydration in the resurrection plant Craterostigma plantagineum. Hochst. Plant Physiol. 115, 113-121. Kamalay, J.C., and Goldberg, R.B. (1980) Regulation of structural gene expression in tobacco. Cell 19, 935-946. Kamalay, J.C., and Goldberg, R.B. (1984) Organ-specific nuclear RNAs in tobacco. Proc. Natl. Acad. Sci. USA 81, 2801-2805. Koltunow, A.M., Truettner, J., Cox, K.H., Wallroth, M., and Goldberg, R.B. (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2, 1201-1224. Lukowitz, W., Mayer, U., and Jürgens, G. (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin- related KNOLLE gene product. Cell 84, 61-71. Luo, M., Bilodeau, P., Dennis, E.S., Peacock, W.J., and Chaudhury, A. (2000) Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds. Proc. Natl. Acad. Sci. USA 97, 10637-10642. Martin, C., and Gerats, T. (1993) Control of pigment biosynthesis genes during petal development. Plant Cell 5, 1253-1264. Mascarenhas, J.P. (1990) Gene activity during pollen development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 317-338. Mitsuda, N., Takeyasu, K., and Sato, M.H. (2001) Pollen-specific regulation of vacuolar H+-PPase expression by multiple cis-acting elements. Plant Mol. Biol. 46, 185-192. Mu, J.H., Stains, J.P., and Kao, T. (1994) Characterization of a pollen-expressed gene encoding a putative pectin esterase of Petunia inflata. Plant Mol. Biol. 25, 539-544. Nadeau, J.A., Zhang, X.S., Li, J., and O’Neill, S.D. (1996) Ovule development: Identification of stage-specific and tissue-specific cDNAs. Plant Cell 8, 213-239. Ohad, N., Yadegari, R., Margossian, L., Hannon, M., Michaeli, D., Harada, J.J., Goldberg, R.B., and Fischer, R.L. (1999) Mutations in FIE, a WD Polycomb group gene, allow endosperm development without fertilization. Plant Cell 11, 407-415. Porceddu, A., Reale, L., Lanfaloni, L., Moretti, C., Sorbolini, S., Tedeschini, E., Ferranti, F., and Pezzotti, M. (1999) Cloning and expression analysis of a Petunia hybrida flower specific mitotic-like cyclin. FEBS Letters 462, 211-215. Reale, L., Porceddu, A., Lanfaloni, L., Moretti, C., Zenoni, S., Pezzotti, M., Romano, B., and Ferranti, F. (2002) Patterns of cell division and expansion in developing petals of Petunia hybrida. Sex. Plant Reprod. 15, 123-132. Rubinelli, P., Hu, Y., and Ma, H. (1998) Identification, sequence analysis and expression studies of novel anther- specific genes of Arabidopsis thaliana. Plant Mol. Biol. 37, 607-619. Sambrook, J., Fritsch, E.J., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y. bw-cnudde 23-08-2004 14:49 Pagina 72

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Sanders, P.M., Bui, A.Q., Weterings, K., McIntire, K.N., Hsu, Y.-C., Lee, P.Y., Truong, M.T., Beals, T.P., and Goldberg, R.B. (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex. Plant Reprod. 11, 297-322. Schneitz, K., Hülskamp, M., and Pruitt, R.E. (1995) Wild-type ovule development in Arabidopsis thaliana- a light microscope study of cleared whole-mount tissue. Plant J. 7, 731-749. Schneitz, K., Balasumbramanian, S., and Schiefthaler, U. (1998) Organogenesis in plants: the molecular and genetic control of ovule development. Trends Plant Sci. 3, 468-472. Scott, R., Dagless, E., Hodge, R., Paul, W., Soufleri, I., and Draper, J. (1991a) Patterns of gene expression in developing anthers of Brassica napus. Plant Mol. Biol. 17, 195-207. Scott, R., Hodge, R., Paul, W., and Draper, J. (1991b) The molecular biology of anther differentiation. Plant. Sci. 80, 167-191. Tsuchiya, T., Toriyama, K., Nasrallah, M.E., and Ejiri, S. (1992) Isolation of genes abundantly expressed in rice anthers at the microspore stage. Plant Mol. Biol. 20, 1189-1193. Willing, R.P., Bashe, D., and Mascarenhas, J.P. (1988) An analysis of the quantity and diversity of messenger RNAs from pollen and shoots of Zea mays. Theor. Appl. Genet. 75, 751-753. Willing, R.P., and Mascarenhas, J.P. (1984) Analysis of the complexity and diversity of mRNAs from pollen and shoots of Tradescantia. Plant Physiol. 75, 865-868. Wing, R.A., Yamaguchi, J., Larabell, S.K., Ursin, V.M., and McCormick, S. (1990) Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia. Plant Mol. Biol. 14, 17-28. Wood, A.J., Saneoka, H., Rhodes, D., Joly, R.J., and Goldsbrough, P.B. (1996) Betaine aldehyde dehydrogenase in sorghum. Plant Physiol. 110, 1301-1308. Yoshiba, Y., Nanjo, T., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) Stress-responsive and developmental regulation of delta(1)-pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Arabidopsis thaliana. Biochem. Biophys. Res. Commun. 261, 766-772. Zhang, H.Q., and Croes, A.F. (1983) Proline metabolism in pollen: degradation of proline during germination and early tube growth. Planta 159, 46-49. Zhang, H.Q., Croes, A.F., and Linskens, H.F. (1982) Protein syntheiss in germinating pollen of Petunia: role of proline. Planta 154, 199-203. bw-cnudde 23-08-2004 14:49 Pagina 73

Chapter III

Expression analysis during meiosis in Petunia hybrida anthers using cDNA-AFLP-based transcript profiling

Filip Cnudde, Liesbeth Pierson, Hans de Jong1, Marc Zabeau2, Tom Gerats

1 Department of Plant Sciences, Laboratory of Genetics, Dreijenlaan 2, 6703 HA Wageningen 2 Department of Plant Systems Biology, VIB/Ghent University Technologiepark 927, B-9052 Ghent, Belgium

A part of this chapter is submitted to Plant Physiology bw-cnudde 23-08-2004 14:49 Pagina 74

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Abstract

We have developed a strategy to follow gene expression during male meiosis in Petunia hybrida. Combining a cytology-based sampling on developing anthers with cDNA-AFLP transcript profiling, we were able to analyse changes in gene expression during meiosis. Pollen mother cells of the anthers derived from a single bud develop relatively synchronously through meiosis, except for the smallest anther, which therefore was excluded from sampling. Of the four remaining anthers, one was used for the cytological staging of meiotic chromosomes, based on the 4’,6-diamidino-2-phenylindole (DAPI) staining of enzyme-digested spread pollen mother cells. The remaining three anthers were used for RNA-extraction and subsequent cDNA preparation. The transcript profiling experiments focused on the identification of genes with a modulated expression profile during meiosis, while premeiotic archesporial cells and postmeiotic microspores served as a reference. About 8,000 transcript tags, estimated to represent 35% of the total transcriptome, were generated, 475 of which exhibited a modulated gene expression pattern. Among these, we find many components of meiotic processes, indicating that our screening was effective.

Introduction

Meiosis is a special type of cell division that produces haploid gametes from diploid parental cells. The chromosome number is halved during meiosis because a single round of DNA replication is followed by two consecutive rounds of chromosome segregation. Meiosis has been studied extensively at the cytological level using a range of plants as attractive model systems because of their large chromosomes and the abundance of male meiocytes in the anthers. Until recently however, chromosome cytology has been regarded as difficult in the model species Arabidopsis thaliana because of the small size of its chromosomes (Ross et al., 1996). Second, difficulties in purifying stage-specific meiotic cells from tissue have hampered gene expression analysis studies. Cell culture systems, such as the synchronised tobacco BY-2 cultures used for transcriptome analysis during the mitotic cell cycle by Breyne et al. (2002), cannot be used to propagate large numbers of meiotic cells. As a consequence, the molecular and functional analysis of plant meiotic genes is relatively underdeveloped. Most molecular and genetic studies on meiosis have been performed on yeast. The budding yeast Saccharomyces cerevisiae is an ideal model organism because it proceeds into meiosis fairly synchronously upon nutritional starvation, enabling a temporal analysis of meiotic processes. The availability of the full genomic sequence (Goffeau et bw-cnudde 23-08-2004 14:49 Pagina 75

Transcript profiling of male meiosis 75

al., 1997) allowed large-scale gene expression studies. A genome-wide transcriptome analysis during meiosis in S. cerevisiae using DNA microarrays (Primig et al., 2000) led to the identification of 1600 genes (25% of the total number of genes), exhibiting a modulated expression pattern during meiosis. These genes could be grouped in seven clusters with unique expression patterns. Based on these expression data, Rabitsch et al. (2001) carried out a targeted mutagenesis program for a further characterisation of genes required for meiosis. A similar analysis of the transcriptional program of meiosis in Schizosaccharomyces pombe revealed an unexpectedly small shared meiotic transcriptome (Mata et al., 2002), especially at the level of transcriptional regulation. Recently, microarrays have also been used in studies on gene expression profiles during mouse spermatogenesis, which may lead to the identification of genes with potential relevance to mammalian meiosis (Yu et al., 2003; Schultz et al., 2003). In a comparative study, Hwang et al. (2001) performed a computer-based search for homologues of meiosis-induced yeast genes in Caenorhabditis elegans, Drosophila melanogaster, and mammals. The results revealed a high degree of evolutionary conservation between yeast sporulation and meiosis in higher eukaryotes. However, intriguing differences exist, reflecting that distinctive mechanisms govern progression of meiosis in each organism. We employed cDNA-AFLP transcript profiling for the identification of genes that are modulated in their expression during male meiosis in Petunia hybrida. Earlier, we used the same technique to analyse gene expression during gametogenesis in Petunia (Cnudde et al., 2003). This sensitive and reproducible method does not require prior sequence information, it allows the identification of as yet unidentified genes, and it provides quantitative expression profiles (Breyne et al., 2003). The large chromosomes of Petunia allow a straightforward cytological analysis for the selection and staging of the meiotic samples. In contrast to Arabidopsis, where only about 30 pollen mother cells are present in each anther locule (Armstrong et al., 2001), the bigger anthers of Petunia produce each approximately 10.000 relatively synchronously dividing meiocytes. Sufficient mRNA starting material can be obtained from three anthers derived from a single flower bud, allowing a reproducible gene expression analysis by cDNA-AFLP. Direct comparison of 8,000 amplicons yielded 475 candidate cDNA fragments displaying a modulated and/or meiosis-specific gene expression pattern. 293 Fragments were further analysed by direct sequencing. Homologies with diverse classes of known meiosis genes were retrieved, indicating that our screening was basically effective. Further characterisation of these fragments and their functional classification based on literature datasearches is described in Chapter IV. A thorough analysis of the corresponding gene expression patterns is provided in Chapter V. bw-cnudde 23-08-2004 14:49 Pagina 76

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Experimental procedures

Plant material

Petunia hybrida (line W115) plants were grown under standard greenhouse conditions (21°C on average; 16h photoperiod). Immature flower buds ranging in size up to 5 mm in overall length were collected and the five anthers were immediately dissected using a small forceps and a binocular. The smallest anther of each flower bud was discarded from sampling. Of the remaining four anthers, one was placed in Carnoy’s fixative [ethanol-glacial acetic acid (3:1)] and was allowed to fix at room temperature for 12- 18h. The remaining three anthers of one flower bud were put in a 2.2ml eppendorf, frozen in liquid N and stored at –80ºC for subsequent RNA isolation. 2,

Staining of enzyme-digested spread pollen mother cells

For the determination of the meiotic stage, the fixed anther was used for cytological analysis using 4’,6-diamidino-2-phenylindole (DAPI)-staining. Washing steps, enzymatic digestion, spreading on the slide and DAPI-staining were performed as described by Ross et al. (1996). The washing steps and enzyme treatments were carried out in eppendorfs. Digital photographs of DAPI-stained cells were taken with a Leitz Orthoplan microscope (LSM, Wetzlar, Germany) with XBO-150 Watt lamp (Osram GmbH, Munich, Germany) and a CoolSNAP CCD camera (Roper Scientific, USA). The images were enhanced using Adobe Photoshop 5.0 (San Jose, CA).

Total RNA isolation and cDNA synthesis

For each sample total RNA was isolated from three anthers derived from a single flower bud. Tissue homogenisation occurred in 2.2ml eppendorfs in a laboratory mixer mill MM300 bead homogenisation system (Retsch GmbH and Co, Haan, Germany) for 40” at speed 20Hz. These parameters have been optimised in several experiments; longer homogenisation at higher speeds resulted in a partial RNA degradation. Eppendorfs, beads and eppendorf tube holders were cooled beforehand in liquid N2. Immediately after homogenisation RNeasy-buffer was added and total RNA was isolated using the “RNeasy Kit” according to the manufacturer’s instructions (Qiagen). The total RNA was treated with DNase (Promega, Madison, WI) for 30’ at 37ºC and purified by phenol-chloroform-extraction and ethanol precipitation. The concentration bw-cnudde 23-08-2004 14:49 Pagina 77

Transcript profiling of male meiosis 77

of the RNA was determined spectrophotometrically. 3µg of total RNA was used for cDNA synthesis. First and second cDNA strands were synthesised according to standard protocols (Sambrook et al., 1989). The resulting double stranded cDNA was subsequently purified by phenol extraction and ethanol precipitation.

cDNA-AFLP analysis

The cDNA-AFLP-based transcript profiling procedure was performed according to Breyne et al. (2003). Double-stranded cDNA was used for cDNA-AFLP analysis. The restriction enzymes used were BstYI and MseI (New England Biolabs, Beverly, Mass.). For preamplifications, an MseI-primer without selective nucleotides was combined with a BstYI-primer containing a T at the 3’-end. The amplification mixtures obtained were diluted 600-fold and 5µl was used for final selective amplifications following the described procedure (Breyne et al., 2002). BstT- and MseI-primers with two selective nucleotides each were used for the cDNA-AFLP analysis, and all 256 possible primer 33 combinations were performed. The P-ATP-labeled fragments were separated on a 5% polyacrylamide gel. Dried gels were exposed to Kodak Biomax films as well as scanned in a Phospho-Imager (Molecular Dynamics).

Reamplification of AFLP fragments and direct sequencing

Fragments corresponding to differentially expressed transcripts were excised from the dried polyacrylamide gel and suspended in 150µl H O for 3h at room temperature. A 2 small aliquot (5µl) of the eluted DNA suspension was used for the reamplification reaction under the same PCR conditions and using the same primer combination as for selective amplification. Reamplified products were checked on a 1% agarose gel. Sequence information was obtained by direct sequencing of one strand using the selective BstYI-primer as a sequencing primer. As a control, we checked whether the sequences corresponded to the excised fragments in size and selective nucleotides of the MseI-primer. The whole procedure was repeated when no good quality sequence could be obtained for a given fragment. The sequences obtained were compared to those in the GenBank database using BLAST sequence alignments (Altschul et al., 1997). bw-cnudde 23-08-2004 14:49 Pagina 78

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Results

Determination of meiotic stages

For the dissection of the process of meiosis in anthers of Petunia hybrida a reliable staging strategy is a first prerequisite. A developmental staging has been described for Petunia flowers, based on developmental landmarks linked with morphometric data (Porceddu et al., 1999); at the level of meiosis however, no further details were presented. We have found that it was not possible to correlate bud and/or anther size with the cytological stage of male meiosis in Petunia, as there is too much size variation, dependent on environmental and developmental factors. We used bud and anther size as criteria for a rough preselection. During male meiosis, Petunia flower buds are 2- 4mm in size (stage 3 in Porceddu et al., 1999), which is about ten times larger than Arabidopsis flower buds at the same stage (Armstrong and Jones, 2003). The anthers are approximately 1mm in size. Cytological analysis of DAPI-stained enzyme-digested spread pollen mother cells (Ross et al., 1996) proved to be a valuable method for the determination of meiotic stages. Only leptotene stages were sometimes hard to recognise in stained preparations, because of the gradual and subtle changes in the appearance of the chromatin. Fig. 1 shows an overview of the meiotic stages that were determined cytologically.

Figure 1 Overview of the meiotic stages determined by DAPI-staining in microsporocytes of Petunia hybrida. A leptotene, B zygotene, C pachytene, D early diplotene, E late diplotene, F early metaphase I, G metaphase I, H early anaphase I, I anaphase I, J telophase I, K dyad stage, L metaphase II, M telophase II, N tetrad, O end of tetrad (see also appendix) bw-cnudde 23-08-2004 14:49 Pagina 79

Transcript profiling of male meiosis 79

Synchronicity of male meiosis in Petunia hybrida

A second important prerequisite for obtaining representative gene expression profiles with cDNA-AFLP transcript profiling is the homogeneity of the samples. In contrast to female meiosis, pollen mother cells develop rather synchronously through meiosis in most plant species such as Arabidopsis (Ross et al., 1996) and lily (Riggs, 1997). Since in Petunia large amounts of meiotic cells can be examined, a cytological analysis can easily be performed on one anther. Within one anther, we found that the synchronicity of meiotic development is particularly high during the long prophase I. The level of synchronicity decreases in later meiotic stages. Especially during meiosis II often three subsequent meiotic stages could be observed in one anther preparation. In most cases, however, one meiotic stage prevailed (data not shown). In a next step, the five anthers derived from one Petunia flower bud were analysed cytologically. The progression through meiosis occurred synchronously, except for the abaxial anther derived from the stamen alternating with the two big petals at the ventral side of Petunia hybrida flowers. Compared to the two large dorsal stamens and the two intermediate lateral stamens, this stamen is less well developed: it has the shortest filament that carries the smallest anther (85% of the size of the other anthers) (Fig. 2 and Fig. 3). Frequently, meiosis in this smallest anther was not synchronous with the other anthers; most often, meiosis had progressed further in this anther.

Figure 2 Five stamens of a Petunia hybrida flower. The abaxial anther is the smallest and has the shortest filament. Frequently, meiosis in this anther is not synchronous with the four remaining anthers, (see also appendix) bw-cnudde 23-08-2004 14:49 Pagina 80

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dorsal Figure 3 Floral diagram of Petunia hybrida inflorescence axis

sepal

petal

stamen

carpel ventral bract

Sampling strategy

Our sampling strategy is shown in Fig. 4. Flower buds were collected in the greenhouse and the five anthers were dissected. Because of its lack of synchronicity in meiotic progression the smallest anther was discarded. One anther was put in Carnoy’s fixative [ethanol-glacial acetic acid (3:1)] for fixation overnight and subsequent cytological analysis, while the three remaining anthers were frozen in liquid nitrogen and stored at -80ºC. When an interesting meiotic stage was determined with DAPI-staining, the three anthers derived from the same flower bud were selected for total RNA extraction and cDNA synthesis. To ensure that our sampling was as homogenous as possible, pooling samples from different flower buds was avoided. Three Petunia anthers undergoing meiosis yield approximately 3µg of total RNA, a fairly high quantity for such a small amount of tissue (2mg). Since the flower buds corresponding to premeiotic stages were considerably smaller in size, anthers of two flower buds were pooled to obtain this quantity of total RNA. Eight meiotic samples were assembled based on the meiotic stages identified by microscopical analysis of DAPI-stained spread pollen mother cells. Hereby, we focused on prophase I stages to investigate the meiosis-specific processes of synapsis and recombination. Coincidentally, the higher level of synchronicity of prophase I stages is an important advantage for gene expression analysis. Furthermore, two premeiotic samples were pooled from anthers derived from two flower buds each of 1.8 and 2.2mm in size, respectively (stage 2 in Porceddu et al., 1999). Two postmeiotic samples were bw-cnudde 23-08-2004 14:49 Pagina 81

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Figure 4 Overview of our sampling strategy

taken from anthers derived from single flower buds of 4.75 and 5mm, respectively (stage 4 in Porceddu et al., 1999). We confirmed the pre- and postmeiotic nature of these samples by DAPI-staining.

cDNA-AFLP Transcript Profiling

We used the transcript profiling technique, an improved cDNA-AFLP method developed by Breyne et al. (2003), for gene expression analysis. An overview of the protocol is shown in Fig. 5. We used BstT and MseI primers with two additional selective nucleotides each, providing a total of 256 possible primer combinations. We

cDNA 5’poly A 3’ Figure 5 Overview of the cDNA- Biotin AFLP transcript profiling protocol First restriction digestion

3’ end capturing

Second restriction digestion

Adapter ligation

One diagnostic fragment per transcript bw-cnudde 23-08-2004 14:49 Pagina 82

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have chosen to work with +2/+2 primer combinations because of the higher level of sensitivity and the smaller number of generated fragments: around 35 AFLP tags per primer combination, allowing an efficient quantitative analysis using the AFLP- QuantarPro image analysis software. cDNA-AFLP transcript profiling on the samples described above showed consistent gene expression patterns, indicating that the small amount of starting material was sufficient (Fig. 6). While the majority of signals on the polyacrylamide gel was constitutive, cDNA fragments exhibiting a modulated gene expression pattern were well represented. These fragments were excised from gel, reamplified and sequenced. Homologies with known meiotic genes from yeast and Arabidopsis showed that our approach was effective (see Chapter IV) and that it was possible to screen for meiotic genes in our samples, consisting of both meiocytes and often mitotically dividing somatic cells derived from different anther tissues.

Figure 6 cDNA-AFLP transcript profiling during male meiosis, using BstT/Mse +2/+2 primer combinations. Expression levels are compared during 6 stages, which are determined by cytological analysis. Arrows indicate fragmens with a modulated expression pattern during meiosis. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene-metaphase I, 5 dyad stage-telophase II, 6 postmeiotic

Limitations of cytological staging

As shown in Fig. 6, meiosis-specific cDNA fragments appeared characteristically in two consecutive lanes on the polyacrylamide gel. Early meiotic fragments were present from leptotene up to zygotene, while late meiotic fragments were expressed from pachytene up to telophase II (see also Chapter V). The abrupt transition between the zygotene and pachytene stages observed on gel putatively indicated a need for a more refined cytological staging. Therefore, we repeated our transcript profiling experiment on an extended set of 36 samples with partially overlapping, cytologically determined meiotic bw-cnudde 23-08-2004 14:49 Pagina 83

Transcript profiling of male meiosis 83

stages. We could confirm the expression profiles from the first experiment, including the abrupt transition between the zygotene and pachytene stages (data not shown). This suggests that progression through the meiotic cell cycle is accompanied by sudden changes in the transcriptome. In further experiments, intended to cover a significant part of the total transcriptome, the initial number of 12 samples was halved by pooling two consecutive samples in order to reduce the number of gels in our screening. In total, all 256 primer combinations have been performed, generating approximately 8,000 transcript tags. We estimate that 35% of the total Petunia transcriptome has been screened. 475 transcript tags showed a modulated gene expression pattern during meiosis and were selected for further characterisation.

Discussion

Expression analysis during meiosis in plants

We have developed a strategy to follow gene expression during male meiosis in Petunia hybrida. Combining a cytology-based sampling on developing anthers with cDNA- AFLP transcript profiling, we were able to analyse gene expression during meiosis in developing anthers. The abundance of synchronous microsporocytes favours the analysis of male meiosis, while a similar approach to study female meiosis is not apparent due to the paucity and asynchronous behaviour of the megasporocytes in the ovules. Substantial differences may exist at the molecular level between male and female meiosis, as indicated by the identification of several Arabidopsis meiotic mutants that are affected solely in male or female meiosis (see Chapter I, Table 1). Therefore, our results cannot be readily generalised and refer strictly to male meiosis. To analyse male meiosis, we used cytologically-staged anthers that were dissected from a single flower bud. By relying on the inherent synchronicity of meiotic development, our experimental setup allowed us to analyse gene expression in a ‘natural’ situation. In contrast, transcriptome analysis during cell division was performed on a cell suspension culture that was synchronised by the use of a chemical blocking agent, aphidicolin, whereby the induction of stress-related genes cannot be excluded (Breyne et al., 2002). The factors that trigger meiosis are different in plants and yeasts, which is also reflected in the experimental setup and the transcriptional program. Diploid yeast cells undergo sexual differentiation under conditions of nutritional stress, notably nitrogen starvation, triggering the rapid, transient induction of metabolic and stress-related genes bw-cnudde 23-08-2004 14:49 Pagina 84

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after transfer to sporulation medium (Chu et al., 1998). In contrast, meiosis in flowering plants is largely determined within the floral developmental program (Bhatt et al., 2001) and marks the transition from the sporophytic to the gametophytic generation in the life cycle of all plants (Dickinson, 1994). A comparison of the transcriptional programs in yeast and plants is provided in Chapter V.

Cytological analysis to determine meiotic stages in Petunia anthers

The large chromosomes of Petunia and the abundant microsporocyte production allow a straightforward cytological determination of meiotic stages by DAPI staining. However, a difficult issue appears to be how best to define the onset of leptotene. Since the transition from the premeiotic phase to leptotene is gradual and difficult to define precisely simply on the appearance of chromatin in stained preparations, our staging is probably less efficient for these samples. Recently, a precise strategy to mark early leptotene stages was reported by Armstrong et al. (2003) in Arabidopsis, based on the immunolocalisation pattern of a well-known protein involved in synapsis, ASY1. Pollen mother cells develop rather synchronously through meiosis in Arabidopsis (Ross et al., 1996) and lily (Riggs, 1997), in contrast to female meiosis. Using cytological analysis, we observed a reasonable degree of synchrony within anthers and also between anthers in single flower buds. The synchronicity in progression through meiosis is particularly high during prophase I, but decreases later in meiosis. This is probably related to the differences in duration of the meiotic stages. Recently, a meiotic time-course has been established for Arabidopsis pollen mother cells (Armstrong et al., 2003). It showed that leptotene and zygotene/pachytene stages take relatively long, with durations of 6h and 15.3h, respectively, while the remaining meiotic divisions stages are completed very rapidly, within 3h. Therefore, we assume that our samples are more homogeneous in timing in prophase I, while later in meiosis a larger sample heterogeneity cannot be avoided. Fortunately, key meiotic events, such as recombination and synapsis, take place during the well-covered prophase I. Unexpectedly, we had to discard the smallest anther from sampling after frequent observations that it did not progress synchronously through meiosis with the four remaining anthers in a single Petunia flower bud. Often, meiosis had progressed further in this anther. However, this lead does not extend to anthesis, since we could not observe differences in timing of dehiscence. The special nature of the abaxial anther is not uncommon in the family of the Solanaceae. For example, while this anther is weaker developed in Petunia, it is completely aborted in its close relative Salpiglossis (Fries, 1911). bw-cnudde 23-08-2004 14:49 Pagina 85

Transcript profiling of male meiosis 85

cDNA-AFLP transcript profiling

We employed cDNA-AFLP transcript profiling for the identification of genes that are modulated in their expression during male meiosis in Petunia hybrida. To ensure that our sampling was as homogeneous as possible, pooling of anthers derived from different Petunia flower buds was avoided and the smallest anther was excluded from sampling. Since we had to sacrifice one more anther for the cytological staging, only three anthers remained for RNA-extraction and cDNA preparation. These yielded approximately 3µg of total RNA, about 10 times less than the amount of starting material used in a similar transcript profiling experiment during gametogenesis (Cnudde et al., 2003) and 200 times less than used by Primig et al. (2000) in a DNA microarray experiment to study sporulation in yeast. Therefore, the poly(A)+ isolation step was omitted to avoid further RNA losses. Despite the lower RNA amounts, reproducible gene expression patterns were generated by cDNA-AFLP transcript profiling, stressing the high level of sensitivity of this technique. Since complete anthers were used, our sampling was not homogeneous. In fact, apart from the meiocytes in the pollen sac, cells from a variety of anther tissues were sampled, such as the epidermis, the endothecium, the connective tissue and the biologically active tapetum. Often these cells are undergoing mitotic cell divisions, as observed in our DAPI-stained preparations. As a consequence, further analysis is required to investigate whether a specific transcript tag is (specifically) expressed in the meiocytes. This extra spatial information can be provided by in situ hybridisation (see Chapter VI). Exclusive sampling of microsporocytes is technically difficult and cannot be performed by mere anther squashing since, at the initiation of meiosis, the anther is still a compact structure. As an alternative to a further microdissection of the anther, certain mutations can be helpful to increase the homogeneity of the sampled cells and to enrich for meiocytes. For example, the Arabidopsis excess microsporocytes1 (ems1) mutant lacks tapetal cells and produces excess microsporocytes that progress normally through meiosis, but fail to undergo cytokinesis (Zhao et al., 2002). However, such mutations have not yet been identified in Petunia hybrida.

Towards a compendium in Petunia?

By linking expression experiments in the future, a reference database or compendium of expression profiles can be constructed, corresponding to diverse treatments or developmental studies (Hughes et al., 2000). Such a transcriptome map can also be bw-cnudde 23-08-2004 14:49 Pagina 86

86 CHAPTER III

developed based on the expression profiles generated by cDNA-AFLP transcript profiling, whereby a particular transcript tag can be identified by fragment size and primer combination. In Petunia, such an approach could be used to link this experiment with our previous transcript profiling screening for gametogenesis-related genes (see Chapter II). In fact, both experiments are perfectly in line, visualising gene expression in stage 3 anthers (meiosis) and stage 4-5-6-7-8 anthers (gametogenesis) (Porceddu et al., 1999). This is confirmed by the matching expression profiles of fragments that are upregulated postmeiotically (see Chapter V) with anther-specific fragments in the gametogenesis screening (data not shown). Furthermore, a Petunia compendium could be used to enrich our data set for meiocyte-specific transcripts. Therefore, our set of expression profiles modulated during meiosis in anthers could be systematically trimmed of transcripts found in postmeiotic anthers or vegetative floral organs. Notably, transcripts of many crucial meiotic genes in Arabidopsis are also detectable in vegetative tissues, which could prevent them from being selected (Mercier et al., 2003).

Conclusions

In conclusion, we can state that cDNA-AFLP transcript profiling on developing anthers in Petunia hybrida is a valuable method to analyse the meiotic transcriptome in relation to cytologically defined landmarks. Based on their modulated gene expression pattern, candidate meiosis-related AFLP tags can be selected that can be analysed further on the basis of their sequence homology and/or their clustering with known meiotic genes. On a smaller scale, cDNA-AFLP transcript profiling is an excellent tool for gene discovery, generating new candidates for reverse genetics approaches. In a genome-wide screening (e.g. Breyne et al., 2002) transcriptome analysis can be used to create a complete database as a starting point for the unravelment of an entire biological process. bw-cnudde 23-08-2004 14:49 Pagina 87

Transcript profiling of male meiosis 87

References

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W., and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acid Res. 25, 3389- 3402. Armstrong, S.J., Christopher, F., Franklin, H., and Jones, G.H. (2001) Nucleolus-associated telomere clustering and pairing precede meiotic chromosome synapsis in Arabidopsis thaliana. J. Cell Sci. 114, 4207-4217. Armstrong, S.J., and Jones, G.H. (2003) Meiotic cytology and chromosome behaviour in wild-type Arabidopsis thaliana. J. Exp. Botany 54, 1-10. Armstrong, S.J., Franklin, F.C.H., and Jones, G.H. (2003) A meiotic time-course for Arabidopsis thaliana. Sex. Plant Reprod. 16, 141-149. Bhatt, A.M., Canales, C., and Dickinson, H.G. (2001) Plant meiosis: the means to 1N. Trends Plant Sci. 6, 114- 121. Breyne, P., Dreesen, R., Vandepoele, K., De Veylder, L., Van Breusegem, F., Callewaert, L., Rombauts, S., Raes, J., Cannoot, B., Engler, G., Inzé, D., and Zabeau, M. (2002) Transcriptome analysis during cell division in plants. Proc. Natl. Acad. Sci. USA 99, 14825-14830. Breyne, P., Dreesen, R., Cannoot, B., Rombaut, D., Vandepoele, K., Rombauts, S., Vanderhaeghen, R., Inzé, D., and Zabeau, M. (2003) Quantitative cDNA-AFLP analysis for genome-wide expression studies. Mol. Gen. Genomics 269, 173-179. Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown, P.O., and Herskowitz, I. (1998) The transcriptional program of sporulation in budding yeast. Science 282, 699-705. Cnudde, F., Moretti, C., Porceddu, A., Pezzotti, M., and Gerats, T. (2003) Transcript profiling on developing Petunia hybrida floral organs. Sex. Plant Reprod. 16, 77-85. Dickinson (1994) The regulation of alternation of generation in flowering plants. Biol. Rev. 69, 419-422. Fries, R.E. (1911) Die Arten der Gattung Petunia. Kungl. Svenska Vetenskapsakademiens Handlingar 46. Almqvist & Wiksells, Uppsala. Goffeau, A., et al. (1997) The yeast genome directory. Nature 387, (suppl.) 1-105. Hughes, T.R., Marton, M.J., Jones, A.R., Roberts, C.J., Stoughton, R., Armour, C.D., Bennett, H.A., Coffey, E., Dai, H., He, Y.D., Kidd, M.J., King, A.M., Meyer, M.R., Slade, D., Lum, P.Y., Stepaniants, S.B., Shoemaker, D.D., Gachotte, D., Chakraburtty, K., Simon, J., Bard, M., and Friend, S.H. (2000) Functional discovery via a compendium of expression profiles. Cell 102, 109-126. Hwang, S.Y., Oh, B., Knowles, B.B., Solter, D., and Lee, J.-S. (2001) Expression of genes involved in mammalian meiosis during the transition from egg to embryo. Mol. Reprod. Dev. 59, 144-158. Mata, J., and Bähler, J. (2003) Relations between gene expression and gene conservation in fission yeast. Genome Res. 13, 2686-2690. Mata, J., Lyne, R., Burns, G., and Bähler, J. (2002) The transcriptional program of meiosis and sporulation in fission yeast. Nat. Genet. 32, 143-147. Mercier, R., Armstrong, S.J., Horlow, C., Jackson, N.P., Makaroff, C.A., Vezon, D., Pelletier, G., Jones, G.H., and Franklin, F.C.H. (2003) The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis. Development 130, 3309-3318. Porceddu, A., Reale, L., Lanfaloni, L., Moretti, C., Sorbolini, S., Tedeschini, E., Ferranti, F., and Pezzotti, M. (1999) Cloning and expression analysis of a Petunia hybrida flower specific mitotic-like cyclin. FEBS Letters 462, 211-215. Primig, M., Williams, R.M.,Winzeler, E.A., Tevzadze, G.C., Conway, A.R., Hwang, S.Y., Davis, R.W., and Esposito, R.E. (2000) The core meiotic transcriptome in budding yeasts. Nat. Genet. 26, 415-423. Rabitsch, K.P., Toth, A., Galova, M., Schleiffer, A., Schaffner, G., Aigner, E., Rupp, C., Penkner, A.M., Moreno- Borchart, A.C., Primig, M., Esposito, R.E., Klein, F., Knop, M., and Nasmyth, K. (2001) A screen for genes required for meiosis and spore forma Riggs, C.D. (1997) Meiotin-1: the meiosis readiness factor? BioEssays 19, 925-931. bw-cnudde 23-08-2004 14:49 Pagina 88

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Ross, K.J., Fransz, F., and Jones, G.H. (1996) A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosome Res. 4, 507-516. Sambrook, J., Fritsch, E.J., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y. Schultz, N., Hamra, F.K., and Garbers, D.L. (2003) A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc. Natl. Acad. Sci. USA 100, 12201-12206. Yu, Z.R., Guo, R., Ge, Y.H., Ma, J., Guan, J.K., Li, S., Sun, X.D., Xue, S.P., and Han, D.S. (2003) Gene expression profiles in different stages of mouse spermatogenic cells during spermatogenesis. Biol. Reprod. 69, 37-47. Zhao, D.-Z., Wang, G.-F., Speal, B., and Ma, H. (2002) The EXCESS MICROSPOROCYTES1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes Dev. 16, 2021-2031. bw-cnudde 23-08-2004 14:49 Pagina 89

Chapter IV

cDNA-AFLP-based transcript profiling enables the assembly of a comprehensive catalogue of genes modulated during male meiosis

Filip Cnudde, Tom Gerats

A part of this chapter is submitted to Plant Physiology bw-cnudde 23-08-2004 14:49 Pagina 90

90 CHAPTER IV

Abstract

Combining a cytology-based sampling of developing Petunia anthers with cDNA- AFLP-based transcript profiling, we were able to build a comprehensive inventory of genes with a modulated expression pattern during male meiosis. Our collection comprises 475 unique transcript fragments, isolated on the basis of their differential gene expression pattern. 293 Of these (62%) could be characterised by direct sequencing; 90 fragments (30%) showed homology with known genes in a database search. They belong to a wide range of gene classes and include previously characterised meiotic genes in Arabidopsis, yeast and mammals. A functional classification of these tagged genes, based on literature datasearches, demonstrated that many components of meiosis have been encountered, overall covering the complete process of meiosis. Other tags match genes without an allocated function (8%) or share no homology (62%) in a database search either because the tags are too short or because they represent sequences of previously uncharacterised genes. Although incomplete, our data set contributes to the further unraveling of meiosis at a molecular level in plants.

Introduction

Meiosis has been studied extensively at the cytological level using plants as attractive model systems because of their usually large chromosomes and the availability of a large collection of meiotic mutants. In particular, the abundance of male meiocytes in anthers and the relatively prolonged prophase in plants have enabled cytologists to carry out highly detailed studies on chromosome behaviour during meiosis (e.g. Cleland, 1923; Newton and Darlington, 1929). Despite this long history of research in flowering plants, the molecular and functional analysis of meiotic genes is relatively underdeveloped in plants, in contrast to, for example, cell cycle research. Strikingly, the term ‘meiosis’ was not even mentioned in the presentation of the Arabidopsis thaliana genome sequence (Arabidopsis Genome Initiative, 2000). Upon the entry in the functional genomics era, meiotic studies in plants started to benefit from the immense amount of data generated by large-scale gene expression studies on meiosis in yeast. A genome-wide transcriptome analysis during meiosis in Saccharomyces cerevisiae using DNA microarrays (Primig et al., 2000) led to the identification of 1600 genes (25% of the total genome), exhibiting a modulated expression pattern during meiosis. These genes could be grouped in 7 clusters with similar expression patterns. A comparable analysis of the transcriptional program of meiosis was carried out in Schizosaccharomyces pombe (Mata et al., 2002). Using bioinformatics tools, bw-cnudde 23-08-2004 14:49 Pagina 91

Homologies 91

homologues of sporulation-induced yeast genes were searched in other eukaryotes (Hwang et al., 2001), often revealing intriguing differences in the meiotic transcriptome. The combination of the flood of genomics data from yeast research with the availability of the full genomic sequence and large mutant collections of Arabidopsis is a major reason for the growing interest in molecular studies on meiosis in Arabidopsis. Today, the scientific input in the area of meiotic research in plants is rapidly expanding and the stream of recent publications is probably the best example for the high level of competitiveness in this dynamic field. In plants, almost all meiotic genes cloned so far have been isolated from Arabidopsis thaliana via insertion tagging. Several mutants, such as AtMRE11 (Bundock et al., 2002) AtRAD51, AtDMC1 (Doutriaux et al., 1998), and AtRAD50 (Gallego et al., 2001) were the result of a reverse genetics strategy, based on sequence information of known meiotic genes in yeast. However, for most yeast meiotic genes no Arabidopsis sequences with significant similarity can be found. Therefore, the number of target genes for reverse genetics is relatively limited (Mercier et al., 2001). We employed cDNA-AFLP transcript profiling for the identification of genes that are modulated in their expression during male meiosis in Petunia hybrida. Earlier, we used the same technique to analyse gene expression during gametogenesis in Petunia (Cnudde et al., 2003). This chapter presents the results of our screening: a comprehensive collection of meiosis-related gene fragments in plants. These tags were characterised by sequencing and classified according to their estimated functions, based on extensive literature data, often derived from mammalian research. In this way, a basis to unravel the basic mechanisms of meiosis in plants is provided.

Experimental procedures

cDNA-AFLP transcript profiling to analyse gene expression during male meiosis

We have used cDNA-AFLP transcript profiling to analyse changes in gene expression during male meiosis in Petunia hybrida. Our sampling strategy, based on a cytological determination of the meiotic stages, is described in the experimental procedures of Chapter III. The cDNA-AFLP-based transcript profiling procedure is also explained in Chapter III. Protocols for the quantitative measurement of the expression profiles and cluster analysis are provided in Chapter V. bw-cnudde 23-08-2004 14:49 Pagina 92

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Reamplification of AFLP fragments and direct sequencing

Fragments corresponding to differentially expressed transcripts were excised from the dried polyacrylamide gel and suspended in 150µl H O for 3h at room temperature. A 2 small aliquot (5µl) of the eluted DNA suspension was used for the reamplification reaction under the same PCR conditions and using the same primer combination as for selective amplification. Reamplified products were checked on a 1% agarose gel. Sequence information was obtained by direct sequencing of one strand using the selective BstYI-primer as a sequencing primer. As a control, we checked whether the sequences corresponded to the excised fragments in size and selective nucleotides of the MseI-primer. The whole procedure was repeated when no good quality sequence could be obtained for a given fragment.

Homology search and functional classification

After the removal of the adapter sequences on both edges of the raw DNA sequence, each tag was subjected to a homology search for further characterisation. Therefore, the sequences obtained were compared with those in the publicly available GenBank database using BLAST sequence alignments (Altschul et al., 1997). More precisely, the nucleotide sequence was compared with those present in DNA sequence databases (BLASTN) and with a translated sequence databases in 6 frames (TBLASTX). The Expect-value (E) was used as a parameter to measure the level of homology. Homologies were considered significant when the E-value was not greater than 0.001. When a homology was obtained with an unknown sequence, this sequence was in turn subjected to a BLAST search. The functional classification of the tagged genes was based on literature data. Information obtained by gene expression studies and mutant analyses on homologous genes in yeast, other plants and mammals, proved to be particularly useful to investigate a possible link with meiotic mechanisms. In doing so, we used diverse information resources, such as GOOGLE (http://www.google.com) and the NCBI PubMed database (http://www.ncbi.nlm.nih.gov). bw-cnudde 23-08-2004 14:49 Pagina 93

Homologies 93

Results

The majority of the sequences shows no significant homology

In total, 475 cDNA-AFLP fragments were selected for further characterisation on the basis of their modulated gene expression pattern during male meiosis in Petunia hybrida. All fragments were isolated from the dried polyacrylamide gel and were successfully reamplified by PCR. Direct sequencing of PCR products gave sequence information for 293 cDNA fragments (62%). For the remaining transcript tags no clear sequence information was obtained, probably due to the occurrence of co-migrating fragments in the sample. This success rate for direct sequencing is comparable to the ones obtained in earlier transcript profiling experiments: 55% (Cnudde et al., 2003) and 65% (Breyne et al., 2003), despite the additional selective nucleotide used in our experiments. In theory, the use of a cDNA-AFLP primer containing an additional selective nucleotide reduces the complexity of the amplification mixture 4-fold and will lead to less co-migrating fragments. As an alternative, PCR products can be cloned and individual colonies can be sequenced (Breyne et al., 2002). However, since we never aimed for a complete, genome-wide screening, we did not pursue characterising fragments that could not be directly sequenced. The sequences obtained were compared to those present in the GenBank database using the BLAST program (Altschul et al., 1997); 90 fragments (30%) showed a significant homology (maximum E-value 0.001) to genes with known function, while another 25 tags (8%) match genes without an allocated function (ESTs or putative proteins). The remaining 178 fragments (62%) either represent sequences of previously uncharacterised genes, or share no homology. These figures are comparable to earlier cDNA-AFLP analyses in Petunia (Cnudde et al., 2003) and Arabidopsis (Breyne et al., 2003). This could be a consequence of the fact that the produced AFLP tags are too short. Fig. 1 shows a clear correlation between fragment size and the chance to find a significant homology in the database. Furthermore, a clear difference in average length can be observed between the fragments displaying a significant homology (205bp) and the ones without significant homology (143bp) in a database search. The overall average length of the 475 isolated fragments is 168bp. Another explanation might be that these tags are derived from the 3’-end region of the transcripts, which might be too diverged to identify homologous sequences. However, we observed that the majority of the tags at least partially reside within the protein coding regions, in agreement with the in silico analysis of AFLP fragments performed in Arabidopsis (Dreesen, 2002). An overview of all transcript tags with significant homologies is presented in Table 1. bw-cnudde 23-08-2004 14:49 Pagina 94

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Table 1 Relevant homologies of sequences of AFLP fragments to sequences in the database

AFLP Length Expression Homologyb BLAST tag (bp) patterna score

1 268 2 Unknown protein – (AB026643) 2e-21 4 351 3-4-5,+ Unknown protein – (AC007508.6) 3e-24 9 143 1-2-3 Putative cytochrome P450 – (AC005315) 4e-5 11 88 3-4,+ Oxygen-evolving enhancer protein 3 – 8e-4 (AB043962) 12 212 2-3 Meiotic recombination protein DMC1-homolog; RecA/Rad51/DMC1- 1e-23 like protein – (U66836) 14 168 2-3,+ Transketolase 1 chloroplast - (Y15781) 4e-62 16 343 1-2-3,+ Hypersensitive induced response protein - (AF236374) 1e-54 17 290 2-3,+ dTDP-D-glucose-4,6-dehydratase – (AC024260) 2e-44 18 350 3-4-5 Hypothetical regulatory protein – (AL161542) 9e-19 19 204 2,3 60S ribosomal protein L10 - (AB001891) 5e-59 21 296 1-2-3,+ Mitotic checkpoint protein – (AP000417) 2e-46 BUB3 kinetochore protein - (AF081496) 7e-28 22 226 2-3-4-5,+ Putative protein kinase - (AC003974) 0.005 23 197 1-2-3 Methionine synthase – (AF082893) 3e-63 25 295 4-5,+ Unknown protein – (AY035044) 3e-21 Ran-binding protein 5 – (AF294327) 5e-10 30 161 1-2-3 Dolichyl-di-phospho-oligosaccharide-protein glycotransferase-like - 1e-19 (AB018119) 33 191 3-4-5 Ribosomal protein L29 – (AB042860) 0.009 36 140 1-2-3 Putative phosphatidylinositol-4-phosphate-5-kinase - 0.014 (AF019380) 37 250 1-2-3 Ubiquitin – (U84968) 4e-27 40 130 1-2-3 Trehalose-6-phosphate-synthase – (AF370287) 9e-8 44 173 1-2-3 Cytochrome-b5 reductase-like protein – (NC_003076) 3e-19 46 217 2-3,+ RecA/Rad51/DMC1-homolog - (U66836) 5e-23 47 125 2-3 Protein Phosphatase 2C – 9e-6 (AB083482) 48 110 2-3 Fructose 1,6 biphosphatase-precursor – 7e-25 (AF134051) 51 290 1-2-3 Ran-binding protein 5 - (AF294327) 2e-09 54 119 1-2-3,+ Translation initiation factor - (AF347653) 3e-41 56 339 2-3 RecA/Rad51/DMC1-like protein - (U66836) 3e-24 59 195 2-3,+ Unknown protein - (AB023046) 6e-28 60 202 2-3-4-5 Hypothetical protein - (AL138659) 8e-7 homology with : (6e-44) putative leucine-rich repeat disease resistance protein - (AC007267) 66 119 4-5,+ Putative protein , contains F-box – (AL132969) 9e-7 71 395 4-5-6,+ 4-coumarate-CoA ligase 2 - Arabidopsis (AC011000) 2e-22 76 129 2-3-4-5- Meiotic serine protease – (AF181496) 1e-31 6,+ 85 148 1,2,3,+ Mitochondrial nad5 gene for NADH dehydrogenase subunit 5 - 1e-34 (X600491) 89 161 1-2-3 Dolichyl-di-phospho-oligosaccharide protein glycotransferase 1e-16 – (AY034944) 90 218 1-2-3,+ 40S ribosomal protein S8 - (U64436) 1e-26 91 256 1-2-3 Arginine decarboxylase 1 - (AF127240) 1e-61 93 192 1-2-3 Unknown protein – (AC007945) 2e-10 98 93 1-2-3,+ Putative myo-inositol-1-phosphatase (INS-1P) – 2e-23 (AY004246) 99 367 1-2,+ Elongation factor–1 alpha - (AB019427) 8e-85 106 170 1-2-3,+ Ubiquitin conjugating enzyme - (X73419) 2e-46 bw-cnudde 23-08-2004 14:49 Pagina 95

Homologies 95

AFLP Length Expression Homologyb BLAST tag (bp) patterna score

111 172 1-2-3,+ Lipoamide dehydrogenase - (AF228638) 1e-10 114 70 1-2-3,+ Cr14 –Cytochrome c reductase 14kDa protein – 3e-9 (X79276) 115 133 1-2-3,+ Outer envelope membrane protein homolog – (T12975) 1e-4 121 254 1-2-3,+ Ubiquitin conjugating enzyme - (AF034946) 3e-43 122 131 4-5,+ Unknown protein - (AC008153) 9e-5 139 371 1-2-3 Chloroplast rbcL gene for ribulose biphosphate 4e-61 carboxylase/oxygenase large subunit – (X04976) 141 323 4-5-6,+ Catalase - (Z36977) 3e-43 172 124 3-4-5 Thiol protease homolog P21 - (U31094) 3e-41 173 123 3-4-5 Thiol protease homolog P21 - (U31094) 8e-39 183 116 1-2-3,+ Small nuclear ribonucleoprotein polypeptide F (snrpF) – 9e-8 (AK008240) 184 114 1-2-3 tRNA isopentenyl transferase – (AF109376) 5e-7 203 309 1-2,+ Meiotic asynaptic mutant – (AF157556) 1e-13 204 306 1-2 Meiotic asynaptic mutant – (AF157556) 6e-13 205 200 1-2-3-4-5 Mitochondrial gene for NADH dehydrogenase subunit 3 (nad3) and 7e-22 ribosomal protein S12 – (X84008) 206 171 4-5,+ Photoperiod responsive protein - (AJ306423) 5e-8 homology with: receptor-like kinase gene – (AB026638) 209 156 1-2-3,+ Ribosomal protein L19 – (Z31720) 6e-28 223 141 1-2-3,+ Strong similarity to histone 2B-protein – (AC007583) 7e-9 227 107 1-2-3-4- Putative cytosolic NADP-malic enzyme – 2e-11 5-6,+ (AF288920) 228 108 3-4-5-6,+ Putative cytosolic NADP-malic enzyme – 2e-8 (AF288920) 237 337 2-3 Histone H2B protein – (AC007583) 3e-41 242 125 2-3 Unknown protein – (AB015469) 8e-9 245 380 1-2-3 Poly(A)- specific ribonuclease (PARN) - (NP_175983) 9e-7 246 343 2-3,+ Histone H2B – (AL162459) 3e-23 251 378 4-5,+ Hydroxy-methylglutaryl coenzyme A synthase – 3e-17 (AF396829) 253 117 1-2-3,+ Kinesin-like protein – (AL035679/AL161594) 0.001 256 267 4-5,+ Malate dehydrogenase – (AF149413) 1e-10 257 209 4-5-6 Putative cytosolic NADP-malic enzyme – 3e-13 (AF288920) 258 228 1-2-3 40S ribosomal protein S19 - (AB006696) 5e-16 261 107 1-2-3,+ Mitochondrial DNA for orf224 - (X78038) 2e-14 265 64 1-2-3 UFO-binding protein (UIP2) - (U97021) 2e-9 266 51 4-5,+ Zinc finger protein – (XM 040906.1) 0.025 271 64 4-5 Putative elicitor response protein - (AC051630) 0.002 272 335 1-2-3 t-complex polypeptide 1-homolog - (D11351) 1e-10 279 136 2-3-4-5 Flavonoid 3’ hydroxylase (Ht1) - (AF155332) 8e-4 280 111 1-2-3 UV-damaged DNA binding factor-like protein – 8e-7 (AL161503) 282 75 4-5 cAMP-dependent protein kinase catalytic subunit alpha – 0.003 (XLA413218) 285 53 1-2-3,+ UFO-binding protein UIP - (U97021) 3e-8 287 95 3-4-5-6,+ Putative Ruv DNA-helicase - (AJ276264) 0.020 cancer-testis antigen, meiosis-related (MAGEE1) – 6e-5 (XM 48421.1) 294 254 1-2-3,+ Yippee-like protein – (AC009326) 3e-30 295 205 2-3-4-5,+ Matrix-associated, actin-dependent regulator of chromatin SWI/SNP- 78 (NP 003070.1) 296 145 3-4-5 Unknown protein - (NP 189301) 5e-9 298 118 4-5 Thiol protease homolog P21 - (U31094) 2e-52 304 299 1-2-3-4,+ RAc3 gene for actin – (X15862) 4e-9 bw-cnudde 23-08-2004 14:49 Pagina 96

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AFLP Length Expression Homologyb BLAST tag (bp) patterna score

309 397 1-2-3,+ Putative protein – (NP 197011) 7e-31 homology with DNA-directed RNA-polymerase 311 81 1-2-3 Hypothetical protein – (AC069251) 9e-7 homology with: DAXD12/DEAD-like helicase superfamily, BRCA1-interacting protein, helicase-like 315 106 4-5 IMS1- (AF327647) 0.016 317 353 1-2-3 Similar to N-ethyl maleimide-sensitive fusion protein, contains 7e-5 similarity to ATPases - (AF128393) 318 133 1-2-3 Putative protein - (NP 191365) 3e-16 341 305 2-3,+ Glycine-rich protein - (U74325) 5e-27 342 195 2-3,+ Serine/threonine-protein kinase Bub1, checkpoint-associated-like 2e-16 protein - (BAC79595) 343 177 2-3,+ Aspartyl aminopeptidase - (AF005051) 5e-4 348 307 2-3-4-5-6 Glycine-rich protein – (U74325)2e-29 356 274 3-4-5 Genomic clone – (AB0268616), 7e-4 homology with integral membrane protein/nodulin 358 173 4-5 Hypothetical protein – (AC061957) 1e-14 363 152 4-5-6 Putative integral membrane protein - (AC006284) 2e-9 373 213 1-2-3 Putative acyl-CoA synthetase – (AC006951)1e-14 376 150 4-5-6 Putative integral membrane protein - (AC006284) 7e-8 384 138 4-5 Vacuolar invertase – (Z12027)4e-7 390 214 4-5 Style development-specific protein 9612 precursor; pectate lyase - 3e-20 (X55193) 393 222 3-5,+ Differentially expressed osmotic protein (ODE2) – 3e-9 (AF169204) Auxin response factor 4 - (AAD01512) 1e-5 397 146 4-5,+ mRNA for enolase - (X58108) 1e-32 399 358 2-3,+ Non-photosynthetic ferredoxin - (AB038037) 6e-28 407 272 4-5,+ Putative C3HC4-type RING zinc finger protein – 9e-15 (U93215) 412 75 4-5 Genomic clone – (AC015911.7) 5e-4 414 150 2-3,+ Histone H2A – (AL138656) 3e-14 428 139 2-3 Pyruvate dehydrogenase – (AF209924)3e-20 430 245 3-4-5,+ ADP-ribosylation factor – (AC009398) 2e-10 434 130 3-4-5,+ DNA-damage-repair/toleration protein DRT100 precursor, RecA- 1e-4 homolog - (X66482) 440 288 4-5 Unknown protein – (AC069273) 2e-26 445 334 1-2-3,+ Ripening regulated protein DDTFR8 – 5e-9 (AF204783); homology with : (6e-37) p23 - (AF153128), 446 283 4-5-6,+ Catalase (cat3 gene) - (Z36977) 3e-34 449 141 1-2-3 Putative 40S ribosomal protein S12 - (U19940) 1e-7 450 213 2-3-4-5 Putative protein - (AB016893) 8e-11 homology with testis-specific transcription elongation factor (TFIIS) – (XM 031297) (0.28) Dst1p (DNA Strand Transferase 1), meiotic DNA recombination factor – (NC 001139) 452 81 4-5 Unknown protein – (AC012193) 2e-5 461 119 2-3-4-5 Mitochondrial aldehyde dehydrogenase – 1e-9 (Y09876) 465 417 1-2-3 Histone h2B-3 - (AJ224932) 1e-40 466 414 1-2-3,+ Leucine-rich repeat protein LRP - (X95269) 4e-39 470 102 2-3-4-5 Extensin-like protein - (AF104327) 8e-4 bw-cnudde 23-08-2004 14:49 Pagina 97

Homologies 97

Figure 1 Correlation between fragment size and the chance to find a significant homology in the databases

Homologies with diverse classes of known meiotic genes indicate that our screening is effective

Homologies with known meiotic genes from plants, yeast and mammals showed that our screening was effective. This is particularly true for the retrieval at an early stage in our screening of cDNA tags homologous to well-studied Arabidopsis genes involved in meiosis-specific processes, such as meiotic recombination (DMC1) and synapsis (ASY1) (see further). Both tags have been identified on the basis of their modulated gene expression pattern, demonstrating that our cDNA-AFLP approach can be used successfully to screen for Petunia genes involved in male meiosis.

Table 2 Functional classification of transcript tags Only fragments with a significant level of homology (E-value <0,001) were classified. bw-cnudde 23-08-2004 14:49 Pagina 98

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Overview of the different functional groups of isolated gene fragments that are modulated during male meiosis

In a next step, the successfully sequenced transcript tags were classified according to their estimated functions, based on extensive literature datasearches. An overview is provided in Table 2. The related expression profiles of the tags presented here will be discussed thoroughly in Chapter V.

DNA replication and modification

Extensive and programmed changes occur in chromatin structure and gene expression during meiosis. Despite significant progress in our understanding of molecular and biochemical aspects of chromatin remodeling in yeast in animal systems, very little information exists in plants. In early yeast experiments, it has been shown that histone genes are most abundantly expressed during premeiotic DNA synthesis, where they are responsible for the chromatin organisation of the freshly duplicated DNA, followed by a gradual decline in transcript levels up to sporulation (Kaback and Feldberg, 1985). In a transcriptome analysis of the mitotic cell cycle in tobacco BY2 cells, transcripts of almost 50 different histone genes were found to accumulate specifically during S-phase of the mitotic cell cycle in tobacco BY2 cells (Breyne et al., 2002). In our study, high levels of histone H2A (M414) and H2B (M223, M237, M246 and M465) gene expression were also found during leptotene and zygotene, in agreement with their proposed role in altering the chromatin structure during DNA repair and homologous recombination (Shen et al., 2000). The notion that individual histones play specific roles in DNA damage repair is further supported by an Arabidopsis histone H2A mutant that is deficient in T-DNA integration (Mysore et al., 2000) and by the observation that double strand breaks (DSBs) in mammalian somatic and meiotic cells induce phosphorylation of H2AX, a member of the histone H2A family (Rogakou et al., 1998). This phosphorylation occurs rapidly in regions surrounding the damaged sites, suggesting a disruption of the chromatin structure to facilitate DNA repair, by directly altering histone-DNA interactions or by recruiting chromatin remodeling factors (Rogakou et al., 1998; Paull et al., 2000). Antibodies recognising this specific histone modification at DSB sites have been used successfully by Mahadevaiah et al. (2001) and Jang et al. (2003) in time-course studies focusing on DSB and synaptonemal complex (SC) formation in mouse and Drosophila, respectively. Recently, it has been shown that in S. cerevisiae H2B histones are also involved in the repair of DNA damage (Martini et al., 2002). bw-cnudde 23-08-2004 14:49 Pagina 99

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Histones may, however, fulfill a more specific role in transcriptional regulation during meiosis than anticipated: Hanlon et al. (2003) showed that depletion of the H2A-H2B histone dimer causes major defects in the sporulation pathway in yeast by a specific disruption of the expression of meiotic regulatory genes, while no dramatic changes in chromatin structure could be observed. Also other chromatin remodeling factors were found, such as a SWI/SNF-related, matrix-associated regulator of chromatin (M295), associating with actin (M304) and belonging to the same cluster. SWI/SNF is a large, evolutionarily conserved multiprotein complex involved in many DNA metabolic processes (such as transcription, repair and recombination) through the alteration of chromatin architecture (Wang et al., 1996). Recently, Wagner and Meyerowitz (2002) identified a novel SWI/SNF ATPase homologue that controls ovule development in Arabidopsis. Furthermore, weak homologies were found with a variety of nucleic acid-modifying enzymes, such as ATP- dependent helicases (M222, M230) and topoisomerases (M283, M319). A cDNA-AFLP fragment with homology to a mammalian small nuclear ribonucleoprotein F (snrpF) (M183) was expressed specifically during meiotic prophase I. These proteins act as epigenetic regulators and influence the accessibility of chromosomal regions by modulating the interaction between histones and other proteins (Stern and Hotta, 1984). Finally, a tag from a methionine synthase gene (M23), precursor for S-adenosyl-L-methionine synthase, involved in DNA modification, was found in the histone cluster.

Proteolysis

Protein degradation is an important aspect in the regulation of the cell cycle, both in mitotis and meiosis. The proteolytic machinery responsible for the timely destruction of some of the key regulatory proteins is conserved among all eukaryotes and is mediated by ubiquitination. Genetic and biochemical studies have shown that the ubiquitin pathway is involved in regulation of gene expression, cell cycle progression (Peters, 1998; Zachariae and Nasmyth, 1999), chromatin remodeling (Baarends et al., 1999), and diverse cellular processes (Ciechanover, 1994). However, there is a growing interest in the specific roles of ubiquitin in reproductive processes in mammalian research (Bebington et al., 2001). In our experiments we identified several tags encoding proteins involved in ubiquitin- mediated proteolysis that were induced during early prophase I, such as ubiquitin (M37) and ubiquitin conjugating enzymes or UBCs (M106, M121). Several components of the SCF E3 ubiquitin ligase complex (named after its main components: Skp1, cullin, and an bw-cnudde 23-08-2004 14:49 Pagina 100

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F-box protein) were also retrieved: two F-box proteins (M1 and M66), known to confer substrate specificity by recruiting a particular target to the core ubiquitination machinery, and two tags homologous to Arabidopsis SKP1 (M265 and M285), encoding an SCF subunit that stabilises the complex. Guardavaccaro et al. (2003) showed that the mammalian F-box protein β-Trp1 is important for the control of meiotic and mitotic progression; inactivation of this gene leads to reduced fertility in mouse. In Arabidopsis, F-box proteins are encoded by a large gene family (Kuroda et al., 2002) that is known to be involved in diverse cellular processes, including cell division (Dreesen, 2002) and transcriptional regulation. Undoubtedly, one of the best characterised F-box proteins in plants is UNUSUAL FLORAL ORGANS (UFO) of Arabidopsis, which is required for normal patterning and growth in the floral meristem and binds to Skp1 through a direct interaction with the F-box motif (Samach et al., 1999; Zhao et al., 2001). For this reason, Skp1-homologues are in plant databases also often referred to as UFO-interacting proteins (UIPs) (M265 and M285). Yeast and human SKP1 genes have been found to regulate the mitotic cell cycle (Hoyt, 1997). For example, Skp1 is associated with the kinetochore at M-phase and mediates the degradation of another kinetochore component, allowing sister chromatids to separate (Kaplan et al., 1997). However, the first convincing evidence of the importance of Skp1 for meiosis came from plants. The ask1-1 mutant, carrying a transposon insertion in the ASK1 gene, a homologue of yeast SKP1, was one of the earliest meiotic mutants that were characterised at the molecular level in Arabidopsis (Yang et al., 1999). It is defective in homologue separation in male meiosis I; furthermore, sister chromatid separation at meiosis II seems to be abnormal, suggesting that ASK1 mediates a proteolytic event required for the removal of sister chromatid cohesion along the arms at anaphase I in Arabidopsis male meiosis. Yet other genes involved in protein degradation were differentially expressed during meiosis, such as an AFLP fragment homologous to an aspartyl aminopeptidase (M343) and three tags homologous to a Petunia thiol protease (M172, M173, M298), expressed late in meiosis. They may have functions in common metabolic processes in the meiotic cell. Finally, homology was found with a tomato meiotic serine protease (TMP) gene (M76). TMP transcripts have been detected only in anthers undergoing meiosis, and TMP accumulates as meiotic development proceeds (Riggs et al., 2001), an expression profile that parallels that found for Petunia M76. TMP is the tomato orthologue of LIM9, a serine proteinase from Lilium longiflorum that is synthesised primarily in the tapetum and is secreted into the anther locule during pollen maturation (Taylor et al., 1997). Several proteinases are present at different stages of anther development (DeGuzman and Riggs, 2000; Cnudde et al., 2003). The enhanced proteinase activity can be correlated with morphological and biochemical events of late microsporogenesis that require bw-cnudde 23-08-2004 14:49 Pagina 101

Homologies 101

proteolytic enzymes. The most dramatic of these is the programmed cell death of the tapetal cells and the anther wall cells, which precedes dehiscence. Proteinases could play a general role in the breakdown of the tissues and possibly more specific roles in cleaving specific proteins during pollen maturation (DeGuzman and Riggs, 2000).

Signal transduction

Given the complexity and the multitude of mechanisms involved in progression and control of the meiotic cell cycle, it is not surprising that we identified a relatively large collection of signal transduction components in our screening. Two cDNA-AFLP fragments with varying levels of homology to a protein phosphatase 2C-encoding gene (M47 and M128) clustered closely together and showed peaks in gene expression at the initiation and the end of meiosis. Furthermore, homologies with Arabidopsis protein kinases and receptor-like kinases were found (e.g. M22, M276). In most instances, however, the level of homology was low and a high number of putative or hypothetical protein kinases were encountered in our BLAST database search. This could reflect a backlog in the molecular analysis of signal transduction pathways in plants, which may be caused by their specific nature and low level of expression. Another explanation could be the lower level of evolutionary conservation compared to structural proteins: only certain domains might be conserved. However, several of these less-than-convincing homologies indicating a function in signal transduction were protein kinases involved in human testis-development that are often denoted as testis-specific in the database (M231, M243 and M382). Taken together, these recurrent homologies suggest a weak sequence conservation at the individual gene level, although the overall process apparently has been conserved. A significant homology was found with a phosphatidylinositol-4-phosphate-5- kinase gene of Arabidopsis (M36) and a myo-inositol-1-phosphatase gene of tomato (M98). Several phosphorylated forms of phosphatidylinositol, together with phospholipids, have been proposed to play a role in eukaryotic signal transduction pathways (Divecha and Irvine, 1995; Meijer and Munnik, 2003). In S. cerevisiae, Zheng and Schreiber (1997) demonstrated that phosphatidylinositol kinases are required for signaling pathways leading to regulated protein translation during meiosis, while inositol has been shown to be specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe (Voicu et al., 2002). In Arabidopsis, a well-characterised meiotic protein kinase is AtATM, which belongs to a family of conserved eukaryotic proteins with a C-terminal phosphatidylinositol 3-kinase domain (Garcia et al., 2003). AtATM is a homologue of the human ATM gene. If mutated, ATM causes ataxia bw-cnudde 23-08-2004 14:49 Pagina 102

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telangiectasia, a pleiotropic chromosome stability disorder that causes a neurodegenerative childhood disease (Savitsky et al., 1995). In Arabidopsis, AtATM is involved in cell cycle control and plays a central role in the perception and response to DNA damage, particularly DNA double strand breaks. We also identified gene tags with a possible function in DNA damage response: homologies with Arabidopsis proteins, such as a hypersensitive induced response protein (M16), a putative receptor-like protein kinase involved in disease resistance (M60), a tomato leucine-rich repeat protein (M466), and a putative elicitor response protein (M271) suggest a role in the response to both pathogen stress-induced and developmentally programmed DNA damage. Similarly, a cDNA-AFLP fragment homologous to a photoperiod responsive protein of potato (M206) is induced in late meiosis, suggesting a broader function than a response to light stimuli. Crosstalk between signaling pathways in meiosis has been reported earlier by Honigberg and Purnapatre (2003). They demonstrated that, in yeast, signaling pathways in control of meiotic initiation are integrated into networks, with signaling enzymes regulated by more than one signal acting as nodes in these networks. This does not exclude the possibility that light itself has a direct influence on the meiotic process. It can be hypothesised that a similar regulatory mechanism is used as recently described for the CONSTANS protein involved in photoperiodic flowering in Arabidopsis (Valverde et al., 2004). Plant breeders know by experience that environmental factors can have a considerable impact on the timing of meiosis. Finally, two homologues of known mitotic checkpoint control proteins have been identified: an Arabidopsis checkpoint protein homologous to a human kinetochore protein termed Bub3 (M21) and Bub1, a serine/threonine protein kinase from Oryza sativa (M342). Both Bub1 and Bub3 have been identified by genetic screens in budding yeast as essential components of the mitotic and meiotic spindle checkpoint machinery (Amon, 1999; Yamaguchi et al., 2003). Subsequently, homologues have been identified in higher eukaryotes showing that the spindle checkpoint pathway is highly conserved. When treated with a drug that depolymerises microtubules, Bub mutants (budding uninhibited by benzimidazole) fail to undergo arrest in mitosis and enter anaphase without a functional spindle, leading to aneuploidy and rapid death (Hoyt et al., 1991). In our experiment the expression patterns of both gene tags show an induction during early prophase, supporting a similar function in Petunia hybrida. Remarkably, while high levels of homology have been found with Bub1 genes from organisms as diverse as rice, Drosophila and Schizosaccharomyces pombe, no obvious Bub1 orthologue has yet been identified in Arabidopsis. Since it seems unlikely that Arabidopsis lacks a Bub1 homologue, it might be localised in one of the yet unsequenced regions of the Arabidopsis genome; a Southern blotting experiment could provide more conclusive evidence. bw-cnudde 23-08-2004 14:49 Pagina 103

Homologies 103

Bub1 and Bub3 proteins have been shown to form an attachment-sensitive complex in budding yeast and mammalian cells (Roberts et al., 1994; Taylor et al., 1998). It localises to unattached kinetochores during prophase and promethaphase of mitosis (Taylor and McKeon, 1997) and is lost as kinetochores become properly attached to spindle microtubules (Amon, 1999). Bernard et al. (1998, 2001) showed that, in fission yeast, Bub1 is not only associated with the kinetochores during mitosis, but also during meiosis, where it plays a direct role in ensuring reductional segregation during meiosis I. Recently, Kitagawa et al. (2003) reported biochemical evidence that Bub1 associates with centromere DNA via Skp1 and that Skp1-Bub1 interaction is required by the spindle checkpoint pathway for detecting lack of sufficient tension at kinetochores. Furthermore, Bub1 plays a role in maintaining chromosomal stability. Mutations in human homologues of Bub1 disrupt the spindle checkpoint and have been associated with some types of cancer (Cahill et al., 1998; Langerod et al., 2003), consistent with a link between aneuploidy and cancer.

Cytoskeletal assembly and organisation

A cDNA-AFLP fragment that was induced premeiotically and during early prophase I showed homology at the DNA level with the 3’UTR of a tomato gene encoding a ripening regulated protein (M445), which is homologous to p23, a highly conserved tubulin-binding protein. It has been shown earlier in pea (Woo and Hawes, 1997) and mouse (Gachet et al., 1999) that p23 is upregulated during mitosis and that the p23 protein transiently associates with microtubules during the cell cycle. In S. cerevisiae, p23 interacts with Skp1, a component of the SCF ubiquitin-conjugating complex. Russel et al. (1999) demonstrated that the p23-Skp1 heterodimer is an essential component of CBF3, a centromere-binding complex that initiates kinetochore formation in mitotically dividing yeast cells. Interestingly, in our experiment the AFLP fragment for p23 (M445) and a Skp1- homologue (M265) cluster tightly together with high levels of expression during early prophase I, suggesting a similar interaction during meiosis in Petunia hybrida. Furthermore, homologies have been found with actin (M304), a principal component of the cytoskeleton, and a t-complex chaperonine (M272) that mediates the folding of tubulin and actin polypeptides. Only low levels of homology have been obtained with microtubule-associated proteins (M273, M305 and M316). Baskin (2000) reported earlier that these accessory proteins that bind the protein filaments of the cytoskeleton appear to be less conserved during eukaryotic evolution. Kinesins belong also to this group of microtubule-associated proteins. They bw-cnudde 23-08-2004 14:49 Pagina 104

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constitute one of the superfamilies of mechano-chemical enzymes or ‘motor proteins’ that generate force by converting chemical energy, typically in the form of ATP, into kinetic energy for movement along the microtubules. In our experiment, one cDNA-AFLP fragment that was upregulated early in meiosis showed homology to an Arabidopsis kinesin-like protein (M253). A second early-induced gene tag with a possible motor protein function (M317) was homologous to an Arabidopsis ATPase gene associated with vesicle-mediated transport, and the Mei-1 gene from Caenorhabditis elegans, encoding a meiotic spindle formation protein. So far, the function of molecular motor proteins has mainly been studied during mitosis in yeast and mammals (Hildebrandt and Hoyt, 2000; Mountain and Compton, 2000). Experimental evidence has shown that kinesins move vesicles and organelles within cells and power various aspects of spindle assembly, maintenance and function. Molecular genetic techniques have revealed many novel motor proteins in plants, but relatively little is known about their possible roles (Reddy, 2001). Recently, in Arabidopsis, the ATK1 kinesin-like protein was shown to be required for spindle morphogenesis in male meiosis (Marcus et al., 2002; Chen et al., 2002), while the TETRASPORE mutant, defective in male meiotic cytokinesis, was also characterised as a kinesin mutant (Yang et al., 2003). Furthermore, homology was found with an Arabidopsis outer envelope membrane protein (M115) with a function in nuclear transport. Two cDNA-AFLP fragments that were induced during meiosis showed homology to a murine Ran GTP-binding protein and a human karyopherin or importin beta subunit (M25 and M51). Small GTP-binding proteins regulate diverse processes in eukaryotic cells, functioning as molecular switches that are activated by GTP and inactivated by the hydrolysis of GTP to GDP. The Ran GTPase system is known to mediate the nucleocytoplasmic transport in a cell, feeding metabolic energy into nuclear transport cycles (Azuma and Dasso, 2000; Weis, 2002). In plants, the small GTPase gene superfamily of Arabidopsis has recently been analysed by Vernoud et al. (2003) and there are indications that plant cells use the same basic transport machinery as yeast and animals cells (Arabidopsis Genome Initiative, 2000). In addition to its role in transport, Ran has also been shown to regulate spindle assembly during mitosis in Xenopus egg extracts through an interaction with importin beta, a nuclear transport receptor (Wiese et al., 2001). In organisms such as fungi, in which the nuclear envelope does not disassemble during mitosis, proteins involved in spindle formation must be imported into the nucleus, suggesting a possible evolutionary origin of the relationship between nuclear protein import and spindle assembly (Wiese et al., 2001). Using immunofluorescent labeling, Hinkle et al. (2002) demonstrated the chromosomal association of Ran during meiotic and mitotic divisions in a variety of organisms. Bucciarelli et al. (2003) provided additional evidence that chromosomes play a key role in spindle formation through the generation of Ran-GTP bw-cnudde 23-08-2004 14:49 Pagina 105

Homologies 105

that promotes microtubule growth and stability. Also in Xenopus, Arnaoutov and Dasso (2003) demonstrated the importance of the Ran GTPase system for the regulation of the cell cycle and the correct kinetochore localisation of the spindle checkpoint proteins Bub1 and Bub3. Finally, we found homologies with members of another family of small GTP- binding proteins in Arabidopsis: Adenosine Ribosylation Factors (M422, M430) or Arf GTPases play important roles during membrane trafficking steps in eukaryotic cells (Chavrier and Goud, 1999). The function of ADP-ribosylation factors has also been linked with meiosis: Arf mutants fail to sporulate in Saccharomyces cerevisiae (Rudge et al., 1998) and have a reduced sperm count in male mice (Schurmann et al., 2002). In Arabidopsis, the TITAN5 gene encodes a protein related to the ADP ribosylation factor family of GTP-binding proteins; titan mutants exhibit dramatic alterations in mitosis and cell cycle control during seed development (McElver et al., 2000).

DNA repair and recombination

The process of homologous recombination appears enigmatic: it creates genetic diversity but also provides an important way of repairing DNA damage without errors. The recent cloning and functional analysis of eukaryotic recombination genes suggests that at least part of the intricate mechanism involved is conserved from bacteria to higher eukaryotes (Thacker, 1999) and shares many of its enzymes with those involved in DSB repair in somatic cells (Smith and Nicolas, 1998). In our screening, three cDNA-AFLP fragments have been isolated that showed homology to the DMC1 (disrupted meiotic cDNA) gene (M12, M46 and M56), encoding a well-known, intensively studied meiotic recombination protein. These fragments are nearly identical in sequence, and two tags have been generated using the same primer combination, making it likely that they are derived from the same gene. It is not uncommon that abundantly expressed genes are represented by an extra ‘ghost band’ on the polyacrylamide gel. As yet, it is unknown how many copies of the DMC1 gene are present in the Petunia hybrida genome. As in most other plant species represented in the database, a single copy of the DMC1 gene is present in Arabidopsis (Klimyuk and Jones, 1997), while in rice, two copies of DMC1 have been identified that are differentially expressed during meiosis (Kathiresan et al., 2002). Our three identified DMC1 tags exhibited an identical meiosis-specific gene expression pattern: high mRNA levels were detected in early prophase I from leptotene up to zygotene, in accordance with their proposed function in meiotic recombination. It has been shown in budding and fission yeasts that DMC1 is only required during meiosis for bw-cnudde 23-08-2004 14:49 Pagina 106

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recombination between homologous chromosomes and not for mitotic recombination (Fukushima et al., 2000). Surprisingly, in plants, DMC1 expression has also been detected in dividing vegetative cells from root tips and cell suspensions (Doutriaux et al., 1998; Kathiresan et al., 2002). This caused another setback among plant scientists in their hunt for a meiosis-specific promoter. Together with RAD51, DMC1 is a homologue of the Escherichia coli RecA gene that was identified almost forty years ago in a screening for bacterial mutants that are defective in genetic recombination and exhibit an extreme sensitivity to agents that promote DNA damage (Clark and Margulies, 1965). Today, RecA is one of the most intensively studied proteins and has been shown in vitro to promote recognition of homology and strand exchange between two homologous DNA molecules during both recombination and repair (reviewed in Kowalczykowski and Eggleston, 1994). From prokaryotes to higher eukaryotes, RecA homologues show a strong sequence conservation, suggesting that the fundamental process of homologous recombination is highly conserved in all living cells (Shinohara and Ogawa, 1999). The ancient origin of DMC1 has been further confirmed by Hartung et al. (2002) in a study of the evolution of the eukaryotic recombination machinery, using an approach of intron comparison across kingdom borders. Bishop et al. (1992) characterised the DMC1 gene in S. cerevisiae and demonstrated its involvement in the repair of DSBs at recombination hot spots, in the formation of the synaptonemal complex, and in the progression of the meiotic cell cycle. Genetic analysis of yeast mutants showed that DMC1 has a distinct role in promoting pairing between homologous chromosomes, but not sister chromatids, in the meiotic context (Schwacha and Kleckner, 1997). In plants, the DMC1 gene from yeast was shown to hybridise to a previously characterised meiotic cDNA, LIM15, from Lilium longiflorum microsporocytes (Kobayashi et al., 1994). Since then, DMC1 homologues have been found in every eukaryote examined (for overview, see Stassen et al., 1997), with the intriguing exceptions of Drosophila and C. elegans (Masson and West, 2001), which may reflect an alternative pathway in these organisms (Villeneuve and Hillers, 2001). The first localisation experiments for RecA-like proteins were also performed in lily: Terasawa et al. (1995) identified DMC1 proteins as discrete foci on leptotene and zygotene chromosomes, demonstrating that meiotic recombination begins at the leptotene stage and continues in zygotene. In Arabidopsis, inactivation of DMC1 results in reduced fertility and disturbed meiosis (Couteau et al., 1999). However, whereas DMC1 mutations trigger meiotic arrest in yeast and apoptotic cell death in mouse (Pittman et al., 1998), meiosis is completed in the Arabidopsis mutants, suggesting that the pachytene checkpoint is absent or less stringent in plants. Two other cDNA-AFLP fragments in our screening are related to RecA-like proteins: M452 is a short sequence (81bp) homologous to an unknown Arabidopsis bw-cnudde 23-08-2004 14:49 Pagina 107

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protein that shows some homology with the meiotic recombination protein DMC1 but is expressed later in meiosis, while a BLASTN search for fragment M229 showed homology to the RecA gene from Streptococcus. Besides DMC1, we found another candidate strand-transfer protein that is involved in both recombinational repair of DNA damage and meiotic recombination. cDNA- AFLP fragment M450 is homologous to an Arabidopsis putative protein with high levels of homology to a testis-specific transcription elongation factor (TFIIS) from mouse and DST1 (DNA Strand Transferase1), a meiotic recombination factor with RecA-like activity from S. cerevisiae. Kipling and Kearsey (1991) have pointed out earlier that the DST1 gene product is similar to the TFIIS protein, which is involved in transcription elongation by RNA polymerase II and whose expression is restricted to germ cells during and after meiosis in the course of spermatogenesis (Umehara et al., 1997). Furthermore, homologies were found with DNA repair proteins from Arabidopsis, such as a UV-damaged DNA-binding protein (M280) that might be involved in the recombination pathway. This fragment clusters closely together with a tag homologous to histone 2B (M223) that recently has been shown to be involved in the repair of UV- induced DNA damage in S. cerevisiae (Martini et al., 2002). A second fragment (M343) was induced later in meiosis and showed homology to DRT100 (DNA-damage repair/ toleration), an Arabidopsis cDNA selected for its ability to partially complement Escherichia coli mutants lacking all bacterial defences against UV-light damage to DNA. Pang et al. (1992, 1993) showed that the DRT100 gene product, a leucine-rich protein, complemented in particular RecA- phenotypes related to defective homologous recombination and resistance to DNA-damaging agents. In the light of these results, it is reasonable to assume that DRT100 is also involved in recombinational DNA repair in plants. We found also two homologies with DNA helicases, ATP-dependent DNA- unwinding enzymes with a likely function in DNA repair and meiotic crossing-over. In plants, the molecular mechanism of meiotic crossing-over and its regulation remain elusive. In S. cerevisiae, the process has been unraveled further, and a number of genes have been characterised that are specifically involved in meiotic crossing-over but not required for gene conversion, such as the Mer3 DNA helicase (Nakagawa and Kolodner, 2002). Fragment M287 is induced late in meiosis and shows a homology to a meiosis-related, testis-specific protein known as cancer-antigen in humans and to a plant Ruv DNA- helicase-like protein. Proteins related to the bacterial RuvB DNA helicase have been shown to catalyse branch migration of Holliday junctions (Shen et al., 2000). Fragment M311 is expressed early in meiosis and shows a homology to a hypothetical protein in Arabidopsis, related to a DEAD box-like helicase that interacts with the gene product of the breast cancer susceptibility gene BRCA1. BRCA1 and BRCA2 are two intensively bw-cnudde 23-08-2004 14:49 Pagina 108

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studied tumour suppressor genes, mutations of which are responsible for a substantial proportion of hereditary high-risk breast and ovarian cancer syndromes. Current data from mutant analysis in mice suggest that BRCA genes play an essential role in DNA repair and the maintenance of genome integrity, with a specific contribution to meiotic crossing-over during spermatogenesis (Welcsh et al., 2000, Xu et al., 2003). Remarkably, there are no clear homologues of BRCA genes in yeast, flies or worms, suggesting a unique role of these proteins in the DNA repair in higher eukaryotes, probably as regulators of more conserved repair functions (Scully and Livingston, 2000). However, orthologues of BRCA1 and BRCA2 are present in Arabidopsis (Dray et al., 2003), and recently, Lafarge and Montane (2003) reported that the AtBRCA1 gene is strongly induced by gamma irradiation. The carboxyl-terminal domain (BRCT) of the BRCA1 protein is an evolutionary conserved module (Koonin et al., 1996) that exists in a large number of proteins involved in DNA repair, recombination and cell cycle control (Bork et al., 1997), including the Arabidopsis MEI1 gene (Grelon et al., 2003). BRCT domains have recently been shown to bind phospho-proteins, such as phosphorylated DNA helicases (Yu et al., 2003). Another highly conserved motif in the BRCA1 gene is the C3HC4 RING zinc- finger domain (Machackova et al., 2001). In our screening, we also found homology to an Arabidopsis C3HC4 RING zinc-finger protein (M407). However, one should be cautious to speculate on a specific function since zinc-finger-containing proteins belong to a large superfamily that has been shown to contribute to many distinct cellular processes, including transcriptional regulation, mRNA stability and processing, and protein turnover (Ravasi et al., 2003).

Synapsis and sister chromatid cohesion

Two cDNA-AFLP fragments with an elevated level of gene expression before meiosis and during leptotene (M203 and M204) showed homology in the database to the ASY1 (asynaptic1) gene of Arabidopsis. Since both fragments migrated closely together on the polyacrylamide gel, we assume that they are derived from the same Petunia gene. asy1 was one of the first meiotic mutants to be characterised in Arabidopsis (Ross et al., 1997); it exhibits a clear asynaptic phenotype, lacking conventional zygotene and pachytene stages (Caryl et al., 2000). Armstrong et al. (2002) showed that the ASY1 protein localised along lateral elements of the synaptonemal complex in Arabidopsis, confirming its role in synapsis. ASY1 shows limited similarity to the yeast meiotic gene HOP1 (Hollingsworth et al., 1990), although it has a slightly different spatial and bw-cnudde 23-08-2004 14:49 Pagina 109

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temporal distribution. To date, ASY1 is still the only plant protein known to be associated with the SC. Furthermore, we found two cDNA-AFLP tags (M409 and M468) that show weak homology to members of a large, evolutionarily conserved superfamily of chromosome- associated proteins known as SMCs (structural maintenance of chromosomes). Both fragments are induced late in meiosis, which would be in accordance with a proposed role of SMC proteins in sister chromatid cohesion, chromosome condensation or DSB repair by homologous recombination (Cobbe and Heck, 2000). A low level of homology to SMC-like proteins was also found for two other fragments of our list: M95 and M324 showed weak homology to human RAD50 and RAD50-interacting protein1, respectively. RAD50 forms a protein complex involved in meiotic recombination, DNA replication, telomere maintenance and cellular DNA damage responses (Daoudal-Cotterell et al., 2002). However, more sequence information is required for these fragments to obtain more significant homologies in a database search.

RNA processing and protein biosynthesis

cDNA-AFLP fragment M245, expressed premeiotically and early in meiosis, showed homology to a poorly characterised Arabidopsis PARN (poly[A]-specific ribonuclease) or DAN (deadenylating nuclease) gene. PARN encodes a 3’ exonuclease enzyme that degrades the poly(A)-tail of eukaryotic mRNA molecules, resulting in translational repression. This deadenylation process initiates the major mRNA degradation pathway in eukaryotes (Decker and Parker, 1993) and plays an important role as an alternative regulatory mechanism in the control of gene expression. This particular function of PARN in mRNA silencing has been demonstrated during meiotic maturation in Xenopus laevis oocytes, where a specific class of maternal mRNAs was translationally silenced and destabilised by deadenylation (Korner et al., 1998; Copeland and Wormington, 2001). Generally, it is believed that RNA processing mechanisms are particularly important for the control of gene expression during mitosis and meiosis, when de novo transcription is severely reduced. Breyne et al. (2002) reported an overrepresentation of RNA-processing genes in mitotic M-phase, indicating that post-transcriptional regulation is involved in gene activity during mitosis. Surosky and Esposito (1992) had demonstrated earlier that meiotic transcripts are highly unstable in Saccharomyces cerevisiae and that the levels of meiotic mRNAs are regulated by both transcriptional control and mRNA turnover. This mRNA turnover rate is regulated primarily by the status of the translation bw-cnudde 23-08-2004 14:49 Pagina 110

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initiation complex, coupling both translation and decay (Mitchell and Tollervey, 2000). In our screening, we found two differentially expressed cDNA-AFLP fragments that showed homology to a translation initiation factor (M54) and an elongation factor-1 alpha (M99) in the database. Traditionally, these protein translation factors have been described as the workhorses of protein synthesis on the ribosome, where they assist in initiating and further elongating the polypeptide chain by one amino acid at a time (Andersen et al., 2003). However, there is a growing body of evidence for a role beyond housekeeping. A germ cell-specific elongation factor-1 alpha, identified in Xenopus oocytes (Morales et al., 1991), has been shown to be involved in microtubule reorganisation during the cell cycle (Shiina et al., 1994). Recently, two protein translation factors have been identified as important human oncogenes, suggesting a second, regulatory role for translation initiation and elongation factors in cell growth, apoptosis, and tumorigenesis (Thornton et al., 2003). Furthermore, we found a collection of transcripts encoding ribosomal proteins (M19, M33, M90, M209, M258, and M449) that are modulated during meiosis. Transcriptional regulation has been demonstrated before in plants for some isolated ribosomal proteins (Lee et al., 1999; Moran et al., 2000) and protein translation factors (Pokalsky et al., 1989), and seems to correlate with increased translation rates during meiosis. In a similar way, a large number of transcripts involved in protein synthesis have been detected during cell division in plants (Breyne et al., 2002).

Can Petunia get Prader-Willi?

An unexpected result of the database screening for homologies to our Petunia transcript fragments was the repeatedly encountered association with human genes involved in the Prader-Willi syndrome (PWS). PWS is a neurological disorder caused by a deficiency of a gene or a group of genes, expressed exclusively from the paternal chromosome 15 (Jiang et al., 1998). Perhaps it represents one of the best examples for the involvement of genomic imprinting in human genetic diseases (Sapienza and Hall, 1995). Genomic imprinting is a mechanism by which one copy of a gene is preferentially silenced according to its parental origin (Barlow, 1995). This epigenetic phenomenon has also been described in plants (for review, see Alleman and Doctor, 2000), with the Arabidopsis MEDEA gene, active in early seed development, as the best-known example (Grossniklaus et al., 1998). Currently, imprinting has been documented in more than 30 mouse and human genes (Brannon and Bartolomei, 1999; Kelsey, 1999), many of which are crucial for early development. Recently, it has been shown in mouse germ cells that imprinting by differential DNA methylation is established prior to meiosis in bw-cnudde 23-08-2004 14:49 Pagina 111

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the male germline, while maternal imprints are established later, during meiotic maturation in the oocytes (Kerjean et al., 2000; Lucifero et al., 2002). In our screening, no fragment encoding a DNA methyltransferase, the key enzyme in genomic imprinting, could be retrieved. However, we found homologies to three mammalian imprinted genes that have been reported to play a role in the Prader-Willi syndrome: PARN (M245) (Buiting et al., 1999), SNRPN (M183) (Toth-Fejel et al., 1996) and a gene encoding a zinc-finger polypeptide with a C3HC4-type RING domain (M407) (Jong et al., 1999a, 1999b). Normally, these genes are expressed only from the paternal allele. Loss of imprinting – that is, biallelic expression – of these genes results in an inappropriate epigenotype, leading to the PWS phenotype. As yet it is unknown whether their orthologues in plants are also imprinted during gametophyte development. What is the connection between transcripts expressed during meiosis in the anthers of Petunia hybrida and the Prader-Willi syndrome? Despite intensive research on imprinting in mammals, the underlying mechanisms that are involved in establishing and maintaining a parental imprint are not well understood. In plants, almost nothing is known about the genetic regulation of imprinting. However, we know that our transcripts are derived from genes expressed at stages where imprints are established. Hence, it is tempting to speculate that expression in the male germline contributes to the setting of the paternal-specific gametic imprint. A similar idea has been proposed by Jong et al. (1999a) when he observed that the imprinted RING zinc-finger gene associated with PWS was also expressed at high levels in testis, in meiotic and post-meiotic germ cells. The early developmental monoallelic expression of the X-chromosome-linked Xist gene in humans provides additional support for this theory (Monk, 1995). Currently it is unknown how the process of setting the imprint is related to the transcriptional activity of a gene. However, it can be speculated that gene expression from a specific allele prevents it from being methylated. For the RING zinc-finger gene it has been demonstrated that only the maternal allele is methylated (Jong et al., 1999b), consistent with a role for paternal gene expression protecting the paternal allele from methylation. Further research will be required to determine if the corresponding genes are also imprinted in Petunia. In this regard, our staging strategy for the dissection of male meiosis could also be used to determine the exact mechanisms involved in the establishment of the genomic imprinting pattern for each gene at each stage during germ cell development. Arabidopsis T-DNA insertion mutants for these genes have also been requested from the SALK collection (http://signal.salk.edu/cgi-bin/tdnaexpress) and will be examined for imprinting defects. bw-cnudde 23-08-2004 14:49 Pagina 112

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Metabolism

A significant amount of the cDNA-AFLP fragments that were preferentially expressed during meiosis, match with genes coding for enzymes involved in the basic metabolism of the cell. This is not surprising since our samples are derived from anther tissue undergoing cell growth and microsporogenesis, and thus require the synthesis of new molecules. Approximately thirty fragments are derived from genes encoding enzymes that support diverse metabolic processes, such as photosynthesis, cell wall synthesis, respiration, and the synthesis of sugars, lipids, fatty acids, nucleotides and amino acids. However, it cannot be excluded that some of these enzymes have a more specific role during meiosis. Cytochrome P450 (M9), for example, belongs to a large superfamily that participates in numerous biochemical pathways. Some P450 genes with developmentally regulated patterns of expression have been shown to have highly specific functions, for example during pollen tube growth (Nadeau et al., 1996).

Overlap with tobacco cell cycle database

In an analogous transcript profiling experiment, Breyne et al. (2002) assembled an inventory of 1500 tobacco cDNA-AFLP fragments that are modulated during cell division. Since Petunia hybrida and Nicotiana tabacum are closely related Solanaceae species, and since identical primer combinations have been used for the amplification reactions, it is possible to compare the individual tags of both databases. We used a BLASTN-search (Altschul et al., 1997) to screen for sequences that are modulated during both mitotic and male meiotic processes. Of the 293 successfully sequenced gene tags present in our collection, 59 (20%) were homologous to sequences in the tobacco cell cycle database. Not surprisingly, these fragments mostly encoded general housekeeping proteins (e.g. cytochrome P450), and ubiquitous proteins belonging to diverse functional classes (e.g. histones, ribosomal proteins and ubiquitin conjugating enzymes). On the other hand, gene fragments with a proposed meiosis-specific function (e.g. DMC1, ASY1 and a meiotic protease-encoding gene) were absent in the cell cycle database, in line with their unique role in the meiotic process.

Discussion

Combining a cytogenetically-based sampling of developing Petunia anthers with cDNA-AFLP-based transcript profiling, we were able to build a comprehensive bw-cnudde 23-08-2004 14:49 Pagina 113

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inventory of genes with a modulated expression during meiosis. Our collection comprises 475 unique transcript fragments corresponding to genes with fluctuating expression levels during male meiosis. Among these, we identified many components of meiotic processes, indicating that our screening was basically effective. Of the 293 successfully sequenced gene tags present in our collection, only 90 fragments (30%) displayed a significant level of homology in a GenBank database search using the BLAST algorithm. This is partly due to the relatively small size of the generated AFLP tags, which range between 50 and 450bp, with an average length of 168bp. In addition, the molecular and genetic dissection of meiotic genes is underdeveloped in plants and the list of known meiotic genes in Arabidopsis is still short, although growing fast. Furthermore, there are indications that organism-specific genes are overrepresented among genes induced during sexual development. For example, Mata and Bahler (2003) recently reported a particular enrichment of S. pombe-specific genes induced during meiotic prophase in an experiment studying gene conservation in fission yeast. In many cases, however, weak homologies could be found with known meiotic genes from Arabidopsis or Saccharomyces. The high incidence of low-homology hits with meiotically related genes in our database search suggests an actual function during meiosis for the tagged gene, as can be expected on the basis of its modulated expression profile. On the other hand, there is a constant danger of over-interpreting the results of a homology search and, often, expert knowledge of the biological process is required for a correct cross-species gene annotation. For example, in some cases, it might be more relevant to look for the presence of conserved sequence motifs that are essential to the biological function than to take the overall sequence similarity into account [e.g. the BRC repeats in different species are well conserved, in contrast with the rest of the BRCA2 gene (Bignell et al., 1997)]. In the end, only a functional analysis, for example by mutant studies, can provide sufficient evidence for a true meiosis-specific function, while a thorough homology search and cluster analysis are extremely helpful tools to make a well-educated guess for such a further analysis. A functional classification of the tagged genes, based on literature datasearches, demonstrated that components of many different meiotic processes could be found. Conversely, one can also start off from the genes or processes that were not represented in our database. Several explanations are possible to explain their absence. First of all, our screening is not genome-wide; we estimate that 35% of the Petunia transcriptome was analysed and our direct sequencing approach was successful in 62% of these cases. This could explain the absence in our database of homologues of well- known, conserved meiotic genes, such as SPO11 and RAD51. Second, our experimental setup possibly excluded the visualisation of scarcely bw-cnudde 23-08-2004 14:49 Pagina 114

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expressed genes or genes whose expression is restricted to a narrow timeframe, despite our choice of +2/+2 primer combinations that ensure a high level of sensitivity in the cDNA-AFLP screening. Namely, since complete anthers were collected, our samples were not fully homogeneous and consisted not only of meiocytes, but also contained cells belonging to the tapetum, the epidermis, the endothecium, the connective tissue and the vascular bundle. This heterogeneity in the sampled tissues could be an explanation for the underrepresentation of transcription factors, which typically have a low cellular concentration, in our database, compared to the tobacco cell cycle experiment of Breyne et al. (2002). Finally, we may assume that our functional classification is biased towards the more conserved biological processes, while cDNA-AFLP tags corresponding to less conserved genes and processes might well be present in our collection but cannot be recognised on the basis of a homology search. It has been reported earlier that between meiotic genes with potentially similar functions often little sequence similarity can be found across species (Dresser, 2000). For example, gene sequences encoding structural components of the synaptonemal complex, such as zip1 (Dong and Roeder, 2000), are notoriously poorly conserved. This lack of sequence similarity is surprising, considering the striking morphological and structural similarities among SCs from different species (von Wettstein et al., 1984). Similarly, genes involved in chromatid cohesion and segregation in yeast often lack putative homologues in Arabidopsis (Mercier et al., 2001), which could provide an explanation for the underrepresentation of both functional classes in our database. In contrast, genes involved in meiotic recombination and DNA repair are highly conserved during evolution and are well represented in our collection. During the last few years there has been an exponential growth in the amount of biological information on meiosis in public databases, mainly derived from research on sporulation in budding yeast. Therefore, the results of our homology search generally reflect the shared processes governing progression of meiosis in plants and yeast. For example, components of the SCF proteolytic complex are well represented in our screening, in contrast to the Anaphase Promoting Complex (APC) that is known to promote exit from the meiotic metaphase in yeast. Interestingly, this lack of APC- related factors has also been reported in mammalian meiosis (Hwang et al., 2001), suggesting that a different mode of metaphase-anaphase transition might be at play. The identification of Bub1 and Bub3 checkpoint control genes in our experiment indicates that the basic mechanism of meiotic checkpoint control is conserved. This is confirmed by the identification of homologues for these genes in mammals, C. elegans and Drosophila in a computer-based search by Hwang et al. (2001). Furthermore, the spindle checkpoint is an excellent example of a conserved mechanism between mitosis and meiosis, since it also controls the progression during the mitotic cell cycle. bw-cnudde 23-08-2004 14:49 Pagina 115

Homologies 115

Most frequently, we found homologies with genes from Arabidopsis thaliana, which is not surprising since it is the closest relative of Petunia hybrida that has been fully sequenced. However, some fragments matched closer with conserved meiotic genes from other organisms, e.g. tag M342 with Bub1 from Drosophila melanogaster. Furthermore, several links were found with mammalian testis development, for example by protein kinases that are denoted as testis-specific in the database. These similarities between microsporogenesis and spermatogenesis must have been conserved during millions of years of evolution. Another striking homology was found with a RecA- homologue of Streptococcus (M229), adding further proof to the high level of evolutionary conservation of meiotic recombination proteins. The processing and interpretation of homology data on large numbers of genes is a laborious and time-consuming task that requires expert knowledge of the relevant biological research field. There is now extensive information about genes involved in sexual reproduction in many species, and comparison of data from different organisms can be extremely useful. Furthermore, the inclusion of relevant high-throughput expression profiling and proteome data should enhance our understanding of the underlying mechanisms. Therefore, knowledge bases such as GermOnline (Primig et al., 2003) are becoming increasingly important platforms for the scientific community. GermOnline (http://www.germonline.org) is a new cross-species database on germline development and gametogenesis that allows researchers to directly contribute and update knowledge about genes of interest. Today, GermOnline Release 2.0 contains information on 684 genes from Saccharomyces cerevisiae involved in meiosis, spore formation and germination. When contributions from other species start to grow, this database will facilitate comparative analysis of meiotic processes across species.

In conclusion, cDNA-AFLP transcript profiling on developing anthers in Petunia hybrida is a valuable method to generate transcript tags modulated during male meiosis. A thorough characterisation of these fragments based on sequence homology enabled us to classify them into functional groups. Homologies with diverse classes of known meiosis genes were retrieved, indicating that our screening was basically effective. Although incomplete, our comprehensive data set provides a starting point for the further unraveling of meiosis at a molecular level in plants, allowing a knowledge-based selection of tags for further characterisation.

Acknowledgements

The sequencing team from Ghent provided indispensable support in the processing of bw-cnudde 23-08-2004 14:49 Pagina 116

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the high number of reactions: Wilson Ardiles-Diaz, Raimundo Villarroel and Jan Gielen, your help was greatly appreciated. Bernard Cannoot also devoted some of his precious time to a couple of tedious reamplification reactions. Finally, the stimulating discussions with Janny Peters and Peter de Boer were a good guide to find our way in the resulting gene collection.

References

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Umehara, T., Kida, S., Hasegawa, S., Fujimoto, H., and Horikoshi, M. (1997) Restricted expression of a member of the transcription elongation factor S-II family in testicular germ cells during and after meiosis. J. Biochem. 121, 598-603. Valverde, F., Mouradov, A., Soppe, W., Ravenscroft, D., Samach, A., and Coupland, G. (2004) Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303, 1003-1006. Vernoud, V., Horton, A.C., Yang, Z., and Nielsen, E. (2003) Analysis of the small GTPase gene superfamily of Arabidopsis. Plant Physiol. 131, 1191-1208. Villeneuve, A.M., and Hillers, K.J. (2001) Whence Meiosis? Cell 106, 647-650. Voicu, P.M., Poitelea, M., Schweingruber, M.E., and Rusu, M. (2002) Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe. Archives of Microbiology 177, 251-258. von Wettstein, D., Rasmussen, S.W., and Holm, P.B. (1984) The synaptonemal complex in genetic segregation. Annu. Rev. Genet. 18, 331-413. Wagner, D., and Meyerowitz, E.M. (2002) SPLAYED, a novel SWI/SNF ATPase homolog, controls reproductive development in Arabidopsis. Curr. Biol. 12, 85-94. Wang, W., Cote, J., Xue, Y., Zhou, S., Khavari, P.A., Biggar, S.R., Muchardt, C., Kalpana, G.V., Goff, S.P., Yaniv, M., Workman, J.L., and Crabtree, G.R. (1996) Purification and biochemical heterogeneity of the mammalian SWI-SNF complex. EMBO J. 15, 5370-5382. Weis, K. (2002) Nucleocytoplasmic transport: cargo trafficking across the border. Curr. Opin. Cell Biol. 14, 328- 335. Welcsh, P.L., Owens, K.N., and King, M.C. (2000) Insights into the functions of BRCA1 and BRCA2. Trends Genet. 16, 69-74. Wiese, C., Wilde, A., Moore, M.S., Adam, S.A., Merdes, A., and Zheng, Y. (2001) Role of importin-β in coupling Ran to downstream targets in microtubule assembly. Science 291, 653-656. Woo, H.H., and Hawes, M.C. (1997) Cloning of genes whose expression is correlated with mitosis and localised in dividing cells in root caps of Pisum sativum L. Plant Mol. Biol. 35, 1045-1051. Xu, X., Aprelikova, O., Moens, P., Deng, C.X., and Furth, P.A. (2003) Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice. Development 130, 2001-2012. Yamaguchi, S., Decottignies, A., and Nurse, P. (2003) Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint. EMBO J. 22, 1075-1087. Yang, M., Hu, Y., Lodhi, M., McCombie, W.R., and Ma, H. (1999) The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation. Proc. Natl. Acad. Sci. USA 96, 11416-11421. Yang, C.-Y., Spielman, M., Coles, J.P., Li, Y., Ghelani, S., Bourdon, V., Brown, R.C., Lemmon, B.E., Scott, R.J., and Dickinson, H.G. (2003)TETRASPORE encodes a kinesin required for male meiotic cytokinesis in Arabidopsis. Plant J. 34, 229-240. Yu, X., Chini, C.C., He, M., Mer, G., and Chen, J. (2003) The BRCT domain is a phospho-protein binding domain. Science 302, 639-642. Zachariae, W., and Nasmyth, K. (1999) Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev. 13, 2039-2058. Zhao, D., Yu, Q., Chen, M., and Ma, H. (2001) The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis. Development 128, 2735-2746. Zheng, X.F., and Schreiber, S.L. (1997) Target of rapamycin proteins and their kinase activities are required for meiosis. Proc. Natl. Acad. Sci. USA 94, 3070-3075. bw-cnudde 23-08-2004 14:49 Pagina 123

Chapter V

Clustering genes expressed during male meiosis

Filip Cnudde, Tom Gerats

A part of this chapter is submitted to Plant Physiology bw-cnudde 23-08-2004 14:49 Pagina 124

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Abstract

Using cDNA-AFLP transcript profiling, we have visualised approximately 35% of the transcriptome of Petunia hybrida anthers during male meiosis (see Chapter III). Further quantification of the gene expression profiles resulted in a data matrix that allowed a detailed statistical analysis of modulated gene expression during male meiosis. Significant transcriptional changes could be measured for many transcripts at the initiation and at the end of meiosis, emphasising the unique nature of the meiotic process during sexual development and the involvement of a specialised transcriptional program. Cluster analysis showed that a transcriptional cascade governs the initiation and progression through the meiotic cell cycle and revealed a high level of coordination and a precise temporal activation of meiotic genes. We identified five large clusters of co-expressed genes and could correlate these with major biological processes during meiosis. A disproportionate number of Petunia transcript fragments induced during pachytene and later meiotic stages show no homology in a database search, suggesting a backlog in the molecular analysis of meiosis-specific genes. This first large-scale examination of the temporal ordering of gene expression patterns during plant meiosis opens new perspectives for gene discovery, cross-species comparisons with well-studied model systems such as yeast, and the elucidation of underlying transcriptional regulatory networks that control the complex and highly regulated processes that occur during meiosis.

Introduction

The rapid advances in large-scale sequencing and the development of functional genomics tools for gene expression studies have revolutionised our approach to characterise biological processes. Technologies, such as microarray analysis (Schena et al., 1995; Lockhart et al., 1996), serial analysis of gene expression (SAGE) (Velculescu et al., 1995) and cDNA-AFLP transcript profiling (Breyne and Zabeau, 2001; Cnudde et al., 2003), allow the high-throughput measurement of expression of thousands of genes. These experiments generate an immense amount of data points, which in itself poses new challenges for an adequate extraction of the inherent significant biological information and statistical interpretation. Clustering - the grouping of genes with similar expression profiles - is frequently used in microarray experiments as one of the first steps in data analysis. A number of clustering algorithms has been developed to reorganise the complex data matrix in a more informative way (Tamayo et al., 1999). A common computational approach is hierarchical clustering (Eisen et al., 1998). This algorithm sorts through all the data to bw-cnudde 23-08-2004 14:49 Pagina 125

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identify pairs of genes that behave most similarly and then progressively adds other genes to the initial pairs to form clusters of genes with a correlated expression profile. The software tools, developed by Michael Eisen and co-workers, employ a hierarchical clustering algorithm to organise gene expression patterns into a kind of phylogenetic tree, reflecting similarity in expression. The resulting graphical presentation allows a visual exploration of the high-dimensional data and has become a familiar appearance in many papers on large-scale gene expression studies (e.g. Spellman et al., 1998; Maleck et al., 2000; Breyne et al., 2002). A second clustering method that allows an easy comparison of gene expression patterns is adaptive quality-based clustering (De Smet et al., 2002). This software program is based on the K-means clustering algorithm (Tamayo et al., 1999) and defines separate groups of significantly co-expressed genes without the requirement to specify the number of clusters a priori. We have used both clustering methods to analyse our set of gene expression data, generated by cDNA-AFLP transcript profiling during male meiosis in Petunia hybrida. This transcript profiling experiment focused on the analysis of modulated gene expression during meiosis, and resulted after visual inspection in a collection of 475 gene fragments with clear periodical variation in their transcript levels during meiosis. Of these, 293 tags could be successfully sequenced and were subsequently classified into functional groups, based on homology searches (see Chapter IV). We performed a cluster analysis on this data set to identify groups of genes with highly similar expression profiles. Since co-expression of genes is a strong indication of their involvement in the same process (Holter et al., 2000), our first goal is to check whether a general correlation can be found between specific clusters and the functional classes they represent. Furthermore, cluster designations of unknown genes may be useful in providing clues about their possible function during meiosis. In this way, a targeted selection can be carried out to identify candidate genes that might be involved in a particular meiotic process. Examination of the temporal ordering of gene expression patterns can also provide insights into the mechanism of transcriptional regulation and can be a first step in the identification of new transcription factors. This information can be used to unravel the underlying transcriptional regulatory networks that control the complex and highly regulated processes that occur during meiosis. Moreover, cluster analysis allows us to make cross-species comparisons of gene expression patterns during meiosis between Petunia hybrida and yeasts. The transcriptional program of meiosis and sporulation has been extensively studied using microarrays in Saccharomyces cerevisiae (Chu et al., 1998; Primig et al., 2000) and Schizosaccharomyces pombe (Mata et al., 2002). The level of conservation of the bw-cnudde 23-08-2004 14:49 Pagina 126

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regulatory machinery can provide more insight in the evolution of the mechanisms of meiotic control, including transcriptional regulation.

Experimental procedures

cDNA-AFLP transcript profiling to analyse gene expression during male meiosis

We have used cDNA-AFLP transcript profiling to analyse changes in gene expression during male meiosis in Petunia hybrida. Our sampling strategy, based on a cytological determination of the meiotic stages, is described in the experimental procedures of Chapter III. The cDNA-AFLP-based transcript profiling procedure is also explained in Chapter III. Protocols for the reamplification of cDNA-AFLP fragments, sequencing and characterisation by homology searching can be found in Chapter IV.

Quantitative measurements of the expression profiles and data analysis

The following methods have been described earlier by Breyne et al. (2003) for the quantitative cDNA-AFLP analysis during cell division in plants. Dried cDNA-AFLP gels were scanned by means of Phospho-Imager technology (Molecular Dynamics). Gel images were quantitatively analysed using the AFLP-QuantarPro image analysis software (Keygene, Wageningen, The Netherlands). This software has been designed for accurate lane definition, fragment detection and quantification of band intensities. All visible AFLP fragments were scored and individual band intensities were measured per lane. The data obtained were used to determine the quantitative expression profile of each transcript. The raw data were first corrected for differences in total lane intensities, which may arise due to loading errors or differences in the efficiency of PCR amplification with a given primer combination for one or more time points. For each primer combination, the intensity values of all scored bands were summed per lane. Each of the summed values was divided by the maximal summed value to give the correction factors. Finally, all raw values generated by QuantarPro were divided by these correction factors. Subsequently, each individual gene expression profile was variance-normalised by standard statistical approaches as used for microarray-derived data (Tavazoie et al., 1999). For each transcript, the mean expression value across the time course was subtracted from every individual data point, after which the value obtained was divided bw-cnudde 23-08-2004 14:49 Pagina 127

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by the standard deviation. A coefficient of variation (CV) was calculated by dividing the standard deviation by the mean.

Cluster analysis

Cluster analysis of the expression data was executed using two methods according to Dreesen (2002). In the first method, a hierarchical average linkage clustering was performed using the Cluster software developed by Michael Eisen and co-workers (1998). Hierarchical clustering is a pairwise average-linked method that uses the standard correlation coefficient as a measure of similarity in the behaviour of two genes. While this definition of co-expression is particularly suitable to capture similarity ‘in shape’, it places no emphasis on the magnitude of changes in expression. For the visualisation of the results, the Treeview software (Eisen et al., 1998) was applied. Both software packages can be downloaded for free at http://www.microarrays.org/software. html. In the output file, measured changes in mRNA levels are shown in a compact graphical format, with rows corresponding to individual genes and columns corresponding to (in our case) the sampled meiotic stages. Furthermore, different clusters are shown in a hierarchical tree. By clicking on a node, the corresponding cluster can be viewed in more detail. The expression values are presented with colour- codes for each measured time point, where bright red stands for the highest expression value and bright green for the lowest expression value. Increases in expression are represented as graded shades of red, and decreases as graded shades of green. Black represents a mean expression value of a given expression profile. Missing data are represented in grey. The second method is called adaptive quality-based clustering (De Smet et al., 2002) and is accessible at http://www.esat.kuleuven.ac.be/~thijs/Work/Clustering.html. This program is similar to K-means clustering, except that the number of clusters need not be defined in advance and that expression profiles that do not fit in any cluster are rejected. As an output, genes are grouped in different clusters that are presented in graphs, showing the average expression pattern in function of time. The minimal number of tags in a cluster and the acquired probability of genes belonging to a cluster were set to 2 and 0.95, respectively, a stringent condition for inclusion into a cluster. Both methods require the input expression data to be submitted in a text-format (.txt). bw-cnudde 23-08-2004 14:49 Pagina 128

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Results

Genes modulated during meiosis are regulated in successive waves of transcription

The transcript profiling experiment focused on the analysis of modulated gene expression during meiosis, while premeiotic archesporial samples and postmeiotic microspores served as a reference. The meiotic process itself was subdivided into four stages, based on cytological analysis: leptotene, zygotene, pachytene-metaphase I, dyad stage-telophase II. In a large-scale screening for genes modulated during male meiosis in Petunia hybrida, 256 primer combinations have been performed, generating approximately 8,000 transcript tags. We estimate that 35% of the total Petunia transcriptome has been visualised on gel. After visual inspection, 475 transcript tags with a modulated gene expression pattern were selected for further characterisation. In a next step, Phospho-Imager gels were analysed using the AFLP-QuantarPro image analysis software for the quantification of band intensities. All visible AFLP fragments were scored and the raw data were first corrected for differences in total lane intensities. Subsequently, each individual gene expression profile was variance- normalised by standard statistical approaches as used for microarray-derived data (Tavazoie et al., 1999). This initial data set comprised 7,408 quantified expression profiles. Approximately 500 expression profiles were not included because of insufficient quality for cluster analysis or because the corresponding Phospho-Imager scans were not available. Further, we arranged our data set according to the coefficient of variation (CV), a parameter defining the significance of differential expression. This CV was used to establish a cut-off value, and only expression profiles with a CV greater than 0.7 were selected for further cluster analysis. This objective scoring method based on statistical analysis resulted in a data set of 978 fragments exhibiting a significantly modulated expression profile, which included most of the 475 gene fragments that were previously selected after visual inspection. This treshold level corresponds to a 3-fold transcriptional change, a stringent condition for inclusion into our data set to minimize false-positive assessments. In a first approach, we performed hierarchical average linkage clustering according to Eisen et al. (1998). Fig. 1 presents an overview of the resulting clusters of genes with similar expression profiles, while the matching dendrogram shows the relationships between these clusters. Roughly, five different subnodes can be discriminated in the clustering tree, the corresponding temporal patterns are visualised by a colour code. The modulated expression patterns show considerable changes in mRNA abundance, with peak levels represented by bright red colour. The five groups of co-expressed genes are bw-cnudde 23-08-2004 14:49 Pagina 129

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Figure 1 Hierarchical average linkage clustering of the expression patterns of 978 significantly modulated cDNA- AFLP fragments according to Eisen et al. (1998). The matching dendrogram shows five different clusters of co- expressed genes, with maximal expression level at different stages during meiosis. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 postmeiotic (see also appendix)

tightly clustered and reach their maximal expression level at different stages during meiosis. As a result, a transcriptional cascade can be observed that governs initiation and progression through the meiotic cell cycle. These successive waves of transcription reveal a high level of coordination and a precise temporal activation of meiotic genes, indicating a strict regulation at the transcriptional level. Large clusters of coregulated genes were expressed exclusively premeiotically or postmeiotically, while other clusters harboured meiosis-specific genes, highly expressed during early, middle or late meiosis (see Fig. 1). The highest levels of gene induction were found in the cluster with postmeiotically expressed genes; up to 60-fold increases in mRNA abundance could be observed. In contrast, a majority of the premeiotically expressed genes is 20-to 30-fold downregulated upon meiotic initiation. A second group of premeiotically expressed genes is less repressed and remains transcriptionally active during early prophase. However, we mainly focused on genes modulated during meiosis: most of the 475 transcript tags selected for further characterisation (see Chapter IV) belonged to a cluster with meiosis-specific gene expression patterns. Two major clusters harbour genes expressed in early prophase (leptotene-zygotene) or later in meiosis (from pachytene to telophase II). Finally, a third, smaller cluster contains ‘middle’ genes reaching their peak expression level at the zygotene stage. bw-cnudde 23-08-2004 14:49 Pagina 130

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High-resolution clusters defined by adaptive quality-based clustering

To confirm the results obtained by hierarchical average linkage clustering, the same data set of strongly modulated fragments was subjected to a second clustering algorithm, called adaptive quality-based clustering (De Smet et al., 2002). As a result, 11 clusters were generated, representing the expression profiles of 826 genes (85% of the total data set). Of these, five major clusters harbour 87% of all expression patterns, while the remaining six smaller clusters show variations on this theme (see Fig. 2). In general, adaptive quality- based clustering confirmed the clustering tree of Fig. 1. Moreover, genes sorted close to each other by hierarchical clustering were assigned to the same cluster in the adaptive quality-based method. However, the substantial discriminatory power of the latter method allows a further refinement in the classification of co-expressed genes, compared to hierarchical average linkage clustering. This high-resolution unraveling of gene expression patterns can provide helpful clues for the identification of underlying regulatory networks and the elucidation of overall meiotic gene functions.

Figure 2 Adaptive quality-based clustering of the expression patterns of 978 significantly modulated cDNA-AFLP fragments according to De Smet et al. (2002). A-E Five major clusters harbouring 87% of all expression patterns. A premeiotic, B downregulated, C early meiotic, D late meiotic, E postmeiotic. 1-6 Sampled meiotic stages. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 postmeiotic bw-cnudde 23-08-2004 14:49 Pagina 131

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Towards the borders of statistical significance and beyond

In a next step, we applied both clustering techniques on our entire data set of 7,408 expression profiles to screen for clusters that were not defined previously and that might have been concealed by the setting of our treshold level. Using adaptive quality-based clustering, with the parameters still fixed at a minimum limit of 2 genes and a probability of 0.95 for a gene to belong to a cluster, no new clusters could be observed (data not shown). In fact, the total number of gene expression patterns put into a cluster (866) hardly exceeded the 826 strongly modulated profiles that were clustered in the previous experiment. Since hierarchical clustering defines similarity ‘in shape’, without emphasising the magnitude of changes in expression, it provides a more suitable method for an in-depth analysis of gene expression profiles. For example, two groups of co-expressed genes with interesting profiles were selected from the overview picture (see Fig. 3). Cluster A contains genes that are downregulated during the complete process of meiosis; transcriptional activity is restricted to pre-and post-meiotic stages. In contrast, genes belonging to cluster B (Fig. 3) show elevated levels of gene expression during meiosis. In particular, a subgroup of co-expressed genes (indicated by an arrow, Fig. 3) is strongly downregulated in the last sample, taken after meiosis, as can be deduced from the bright green colour allocation. As yet, no cDNA-AFLP fragments belonging to these specific clusters have been characterised further by sequencing.

Figure 3 Hierarchical average linkage clustering of entire dataset of quantified expression patterns; selected clusters. A Cluster A contains genes that are downregulated during the complete process of meiosis with transcriptional activity restricted to pre-and post- meiotic stages. B Cluster B contains genes with elevated levels of gene expression during meiosis. Black arrow: indicates a subgroup of co- expressed genes strongly down- regulated in the postmeiotic sample. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 postmeiotic (see also appendix) bw-cnudde 23-08-2004 14:49 Pagina 132

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In the light of these results, it seems reasonable to assume that our treshold level, which was in fact chosen arbitrarily, is an adequate quantitative measure for the overall clustering of modulated gene expression patterns. However, it is quite clear that low- magnitude transcriptional changes can be biologically significant (Hughes et al., 2000). Therefore, additional experiments would be required to analyse subtle differences in gene expression during plant meiosis.

Correlation of transcriptional activity with major biological events during meiosis

The results of our cluster analysis showed that meiotic genes are expressed in a transcriptional cascade. In a next step, we checked if the proposed biological function of the characterised fragments (see Chapter IV) fits with the gene expression profiles that were generated by our cDNA-AFLP approach. Therefore, each functional class – defined on the basis of sequence homology – was analysed separately by hierarchical clustering. Since genes involved in the same biological processes are more likely to be co-expressed, we cannot only evaluate the efficiency of our functional classification, but we can also extend expression profiling to gene discovery.

Figure 4 Hierarchical average linkage clustering of expression profiles belonging to sequenced cDNA- AFLP fragments modulated during meiosis. A-C Functional classes defined in Chapter IV, (see also appendix) bw-cnudde 23-08-2004 14:49 Pagina 133

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DNA replication and modification

As shown in Fig. 4A, genes involved in DNA replication and modification are most abundantly expressed in premeiotic and early prophase I stages. This correlates well with the extensive changes in chromatin structure that occur at early meiosis. In comparison, Breyne et al. (2002) reported high levels of histone gene expression during S-phase of plant mitosis, in agreement with their role in chromatin remodeling. We observed two subgroups in the hierarchical clustergram, differing in premeiotic expression. The largest group is broadly expressed from the premeiotic stage up to zygotene, possibly suggesting a function during premeiotic DNA synthesis as well as during meiosis. A second group of genes shows transcript accumulation during leptotene and zygotene, in agreement with a proposed role in altering the chromatin structure during DNA repair and homologous recombination (Shen et al., 2000). Fragments homologous to different histone H2B- encoding genes can be found in both subclusters, suggesting different regulatory mechanisms at the transcriptional level. For transcript tags with low levels of homology in a database search, such as topoisomerase II (M283), a chromatin boundary protein (M416) and a SWI/SNF-related actin-dependent regulator of chromatin (M295), their co-expression with well-characterised, DNA replication/modification-associated genes provides extra evidence for a genuine function in chromatin remodeling.

Proteolysis

Protein degradation is, besides transcriptional control, an important regulatory mechanism governing a coordinated progression through meiosis. The expression patterns of identified proteolytic genes were clustered (Fig. 4B), allowing a further subdivision into an ‘early’ group of tags encoding proteins involved in ubiquitin- mediated proteolysis and a ‘late’ group of protease-encoding genes. The significant representation and coordinated expression of ubiquitin-related genes and components of the SCF proteolytic complex reveals a global pathway activation at the initiation of meiosis, suggesting an important regulatory role of the proteolytic machinery, like in mitosis (Peters, 1998; Zachariae and Nasmyth, 1999). However, the presence of a second group of protease-encoding genes, including a tomato meiotic serine protease gene (M76) is an indication that additional proteolytic mechanisms come into play later during plant meiosis. bw-cnudde 23-08-2004 14:49 Pagina 134

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Signal transduction

We identified a large collection of signal transduction components in our screening, reflecting the complexity and the multitude of mechanisms involved in progression and control of the meiotic cell cycle. In most instances, only low levels of homology were encountered in a BLAST database search, restricting our ability to deduce a specific function in the meiotic regulatory machinery for a particular transcript tag. However, the extra information provided by the temporal expression patterns of these candidate signal transduction genes (Fig. 4C) often allows us to speculate more extensively on the definition of a possible gene function. For example, a tag with a low level of homology to a yeast MAP-kinase (M259) is upregulated towards the end of meiosis, making a crucial role during meiosis unlikely, while a fragment homologous to protein phosphatase 2C (M45) seems only active before and after meiosis, where it can possibly act as an inhibitor for meiotic gene expression. In contrast, an Arabidopsis putative protein kinase (M22) is expressed during the whole meiotic process, making it an eligible target for further characterisation. Of particular interest are the kinases that are activated during the meiosis I-to-II transition. These fragments are candidates for the characteristic repression of the S- phase after exit from meiosis I. The regulation of this transition has been unraveled in Xenopus and starfish oocytes, revealing a complex signaling network (Kishimoto, 2003). Furthermore, some cDNA-AFLP fragments have been found to represent genes that play a role in the perception and response to DNA damage. A putative phosphatidylinositol-4-phosphate-5-kinase (M36), a tomato myo-inositol-1- phosphatase (M98) and a hypersensitive-induced response protein (M16) are expressed early during meiosis and show peak expression levels at leptotene, suggesting a specific function in the response to DNA double strand breaks initiating the recombination pathway. This proposed role is further supported by the characterisation of the Arabidopsis AtATM protein kinase (Garcia et al., 2003), which is also a member of the phosphatidylinositol signaling pathway whose activity is induced by DNA damage, particularly DSBs. Other protein kinases and a putative elicitor response protein (M271) most likely have a different function, since they are expressed later in meiosis, from pachytene up to the end of the second meiotic division. Finally, it is remarkable that the two fragments representing the checkpoint control proteins Bub1 and Bub3 (M342 and M21, respectively) are expressed well in advance of the meiosis I division. Both proteins have been identified as essential components of the mitotic and meiotic spindle checkpoint machinery (Amon, 1999; Yamaguchi et al., 2003), controlling the proper attachment of the kinetochores to the spindle microtubules at metaphase I. Recently, it has been shown by Kitagawa et al. (2003) that bw-cnudde 23-08-2004 14:49 Pagina 135

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Bub1 associates with centromere DNA via Skp1, a component of the SCF ubiquitin- conjugating complex, during mitosis in budding yeast. In our screening, we found upregulated transcript fragments for both proteins, supporting a similar interaction during meiosis in Petunia hybrida.

Figure 5 Hierarchical average linkage clustering of expression profiles belonging to sequenced cDNA-AFLP fragments modulated during meiosis. A-C Functional classes defined in Chapter IV, (see also appendix)

Cytoskeletal assembly and organisation

Interestingly, the same ‘early’ expression profile can be found for the cDNA-AFLP fragment representing p23 (M445) (Fig. 5A), a tubulin-binding protein that also associates with Skp1 to initiate kinetochore formation in mitotically dividing yeast cells (Russel et al., 1999). Thus, coupling the yeast protein data (kinetochore localisation) with our transcript profiling data in Petunia (co-expression), we can assume that Bub1, Skp1, and p23 are coregulated during meiosis and act together as components of a centromere-binding complex in plants. Other interesting observations from Fig. 5A are the strong downregulation of actin (M304) after meiosis and the broad expression profiles for ADP-ribosylation factors (M422, M430), which apparently play important roles during membrane trafficking steps in meiotic cells. Furthermore, genes with a presumed function in the assembly of the meiotic spindle, such as MEI-1 (M317), an Arabidopsis kinesin-like protein (M253), bw-cnudde 23-08-2004 14:49 Pagina 136

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a Ran GTP-binding protein (M51) and a microtubule-associated protein (M77) are expressed early in meiosis, well in advance of the actual appearance of the spindle apparatus and the attachment of the bivalents at metaphase I. A possible explanation is that these proteins require some time to promote microtubule growth and stability, as has been shown for Ran-GTP (Bucciarelli et al., 2003). A similar early induction pattern for cytoskeletal genes was observed during mitotic cell division in tobacco (Breyne et al., 2002). Finally, the weak homology with the C. elegans MEI-1 gene is further supported by co-expression with an Arabidopsis kinesin-like protein, indicating a common motor protein function for movement along the microtubules.

DNA repair and recombination

As shown in Fig. 5B, the identified fragments representing genes involved in DNA repair and meiotic recombination can be further subdivided into an early and a late group. A tag homologous to the well-known, meiosis-specific DMC1 gene (M56) exhibited the expected expression pattern: high mRNA levels were detected in early prophase I from leptotene up to zygotene, in agreement with the proposed function of single-strand invasion in the DSB repair model of meiotic recombination (Szostak et al., 1983). According to this model, the strand invasion step is followed by the formation of a joint molecule containing two Holliday junctions. In our cDNA-AFLP screening, we isolated a short fragment (M115) homologous to an Arabidopsis outer envelope membrane protein, but also showing a low level of homology to a bacterial Holliday junction RuvB DNA helicase. The expression pattern of this tag further supports a possible function in the formation of a joint molecule at the pachytene stage. Apart from DMC1, another RecA-like gene was identified in our screening (M229). However, this fragment is maximally expressed at the pachytene stage, indicating a different function during meiotic recombination. Known DNA repair genes such as DRT100 (DNA-damage repair/toleration) (M343), a Rad50-homologue (M95) and a cDNA fragment encoding a putative nucleotide repair protein (M458) are expressed later in meiosis, together with several helicase genes (M222, M230, M287). They are likely involved in the last steps of the recombination pathway: Holliday junction resolution leading to the formation of heteroduplexes and the correction of mismatched basepairs. bw-cnudde 23-08-2004 14:49 Pagina 137

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Synapsis and sister chromatid cohesion

A Petunia homologue of the Arabidopsis ASY1 gene is nearly the only link with synapsis that could be identified in our screening. To date, ASY1 is still the only plant protein known to be associated with the SC. It is also one of the first plant meiotic proteins of which the expression has been investigated in immunolocalisation studies (Armstrong et al., 2002), providing the unique opportunity of comparing the expression of a meiotic gene at the transcriptional and the translational level. As shown in Fig. 5C, the two ASY1 fragments (M203 and M204) are expressed in the premeiotic stage and during leptotene. This timing corresponds to the initiation of axial element development that precedes the start of chromosome synapsis at zygotene. The ASY1 protein appears in Arabidopsis as punctate foci during premeiotic interphase. As prophase I proceeds, the signal becomes increasingly more continuous and is closely associated with the axial elements of unsynapsed chromosomes and the synaptonemal complexes of synapsed chromosomes, persisting until the diplotene stage (Armstrong et al., 2002). This indicates that the ASY1 protein remains relatively stable for 24h after transcription ceases at leptotene. The appearance of continuous ASY1 antibody signals has also been used in a meiotic time-course study for Arabidopsis thaliana to define the onset of leptotene, which is difficult to determine in DAPI-stained nuclei (Armstrong et al., 2003). Furthermore, the expression pattern of two cDNA-AFLP tags (M409 and M468) is in agreement with their homology to SMC-proteins. Both fragments are induced late in meiosis, which would be in accordance with a proposed function in sister chromatid cohesion and chromosome segregation (Cobbe and Heck, 2000).

RNA processing and protein biosynthesis

In our screening, we identified a collection of transcripts representing ribosomal proteins (M19, M33, M90, M209, and M449) and translation initiation (M54) and elongation (M99) factors that are upregulated premeiotically and early in meiosis (data not shown). This may be correlated with increased translation rates during meiosis, required for the synthesis of a whole set of meiosis-specific proteins and the high levels of protein synthesis during meiosis.

An overview of the distribution of these functional classes over the five major clusters is presented in Fig. 6. bw-cnudde 23-08-2004 14:49 Pagina 138

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ClusterCluster 1 UnknowUnknown CClusterluster 2 GametogenesiGametogenesis CClusterluster 3 DDNNA rreplicationeplication, ubiiquitiquitin-mediate-mediated pproteolysisroteolysis, ssynaptonemaynaptonemal ccompleomplex fformationormation, spindlspindle aassemblyssembly, RRNNA processingprocessing, pproteirotein bbiosynthesiiosynthesis CClusterluster 4 PProteasesroteases, signasignal ttransductioransduction (kkinaseinases, pphosphotasehosphotases), cchromosomhromosome ssegregationegregation, DNDNA repairepair ClusterCluster 5 DDNNA modificationmodification, checkpoincheckpoint controlcontrol, recombinationrecombination, mmembranembrane transportransport

Figure 6 Distribution of functional classes over the 5 major clusters (see also appendix)

Correlations between gene expression and homology search results

In a next step, we analysed whether a correlation could be found between the expression profile of an isolated gene fragment and our success rate of finding a significant match in a homology search. Therefore, we looked at the distribution of the transcript tags for which a significant homology to a known gene was found in a BLAST database search over the different clusters. Using the same settings as stated above, we applied the adaptive quality-based clustering algorithm to our data set of 413 gene expression profiles of the fragments that were characterised by sequencing. In total, 475 tags have been isolated and directly sequenced. However, the expression patterns of 62 tags are missing in our database because they have not been quantified by the AFLP-QuantarPro image analysis software. Three major clusters could be identified (Fig. 7), similar to clusters B, C and D in Fig. 2. Since this data set is comprised of gene fragments modulated specifically during meiosis, fragments expressed exclusively premeiotically and postmeiotically (Fig. 2, clusters A and E, respectively) were absent. As shown in Fig. 7, transcript tags showing a significant homology in a BLAST database search are unequally distributed over these clusters. In particular, fragments expressed late during meiosis (cluster 1) have a considerable smaller chance of finding a match in the BLAST database than early meiotic fragments (clusters 2 and 3). Of the 100 fragments belonging to cluster 1, only 8 showed a significant homology (8%), bw-cnudde 23-08-2004 14:49 Pagina 139

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compared to 18 of the 66 cluster 2 fragments (27%) and 16 of the 50 cluster 3 fragments (32%). This disproportionate number of Petunia genes without homology may indicate that highly specialised processes are functioning towards the end of prophase I and during the meiotic divisions. Furthermore, our data suggest that genes belonging to this cluster are poorly represented in public databases, probably because their expression is restricted to a short timeframe during meiosis.

Figure 7 Distribution of sequenced cDNA-AFLP fragments over the different clusters and their chance to yield a significant homology to a known gene in a database search

Discussion

Identification of meiotically regulated transcripts

Quantification of the gene expression data generated by cDNA-AFLP transcript profiling opens up new possibilities for employing computational analysis in the study of gene expression. For example, it provides an objective scoring method for modulated expression profiles based on statistical analysis. In our screening, we used a threshold level corresponding to a 3-fold transcriptional change, resulting in a data set of 978 modulated expression profiles. This collection included nearly all 475 transcript tags that were isolated for direct sequencing (see Chapter IV) after visual inspection, confirming the efficiency of our previous selection of genes modulated specifically during meiosis. For the rest, it contained the profiles of many genes that are expressed exclusively premeiotically and postmeiotically and often show drastic transcriptional changes. bw-cnudde 23-08-2004 14:49 Pagina 140

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In total, 7,408 expression profiles have been quantified, 978 of which were significantly modulated. In other words, in the sampled anthers of Petunia hybrida, 13% of the visualised transcriptome shows significant differential gene expression during meiosis. Curiously, a similar percentage of the transcript tags were significantly modulated during mitotic cell division in tobacco (Breyne et al., 2002). In comparison, microarray- based genome-wide expression studies revealed dramatic global transcriptional changes in yeast meiosis: almost 2,000 genes were significantly upregulated at least twofold during meiosis in Schizosaccharomyces pombe (41% of all genes) (Mata et al., 2002), while Primig et al. (2000) reported 1,600 meiotically regulated genes with at least a 4-fold induction (26% of all genes) in Saccharomyces cerevisiae. Our identification of a gene as meiotically regulated was objective in the sense that it was entirely quantitative. Nevertheless, it should be clear that the setting of the threshold is in fact arbitrary and it cannot be excluded that certain key regulators of meiosis do not meet the criteria for inclusion in our data set of modulated genes, for example because they are also expressed outside the meiotic process. Interestingly, many Arabidopsis genes, including DMC1 (Doutriaux et al., 1998), MS5 (Glover et al., 1998), SYN1/DIF1 (Bai et al., 1999; Bhatt et al., 1999), ASY1 (Caryl et al., 2000), SPO11 (Hartung and Puchta, 2000), SWI1 (Mercier et al., 2001) and AHP2 (Schommer et al., 2003) encode proteins that are on current evidence entirely specific to meiosis, yet transcripts from them are detected in other vegetative tissues, in contrast with their counterparts in yeast. This suggests that a substantial amount of meiotic gene expression is regulated at a post- transcriptional level in Arabidopsis, and probably in other plants (Mercier et al., 2003), which could prevent them from being selected in our screening. Furthermore, low- magnitude transcriptional changes may actually be biologically significant.

Analysis of the transcriptional cascade

We performed a cluster analysis on our data set to identify groups of genes with highly similar expression profiles. We used two complementary clustering algorithms: hierarchical average linkage clustering (Eisen et al., 1998) for the generation of an overview picture and adaptive quality-based clustering (De Smet et al., 2002) for a classification of co-expressed genes. As a result, a transcriptional cascade can be observed that governs initiation and progression through the meiotic cell cycle, as has been demonstrated in a temporal analysis of the meiotic transcriptome in yeast (Chu et al., 1998; Primig et al., 2000; Mata et al., 2002). These successive waves of transcription reveal a high level of coordination and a precise temporal activation of meiotic genes, stressing the importance of regulation at the transcriptional level. bw-cnudde 23-08-2004 14:49 Pagina 141

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We identified five large clusters of co-expressed genes (Fig. 2): premeiotic, downregulated, early meiotic, late meiotic, and postmeiotic. Subsequently, we checked the correlation with major biological processes during meiosis. Premeiotically expressed genes (235) are strongly repressed upon entry into meiosis. Fragments belonging to this class have not been characterised further by sequencing. However, it can be hypothesised that it contains genes involved in premeiotic DNA replication and mitotic regulatory proteins that must be switched off to allow a normal initiation of the meiotic cell cycle. In comparison, in S. cerevisiae, transcripts from more than 600 genes diminished in the course of sporulation (Chu et al., 1998). However, in this case, initial repression may reflect the cessation of growth upon nitrogen starvation. Further, 80 Petunia cDNA-AFLP fragments were downregulated, showing a delayed decrease in transcript levels up to the end of the zygotene stage. Many of the transcript tags with this expression pattern have roles in ubiquitin-mediated proteolysis, RNA processing and protein biosynthesis, showing the preparations of the meiotic cell for the recruitment of a specialised set of proteins upon commitment to meiosis. Homologies to genes involved in DNA replication, synapsis of homologous chromosomes (ASY1) and spindle assembly may provide additional evidence for the close relationship between the initiation of meiosis and the premeiotic S-phase, as has been shown in yeast (Borde et al., 2000; Watanabe et al., 2001). About 98 fragments showed a pattern of expression characterised by an early induction at leptotene, followed by a decrease in transcript levels at zygotene. Characterised genes with this transient induction pattern are likely to encode meiosis- specific proteins, involved in meiotic recombination (DMC1), DNA modification, checkpoint control and membrane transport. This cluster is comparable to the ‘early gene’ clusters distinguished in budding yeast (Chu et al., 1998; Primig et al., 2000) and fission yeast (Mata et al., 2002). A second cluster of meiosis-specific genes comprises about 108 fragments following a pattern of expression characterised by a ‘late’ induction at pachytene and sustained expression during the meiotic divisions. These transcript tags showed a homology to genes encoding DNA repair proteins that are likely to be involved in Holliday junction resolution and mismatch repair. Further, homologies are found with proteases, signal transduction proteins (kinases, phosphatases) and chromosome segregation proteins (SMCs). Compared to the gene expression patterns of the ‘middle’ genes in yeast (Chu et al., 1998; Primig et al., 2000; Mata et al., 2002), the expression of Petunia fragments belonging to this cluster is more repressed in the postmeiotic stage, probably reflecting differences in the timing of the last sample. Finally, about 200 genes are induced after completion of the meiotic divisions. They putatively represent gametogenesis-related genes, many of which may have unique bw-cnudde 23-08-2004 14:49 Pagina 142

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functions during microspore development. They correspond to the anther-specific transcripts that were identified during a previous cDNA-AFLP transcript profiling experiment on developing Petunia hybrida floral organs (see Chapter II, Cnudde et al., 2003). A similar cluster of postmeiotically induced genes is present in yeast (Chu et al., 1998; Primig et al., 2000; Mata et al., 2000). However, these genes encoding proteins involved in spore maturation and ascus formation show low levels of homology with plant genes (Cnudde et al., 2003).

Identification of coregulated genes

In general, the putative biological function of a gene, as proposed on the basis of sequence homology, corresponded well with the accompanying transcript profile. Transcript fragments that are nearly identical in sequence show homology to the same gene (e.g. genes encoding skp1, ubiquitin conjugating enzyme, thiol protease) are tightly co- expressed (Fig. 4B), suggesting a common transcriptional regulation. Furthermore, because genes with related functions tend to be expressed in similar patterns, cluster analysis may provide a basis for the functional characterisation of as yet unknown co- expressed genes. Accordingly, we found additional support for the proposed functions of several isolated fragments with low levels of homology, based on their temporal association with meiotic genes of known function. Likewise, strict coregulation of genes encoding proteins involved in successive steps of the cell cycle has been demonstrated in plants (Breyne et al., 2002) and gene expression profiling data have been successfully used to infer gene functions in yeast meiosis (Toth et al., 2000). Among the gene fragments identified, those with meiosis-specific expression profiles are of particular interest because they may encode critical proteins involved in unique meiotic processes, such as recombination and synapsis. In this way, a targeted selection can be carried out to identify candidate gene fragments with a function in a particular aspect of meiosis. Future studies, including mutational analysis, is required for a further investigation of their specific role during meiosis. In yeast, this reverse genetics approach using gene expression data has been shown to be an effective method for identifying previously uncharacterised genes involved in specific meiotic processes (Rabitsch et al., 2001). The high potential of a similar approach in plants is shown by the detection of a disproportionately large number of unknown cDNA-AFLP fragments expressed during late meiosis. This may well reflect a backlog in the molecular analysis of a whole group of coregulated genes, resulting in an underrepresentation in public databases. This might be because their expression is restricted to a short timeframe during meiosis, indicative of a function during specialised processes. Such genes would be missed in a majority of the bw-cnudde 23-08-2004 14:49 Pagina 143

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large-scale EST sequencing projects. Furthermore, unknown genes have a greater chance of being wrongly annotated in genome-wide sequencing projects (Wang et al., 2003). Another explanation might be that genes belonging to this cluster show a lower degree of cross-species conservation, as has been shown for meiotic genes in fission yeast by Mata and Bahler (2003). They recently reported a particular enrichment of S. pombe-specific genes induced during meiotic prophase in an experiment studying gene conservation. Since Petunia fragments involved in signal transduction often show a late meiotic induction pattern, it can be hypothesised that a specialised, plant-specific signaling pathway has evolved, mediating progression through the meiotic cell cycle.

Unraveling transcriptional regulation of meiosis in plants

The results of our cluster analysis indicate that meiotic progression in Petunia is propelled by a precise temporal activation of transcription cascades, as has been shown in yeast meiosis (Mitchell, 1994; Kupiec et al., 1997). So far, many meiosis-specific transcription factors have been identified in yeast (Vershon and Pierce, 2000). Especially the immense amount of data generated by large-scale expression studies has contributed to a better understanding of the regulatory network that controls the different stages of meiosis. Examining the promoters of genes in each temporal class has served as a useful method to identify common transcriptional regulatory elements (Chu et al., 1998; Primig et al., 2000). In plants, however, little is known about the promoters, transcription factors and regulation of genes required for meiosis. We may suspect that most of the key regulators in this process were not detected by our search strategy based on modulated expression during meiosis, probably due to the low cellular concentration typical of transcription factors. No plant homologues can be identified for well-known transcription factors in budding yeast, such as Ime1, Ume6, and Ndt80 (Vershon and Pierce, 2000), suggesting the development of a unique regulatory network governing the initiation and progression through meiosis in plants. This would not be surprising since meiosis in flowering plants is largely determined within the floral developmental program (Bhatt et al., 2001) making the overall transcriptional regulatory pathways in yeast and mammalian meiosis irrelevant, since the former is primarily designed to receive signals from nutritional starvation while the latter is tuned for hormonal stimulus (Hwang et al., 2001). The results of our cluster analysis may provide a foundation for unraveling the transcriptional mechanisms of meiotic cell cycle regulation in plants. Since the Petunia hybrida genome has not yet been sequenced, we propose that computational analysis of the promoters of potentially coregulated genes is carried out in Arabidopsis orthologues bw-cnudde 23-08-2004 14:49 Pagina 144

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to screen for common transcription factor binding sites. Furthermore, we identified in a database search several transcript tags homologous to zinc-finger proteins with a potential regulatory role (see Chapter IV). In conclusion, examination of the temporal ordering of gene expression patterns by cluster analysis opens new perspectives for gene discovery and reverse genetics approaches, by correlating transcriptional activity with major biological events during meiosis. Furthermore, it allows us to make cross-species comparisons of the transcriptional cascades of well-studied model systems such as yeast, and may provide a first step towards the elucidation of underlying transcriptional regulatory networks that control the complex and highly regulated processes that occur during meiosis.

Acknowledgements

We wish to thank Rozemarijn Dreesen for useful advice on the statistical analysis of the data obtained. The voluntary assistance of Bernard Cannoot in the QuantarPro analysis and of Etienne and Sabine Cnudde in the setup of the database was greatly appreciated.

References

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Chapter VI

Cellular localisation of a selected subset of modulated Petunia hybrida transcripts by in situ hybridisation

Filip Cnudde, Koen Weterings, Liesbeth Pierson, Tom Gerats

A part of this chapter is submitted to Plant Physiology bw-cnudde 23-08-2004 14:49 Pagina 148

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Introduction

Combining a cytology-based sampling of developing Petunia anthers with cDNA- AFLP-based transcript profiling, we were able to build a comprehensive inventory of genes with a modulated expression pattern during male meiosis (see Chapter III). The use of transcript profiling technology permitted the temporal expression profiles of hundreds of genes to be simultaneously examined, resulting in a collection of 475 differentially expressed transcript fragments. However, we cannot extend this high- throughput approach for gene discovery to the analysis of gene function. In particular, technical limitations and extensive series of cumbersome sample treatments limit the numbers and types of analyses that can be performed. New tools are being developed, such as high-throughput in situ RT-PCR (Koltai and Bird, 2000), but so far, conventional methods are still widely applied. Forced into a reductionist approach, we selected a subset of five modulated fragments for further analysis. Since the majority of primer combinations in the cDNA-AFLP transcript profiling experiment has not been repeated, our first goal is to confirm the observed modulations in mRNA abundance. It has been shown earlier that the kinetics of expression revealed by cDNA-AFLP analysis are comparable to those found by Northern analysis (Bachem et al., 1996). However, we may expect that the expression levels of many meiotically regulated genes are too low to allow detection by Northern blots, as, for example, reported in Arabidopsis thaliana for AHP2 (Schommer et al., 2003). Although semi- quantitative RT-PCR using gene-specific primers could be an alternative, we preferred to do in situ hybridisations. This PCR-independent method allows us to determine both temporal and spatial expression patterns. Hence, we can check whether the localisation of specific gene expression to particular tissue types follows the earlier determined modulation and whether these data can give us a further clue to the gene’s putative function. Since complete anthers were used for our sampling in the transcript profiling experiment, the amplicons detected in our screening reflect only the average transcript level in all cells and tissues that comprise the anther sample. The extra spatial information provided by in situ hybridisation allows us to determine whether the expression of a gene is restricted to the microsporocytes - suggesting a meiosis-specific function -, or whether it is distributed over different cells and tissue types of the anther. Moreover, the use of transverse sections of complete Petunia flower buds allows an immediate comparison with adjacent flower tissues, such as sepals, petals and ovaries. In general, the results of the in situ hybridisation experiments are in line with the gene expression patterns generated by cDNA-AFLP transcript profiling. The observed modulations in mRNA abundance during male meiosis in Petunia could be confirmed for a subset of five selected genes, indicating that our screening was basically effective. bw-cnudde 23-08-2004 14:49 Pagina 149

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Furthermore, the localisation of gene expression to different anther tissues provided useful information for subsequent functional analysis studies.

Experimental procedures

cDNA-AFLP transcript profiling to analyse gene expression during male meiosis

We have used cDNA-AFLP transcript profiling to analyse changes in gene expression during male meiosis in Petunia hybrida. Our sampling strategy, based on a cytological determination of the meiotic stages, is described in the experimental procedures of Chapter III. The cDNA-AFLP-based transcript profiling procedure is also explained in Chapter III. Protocols for the recuperation of cDNA-AFLP fragments, sequencing and characterisation by homology searching can be found in Chapter IV. The procedures for the analysis of the temporal gene expression patterns are presented in Chapter V.

In situ hybridisation

In situ RNA hybridisation experiments were performed as described by Cox and Goldberg (1988) with minor modifications. Immediately after the excision of one anther for the cytological analysis, Petunia flower buds were fixed overnight at 4ºC in 0.25% (w/v) glutaraldehyde, 4% (w/v) paraformaldehyde solution prepared in 0.01M phosphate buffer (pH 6.8), and subsequently dehydrated, cleared and embedded in paraffin. Ten-micrometer sections were hybridised to [33P]-UTP-labeled RNA probes at a specific activity of 4 to 5 x108 dpm/µg. Sense and antisense RNA probes were synthesised using the cDNA-AFLP tags cloned into a pGEM-T vector. After hybridisation and emulsion development, digital photographs were taken with a CoolSNAP CCD camera (Roper Scientific, USA) using either bright-field or dark-field illumination on a Leitz Orthoplan (LSM, Wetzlar, Germany). The images were enhanced using Adobe Photoshop 5.0 (San Jose, CA). bw-cnudde 23-08-2004 14:49 Pagina 150

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Results and discussion

Probe selection and sectioning

We selected five cDNA-AFLP tags for further characterisation by in situ hybridisation on the basis of expression pattern, fragment length and homology in a database search. Of these, transcript tags homologous to two well-known meiotic genes in Arabidopsis, DMC1 (M56) and ASY1 (M203), served as a control. Expression patterns for both genes, involved in meiotic recombination and synapsis, respectively, are well defined at the mRNA and protein level. The remaining three fragments are homologous to genes encoding a poly(A) ribonuclease (PARN) (M245); Bub1, a checkpoint protein (M342) and a C3HC4 zinc finger protein (M407). They were selected on the basis of their gene expression pattern, since they all belong to different clusters as defined in Chapter V. These cDNA-AFLP tags, ranging from 200-400bp in size, were used to make [33P]- UTP-labeled RNA probes for the detection of mRNA species in situ. 10-12 Transverse sections of young Petunia flower buds undergoing male meiosis were mounted on an individual slide. One anther was dissected from a young Petunia flower bud for the cytological determination of the meiotic stage, while the rest of the bud was fixed. We used transverse sections of young Petunia flower buds to see the morphology of the remaining four anthers and their locules. While young anther sacs hold well together, older anthers, at meiosis II and later stages, tend to lose the cells from within the locules during sectioning, because the pollen precursors become progressively more separated from each other and the surrounding cells (Aragon-Alcaide et al., 1998). However, we have obtained good intact sections showing early meiosis I stages. Figures 1 to 5 show a series of in situ images of flower buds at different stages before and during male meiosis. In each case, four anthers are present with locules comprising the central meiocytes (microsporocytes) and the surrounding tapetal cell layer. Furthermore, adjacent flower tissues can be observed, such as sepals, petals and ovaries. Interestingly, no obvious differences in gene expression could be observed on the in situ’s between the smallest anther and the remaining three anthers in the Petunia flower bud. Based upon the cytological staging of samples, we had previously decided to discard the smallest anther because meiosis did not occur synchronously with the other anthers (see Chapter III). Probably this discrepancy can be explained by the prolonged expression over different meiotic stages for these selected genes. bw-cnudde 23-08-2004 14:49 Pagina 151

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Figure 1 Expression analysis of DMC1 homologue M56. Upper panel cDNA-AFLP expression pat- tern. 1,2 premeiotic; 3 leptotene; 4 leptotene – zygotene; 5 early zygotene; 6 zygotene; 7 pachytene; 8 metaphase I; 9 dyad stage; 10 telophase II; 11,12 postmeiotic. Lower panel In situ hybridisation using [33P]-UTP-labeled RNA probe on sections of Petunia flower buds, exposed for 35 days and examined using dark-field illumination. A Sense, B-H Antisense. A,B premeiotic; C leptotene; D zygotene; E late zygotene – pachytene; F pachytene; G diplotene; H tetrad. Bar = 0.5mm.

In situ hybridisation of DMC1

According to the results of our cDNA-AFLP transcript profiling experiment, DMC1 is an early meiotic gene, expressed specifically during leptotene and zygotene (see Fig. 1). Using this fragment as a probe for in situ hybridisation, high levels of DMC1 expression could be measured during early meiosis (signals detectable after 1 day). DMC1 mRNA was detected exclusively in the microsporocytes, in agreement with its proposed meiosis- specific function of single-strand invasion during meiotic recombination. However, while no premeiotic expression was visible in the cDNA-AFLP pattern, the in situ hybridisations showed a clear signal in the pollen mother cells before meiosis (Fig. 1). This difference reflects the difficulty in how best to define the onset of leptotene, as described earlier in Chapter III. Since the transition from G2 to leptotene is gradual and difficult to define precisely simply on the appearance of chromatin in stained preparations, our staging is probably less precise for these samples. Morphometric data are of limited use at this stage, as there is too much variation in bud and anther size, depending on developmental and environmental factors (Chapter III). Therefore, more precise strategies will be required to mark early leptotene stages, for example by relying on immunolocalisation techniques to follow the appearance of continuous ASY1 antibody signals along the entire length of the chromosomes, as used by Armstrong et al. (2003). bw-cnudde 23-08-2004 14:49 Pagina 152

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The strong DMC1 expression in microsporocytes of Petunia hybrida is in agreement with the in situ data and the results of AtDMC1 promoter:GUS protein gene fusions in Arabidopsis thaliana (Klimyuk and Jones, 1997), indicating a meiosis-specific expression pattern. In these experiments, DMC1 expression has been analysed in inflorescence buds at different stages of flower development. Unexpectedly, DMC1 expression has also been reported in dividing vegetative cells from root tips and cell suspensions (Doutriaux et al., 1998; Kathiresan et al., 2002). This ‘promoter leakiness’, seems to be a recurrent theme for plant genes encoding proteins that are on current evidence entirely specific to meiosis (Mercier et al., 2003). Expression and localisation experiments for DMC1 have also been performed at the protein level in Lilium longiflorum. Using Western blot analysis on a range of lily microsporocytes at prophase I stages, Terasawa et al. (1995) demonstrated high protein levels of Lim15, the DMC1 homologue of lily, at leptotene and zygotene, in agreement with our gene expression data. No expression could be detected in premeiocytes and diplotene microsporocytes. Further, immunolocalisation experiments were used to identify DMC1 proteins as discrete foci on leptotene and zygotene chromosomes, demonstrating that meiotic recombination begins at the leptotene stage and continues in zygotene.

Figure 2 Expression analysis of ASY1 homologue M203. Upper panel cDNA-AFLP expression pattern. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 post- meiotic. Lower panel In situ hybridisation using [33P]-UTP-labeled RNA probe on sections of Petunia flower buds, exposed for 35 days and examined using dark-field illumination. 4* diplotene. A Sense, B-H Antisense. A,B premeiotic; C leptotene; D zygotene; E pachytene; F diplotene; G tetrad; H pollen grain. Bar = 0.5mm. bw-cnudde 23-08-2004 14:49 Pagina 153

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In situ hybridisation of ASY1

Using cDNA-AFLP transcript profiling, high levels of ASY1 expression were detected in the premeiotic and leptotene stages (see Fig. 2). From zygotene on, the expression is downregulated. The two indicated fragments are likely to be derived from the same gene. It is not uncommon for abundantly expressed genes to show an extra ‘ghost band’ on the polyacrylamide gel. This expression pattern was confirmed by in situ hybridisation, showing high levels of ASY1 gene expression localised in premeiocytes and leptotene microsporocytes. This timing corresponds to the initiation of axial element development that precedes the start of chromosome synapsis at zygotene. A detailed expression analysis of ASY1 over a range of cytologically determined meiotic samples has not been presented earlier in plants. However, RT-PCR experiments have been performed to investigate the expression of the ASY1 gene in several tissues of Arabidopsis thaliana (Caryl et al., 2000). Again, high levels of gene expression were detected in non-meiotic tissues, in contrast to its yeast homologue, HOP1, which is specifically induced during meiosis (Hollingsworth et al., 1990). Using immunolocalisation, Armstrong et al. (2002) showed that the ASY1 protein begins to accumulate during premeiotic interphase as punctate foci and as meiosis progresses, labeling is clearly associated with chromosome axes as a continuous signal. This is maintained throughout pachytene, but begins to disappear as the homologues desynapse. These observations support the role of ASY1 in synapsis, which was suggested after cytological characterisation of the asy1 T-DNA insertion mutant in Arabidopsis (Ross et al., 1997).

In situ hybridisation of PARN

According to its gene expression profile generated by cDNA-AFLP transcript profiling, PARN is downregulated during meiosis and belongs to the same cluster as ASY1 (see Chapter V). This fragment, homologous to an Arabidopsis poly[A]-specific ribonuclease- encoding gene, was proposed to play a role in RNA processing mechanisms that control gene expression during meiosis (see Chapter IV). As shown in Fig. 3, PARN expression was detected before meiosis and in the leptotene and zygotene stages. In situ hybridisations confirmed the premeiotic and early prophase-specific expression pattern. Signals were first detectable after 35 days, indicating low levels of gene expression. PARN expression was not only measured in microsporocytes, but also in premeiotic ovules (see Fig. 3), indicating a general role during male and female bw-cnudde 23-08-2004 14:49 Pagina 154

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Figure 3 Expression analysis of PARN homologue M245. Upper right panel cDNA-AFLP expression pattern. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 postmeiotic. Upper left panel In situ hybridisation using [33P]-UTP-labeled RNA probe on sections of Petunia carpels, exposed for 67 days and examined using bright-field illumination. A Sense, zygotene; B Antisense, zygotene. Lower panel In situ hybridisation on sections of Petunia anthers. C Sense, D-F Antisense. C leptotene, D premeiotic, E leptotene, F zygotene. Bar = 0.1mm.

meiosis. As our screening for candidate meiotic genes focused on the identification of fragments modulated during male meiosis in the anthers of Petunia hybrida, as yet we have no information about their expression behaviour and possible function during female meiosis. The identification of several Arabidopsis mutants affected solely in male or female meiosis (see Chapter I, Table 2) indicates substantial differences between both meiotic processes. Therefore, generalisations of meiotic functions should always be treated with caution.

In situ hybridisation of Bub1

In our cDNA-AFLP transcript profiling experiment, we observed a modulated gene expression pattern for Bub1, a gene encoding a protein kinase involved in the spindle checkpoint pathway. Bub1 belongs to the ‘early meiotic’ gene cluster (see Chapter V), since the highest levels of gene expression were measured at the leptotene and zygotene stages (see Fig. 4). The results of our in situ experiment also indicated Bub1 gene expression in pollen mother cells before meiosis, in contrast with the faint band on the cDNA-AFLP gel for bw-cnudde 23-08-2004 14:49 Pagina 155

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the premeiotic stage (Fig. 4). In general, Bub1 transcripts seem to accumulate in all cell types of the anther during early meiosis. From pachytene on, signals can no longer be detected.

Figure 4 Expression analysis of Bub1 homologue M342. Upper panel cDNA-AFLP expression pattern. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 post- meiotic. Lower panel In situ hybridisation using [33P]-UTP-labeled RNA probe on sections of Petunia anthers, exposed for 67 days and examined using bright-field illumination. A Sense, B-E Antisense. A,B premeiotic; C leptotene; D zygotene; E late zygotene – pachytene. Bar = 0.1mm.

In situ hybridisation of a C3HC4 RING zinc finger protein-encoding gene

In our screening we identified an upregulated transcript tag homologous to a putative C3HC4-type RING zinc finger protein-encoding gene from Arabidopsis, a member of a large superfamily. According to the expression profile generated by cDNA-AFLP transcript profiling, the highest levels of gene expression were found later in meiosis, from pachytene up to the meiosis II division (see Fig. 5). Interestingly, the results of our in situ hybridisation confirmed the upregulated levels of gene expression of the C3HC4 zinc finger protein, but localised the expression to the tapetum, while no signal could be retrieved in the meiocytes (see Fig. 5). This is in contrast with the expression pattern of TAZ1, another anther-specific zinc finger gene that has been characterised in Petunia (Kapoor et al., 2002). In the premeiotic phase, TAZ1 transcripts were found to accumulate in all cell types of the bw-cnudde 23-08-2004 14:49 Pagina 156

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Figure 5 Expression analysis of RING zinc finger protein encoding-gene M407. Upper right panel cDNA-AFLP expression pattern. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 postmeiotic. Upper left panel In situ hybridisation using [33P]-UTP- labeled RNA probe on sections of Petunia flower buds, exposed for 67 days and examined using bright-field illumination. A Sense, B-C Antisense. A zygotene, B leptotene, C zygotene. Bar = 0.5mm Lower panel In situ hybridisation on sections of Petunia anthers. D Sense, E-H Antisense. D leptotene, E leptotene, F zygotene, G pachytene, H diplotene. Bar = 0.1mm.

anther except the tapetum and the gametophytic tissues, whereas the postmeiotic phase of anther development was characterised by expression exclusively in the tapetal cells. The importance of the tapetum for pollen development has been demonstrated by the observation that selective destruction of the tapetum in tobacco and oilseed rape results in the failure of pollen formation (Mariani et al., 1990, 1992; Denis et al., 1993). This idea is further supported by the finding that mutants that are defective in tapetum development also produce abnormal microsporocytes or pollen grains, e.g. ms3 mutants (Chaudhury et al., 1994). However, it has never been shown that the tapetum also affects male meiosis. In the Arabidopsis excess microsporocytes1 (ems1) mutant, for example, tapetal cells are lacking but microsporocytes progress normally through meiosis up to cytokinesis (Zhao et al., 2002). After differentiation, tapetal cells divide mostly anticlinally in Petunia, forming a single layer of columnar cells that lines the anther locule at the end of meiosis (Kapoor et al., 2002). During meiosis, tapetal cells also undergo nuclear divisions, resulting in an overall increase in DNA content from 2 to 4C (during meiotic prophase) to 7 to 8C by the end of meiosis (Liu et al., 1987). Because they are intensely involved in the replication of DNA, tapetal cells have been shown to lack poly(A) RNA. For this reason, the tapetum bw-cnudde 23-08-2004 14:49 Pagina 157

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has been suggested to play no active role in the nutrition of microsporocytes before and during meiosis (Raghaven, 1989; Kapoor et al., 2002). In the sequence of developmental events, the first known contribution of the tapetum to the developing microspores is the release of callase, which frees individual microspores by digesting the callose wall around tetrads (Frankel et al., 1969; Stieglitz and Stern, 1973). In the light of these results, we may suggest that this zinc finger gene has an important function in microspore development, but the restriction of its expression to the tapetum precludes a specific role in male meiosis.

Conclusions

Using radioactive in situ hybridisations, we could confirm the modulated gene expression pattern for all five selected genes, validating our cDNA-AFLP results. The extra spatial information provided by the in situ experiments allows a further selection of genes with a particular function during male meiosis, based on a meiocyte-specific gene expression pattern.

References

Aragon-Alcaide, L., Beven, A., Moore, G., and Shaw, P. (1998) The use of vibratome sections of cereal spikelets to study anther development and meiosis. Plant J. 14, 503-508. Armstrong, S.J., Caryl, A.P., Jones, G.H., and Franklin, F.C.H. (2002) Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica. J. Cell Sci. 114, 4207- 4217. Armstrong, S.J., Franklin, F.C.H., and Jones, G.H. (2003) A meiotic time-course for Arabidopsis thaliana. Sex. Plant Reprod. 16, 141-149. Bachem, C.W.B., van der Hoeven, R.S., de Bruijn, S.M., Vreugdenhil, D., Zabeau, M., and Visser, R.G.F. (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development. Plant J. 9, 745-753. Caryl, A.P., Armstrong, S.J., Jones, G.H., and Franklin, F.C.H. (2000) A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1. Chromosoma 109, 62-71. Chaudhury, A.M., Lavithis, M., Taylor, P.E., Craig, S., Singh, M.B., Signer, E.R., Knox, R.B., and Dennis, E.S. (1994) Genetic control of male fertility in Arabidopsis thaliana: Structural analysis of premeiotic developmental mutants. Sex Plant Reprod. 7, 17-28. Cox, K.H., and Goldberg, R.B. (1988) Analysis of plant gene expression. In Plant Molecular Biology: A Practical Approach, C.H. Shaw, ed., IRL Press, Oxford, pp 1-34. Denis, M., Delourme, R., Gourret, J.P., Mariani, C., and Renard, M. (1993) Expression of engineered nuclear male sterility in Brassica napus. Plant Physiol. 101, 1295-1304. Doutriaux, M.-P., Couteau, F., Bergounioux, C., and White, C. (1998) Isolation and characterisation of the RAD51 and DMC1 homologs from Arabidopsis thaliana. Mol. Gen. Genet. 257, 283-291. bw-cnudde 23-08-2004 14:49 Pagina 158

158 CHAPTER VI

Frankel, R., Izhar, S., and Nitsan, J. (1969) Timing of callase activity and cytoplasmic male sterility in Petunia. Biochem. Genet. 3, 451-455. Hollingsworth, N.M., Goetsch, L., and Byers, B. (1990). The HOP1 gene encodes a meiosis-specific component of yeast chromosomes. Cell 61, 73-84. Kapoor, S., Kobayashi, A., and Takatsuji, H. (2002) Silencing of the tapetum-specific zinc finger gene TAZ21 causes premature degeneration of tapetum and pollen abortion in Petunia. Plant Cell 14, 2353-2367. Kathiresan, A., Khush, G.S., and Bennett, J. (2002) Two rice DMC1 genes are differentially expressed during meiosis and during haploid and diploid mitosis. Sex. Plant Reprod. 14, 257-267. Klimyuk, V.I., and Jones, J.D.G. (1997) AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression. Plant J. 11, 1-14. Koltai, H., and Bird, D.M. (2000) High throughput cellular localization of specific plant mRNAs by liquid-phase in situ reverse transcription-polymerase chain reaction tissue sections. Plant Physiol. 123, 1203-1212. Liu, X.C., Jones, K., and Dickinson, H.G. (1987) DNA synthesis and cytoplasmic differentiation in tapetal cells of normal and cytoplasmic male sterile lines of Petunia hybrida. Theor. Appl. Genet. 74, 846-851. Mariani, C., De Beuckeleer, M., Truettner, J., Leemans, J., and Goldberg, R.B. (1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347, 737-741. Mariani, C., Gossele, V., De Beuckeleer, M., De Block, M., Goldberg, R.B., De Greef, W., and Leemans, J. (1992) A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357, 384-387. Mercier, R., Armstrong, S.J., Horlow, C., Jackson, N.P., Makaroff, C.A., Vezon, D., Pelletier, G., Jones, G.H., and Franklin, F.C.H. (2003) The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis. Development 130, 3309-3318. Raghaven, V. (1989) mRNAs and a cloned histone gene are differentially expressed during anther and pollen development in rice (Oryza sativa L.). J. Cell Sci. 92, 217-229. Ross, K. J., Fransz, P., Armstrong, S.J., Vizir, I., Mulligan, B., Franklin, F.C.H., and Jones, G.H. (1997) Cytological characterization of four meiotic mutants of Arabidopsis isolated from T-DNA transformed lines. Chromosome Res. 5, 551-559. Schommer, C., Beven, A., Lawrenson, T., Shaw, P., and Sablowski, R. (2003) AHP2 is required for bivalent formation and for segregation of homologous chromosomes in Arabidopsis meiosis. Plant J. 36, 1-11. Stieglitz, H., and Stern, H. (1973) Regulation of β-1,3-glucanase activity in developing anthers of Lilium. Dev. Biol. 34, 169-173. Terasawa, M., Shinohara, A., Hotta, Y., Ogawa, H., and Ogawa, T. (1995) Localization of RecA-like recombination proteins on chromosomes of the lily at various meiotic stages. Genes Dev. 9, 925-934. Zhao, D.-Z., Wang, G.-F., Speal, B., and Ma, H. (2002) The EXCESS MICROSPOROCYTES1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes Dev. 16, 2021-2031. bw-cnudde 23-08-2004 14:49 Pagina 159

Summary and perspectives bw-cnudde 23-08-2004 14:49 Pagina 160

160 SUMMARY AND PERSPECTIVES

Meiosis is a key feature of eukaryotic sexual reproduction. This special type of cell division has been studied for over 100 years at the cytological level using plants as attractive model systems. Upon the entry in the functional genomics era, however, most molecular and genetic studies on meiosis have been performed on yeast. Genome-wide gene expression studies using microarrays led to the identification of many previously unknown meiotic genes. Using bio-informatics tools, homologues of meiotically induced yeast genes were searched in other eukaryotes, often revealing intriguing differences in the meiotic transcriptome. So far, the molecular and functional analysis of meiosis is relatively underdeveloped in plants. However, the flood of genomics data from yeast research and the availability of large mutant collections, mainly in Arabidopsis, have induced a growing interest in molecular studies of plant meiosis, as described in the general introduction in Chapter I. We have developed a strategy to follow gene expression during male meiosis in Petunia hybrida, exploring the limits of the cDNA-AFLP technology. This experiment was a continuation of a previous study, described in Chapter II, in which we have demonstrated that cDNA-AFLP transcript profiling is a suitable method to analyse gene expression during male and female gametogenesis in Petunia. Reproductive and vegetative floral organs were sampled at five developmental stages and gene expression profiles were compared. This allowed us to assemble an inventory of 354 transcript tags, in which stamen-specific transcripts exhibiting an upregulation in gene expression were well represented. Further characterisation of the cDNA fragments showed that they corresponded to diverse classes of known gametogenesis genes, indicating that our screening was effective. In agreement with recent studies, a large number of as yet unknown genes were identified, proving that cDNA-AFLP transcript profiling is an efficient and sensitive technique to identify previously unknown transcripts. Since to our surprise almost no cDNA-fragments homologous to known meiotic genes could be retrieved from the gametogenesis experiments (Chapter II), we embarked on a risky enterprise: the elaboration of a functional genomics strategy aiming at the molecular dissection of meiosis in Petunia, a process described in Chapter III. Using DAPI-staining of enzyme-digested spread pollen mother cells to determine the different meiotic stages, we observed that the meiocytes develop rather synchronously through meiosis, except for the smallest anther, which was therefore discarded from sampling. Of the four remaining anthers, one was used for cytological analysis, while total RNA was extracted from the other three anthers for cDNA synthesis. The transcript profiling experiments focused on the identification of genes exhibiting a modulated expression during meiosis, while premeiotic archesporial cells and postmeiotic microspores served as a reference. About 8,000 transcript tags, estimated to represent 35% of the total transcriptome, were generated, 475 of which exhibited a bw-cnudde 23-08-2004 14:49 Pagina 161

Summary and perspectives 161

modulated gene expression pattern. Among these, we find many components of meiotic processes, indicating that our screening was basically effective and demonstrating the high level of sensitivity of cDNA-AFLP transcript profiling. A thorough characterisation of these fragments based on sequence homology is presented in Chapter IV. Of the 293 satisfactorily sequenced gene tags present in our collection, 90 fragments (30%) displayed a significant level of homology in a GenBank database search using the BLAST algorithm. A functional classification of these tagged genes, based on literature datasearches, demonstrated that many components of meiosis have been encountered, overall covering the complete process of meiosis. We could confirm the high level of evolutionary conservation of genes involved in meiotic recombination and DNA repair, processes well represented in our screening. In contrast, components of less conserved processes, such as synapsis, might well be present in our collection, but cannot be recognised on the basis of a homology search. Although incomplete, our comprehensive data set provides a starting point for the further unraveling of meiosis at a molecular level in plants, allowing a knowledge-based selection of tags for further characterisation. In Chapter V we present the first large-scale examination of the temporal ordering of gene expression patterns during plant meiosis. Quantification of the gene expression data generated by cDNA-AFLP transcript profiling allowed a computational analysis. Cluster analysis showed that a transcriptional cascade governs the initiation and progression through the meiotic cell cycle, stressing the importance of regulation at the transcriptional level. We identified five large clusters of co-expressed genes and could correlate these with major biological processes during meiosis. A disproportionately large number of Petunia transcript fragments induced during pachytene and later meiotic stages show no homology in a database search, suggesting a backlog in the molecular analysis of the genes belonging to this specific cluster. Furthermore, we found additional support for the proposed functions of several isolated fragments with low levels of homology in a database search, based on their temporal association with meiotic genes of known function. In Chapter VI we describe the spatial and temporal expression pattern of five modulated fragments by in situ hybridisation. One fragment homologous to a C3HC4 RING zinc finger protein-encoding gene was expressed exclusively in the tapetum. In general, the results of these experiments are in line with the gene expression patterns as generated by cDNA-AFLP transcript profiling. Furthermore, the localisation of gene expression to different anther tissues provides useful information for subsequent functional analysis studies. Our study shows that cDNA-AFLP transcript profiling is a powerful tool for gene discovery that allows large-scale gene expression analysis in non-model species. bw-cnudde 23-08-2004 14:49 Pagina 162

162 SUMMARY AND PERSPECTIVES

However, since the generated tags are short, they sometimes may be not informative enough for the identification of known homologues. Therefore, the construction of a cDNA library of anthers undergoing meiosis is a necessary next step for the isolation of longer cDNA clones. Our data set, although incomplete, provides a starting point for the further unraveling of meiosis at a molecular level in plants, allowing a knowledge-based selection of tags for further characterisation. This can be performed by in situ analysis to screen for a meiocyte-specific expression pattern, while mutant studies in combination with cytological analysis can provide more information about the possible function. As a first step, ten Arabidopsis T-DNA insertion mutants have been ordered from the SALK Institute (http://www.arabidopsis.org/abrc/tdna_ecker.html), while in Petunia hybrida a transposon insertion library is available to screen for meiotic mutants. Integration of our data into existing databases like GermOnline (http://www.germonline.org) will add to the processing and interpretation of homology data on large numbers of genes and facilitate comparative analysis of meiotic processes across species. bw-cnudde 23-08-2004 14:49 Pagina 163

Samenvatting en perspectieven

Meiose is een belangrijk basiskenmerk van de seksuele voortplanting bij eukaryoten. Deze gespecialiseerde celdeling werd door cytologen reeds ruim 100 jaar geleden bestu- deerd; aanvankelijk werden hierbij vooral planten als modelsysteem gebruikt. Met de snelle opkomst van functional genomics technieken wordt tegenwoordig echter de voorkeur gegeven aan onderzoek op gist bij moleculaire en genetische studies van meio- se. Het gebruik van microarrays voor genoom-wijde genexpressie studies leidde tot de identificatie van vele, voorheen onbekende, meiotische genen in gist. Met behulp van bio-informatica werden homologe genen gevonden in andere eukaryoten; hierbij kon- den vaak intrigerende verschillen in het meiotische transcriptoom worden aangetoond. Tot dusver vormen planten nog relatief onontgonnen werkterrein wat de moleculaire en functionele analyse van meiose betreft. Door de toevloed van genomics data uit het gis- tonderzoek en de beschikbaarheid van grote collecties mutanten, voornamelijk in Arabidopsis, is deze situatie echter snel aan het veranderen. Een uitgebreid overzicht van de groeiende interesse in moleculaire studies op meiose in planten is beschreven in de algemene inleiding in Hoofdstuk I. We hebben een strategie ontwikkeld om genexpressie te bestuderen tijdens de man- nelijke meiose in Petunia hybrida. Dit experiment was een vervolg op een vorige studie, beschreven in Hoofdstuk II, waarbij we hebben aangetoond dat cDNA-AFLP trans- cript profiling een geschikte methode is voor het analyseren van genexpressie geduren- de mannelijke en vrouwelijke gametogenese in Petunia. Vegetatieve en geslachtelijke bloemorganen werden verzameld en genexpressieprofielen werden vergeleken geduren- de vijf ontwikkelingsstadia. Deze werkwijze liet ons toe om een collectie aan te leggen van 354 transcript fragmenten, waaronder vele geïnduceerde helmknop-specifieke frag- menten. Bij een verdere karakterisering van deze cDNA fragmenten konden diverse klassen van gekende gametogenese genen worden teruggevonden, wat de effectiviteit van onze screening bewijst. Voor meer dan de helft van deze fragmenten kon echter geen homologie gevonden worden in de database, een percentage dat vergelijkbaar is met andere transcript profiling experimenten. Dit wijst erop dat de gebruikte methode voldoende gevoelig is om nieuwe transcripten te identificeren. Tot onze verrassing werden er bij deze gametogenese experimenten geen cDNA frag- menten gevonden die homologie vertoonden met gekende meiotische genen. Daarom werden de mouwen opgesloofd voor een gedurfd experiment: het opzetten van een functional genomics strategie gericht op een moleculaire dissectie van het meioseproces in Petunia, zoals beschreven in Hoofdstuk III. Om de meiotische stadia te visualiseren werden helmknoppen met enzymen behandeld om de pollenmoedercellen te spreiden, bw-cnudde 23-08-2004 14:49 Pagina 164

164 SAMENVATTING EN PERSPECTIEVEN

gevolgd door een kleuring met 4’,6-diamidino-2-phenylindole (DAPI). Met deze methode konden we achterhalen dat het meioseproces relatief synchroon verloopt in de Petunia bloemknop. Dit is echter niet het geval voor de kleinste helmknop, die meest- al iets voorop loopt in de meiose en om die reden niet werd weerhouden. Van de ove- rige vier helmknoppen diende er nog een te worden opgeofferd voor de cytologische analyse, zodat slechts drie helmknoppen resteerden voor RNA-extractie en cDNA syn- these. Het transcript profiling experiment was gericht op het identificeren van genen die differentieel tot expressie komen gedurende de mannelijke meiose, waarbij stadia voor en na de meiose (archesporen en microsporen) als referentie werden gebruikt. Ongeveer 8000 transcript fragmenten, naar schatting 35% van het totale transcriptoom, werden gevisualiseerd op gel, waaronder 475 met een gemoduleerde expressie. Vele van die frag- menten vertoonden homologie met gekende meiotische genen, wat de effectiviteit van onze screening bewijst en opnieuw de hoge gevoeligheid van de cDNA-AFLP transcript profiling methode aantoont. Een grondige karakterisering van deze fragmenten aan de hand van sequentie homo- logieën is beschreven in Hoofdstuk IV. Van de 293 gesequeneerde genfragmenten in onze collectie vertoonden 90 (30%) een significante homologie in een GenBank data- base search met het BLAST algoritme. Een functionele klassificatie van deze genen aan de hand van literatuuronderzoek toonde aan dat componenten van verscheidene meio- tische processen konden worden teruggevonden, wat een geslaagde dissectie van het gehele meioseprogramma impliceert. Genen betrokken bij meiotische recombinatie en DNA herstel waren daarbij opvallend goed vertegenwoordigd in onze screening, wel- licht mede dankzij de sterke evolutionaire conservering. Componenten van minder goed geconserveerde processen daarentegen, zoals synapsis, zouden wel degelijk aanwe- zig kunnen zijn in onze collectie, maar kunnen niet als dusdanig worden herkend aan de hand van homologie in de database. Onze collectie, weliswaar onvolledig, betekent een startpunt voor de moleculaire analyse van meiose in planten en voorziet in de spe- cifieke behoefte aan selectiemateriaal voor een verdere karakterisering van meiose. In Hoofdstuk V wordt voor het eerst een grootschalige klassificatie van expressiep- rofielen gedurende de meiose gepresenteerd. Door een kwantificatie van de genexpres- siedata, verkregen via cDNA-AFLP transcript profiling, konden deze bewerkingen met de computer worden uitgevoerd. Clusteranalyse liet een golf van genexpressie zien bij die genen die betrokken zijn bij de initiatie en progressie doorheen het meioseproces. Door het groeperen van genen met gelijkaardige expressieprofielen konden vijf grote clusters worden onderscheiden en werden deze gecorreleerd met meiotische mechanis- men en processen. Uit deze data kon ook worden afgeleid dat een relatief grote groep Petunia fragmenten, geïnduceerd gedurende het pachyteen en latere meiotische stadia, geen homologie vertoonden in de database. Dit kan wijzen op een achterstand in de bw-cnudde 23-08-2004 14:49 Pagina 165

Samenvatting 165

moleculaire analyse van de genen die tot deze specifieke cluster behoren. Verder konden in verschillende gevallen extra bewijzen worden verkregen voor de voorgestelde functies van geïsoleerde fragmenten met weinig significante homologieën, door hun co-expres- sie met gekende meiotische genen. In Hoofdstuk VI, ten slotte, worden de expressiepatronen van vijf geselecteerde genfragmenten verder geanalyseerd via in situ hybridisatie. Daarbij werd voor een frag- ment, homoloog aan een RING zinc finger eiwit, uitsluitend expressie in het tapetum teruggevonden. Over het algemeen bevestigen de in situ resultaten de expressieprofielen verkregen via cDNA-AFLP transcript profiling. Bovendien werd nuttige informatie bekomen over de lokalisatie van genexpressie over de gehele helmknop. Onze studie heeft aangetoond dat cDNA-AFLP transcript profiling een geschikte methode is voor het analyseren van de genexpressie in minder gebruikte labspecies. Aangezien de fragmenten echter relatief kort zijn, kan soms onvoldoende informatie verkregen worden voor het opsporen van gekende homologen. Daarom is de construc- tie van een cDNA library voor de mannelijke meiose in Petunia een belangrijke vol- gende stap voor het isoleren van langere cDNA clones. Onze dataset biedt een startpunt voor de moleculaire analyse van meiose in planten en maakt een gerichte selectie van cDNA fragmenten voor verdere karakterisatie moge- lijk. Verdere analyse kan bijvoorbeeld gebeuren via in situ hybridisaties om meiose-speci- fieke expressiepatronen te identificeren of door het bestuderen van mutanten om met behulp van cytologische technieken meer informatie over een mogelijke functie te ver- krijgen. Tien Arabidopsis T-DNA insertiemutanten zijn alvast verkregen via het SALK instituut (http://www. arabidopsis.org/abrc/tdna_ecker.html) en intussen is ook de trans- poson library in Petunia hybrida gebruiksklaar om meiotische mutanten op te sporen. Het integreren van onze data in bestaande databases zoals GermOnline (http://www.germonline.org) zal het interpreteren van homologiegegevens voor grote aantallen genen in de toekomst vereenvoudigen en betekent een aanmoediging voor het maken van vergelijkende analyses van meiotische processen over de soortgrenzen heen. bw-cnudde 23-08-2004 14:49 Pagina 166

Dankwoord

Het spandoek was al enige tijd in zicht, maar nu is de eindmeet eindelijk bereikt. Wie had in het begin van de rit ooit kunnen voorzien dat die in Nijmegen zou liggen? Ik had ook niet gedacht dat het hele parcours onderweg zo grillig en zwaar zou zijn, en al hele- maal niet dat het zo’n lange wedstrijd ging worden. Maar nu ik wat uitgehijgd ben en het zweet van mijn voorhoofd heb gewist, kan ik terugblikken op een onvergetelijke ervaring, waarvan ik zoveel heb geleerd. Gelukkig kon ik rekenen op een schitterende ploeg met fantastische mensen, stuk voor stuk noeste werkers die teamgeest hoog in het vaandel voeren. En dan was er de ploegleider, die me met goede raad overstelpte en mij uit de wind zette wanneer ik de pedalen kwijt was. Ten slotte waren er de vele trouwe supporters die mij onophoudelijk aanmoedigden en af en toe een duwtje in de rug gaven. Bedankt iedereen, zonder jullie had ik hier vandaag niet op het podium gestaan! Het begon allemaal in het kleine maar dappere Rhodococcus-team in Gent. Aanvankelijk voelde ik me een beetje onwennig in het grote, oude Ledeganckgebouw, maar dankzij de toffe groepssfeer kon ik me al snel helemaal uitleven in het labwerk. Koen, Danny, Wim, Tita, Tania, Karen en Rozemarijn, het was een plezier om met jul- lie te mogen samenwerken. Het waren – achteraf bezien – zorgeloze jaren, over brede wegen en met een stevige rugwind, weliswaar in een strak tempo, maar toch met volop tijd voor ontspanning. En voor dat laatste was er natuurlijk de superactieve en gezellige studentenvereniging ’t KUC met talloze onvergetelijke activiteiten. Toch liep het onderzoek niet zoals verhoopt, de Petunia-Rhodococcus interactie was ongrijpbaarder dan gedacht en de gebruikte techniek bleek onvoldoende gevoelig. Een plotse transfer naar een nieuwe ploeg was het gevolg en ik verhuisde naar het andere eind van de gang, naar de Petunia-bende met Jan, Michiel en Bernard...Op vol- strekt unieke wijze slaagden zij erin om goede wetenschap te combineren met een repu- tatie van feestvierders. Sommige van die drinks zijn ronduit legendarisch geworden. Blij met de geboden kans stortte ik me vol energie op het nieuwe onderwerp en een nieuwe techniek. Ook tijdens de weekends en in de avonden werd soms doorgewerkt, maar d’r was altijd wel iemand te vinden voor een praatje. Bartel, Nathalie, en zovele anderen, bedankt voor het gezelschap en de welkome onderbrekingen. Plots voerde de rit echter over de landsgrenzen heen, weg uit de vertrouwde en geliefde omgeving van het Gentse naar het onbekende Nijmegen. Het was wel even wennen in het begin, vooral ook omdat het aanvankelijk zo stil en rustig was in het nieuwe labo. Gelukkig kon ik rekenen op de steun van Titti en ‘haar jongens’ beneden: Koen, Richard, Marc, Karin, Ivo en Jeroen. Bedankt voor alle hulp om mij ook in Nijmegen uit de startblokken te krijgen en om mijn sociale leven toch nog op een aan- bw-cnudde 23-08-2004 14:49 Pagina 167

vaardbaar peil te houden. Else, Mieke, Huub, Jan, Wim, Peter, Tamara, Jose, Theo, Rinus en alle mensen van de kas, bedankt voor de gezellige babbels op feestjes en recep- ties allerhande. Maar al snel kwam er boven ook al wat meer leven in de brouwerij en begon er een gezellig sfeertje te ontstaan. Janny, Anneke, Marieke, Laurens, Paul, Tanja en natuurlijk Stefan, ‘t was een plezier om met jullie te mogen samenwerken. Dankzij jullie kwam ik altijd met volle goesting naar het lab, zelfs als ‘t niet echt nodig was. Stefan, waarde collega & vriend, bedankt voor alles en vooral voor de vele gesprekken over oosterse wijsheden, Petunia’s en andere schone dingen des levens. Liesbeth, ik blijf je eeuwig dankbaar voor je grenzeloze hulpvaardigheid bij het maken van de plaatjes en de inspirerende gesprekken. In volle eindsprint, tijdens het schrijven van dit proefschrift, heb ik, slingerend van links naar rechts over de weg, nog vele moeilijke momenten moeten doorspartelen. Bedankt aan alle mensen die mij hebben geholpen en aangemoedigd om de eindstreep te halen. Tom, beste ploegleider, met jouw geduldige, menselijke aanpak heb je mij steeds de vrijheid gegeven om zelf mijn onderzoek uit te stippelen. Bedankt voor alle raad en levenswijsheid en het niet aflatende vertrouwen dat je in mij hebt getoond. Lieve vader, moeder en zus, jullie stonden steeds voor mij klaar en slaagden erin om mij telkens opnieuw helemaal “thuis” te laten voelen als ik een weekendje naar België kwam. Ik hou van jullie, Filip bw-cnudde 23-08-2004 14:49 Pagina 168

Curriculum vitae

De auteur van dit proefschrift werd op 10 maart 1973 geboren te Gent. Hij volgde Latijn-Wetenschappen aan het Sint-Hendrikscollege in Deinze, waarna hij in oktober 1991 voor bio-ingenieur ging studeren aan de Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen aan de Universiteit Gent. Zijn thesisonderzoek verrichtte hij van juli 1995 tot mei 1996 in de Hybrid Improvement Systems Group van Plant Genetic Systems in Gent onder begeleiding van Dr. F. Michiels. Dit resul- teerde in een onderzoeksverslag met als titel “Koper-induceerbare transgenen in tran- siënt en stabiel getransformeerde plantencellen”. In juni 1996 studeerde hij met grote onderscheiding af als bio-ingenieur in de cel-en genbiotechnologie, waarna hij als wetenschappelijk onderzoeker aan de slag ging aan het Departement Plantengenetica in de Faculteit Wetenschappen van de Gentse universiteit. In dit FWO-onderzoek, begeleid door Dr. Koen Goethals en Prof. Dr. Tom Gerats, werd een moleculaire analyse uitgevoerd van de interacties tussen het fytopathogen Rhodococcus fascians en geïn- fecteerde planten bij de vorming van bladgallen. In oktober 1999 werd het eigenlijke promotieonderzoek aangevat, waarvan de resultaten staan beschreven in dit proefschrift. Dit onderzoek werd aanvankelijk uitgevoerd in de Petunia groep van het Departement Plantengenetica, verbonden aan het Vlaams Instituut voor Biotechnologie (VIB). In oktober 2001 werd evenwel met de promotor mee verhuisd naar Nijmegen, waar het werk werd verdergezet aan het Departement Experimentele Plantkunde aan de Faculteit der Natuurwetenschappen, Wiskunde en Informatica aan de Katholieke Universiteit Nijmegen (KUN). Naast zijn promotieonderzoek was hij ook actief op het terrein van wetenschap en communicatie, o.a. als vrijwillig medewerker op de VIB-tentoonstelling “Eet eens genetisch” of als deelnemer van de masterclass Licence to Life Sciences in Amsterdam. Verder volgde hij in het voorjaar van 2004 een cursus wetenschapsjournal- istiek in Wageningen. Sinds juli 2004 is hij werkzaam bij de groep Marktkunde en Consumentengedrag aan de Universiteit Wageningen, als communicator en trainer bij SAFE FOODS, een Europees programma rond voedselveiligheid. bw-cnudde 23-08-2004 14:49 Pagina 169

Appendix bw-cnudde 23-08-2004 14:50 Pagina 170

170  

Figure 2 Schematic representation of the mitotic (left) and meiotic (right) cell cycle

Figure 3 Schematic overview of meiotic stages (after Bhatt et al., 2001) bw-cnudde 23-08-2004 14:50 Pagina 171

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Figure 5 Double Strand Break Repair Model of meiotic recombination (Szostak et al., 1983); (after Roeder, 1997)

Figure 7 Schematic representation of the regulation of sister chromatid cohesion in mitosis (A) and meiosis (B); (after Dej and Orr-Weaver, 2000) bw-cnudde 23-08-2004 14:50 Pagina 172

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Figure 1 Overview of the meiotic stages determined by DAPI-staining in microsporocytes of Petunia hybrida. A leptotene, B zygotene, C pachytene, D early diplotene, E late diplotene, F early metaphase I, G metaphase I, H early anaphase I, I anaphase I, J telophase I, K dyad stage, L metaphase II, M telophase II, N tetrad, O end of tetrad bw-cnudde 23-08-2004 14:50 Pagina 173

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Figure 2 Five stamens of a Petunia hybrida flower. The abaxial anther is the smallest and has the shortest filament. Frequently, meiosis in this anther is not synchronous with the four remaining anthers.

Cluster 1 Unknown Cluster 2 Gametogenesis Cluster 3 DNA replication, ubiquitin-mediated proteolysis, synaptonemal complex formation, spindle assembly, RNA processing, protein biosynthesis Cluster 4 Proteases, signal transduction (kinases, phosphotases), chromosome segregation, DNA repair Cluster 5 DNA modification, checkpoint control, recombination, membrane transport bw-cnudde 23-08-2004 14:50 Pagina 174

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Figure 1 Hierarchical average linkage clustering of the expression patterns of 978 significantly modulated cDNA-AFLP fragments according to Eisen et al. (1998). The matching dendrogram shows five different clusters of co-expressed genes, with maximal expression level at different stages during meiosis. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 postmeiotic bw-cnudde 23-08-2004 14:50 Pagina 175

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Figure 3 Hierarchical average linkage clustering of entire dataset of quantified expression patterns; selected clusters. A Cluster A contains genes that are downregulated during the complete process of meiosis with transcriptional activity restricted to pre-and post-meiotic stages. B Cluster B contains genes with elevated levels of gene expression during meiosis. Black arrow: indicates a subgroup of co-expressed genes strongly downregulated in the postmeiotic sample. 1 premeiotic, 2 leptotene, 3 zygotene, 4 pachytene – metaphase I, 5 dyad stage – telophase II, 6 postmeiotic bw-cnudde 23-08-2004 14:50 Pagina 176

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Figure 4 Hierarchical average linkage clustering of expression profiles belonging to sequenced cDNA- AFLP fragments modulated during meiosis. A-C Functional classes defined in Chapter IV.

Figure 5 Hierarchical average linkage clustering of expression profiles belonging to sequenced cDNA- AFLP fragments modulated during meiosis. A-C Functional classes defined in Chapter IV. bw-cnudde 23-08-2004 14:49 Pagina 2 UITNODIGING

voor het bijwonen van

de openbare verdediging van mijn

Transcript Profiling during male meiosis in Petunia hybrida proefschrift

Transcript Profiling to analyse gene expression during male meiosis in Petunia hybrida

Op woensdag 6 oktober 2004 om 15.30 u precies in de Aula van de Radboud Universiteit Nijmegen, Comeniuslaan 2

Transcript profiling to analyse gene expression during male meiosis in Receptie na afloop van Petunia hybrida de promotie in de aula

Filip Cnudde

Filip Cnudde [email protected]

Paranimfen:

Janny Peters ([email protected])

Filip Cnudde Stefan Royaert ([email protected])