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Please be advised that this information was generated on 2021-10-11 and may be subject to change. Aspects of ethylene-mediated communication in plants

Aspects of ethylene-mediated communication in plants

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

Proefschrift ter verkrijging van de graad van doctor aan de Katholieke 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 donderdag 5 februari 2004, des namiddags om 1.30 uur precies door

Ivo Rieu geboren op 25 oktober 1975 te Breda Promotor: Prof. Dr. C. Mariani Copromotor: Dr. K.A.P. Weterings

Manuscriptcommissie: Prof. Dr. G.C. Angenent Dr. D. van der Straeten (Rijks Universiteit Gent, België) Dr. E.J. Woltering (ATO Wageningen UR)

This research was funded by the Netherlands Organization for Scientific Research (NWO) Il faut regarder toute la vie avec des yeux d’enfants

Henri Matisse

Contents

Chapter 1 9 General introduction

Chapter 2 23 The Arabidopsis ethylene receptor ERS1 accumulates in the endoplasmic reticulum

Chapter 3 37 Expression analysis of five tobacco EIN3 family members in relation to tissue-specific ethylene responses

Chapter 4 47 Ethylene regulates the timing of anther dehiscence in tobacco

Chapter 5 59 Evolution of unitegmy in the euasterids

Chapter 6 77 Summary and concluding remarks

Nederlandse samenvatting 81

Dankwoord 85

Curriculum vitae 87

Appendix 89

Chapter 1

General introduction

I. ANGIOSPERM EVOLUTION AND DEVELOPMENT

On its way to chaos, a tiny part of the energy radiating from the sun, encounters an obstacle on Earth. It is temporarily used to support a unique, highly structured chemical environment that occurs on our planet in the form of life. At this moment in evolutionary time, plants are the pillars of Earth’s life. Their photosynthetic system captures the energy from photons to support their own growth and maintenance, and thereby feeds, directly or indirectly, an ecosystem’s various consumers, including animals. Although most humans nowadays live far from natural ecosystems and farms, our dependence on plants is evident in our lumber, paper, medicines, and most important, in the food that we eat and the oxygen that we breathe.

PLANT EVOLUTION The theory that dominates biology ever since its postulation is Darwin’s theory of evolution (Darwin, 1859). It explains how organisms may change over generations due to the force of natural selection and, when pursued, it can be used to explain how today’s life with all its diversity has evolved from the most primitive and earliest forms. This primitive form of life is thought to have originated in the watery environments on Earth about 4 billion years ago (Schopf et al., 1983). The simplest form that still exists today are the prokaryotes or bacteria. An important step in the evolution of life, and plants in particular, which took place about 3 billion years ago, was the evolution of bacteria that had the machinery to capture the energy from sun light and carry out one of the most important chemical reactions that we know of: photosynthesis, or light + CO2 + water ^ O2 + sugars (Summons et al., 1999). Another important step, at least 2.7 billion years ago, was the evolution of eukaryotes, which amongst other things can be discerned by the presence of organelles (Summons et al., 1999). Some of these intracellular structures are thought to have derived from smaller bacterial cells that came to live inside a larger cell. In this way, “endosymbiosis” of a photosynthetically active bacterium gave rise to the chloroplast (Margulis, 1981; Cattolico, 1986). While other ancestral eukaryotic organisms evolved into the animals and the fungi, the eukaryotic organisms carrying chloroplasts, similar to algae, evolved into the plants about 450 million years ago. This step was accompanied by a transition from the aquatic environment to the terrestrial environment (Gray and Shear, 1992, Willis and McElwain, 2002). Three of the major evolutionary novelties that shaped the plants during further

9 Chapter 1 evolution are the evolution of vascular tissue, the evolution of ovules and seeds and, most recently, the evolution of flowers. All of these features are present in the phylum angiosperms, or flowering plants, and today, with at least 250.000 different species, they constitute about 90% of all plant species that inhabit the Earth (Willis and McElwain, 2002).

THE ANGIOSPERM LIFE CYCLE The life cycle of angiosperms consists of an alternation of generations, in which a haploid and a diploid generation produce each other. The diploid, or sporophytic, generation is dominant in the angiosperms. It starts with a fertilized egg cell, or zygote, located in the well-protected environment of a mature ovule. During embryogenesis the basic features of the plant's body plan are established, and when a seedling emerges from the mature seed, it consists of a root system and a shoot system. These two systems each have their unique functions: the root inhabits the soil and primarily absorbs nutrients and water, while the shoot inhabits the air and is the site of photosynthesis. Efficient exchange of products between the two systems takes place via the vascular tissue. Unlike an animal, a plant continues to grow and produces new organs throughout its life, often in an indeterminate fashion. This growth is initiated by cell divisions in the meristems that are located at both ends of the root-shoot axis. For a period of time the plant continues this so- called vegetative growth, during which it produces root and stem tissue, possibly with branches, and leaves. At a certain point, however, a shoot meristem can make a shift from vegetative growth to reproductive growth, which means that it no longer produces sterile leaves, but fertile flowers. Inside the angiosperm flower the male and female haploid, or gametophytic, generations are produced. Meiosis of sporophytic cells inside the anthers and ovules generates haploid spores, which by a very limited number of cell divisions form the male and female gametophytes, or the pollen and embryo sac. Pollination and pollen tube growth brings one of the sperm cells, the male gametes in the pollen, in contact with the egg cell, the female gamete in the embryo sac, and upon fertilization they form the new, diploid, zygote.

REGULATION OF PLANT DEVELOPMENT AND THE ROLE OF PLANT HORMONES Although plants have a less fixed body plan than animals, they are still highly structured. This became evident after the invention of the microscope and the discovery of cells as the basic building units of plants. The shape of a plant is dictated by the oriented division and growth of these cells, mainly near its meristems (Meyerowitz, 1997). However, cells not only differ in their size and form, but also in their function. It has become clear, that differences between cells reflect differences in the set of proteins that is synthesized in these cells. So, although the cells of a plant all have the same genome, they follow different developmental pathways because they selectively express certain genes at certain times. Therefore, the developmental pathway is regulated largely via the control of gene expression by intracellular messengers. But what kind of information modulates the activity of these intracellular messengers so that cells differentiate from each other? One source of information comes

10 Introduction from the plants environment. Light, temperature, water availability and compounds from pathogens and feeding herbivores can all modulate the set of active genes of the cells (Smith, 2000; Shinozaki and Yamaguchi-Shinozaki, 2000; Halitschke et al., 2001; Bonas and Lahaye, 2002). Yet, it is clear that it is endogenous information that primarily directs the particular pathway along which each plant cell develops. Because plant cells are constrained by rigid cell walls and are generally non-motile, it has long been thought that the developmental program of a cell is determined by lineage (Steeves and Sussex, 1989). However, evidence is accumulating that intercellular communication has a much more important role in this process and complex communication networks have been revealed that provide the cells with information on their position and on the developmental state of other cells of the plant (Howell, 1998). Intercellular communication occurs mainly via two routes. Small molecules and sometimes even mRNA and proteins may pass directly from cell to cell via the plasmodesmata (Haywood et al., 2002). The main form of intercellular communication, however, is via molecules that are released by a cell to the apoplast and then translocated to other cells, where low concentrations are perceived through receptor proteins with high- affinity binding sites (Cleland, 1999). These molecules are called plant hormones. The five classical plant hormones encompass auxin, gibberellin, cytokinin, ABA and ethylene, but also more recently identified molecules including brassinosteroids, jasmonic acid, salicylic acid and some sugars, oligosaccharins and polypeptides meet the definition of a plant hormone (Bishop and Koncz, 2002; Turner et al., 2002; Raskin, 1992; Smeekens, 2000; Fry, 1999; Ryan et al., 2002). Upon binding to receptors, these molecules modulate the activity of intracellular messengers and thereby induce a change in the developmental program of the target cells.

II. Intercellular communication via ethylene

One of the most-studied and best-understood hormonal communication systems in plants is communication via ethylene. Ethylene responses have been documented throughout the plant kingdom (Abeles et al., 1992), suggesting that plants use ethylene for communication ever since they colonized land. In angiosperms, ethylene biosynthesis is developmentally regulated and ethylene is thus used to communicate information on the developmental state of a plant cell to its surroundings and even to itself. Furthermore, ethylene is synthesized in response to external factors like physical wounding or stress and pathogen infection and as such spreads information on these environmental conditions to other cells of the plant (Imaseki, 1999). The ethylene biosynthetic pathway has been substantially characterized in several plant species (Yang and Hoffman, 1984; Kende, 1993). Ethylene is derived from the amino acid methionine, which in a first step is converted to S-adenyl-methionine (AdoMet) by AdoMet synthetase. AdoMet serves as an intermediate in a number of biosynthetic pathways, including the production of polyamines. AdoMet is converted to 1-aminocyclopropane-a-carboxylic acid (ACC) by ACC synthase.

11 Chapter 1

The final step is the conversion of ACC to ethylene by the enzyme ACC oxidase (ACO). Generally, the conversion mediated by ACS is the rate-limiting step in ethylene biosynthesis, although under conditions of high ethylene production also the ACO- mediated step may be rate-limiting (Fluhr and Mattoo, 1996). The endogenous signals and environmental signals that control the level of ethylene biosynthesis act via modulation of ACS and ACO gene expression and via modulation of ACS activity (Imaseki, 1999; Wang et al., 2002; Vogel et al., 1998). No mechanisms are known for active, directed, translocation of ethylene and the general concept is that once it is produced, the gaseous ethylene spreads by simple diffusion through the tissue and into the air. Entrapment in the plant tissues, however, can result in substantial translocation (Woltering et al., 1995, 1997). In addition, the ethylene precursor ACC has been shown to be translocated over long distances via vascular tissue (Bradford and Yang, 1980; Else and Jackson, 1998). Most data on ethylene perception and signal transduction come from studies of Arabidopsis, although the pathway seems to be conserved in all plant species studied to date. In Arabidopsis, ethylene is perceived by a family of five membrane-localized receptors - ETR1, ETR2, ERS1, ERS2, and EIN4. Synthesis of the results from biochemical and genetic studies has led to the conclusion that these ethylene receptors actively repress the ethylene response in the absence of ethylene and are inactivated by ethylene binding (fig. 1; Hua and Meyerowitz, 1998; Wang et al., 2002). The input domain of the receptors is located at the N-terminus. This domain contains hydrophobic stretches that function as transmembrane segments and form a membrane-located high-affinity ethylene-binding site, which contains a copper ion (Johnson and Ecker, 1998; Schaller and Bleecker, 1995; Rodriguez et al., 1999). Furthermore, the N-terminal domain contains the dimerization site, and ETR1 and ERS1 have been shown to form homodimers in plants (Schaller et al., 1995; Hall et al., 2000). Binding of ethylene to the input domain is presumed to result in a conformational change that is propagated to the transmitter domain (Bleecker et al., 1998). The transmitter domain consists of signaling modules common to many signaling pathways, and a structure that is homologous to that of bacterial two- component histidine kinases involved in sensing environmental changes (Chang et al., 1993; Johnson and Ecker, 1998). The first module is a GAF domain, which has been associated with cyclic nucleotide binding (Aravind and Ponting, 1997). Its function in the ethylene receptors is still unknown, but it has been suggested to be involved in receptor multimerization or propagation of a conformational change in response to the signal (Mount and Chang, 2002). Downstream of the GAF domain a histidine-kinase domain is located. This domain is slightly divergent between the receptor family members. Based on homology and intron positions, the ethylene receptor family can be divided into two subfamilies (Hua et al., 1998; Bleecker, 1999). Subfamily I includes ETR1 and ERS1 and subfamily II includes ETR2, ERS2 and EIN4. In subfamily I, all the residues that are essential for histidine-kinase activity of the bacterial histidine-kinase domains are conserved (Chang et al., 1993), and ETR1 was indeed shown to be capable of autophosphorylation on the conserved histidine when expressed in yeast (Gamble et al.,

12 Introduction

Figure 1. Model for ethylene signal transduction in Arabidopsis (modified from Schaller and Kieber, 2002).

1998). However, histidine-kinase activity of the subfamily I receptors is not essential for ethylene receptor signaling (Wang et al., 2003). The subfamily II receptors lack a number of essential residues in the histidine-kinase domain and recently a subfamily II ethylene receptor from tobacco has been shown to exhibit serine/threonine kinase activity instead of histidine-kinase activity (Xie et al., 2002). Finally, a subset of the ethylene receptor family of Arabidopsis, i.e. ETR1, ETR2 and ERS1, contain a receiver domain that shows similarity to bacterial response regulators. The presence of such a domain adjacent to the histidine- kinase domain also occurs in some bacterial systems, where it might function as a competing substrate for phosphotransfer, or as a relay station in a series of phosphotransfer steps (Wurgler-Murphy and Saito, 1997). Despite the thorough knowledge about the structure and biochemical properties of the receptors, the mechanisms by which

13 Chapter 1 they function and which modules of the receptors are used for signaling are unknown. The absence of mutant phenotypes in seedlings of single-receptor null mutants suggested that in spite of structural differences there is functional redundancy among the receptors at this stage of development (Hua and Meyerowitz, 1998). The ethylene signal seems to transmitted to the nucleus via a MAPkinase pathway (fig. 1; Novikova et al., 2000; Ouaked et al., 2003). In the absence of ethylene, the Raf MAPKKK homolog CTR1 represses the ethylene response (Kieber et al., 1993), probably via inactivation of a downstream MAPKK protein (Ouaked et al., 2003). The histidine- kinase domains of ETR1 and ERS1 can directly interact with CTR1, but the mechanism of CTR1 activation is not known (Clark et al., 1998). Presence of ethylene would relieve the MAPKK inhibition by CTR1, resulting in the activation of the MAPkinase pathway. The signal is further transduced via EIN2, which is a membrane bound protein with partial homology to NRAMP metal-ion transporters (Alonso et al., 1999). The endpoint of the primary ethylene-signaling pathway is the EIN3 family of transcription factors (Chao et al., 1997; Solano et al., 1998). The EIN3 and EIN3-like proteins bind to the primary ethylene response element that is present in the promoters of several ethylene-regulated genes. Some of the proteins induced by EIN3 are themselves transcription factors, indicating the existence of a transcriptional cascade that induces the expression of the appropriate target genes in response to ethylene. However, still very little is known about this connection between the ethylene signal transduction pathway and the ethylene responses.

It is remarkable that single hormones can induce a diversity of responses. Interaction of the ethylene signal with other signals has been described extensively, but at which stage of the signal transduction these interactions take place is largely unknown (Wang et al., 2002; Gazzarrini and McCourt, 2001). One possibility is that other signals regulate the composition of the ethylene signal transduction pathway so that it is not completely identical in all cells of the plant. Indeed, the ethylene receptors are differentially expressed during development in Arabidopsis and in tomato (Hua et al., 1998; Tieman and Klee, 1999; Lashbrook et al., 1998). It has been shown that the different tomato ethylene receptors have equivalent affinities for ethylene when they are expressed in yeast (Ciardi and Klee, 2001). However, heterodimerization and oligomerization of receptors can have a strong effect on the affinity to a ligand and on the receptor output signal (Heldin 1995; Bray et al., 1998). Because ETR1 and ERS1 have been shown to form homodimers, it has been suggested that also heterodimers between the receptors may form and that they may even exist in larger multimeric complexes. To obtain more insight in possible regulation of the ethylene response at the level of the receptors, knowledge about the localization of the different receptors and their possible interaction would be informative. Chapter 2 describes that ERS1 is localized in the ER. Because a similar localization has been reported for ETR1, physical interactions between these two receptors are possible (Chen et al., 2002). Such direct interactions could not be shown, because the expression of the GFP tagged receptors was too low for fluorescence-based interaction studies.

14 Introduction

The existence of a gene family at the level of EIN3 offers another possibility to modulate the composition of the ethylene-signaling pathway. If the EIN3 family members have different target genes, differential expression or activation may result in different responses to ethylene. Chapter 3 describes the characterization of the tobacco EIN3 family and shows that the members have an identical expression pattern. Although this study does not preclude the possibility of differences in protein quantity and activity, it suggests that there is no regulation of the ethylene response at the EIN3 level. Finally, chapter 4 of this thesis describes a novel response to ethylene. In tobacco, ethylene ensures that dehiscence of the anthers occurs at the time of flower opening. The origin of the ethylene signal in planta is not known, but cells of the anther itself are the direct target, as dehiscence of detached anthers can be accelerated by treatment with ethylene and delayed by inhibition of ethylene perception.

III. OVULE DEVELOPMENT AND EVOLUTION

Another example of hormonal regulation of development was given by De Martinis and Mariani (1999). By studying tobacco transformants in which the synthesis of ethylene was reduced, they showed that ethylene is involved in the progression of ovule and integument development in this plant species. Initially, the aim of the project described in this thesis was to clarify the mechanism behind this function of ethylene. However, a new generation of the transformants that were used in the original study had a weaker phenotype than described for the first generation. Due to these complications and due to possible complexities in the role of ethylene in tobacco ovule development (see chapter 4), the experiments on ovule development were continued independently.

As has been mentioned in the previous section, one of the features that contributed to the enormous success of the angiosperms was the possession of ovules, which form a seed after fertilization. Evolution of these structures revolutionized the plant life cycle by freeing plants from the necessity of external water for sexual reproduction and by affording protection and providing nutrients to the developing embryo. These functions enabled seed plants to expand their geographical distribution beyond the confines of streamsides and sources of water, and enabled progeny to be dispersed away from the parent plant. Evidence from the fossil record indicated that about 350 million years ago, plant evolution had progressed to the point that the female spore, or megaspore, was retained at the sporophyte in the megasporangium and revealed megaspores with an outer protective coating, or integument (Willis and McElwain, 2002). These characters are considered to be the hallmarks of an ovule. The integument probably resulted from the envelopment of the megasporangium by telome trusses that fused around the megasporangium to encase it (fig. 2; De Haan, 1920; Herr, 1995). The number of integuments has varied during further evolution, but the mechanistic origin of this fluctuation is not known. While most extant gymnosperms are unitegmic (they have one integument), the ancestor of the angiosperms

15 Chapter 1

was probably bitegmic (it had two integuments), based on the observation that most basal angiosperms are bitegmic (Herr, 1995; Endress and Igersheim, 2000). Genetic and morphological studies in the angiosperm model species Arabidopsis thaliana have generated a model for ovule and integument development (Schneitz et al., 1995; Gasser et al., 1998). After an ovule primordium has initiated, it grows into a finger­ like structure. Patterning of this structure in the proximal-distal direction results in three zones: the funiculus, chalaza and nucellus. At least in the proximal and central zones, a second pattern is generated in the adaxial-abaxial direction. Two integuments initiate from the abaxial side of chalaza, and subsequently spread around the primordium in a circular manner. Both integuments grow to envelop the nucellus and embryo sac, but their morphogenesis is different. The inner integument grows symmetrically and will differentiate an endothelium. The outer integument grows asymmetrically - more at the abaxial side than at the adaxial side - resulting in curvature of the ovule. Furthermore, the cells of the outer integument are more strongly vacuolated. A number of genes have been implicated in the process of chalaza and integument development, although exact separation of functions has proven difficult (fig. 3). The genes ANT and HLL seem to have overlapping functions in regulating the outgrowth of the proximal two-thirds of the ovule primordium (Schneitz et al., 1998). Ovules of ant hll double mutants are shortened and fail to form a funiculus and sometimes also a chalaza. Proximal-distal patterning and establishment of chalazal identity requires NZZ and BEL1, as the central region has a funiculus identity in nzz bel1 mutants (Balasubramanian and Schneitz, 2000). NZZ, ATS and SUP seem to have a function in abaxial-adaxial patterning of the central zone or maintenance of this pattern (Balasubramanian and Schneitz, 2002; Gaiser et al., 1995). The outer integument grows symmetrically in the nzz ats mutant and in the sup mutant. ATS also has a function in the separation of the two integuments (Leon-Kloosterziel et al., 1994). In ats mutants, the spacing between the integument primordia is reduced, as a result of which they fuse and develop as a single integument. Outgrowth of the outer integument depends on the activity of INO (Villanueva et al., 1999). Based on the function of genes similar to INO, INO is suggested to be necessary for adaxial-abaxial patterning of the outer integument, which would be essential for its outgrowth (Balasubramanian and Schneitz, 2002; Meister et al., 2002). Indeed, other mutants that affect adaxial-abaxial patterning also have reduced outer

16 Introduction

Figure 3. A genetic model for ovule development in Arabidopsis. Parentheses indicate unidentified loci (modified from Gasser et al., 1998).

integument growth (McConnell and Barton, 1998; Eshed et al., 2001). In contrast to INO, HLL and ANT affect initiation of both integuments, probably because they have a general role in cell proliferation during ovule development (Schneitz et al., 1998). Several other genes are necessary for morphogenesis, rather than initiation of the integuments. Defects at the UNC, SUB or LAL loci result in aberrations of the outer integument, and a defect LUG locus results in an abnormal inner integument (Schneitz et al., 1997). With exception of lug, the phenotypes of these mutants are restricted to the ovule. By contrast, mutations at the MOG, BAG, TSL, SIN1, NZZ or TSO1 loci result in aberrations in both integuments and in other parts of the plant (Schneitz et al., 1997; Roe et al., 1997; Lang et al., 1994; Robinson-Beers et al., 1992; Balasubramanian and Schneitz, 2000; Hauser et al., 1998). This is compatible with a more general role for some of these genes in cell growth and division.

17 Chapter 1

While most angiosperms have a bitegmic phenotype, as described in the above model, the number of integuments has been reduced from two to one on multiple occasions during angiosperm radiation (Bouman, 1984). So far, no explanation for this trend has been given in terms of fitness advantage. Four different pathways that can lead from two to one integument have been recorded; two pathways include the reduction of one of the two integuments and two pathways include a form of fusion between the two integuments (Bouman and Callis, 1977; Boesewinkel and Bouman, 1991). Recent advances in molecular phylogeny have provided increasingly detailed information on the evolutionary relations between the angiosperm plant species (APG, 2003). When this information is combined with data on the state of a character in different species, it is possible to trace back the time point in angiosperm evolution at which the character state changed. Using this strategy, Albach et al. (2001) have shown that one of the time points at which the number of integuments was reduced to one, was in the ancestor of the clade of the euasterids. Several morphological studies have addressed the question of which evolutionary pathway led to the reduction in integument number in this clade, but no consensus has been achieved. Comparison of gene expression data between species can be informative in determining homology between organs (Shindo et al., 1999, 2001; Telford and Thomas, 1998). Chapter 5 describes a study aimed at determining the homology between the single euasterid integument and the two ancestral integuments. A general strategy and the use of molecular biological tools for future studies are discussed.

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18 Introduction

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Gray J, Shear W (1992) Early life on land. American Scientist 80: 444-456 Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. Plant Physiology 125: 711-717 Hall AE, Findell JL, Schaller GE, Sisler EC, Bleecker AB (2000) Ethylene perception by the ERS1 protein in Arabidopsis. Plant Physiology 123: 1449-1457 Hauser BA, Villanueva JM, Gasser CS (1998) Arabidopsis TSO1 regulates directional processes in cells during floral organogenesis. Genetics 150: 411-423 Haywood V, Kragler F, Lucas WJ (2002) Plasmodesmata: pathways for protein and ribonucleoprotein signaling. The Plant Cell 14 Suppl: S303-S325 Heldin CH (1995) Dimerization of cell surface receptors in signal transduction. Cell 80: 213-223 Herr JM (1995) The origin of the ovule. American Journal of Botany 82: 547-564 Howell SH (1998) Molecular genetics of plant development. Cambridge University Press, Cambridge Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR, Meyerowitz EM (1998) EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. The Plant Cell 10: 1321-1332 Hua J, Meyerowitz EM (1998) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94: 261-271 Imaseki H (1999) Control of ethylene synthesis and metabolism. In PJJ Hooykaas, MA Hall, KR Libbenga, eds, Biochemistry and molecular biology of plant hormones. Elsevier Science, Amsterdam, pp 209-245 Johnson PR, Ecker JR (1998) The ethylene gas signal transduction pathway: a molecular perspective. Annual Review Of Genetics 32: 227-254 Kende H (1993) Ethylene biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 44: 283-307 Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72: 427-441 Lang JD, Ray S, Ray A (1994) sin1, a mutation affecting female fertility in Arabidopsis, interacts with mod1, its recessive modifier. Genetics 137: 1101-1110 Lashbrook CC, Tieman DM, Klee HJ (1998) Differential regulation of the tomato ETR gene family throughout plant development. The Plant Journal 15: 243-252 Léon-Kloosterziel KM, Keijzer CJ, Koornneef M (1994) A seed shape mutant of Arabidopsis that is affected in integument development. The Plant Cell 6: 385-392 Margulis L (1981) Symbiosis in cell evolution. W.H. Freeman, San Francisco McConnell JR, Barton MK (1998) Leaf polarity and meristem formation in Arabidopsis. Development 125: 2935-2942 Meister RJ, Kotow LM, Gasser CS (2002) SUPERMAN attenuates positive INNER NO OUTER autoregulation to maintain polar development of Arabidopsis ovule outer integuments. Development 129: 4281-4289 Meyerowitz EM (1997) Genetic control of cell division patterns in developing plants. Cell 88: 299­ 308 Mount SM, Chang C (2002) Evidence for a plastid origin of plant ethylene receptor genes. Plant Physiology 130: 10-14 Novikova GV, Moshkov IE, Smith AR, Hall MA (2000) The effect of ethylene on MAPKinase-like activity in Arabidopsis thaliana. FEBS Letters 474: 29-32 Ouaked F, Rozhon W, Lecourieux D, Hirt H (2003) A MAPK pathway mediates ethylene signaling in plants. EMBO Journal 22: 1282-1288 Raskin I (1992) Role of salicylic acid in plants. Annual Review of Plant Physiology and Plant Molecular Biology 43: 439-463 Robinson-Beers K, Pruitt RE, Gasser CS (1992) Ovule development in wild-type Arabidopsis and two female-sterile mutants. The Plant Cell 4: 1237-1249

20 Introduction

Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB (1999) A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science 283: 996-998 Roe JL, Nemhauser JL, Zambryski PC (1997) TOUSLED participates in apical tissue formation during gynoecium development in Arabidopsis. The Plant Cell 9: 335-353 Ryan CA, Pearce G, Scheer J, Moura DS (2002) Polypeptide hormones. The Plant Cell 14 Suppl: S251-S264 Schaller GE, Bleecker AB (1995) Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene. Science 270: 1809-1811 Schaller GE, Ladd AN, Lanahan MB, Spanbauer JM, Bleecker AB (1995) The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer. The Journal of Biological Chemistry 270: 12526-12530 Schaller GE, Kieber JJ (2002) Ethylene. In C Somerville, EM Meyerowitz, eds, The Arabidopsis book. American Society of Plant Biologists, Rockville, doi/10.1199/tab.0071, http://www.aspb.org/publications/arabidopsis/ Schneitz K, Huelskamp M, Pruitt RE (1995) Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. The Plant Journal 7: 731-749 Schneitz K, Hulskamp M, Kopczak SD, Pruitt RE (1997) Dissection of sexual organ ontogenesis: a genetic analysis of ovule development in Arabidopsis thaliana. Development 124: 1367-1376 Schneitz K, Baker SC, Gasser CS, Redweik A (1998) Pattern formation and growth during floral organogenesis: HUELLENLOS and AINTEGUMENTA are required for the formation of the proximal region of the ovule primordium in Arabidopsis thaliana. Development 125: 2555-2563 Schopf JW, Hayes JM, Walter MR (1983) Evolution of earth's earliest ecosystems: recent progress and unsolved problems. In JW Schopf, ed, Earth's earliest biosphere. Princeton University Press, Princeton, pp 361-384 Shindo S, Ito M, Ueda K, Kato M, Hasebe M (1999) Characterization of MADS genes in the gymnosperm Gnetum parvifolium and its implication on the evolution of reproductive organs in seed plants. Evolution and Development 1: 180-190 Shindo S, Sakakibara K, Sano R, Ueda K, Hasebe M (2001) Characterization of a FLORICAULA/LEAFY homologue of Gnetum parvifolium and its implications for the evolution of reproductive organs in seed plants. International Journal of Plant Sciences 162: 1199-1209 Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Current Opinion in Plant Biology 3: 217-223 Smeekens JCM (2000) Sugar-induced signal transduction in plants. Annual Review of Plant Physiology and Plant Molecular Biology 51: 49-81 Smith H (2000) Phytochromes and light signal perception by plants - an emerging synthesis. Nature 407: 585-591 Solano R, Stepanova A, Chao QM, Ecker JR (1998) Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE- FACTOR1. Genes and Development 12: 3703-3714 Steeves TA, Sussex IM (1989) Patterns in plant development. Cambridge University Press, Cambridge Summons RE, Jahnke LL, Hope JM, Logan GA (1999) 2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis. Nature 400: 554-557 Telford MJ, Thomas RH (1998) Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment. Proceedings of the National Academy of Sciences of the United States of America 95: 10671-10675 Tieman DM, Klee HJ (1999) Differential expression of two novel members of the tomato ethylene- receptor family. Plant Physiology 120: 165-172 Turner JG, Ellis C, Devoto A (2002) The jasmonate signal pathway. The Plant Cell 14 Suppl: S153-S164 Villanueva JM, Broadhvest J, Hauser BA, Meister RJ, Schneitz K, Gasser CS (1999) INNER NO OUTER regulates abaxial-adaxial patterning in Arabidopsis ovules. Genes and Development 13: 3160-3169

21 Chapter 1

Vogel JP, Woeste KE, Theologis A, Kieber JJ (1998) Recessive and dominant mutations in the ethylene biosynthetic gene A CS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively. Proceedings of the National Academy of Sciences of the United States of America 95: 4766-4771 Wang KL, Li H, Ecker JR (2002) Ethylene biosynthesis and signaling networks. The Plant Cell 14 Suppl: S131-S151 Wang WY, Hall AE, R OM, Bleecker AB (2003) Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission. Proceedings of the National Academy of Sciences of the United States of America 100: 352-357 Willis KJ, McElwain JC (2002) The evolution of plants. Oxford University Press, New York Woltering EJ, Somhorst D, Van der Veer P (1995) The role of ethylene in interorgan signaling during flower senescence. Plant Physiology 109: 1219-1225 Woltering EJ, De Vrije T, Harren F, Hoekstra FA (1997) Pollination and stigma wounding: same response, different signal? Journal of Experimental Botany 48: 1027-1033 Wurgler-Murphy SM, Saito H (1997) Two-component signal transducers and MAPK cascades. Trends in Biochemical Sciences 22: 172-176 Xie C, Zhang JS, Zhou HL, Li J, Zhang ZG, Wang DW, Chen SY (2003) Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco. The Plant Journal 33: 385-393 Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 88: 553-558

22 Chapter 2

The Arabidopsis ethylene receptor ERS1 accumulates in the endoplasmic reticulum

Ivo Rieu, Marijn Luijten, Michiel Kwantes, Joop Vermeer*, Theodorus W.J. Gadella Jr* and Titti Mariani

* Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands

ABSTRACT

Ethylene acts as a signaling compound during plant development and stress responses. In Arabidopsis, ethylene is perceived by a family of five membrane localized receptors, which can be grouped into two subfamilies. The subfamily II receptors - ETR2, ERS2 and EIN4 - contain a putative signal for transfer to the secretory pathway. By contrast, the subfamily I receptors - ETR1 and ERS1 - do not contain a known signal peptide. Immunological studies have shown that ETR1 localizes to the ER membrane. Using GFP tagging, we show that ERS1 localizes to the ER as well. The study underlines that plants have a tight control over ethylene receptor expression.

INTRODUCTION

Signaling compounds mediate the communication between cells and as such are essential for the existence of multicellular organisms. They are involved in processes like pattern formation, synchronization of developmental programs and stress signaling. As with any form of communication, the signals for intercellular communication need to be produced, translocated and perceived. By definition, perception is achieved through binding of the signaling compound to a protein or protein complex, the receptor. Upon binding, these receptors trigger a cellular response. For a good understanding of receptor functioning, it is important to know where they are located within the cell. The location of receptors is restricted by the ability of the perceived signaling compound to translocate inside cells or not. Steroid hormones, for example, can pass through the cell membrane and this allows the steroid receptors to be present in the cytosol or even inside the nucleus (Tsai and O’Malley, 1994). By contrast, peptide ligands are unable to pass through the

23 Chapter 2 plasma membrane and therefore most peptide receptors are embedded in the plasma membrane (Moody, 1993). In plants, the gaseous compound ethene (C2H4), also known as ethylene, acts as a signaling compound during development and stress responses (reviewed by Abeles et al., 1992; Johnson and Ecker, 1998). Because ethylene is well diffusible in both aqueous and lipid environments (Abeles et al., 1992), its receptor could theoretically be located anywhere in the plant cell. Even before the actual identification of the ethylene receptor, Jerie et al. (1979) described the detection of high affinity binding sites for ethylene in membrane extracts from pea. Nearly a decade later, a forward genetics approach in Arabidopsis yielded a mutant that did not respond to ethylene and showed reduced ability to bind it (Bleecker et al., 1988). This mutant turned out to be mutated in a gene, named ETR1, that encoded a protein with similarity to bacterial two-component histidine kinases (Chang et al., 1993). The presence of saturable, high affinity ethylene-binding sites in yeast expressing ETR1 indicated that this protein was indeed an ethylene receptor (Schaller and Bleecker, 1995). In Arabidopsis, ETR1 is one of the five members of the putative ethylene- receptor family, the others being ERS1, ETR2, EIN4 and ERS2 (Hua et al., 1995; Sakai et al., 1998; Hua et al., 1998). They share extensive sequence similarity, and all the genes that encode these proteins can cause dominant ethylene-insensitivity when mutated. However, besides for ETR1, actual ethylene binding has only been shown for ERS1 so far (Hall et al., 2000). At least during seedling growth, the members of the ethylene-receptor family seem to act redundantly, as single null-mutants do not have a mutant phenotype at this stage of development. Null-mutants in multiple receptors, however, have a constitutive ethylene response phenotype, which showed that the receptors normally act as repressors of the ethylene response (Hua and Meyerowitz, 1998). The dominant ethylene-insensitive phenotype caused by some of the mutant alleles of ETR1 is due to the inability of the protein to bind ethylene, resulting in constitutive repression of the ethylene response, irrespective of the presence of ethylene and irrespective of the presence of other wild-type receptors (Rodriguez et al., 1999; Hall et al., 1999). Despite the thorough characterization of the ethylene receptors in recent years, still very little is known about the location of the receptors inside the cells. Based on gene structure and sequence similarity, the ethylene receptor genes can be divided into two subfamilies: ETR1 and ERS1 belong to subfamily I and ETR2, EIN4 and ERS2 belong to subfamily II (Bleecker, 1999). In conformance with the mentioned observations of Jerie et al. (1979), all five ethylene receptors contain predicted transmembrane segments in their amino-terminal part - three in the subfamily I members, four in the subfamily II members. This first, additional, hydrophobic domain of the subfamily II members is predicted to be a sorting signal for the secretory pathway (Schwacke et al., 2003), although it is still unknown whether these proteins are retained at the ER. A tobacco ethylene receptor homolog, most similar to the members of subfamily II, seems to be targeted to the plasma membrane of tobacco protoplasts in a study using a GFP fusion (Xie et al., 2003). However, the sequences of ETR1 and ERS1 do not reveal into which membrane system in the cell the subfamily I receptors are inserted. Studies using an antibody

24 ERS1 accumulates in the ER indicated that they are membrane associated proteins (Schaller et al., 1995; Hall et al., 2000) and more recently pointed towards the endoplasmic reticulum as the major site of accumulation of the ETR1 ethylene receptor (Chen et al., 2002). Because GFP tagging has been used successfully to determine the localization of various membrane proteins in plants, we used this technique to study the subcellular localization of ERS1. In this paper, we show that the ERS1 protein is associated with an intracellular structure likely to be the endoplasmic reticulum.

MATERIAL AND METHODS

Preparation of fusion constructs pMON-ST-tmdYFP and pMON-GFP-ER were kindly provided by J. E. Carette (University of Wageningen, The Netherlands). pMON-ST-tmdCFP was created by exchanging YFP for CFP using NcoI and SamHI. pMON-CFP-ER was created by exchanging GFP-KDEL by CFP-KDEL using EcoRI and SamHI. pMON-GFP-Zm7hvr was created by cloning the GFP-Zm7hvr fusion, kindly provided by Dr. J.E. Fowler (Oregon State University, Corvallis, USA) into pMON999-35S (Kay et al., 1987)using NcoI and KpnI. The expression vector pMON-EYFP was prepared by removing the CFP coding region from pMON999-35S, using NcoI and SamHI, and inserting the EYFP coding region, amplified from pEYFP-C1 (Clontech, USA) using the primers TACCATGGTGAGCAAGGGCGAGGAG and TCGGATCCTTACTTGTACAGCTCGTCCATGCC. A short adapter, consisting of the self-annealed oligonucleotides ctagagaagccatgggaggagg and catgcctcctcccatggcttct, was inserted between the XbaI and NcoI sites 5’ of the EYFP, thereby losing the original NcoI site. In the final fusion protein, the adapter encoded a Thr- Met-Gly-Gly-Gly peptide, which functioned as a flexible spacer between the receptor and the EYFP. The ERS1 cDNA was kindly provided by E. Meyerowitz, (Caltech, California, USA). Using PCR with primers aatctagaatggagtcatgcgattgttttgag and aaccatggtccagttccacggtctagtttg the ERS1 coding sequence was amplified without the stop codon and an XbaI site was added to the 5’ end and an NcoI site to the 3’ end. The amplified ERS1 coding region was inserted in the pMON-EYFP vector using XbaI and NcoI restriction sites, resulting in pMON-ERSI-EYFP. Using site-directed mutagenesis with oligonucleotides gcttatgcagtttggagcctttttcattctctgtggagctacgc and its complement we introduced the ers1-1 mutation in pMON-ERSI-EYFP, resulting in pMON- ers1-1-EYFP. For plant expression, one of the two CaMV 35S enhancer regions was removed from the pMON-ERS1-EYFP and pMON-ers1-1-EYFP, using Kpn2I restriction and self­ ligation. A cassette containing the 35S promoter, the fused coding regions and the NOS­ terminator was taken out of the pMON vector using PvuII and HindII and inserted in the plant transformation vector pCAMSIA2301, which was pre-digested with Ecl136II and HindIII.

25 Chapter 2

Cowpea protoplast transformation Cowpea (Vigna unguiculata L.) mesophyll protoplasts were prepared and transfected with 10 of plasmid DNA using the polyethylene glycol method as described (Van Bokhoven et al., 1993). Samples were mounted 17 hrs after transfection in 8- chambered cover slides (Nalge Nunc International, Rochester, USA). For FDA staining, FDA (5 mg/mL in acetone) was added to the protoplasts to a final concentration of 50 ^g/mL.

Arabidopsis transformation Arabidopsis thaliana L. ecotype Col-0 plants were grown on soil in a climate chamber at 22oC. The transformation vectors were first transferred to Agrobacterium tumefaciens strain LBA4404 using freeze-thaw transformation (Chen et al., 1994). Arabidopsis plants were transformed by the floral dip method, as described by Clough and Bent (1998). T1 seeds were grown on agar plates containing 0.5X MS salt, including B5 vitamins (pH6.0), 0.8% (w/v) agar and 50 ^g/mL kanamycin. Resistant seedlings were transplanted to soil.

Triple-response assay T2 seeds were sown on agar plates as described. Col-0 and etr1-1 seeds were grown without kanamycin. The seeds were stratified for 4 days at 4oC and then moved to 22oC. After 12 hours in the light, the plates were placed vertically in a desiccator and moved to the dark. For ethylene-treatment, ethylene was added to a concentration of 5 ppm, as confirmed by gas chromatography at the beginning and the end of the treatment. After four days, the plates with seedlings were scanned at high resolution using a flatbed scanner.

Fluorescence imaging Fluorescence microscopy of protoplasts and Arabidopsis seedlings was performed using a Zeiss LSM 510 CLSM (Confocal Laser Scanning Microscope) (Zeiss, Oberkochen, Germany) implemented on an inverted microscope (Axiovert 100). Excitation was provided by the 458, 488 and 514 nm Ar laser lines controlled by an acousto optical tuneable filter (AOTF). For GFP and fluorescein detection the HFT488 dichroic beam splitter was used as a primary dichroic mirror, reflecting excitation and transmitting fluorescence emission, an NFT635 dichroic mirror was used as a secondary splitter. Fluorescence reflected by the NFT635 was additionally filtered through a BP505-550 filter yielding the GFP signal. Fluorescence transmitted through the NFT635 splitter was filtered through an LP650 filter yielding the chlorophyll signal. For triple color imaging of CFP, YFP and chlorophyll, three dichroic beam splitters were used to separate excitation from emission and to divide the fluorescence emission into the CFP, YFP and chlorophyll channels. The HFT 458/514 dual dichroic beam splitter was used as a primary dichroic mirror, a NFT 635 dichroic mirror was used as a secondary splitter and an NFT 515 was used as tertiary dichroic splitter. Fluorescence reflected by both the NFT 635 and NFT515 splitters was filtered through a

26 ERS1 accumulates in the ER

Figure 1. Schematic structural diagram of the used fusion proteins. The constructs encoding these fusions consist of a CaMV 35S promoter, the coding region of ERS1, a five-peptide spacer, the coding region of EYFP and a NOS-terminator. The position of the ers1-1 mutation is indicated with an *.

BP470-500 nm filter yielding the CFP signal. Fluorescence reflected by the NFT635 but transmitted by the NFT 515 splitter was filtered through a BP 530-600 nm filter yielding the YFP channel. Fluorescence transmitted by both the NFT 515 and 635 splitters was additionally filtered by an LP 650 filter to yield the chlorophyll image. Cross-talk free CFP and YFP images were acquired by operating the microscope in the multi-tracking mode, in which the 514 nm excitation was coupled to activation of the YFP-detection channel, and the 458 nm excitation was coupled to activation of the CFP- and chlorophyll-detection channels. A Zeiss water immersion C-Apochromat 40x objective lens (N.A. 1.2) corrected for cover glass thickness (set at 0.16 ^m for Nunc 8-chambered coverslides) was used for scanning. The detection pinholes were set to yield optical sections of approximately 10 ^m for analysis of the level of fluorescence and the FDA staining, and to yield optical sections of approximately 1 ^m for localization analyses. Images were captured and analyzed with the Zeiss LSM510 software (v3.0 SP3).

RESULTS

Cowpea protoplasts transformed with an ERS1-EYFP fusion construct show a low level of fluorescence in the endoplasmic reticulum and in Golgi bodies and aggregates In order to investigate the subcellular localization of the ethylene receptor ERS1, a translational fusion to the 5’ end of the coding region of the green fluorescent protein (spectral variant EYFP) was made and placed under transcriptional control of a double cauliflower mosaic virus (CaMV) 35S promoter (fig. 1). The E R S 1 -E Y F P construct was expressed transiently in cowpea mesophyll protoplasts. As a control for transfection, a similar construct encoding EYFP only was used. Transfected protoplasts were analyzed by CLSM 16 to 24 hours after transfection. More than 80% of the protoplasts transformed with the EYFP construct was highly fluorescent, whereas less than 10% of the protoplasts transformed with the E R S 1 -E Y F P fusion was fluorescent (fig 2). FDA viability staining showed no obvious difference in the ratio between living and dead protoplasts when transformed with the E R S 1 -E Y F P construct or with the EYFP construct (fig. 2), which

27 Chapter 2

Fluorescence analysis FDA viability staining EYFP ERS1-EYFP EYFP ERS1-EYFP

Figure 2. Analysis of fluorescence and viability of protoplasts transformed with EYFP or ERS1- EYFP. Fluorescence in the EYFP and the chlorophyll channel is projected in yellow and red, respectively. Fluorescence of fluorescein is projected in green. The presence of fluorescein indicates that a protoplast is viable. Bar = 100 ^m. Color figure, see appendix.

indicates that the low percentage of fluorescent ERS1-EYFP protoplast was not caused by a lethal phenotype of the protoplasts that strongly expressed the fusion protein. Apparently, the ERS-EYFP fusion protein was expressed at a much lower level than EYFP, resulting in less than 10% of the protoplasts showing enough fluorescence for localization analysis. To localize the fluorescence of the fusion protein, the cowpea protoplasts were analyzed at higher magnification. As references for labeling of the cytoplasm/nucleus, the endoplasmic reticulum (ER), the Golgi bodies and the plasma membrane, protoplasts were transformed with CFP, CFP-ER (Haseloff et al., 1997), ST-tmdCFP (Saint-Jore et al., 2002) or GFP-Zm7hvr (Ivanchenko et al., 2000), respectively. Figure 3 shows that in the protoplasts transformed with ERS1-EYFP, fluorescence was associated with intracellular strands and sometimes with small fluorescent spheres or large aggregates. Because this pattern had similarities to those observed with the cytoplasm/nucleus marker, the ER marker and the Golgi body marker, double transformations with these markers were performed. Figure 4 shows that ERS1-EYFP partially co-localized with the cytoplasmic CFP, but the pattern of the ERS1-EYFP fluorescence was more reticular and fluorescence was also present around the nucleus. By contrast, ERS1-EYFP clearly co-localized with CFP-ER, which indicated that the strands that were labeled with ERS1-EYFP was in fact ER. Most of the small fluorescent spheres co-localized with the Golgi body marker. However, comparison of this marker in protoplasts with and without ERS1-EYFP expression showed that it was mislocalized in the protoplasts with high ERS1-EYFP expression (fig. 4), as it should be highly specific for the Golgi bodies (Saint-Jore et al., 2002; fig. 3). Similarly, the larger aggregates that were sometimes observed were labeled by both the ER marker and the Golgi body marker (data not shown). Taken together, these data indicate that the ERS1-EYFP fusion protein accumulates in the ER in cowpea protoplasts, but if expressed at higher levels, it interferes with proper functioning of the ER-Golgi system resulting in its accumulation in ER/Golgi- derived bodies and aggregates.

28 ERS1 accumulates in the ER

CFP CFP-ER ST-tmdCFP GFP-Zm7hvr

Figure 3. Fluorescence imaging of transformed cowpea protoplast. The reference protoplasts express CFP, CFP-ER, CFP-golgi and GFP-ROP7(HVR), which label the cytoplasm/nucleus, the ER, the Golgi and the plasma membrane respectively. The four examples of protoplasts expressing ERS1-EYFP show different patterns observed in the protoplast population. In the left upper panel, the chloroplasts are projected in red. n, nucleus; c, chloroplast. Bar = 10 ^m. Color figure, see appendix.

The receptor moiety of the ers1-1-EYFP fusion protein is biologically active in Arabidopsis Because of the disturbance of the ER-Golgi system it was not certain that the localization of the ERS1-EYFP protein that was observed in the cowpea protoplasts would reflect the true localization in planta. Therefore the fusion protein was stably transformed into Arabidopsis plants. An advantage of in planta expression was that it provided the possibility to implement a control for intact functioning of the ERS1 protein in the fusion. To this end, a nucleotide change was introduced in the ERS1 coding region in the fusion construct, resulting in a Phe62Ile substitution (construct ers1-1-EYFP; fig.1). This mutation is identical to the ers1-1 mutation, which is a dominant gain-of-function mutation that confers strong ethylene insensitivity in Arabidopsis (Hua et al., 1995). The constructs encoding fusions with wild-type and mutant receptors were transferred to Arabidopsis using Agrobacterium mediated transformation. Thirteen independent kanamycin resistant lines were obtained with ERS1-EYFP and seven with ers1-1-EYFP. Ethylene-sensitivity was tested using the triple-response assay (Guzman and Ecker, 1990). This assay is based on the fact that wild-type seedlings grown in the dark show a markedly different morphology when ethylene is added to the air: the root and hypocotyl stay shorter, the hypocotyl gets thicker and the apical hook becomes accentuated. Figure 4 shows that wild-type Col-0 seedling showed a clear triple response in the assay, whereas etr1-1, an ethylene-

29 Chapter 2

Figure 4. Fluorescence imaging of double transfected protoplast. Protoplasts were analysed for EYFP and CFP fluorescence. The whiter color in the overlay indicates co-localization of EYFP and CFP. Multiple protoplasts were analysed and representative results are shown. In the left upper panel, the chloroplasts are projected in red. n, nucleus; c, chloroplast. Bar = 10 ^m. Color figure, see appendix.

insensitive mutant (Bleecker et al., 1988), was unaffected by the presence ethylene. All plants transformed with ERS1-EYFP showed a normal triple response. However, three out of seven ers1-1-EYFP lines showed a reduced response to ethylene and three lines showed no response to ethylene. The fact that several ers1-1-EYFP expressing plants had an ethylene-insensitive phenotype implies that the receptor moiety of the fusion was signaling properly and that it is likely that the fusion protein was targeted correctly. Therefore, it was concluded that the ERS1-EYFP and ers1-1-EYFP lines were suitable for further analysis.

Arabidopsis transformants expressing ers1-1-EYFP show fluorescence in a dynamic intracellular structure resembling the endoplasmic reticulum Seedlings of the ERS1-EYFP and ers1-1-EYFP lines and wild-type plants were grown in the dark for four days, carefully removed from the plates and mounted in water under a cover slip. First, they were analyzed using a wide-field fluorescence microscope.

30 ERS1 accumulates in the ER

ERS1- ers1-1- wt etrl-1 EYFP EYFP

Figure 5. Analysis of ethylene sensitivity using the triple-response assay. Seedlings of various genotypes were grown in the dark for four days in air or in air supplemented with 5 ppm ethylene. Of the ers1-1-EYFP lines, a line with reduced sensitivity to ethylene and a line with no sensitivity to ethylene are shown.

Cells in the root and hypocotyl of the ethylene-insensitive ers1-1-EYFP lines showed fluorescence that was slightly above background, as determined in wild-type seedlings. The level of fluorescence correlated with the level of ethylene insensitivity of the different lines. In the ERS1-EYFP lines, no fluorescence above background level was detectable. To determine the subcellular localization of the fusion proteins, the roots of the seedlings were analyzed using CLSM. A low level of fluorescence was seen in the ERS1-EYFP lines and ers1-1-EYFP lines, but the level of fluorescence in the former was too low for localization studies (data not shown). Figure 5 shows that in the ers1-1-EYFP lines fluorescence was observed in the nuclear envelope of endodermal cells in the root tip and of epidermal cells of the root body. In these latter cells, reticular and strand-like structures were also observed. This pattern of fluorescence was very similar to that seen in plants expressing GFP-ER (Haseloff et al., 1997), which localizes to the endoplasmic reticulum. Time-series showed that the fluorescent structures in the ers1-1-EYFP lines and in the GFP-ER expressing plants were highly dynamic (data not shown).

DISCUSSION

ERS1 is one of the two subfamily I ethylene receptors in Arabidopsis (Hua et al., 1995, 1998). To better understand the functioning of ERS1, we studied the subcellular

31 Chapter 2

Figure 6. Fluorescence imaging of ers1-1-EYFP and GFP-ER in root endodermis cells and in single root epidermis cells of Arabidopsis transformants. Images of GFP-ER are from Haseloff line Q2500 for the endodermis and line J0631 for the epidermis (Berger et al., 1998). n, nucleus. Bar = 10 ^m. Color figure, see appendix.

localization of this protein in living plant cells using a GFP tagging strategy. Correct localization of the fusion protein was indicated by the observation that the receptor moiety of the fusion was biologically active in Arabidopsis plants (fig. 4). The results obtained with transiently transformed cowpea protoplasts and stably transformed Arabidopsis plants are consistent with localization of the ERS1-EYFP and ers1-1-EYFP fusion proteins in the ER (fig. 3, 5). Accumulation of ERS1-EYFP in bodies or aggregates seems to be an artifact of the cowpea expression system, where high levels of the protein appeared to disturb the ER-Golgi structures. Plant cells seem to have a tight regulation of the level of the ethylene receptors. It has been reported that expression of an ETR1 fusion protein under control of the strong CaMV 35S promoter resulted in low, close to native, levels of protein (Chen et al., 2002) and also in our Arabidopsis transformants, the level of fluorescent protein was much lower than expected from a CaMV 35S promoter driven gene. That this regulation occurs at the transcript level is suggested by the low level of transgene transcript in these plants (preliminary data), and by the finding that in tomato, a decrease in the level of NR mRNA was compensated by slight increase in the level of LeETR4 mRNA (Tieman et al., 2000). However, translational or post-translational control occurs as well, and has been shown to up-regulate the level of ETR1 protein two to three­ fold in case of the presence of ethylene insensitive mutations in the protein (Zhao et al., 2002). This latter mechanism may also explain the higher level of fluorescence in the ers1- 1-EYFP lines than in the ERS1-EYFP lines. Localization of ERS1 at the ER agrees with the findings of Evans et al. (1982a, b), who reported the presence of ethylene binding sites at the ER in Phaseolus vulgaris. Also the second subfamily I ethylene receptor, ETR1, is present at the ER (Chen et al., 2002). Because ETR1 and ERS1 have been shown to form homodimers (Schaller et al., 1995; Hall et al., 2000), it has been suggested that also heterodimers between the receptors may form and they may even exist in larger multimeric complexes. Co-localization of ETR1 and

32 ERS1 accumulates in the ER

ERS1 implies that physical interaction between these two proteins may be possible. For ETR1 it has been shown that the localization signals are present in the N-terminal half of the protein, as this part of the protein is sufficient for ER localization (Chen et al., 2002). That this holds true for ERS1 is suggested by the high level of homology with ETR1 in the N-terminal region. In cowpea protoplasts, removal of the C-terminal cytoplasmic part of the ERS1 protein from the fusion did not have an effect on its localization or the formation of aggregates (data not shown). What the ER targeting and retention signals are remains to be determined. ERS1 does not contain a cleavable signal peptide (Schwacke et al., 2003), so it should contain a signal-anchor, encoded for by the most N-terminal transmembrane region (Lipp and Dobberstein, 1986; Notwehr and Gordon, 1990). However, also in this region no primary sequence homology to known signals is found (Schwacke et al., 2003). Because ERS1 fusion proteins were not transferred further through the secretory pathway to the plasma membrane, the protein must contain a signal to retain it at the ER. The only well-described ER retention signal for membrane proteins, the double lysine motif (Jackson et al., 1990; Benghezal et al., 2000), does not occur in ERS1, indicating the presence of an as yet unidentified retention signal. Remains the question why the ethylene receptors should be located in the ER membrane. Because ethylene is soluble in lipid and aqueous environments and because the C-terminal domain probably resides in the same cytosolic environment, either when anchored at the ER membrane or the plasma membrane (Schaller et al., 1995), there may be no need to transport the receptor further to the cell surface. ER targeting of the ethylene receptors may be due to the cyanobacterial ancestry of these proteins (Mount and Chang, 2002), because there are similarities in the mechanism used for membrane targeting in bacteria and ER targeting in eukaryotes (Garcia et al., 1987). However, keeping a protein from being secreted to the plasma membrane requires additional retention signals. If it would not be essential for the ERS1 protein to be located in the ER membrane, conservation of such retention signals seems to be unlikely. Localization at the ER may allow ERS1 to directly affect ER-based processes such as calcium release, as internal calcium stores have been shown to be involved in ethylene responsiveness (Raz and Fluhr, 1992). Alternatively, the localization at the ER may ensure specificity for the smallest alkene, ethylene, by keeping the ethylene-binding site protected from higher molecular weight alkenes. The weaker effectiveness of these higher molecular weight alkenes in inducing ethylene responses (Burg and Burg, 1967) may be partly due to lower diffusion rates in the cells. Finally, it is still possible that the ER localized fraction of ERS1 that we visualized by GFP tagging is not the active fraction, but merely a fraction in storage. Indeed, preliminary data on the effect of ethylene on one of the ERS1-EYFP lines showed that it resulted in an increased level of fluorescence and its localization along the plasma membrane of epidermal root cells. However, further experiments with ethylene treatment are necessary to verify putative translocation of the ERS1 ethylene receptor. Due to the low expression level, the results obtained with fluorescence imaging are not completely indisputable. It would be useful to confirm that the fusion protein is

33 Chapter 2

membrane associated using western blot analysis, but to unambiguously determine the localization of the fusion protein, it would be necessary to do an immuno-localization study on EM level.

REFERENCES

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34 ERS1 accumulates in the ER

10: 1321-1332 Hua J, Meyerowitz EM (1998) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94: 261-271 Ivanchenko M, Vejlupkova Z, Quatrano RS, Fowler JE (2000) Maize ROP7 GTPase contains a unique, CaaX box-independent plasma membrane targeting signal. The Plant Journal 24: 79-90 Jackson MR, Nilsson T, Peterson PA (1990) Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO Journal 9: 3153-3162 Jerie PH, Shaari AR, Hall MA (1979) The compartmentation of ethylene in developing cotyledons of Phaseolus vulgaris L. Planta 144: 503-507 Johnson PR, Ecker JR (1998) The ethylene gas signal transduction pathway: a molecular perspective. Annual Review Of Genetics 32: 227-254 Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299-1302 Lipp J, Dobberstein B (1986) The membrane-spanning segment of invariant chain (I gamma) contains a potentially cleavable signal sequence. Cell 46: 1103-1112 Moody T (1993) Growth factors, peptides and receptors. Plenum Press, New York Mount SM, Chang C (2002) Evidence for a plastid origin of plant ethylene receptor genes. Plant Physiology 130: 10-14 Notwehr S, Gordon J (1990) Targeting of proteins into the eukaryotic secretory pathway: signal peptide structure/function relationships. BioEssays 12: 479-484 Raz V, Fluhr R (1992) Calcium requirement for ethylene-dependent responses. The Plant Cell 4: 1123-1130 Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB (1999) A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science 283: 996-998 Saint-Jore CM, Evins J, Batoko H, Brandizzi F, Moore I, Hawes C (2002) Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks. The Plant Journal 29: 661-678 Sakai H, Hua J, Chen QG, Chang C, Medrano LJ, Bleecker AB, Meyerowitz EM (1998) ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 95: 5812-5817 Schaller GE, Bleecker AB (1995) Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene. Science 270: 1809-1811 Schaller GE, Ladd AN, Lanahan MB, Spanbauer JM, Bleecker AB (1995) The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer. The Journal of Biological Chemistry 270: 12526-12530 Schwacke R, Schneider A, van der Graaff E, Fischer K, Catoni E, Desimone M, Frommer WB, Flugge UI, Kunze R (2003) ARAMEMNON, a novel database for Arabidopsis integral membrane proteins. Plant Physiology 131: 16-26 Tieman DM, Taylor MG, Ciardi JA, Klee HJ (2000) The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proceedings of the National Academy of Sciences of the United States of America 97: 5663-5668 Tsai MJ, O'Malley BW (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annual Review of Biochemistry 63: 451-486 Van Bokhoven H, Verver J, Wellink J, van Kammen A (1993) Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA. Journal of General Virology 74: 2233-2241 Xie C, Zhang JS, Zhou HL, Li J, Zhang ZG, Wang DW, Chen SY (2003) Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco. The Plant Journal 33: 385-393 Zhao XC, Qu X, Mathews DE, Schaller GE (2002) Effect of ethylene pathway mutations upon expression of the ethylene receptor ETR1 from Arabidopsis. Plant Physiology 130: 1983-1991

35

Chapter 3

Expression analysis of five tobacco EIN3 family members in relation to tissue-specific ethylene responses

Ivo Rieu, Titti Mariani and Koen Weterings

Journal of Experimental Botany (2003) 54: 2239-2244

ABSTRACT

Ethylene induces different sets of genes in different tissues and at different stages of development. To investigate whether these differential responses are caused by differential expression of members of the EIN3 family of transcription factors, five tobacco family members were isolated. They can be divided into three subgroups, which is probably due to the amphidiploid nature of tobacco. In phylogenetic analysis, each of the subgroups clustered with one of the three tomato EIL proteins and all NtEILs proved to be most homologous to Arabidopsis EIN3 and EIL1. Although organ-specific ethylene responses have been observed before, northern blot analysis showed that all NtEILs were expressed in all organs. To study differential NtEIL expression at the cellular level, in situ hybridization was used on the tobacco ovary. It was found that different ovary tissues displayed variable ethylene-induced expression of two ethylene-responsive marker genes. By contrast, no differences were found in expression level or tissue-specificity for any of the NtEILs in the ovary, before or after ethylene treatment. This indicates that the organ and tissue-specific ethylene responses are not caused by differential expression of NtEIL family members. These results support a model in which the developmental signals that regulate the tissue- specific responses are integrated with the ethylene signal downstream of a common primary ethylene-signaling pathway.

INTRODUCTION

The plant hormone ethylene regulates a variety of processes, such as the "triple response” of germinating seedlings, root hair formation, leaf expansion, fruit ripening and petal senescence after fertilization. In addition, ethylene is known to induce defense

37 Chapter 3 responses in reaction to biotic and abiotic stresses (Abeles et al., 1992). Initiation of these processes involves complex regulation of ethylene biosynthesis and depends on the ability of cells to respond in an appropriate manner. During the last decades considerable progress has been made towards the elucidation of both the ethylene-biosynthesis pathway and the ethylene-response pathway. In higher plants, ethylene is synthesized from methionine via formation of S-adenosyl-L-methionine (AdoMet) and 1- aminocyclopropane-1-carboxylate (ACC). The individual reactions of this biosynthetic pathway are catalyzed by the enzymes AdoMet synthase, ACC synthase and ACC oxidase, respectively (Yang and Hoffman, 1984; Kende 1993). Analysis of Arabidopsis thaliana mutants with an aberrant triple response, either absent or constitutive, has led to the identification of a number of components of the primary ethylene-signaling pathway (reviewed by Johnson and Ecker, 1998). Ethylene is perceived by a family of integral membrane receptors that are similar to bacterial two-component histidine kinase receptors and includes ETR1, ERS1, EIN4, ETR2 and ERS2 (Bleecker et al., 1988; Chang et al., 1993; Hua et al., 1995, Sakai et al., 1998; Hua et al., 1998). A Raf-like protein kinase called CTR1 acts downstream of the receptors (Kieber et al., 1993). The receptors and CTR1 actively repress the downstream pathway in the absence of ethylene (Kieber et al., 1993; Hua and Meyerowitz, 1998). The ethylene signal is propagated via a MAPK pathway (Ouaked et al., 2003) to EIN2, which is a membrane protein with homology to Nramp metal-ion transporters (Alonso et al., 1999). The unknown gene products of EIN5, EIN6 and EIN7 also act downstream of CTR1 (Roman et al., 1995). Finally, the signal is transmitted to the EIN3 family of transcription factors. Out of this six-member family, only for EIN3 and EIN3-LIKE1 (EIL1) a function in the ethylene signaling pathway was demonstrated conclusively (Chao et al., 1997; Alonso et al., 2003). EIN3 has been shown to act as a transcriptional activator and bind to the primary ethylene-response element present in the promoter of the ethylene-responsive ERF1 gene (Chao et al., 1997; Solano et al., 1998). Ethylene induces different sets of genes in different tissues, and the response also depends on the developmental stage of the tissue (e.g. Lincoln and Fischer, 1988; Raghothama et al., 1997; Ruperti et al., 2002; Verlinden et al., 2002). At the moment, it is not known how developmental information of the cells is integrated with the ethylene signal to obtain these tissue-specific ethylene responses. One possibility is that the developmental signal acts upstream of the ethylene-signaling pathway and regulates its composition. A potential target for such a regulatory mechanism is represented by EIN3, which is the last component of the primary signaling pathway. Because the EIN3 family members each may act on specific target genes, differential expression of the EIN3 genes could be responsible for the tissue-specific ethylene responses. Although recent silencing experiments suggest redundancy within the tomato (Lycopersicon esculentum) EIN3 family (Tieman et al., 2001), a detailed analysis of gene expression has not been reported so far. In order to investigate the role of the EIN3 family in the ethylene-signaling pathway in tobacco (Nicotiana tabacum), the family members have been isolated, designated NtEILs, and their expression pattern studied at the organ and at the cellular level. Although

38 The tobacco EIN3 family there are organ-specific ethylene responses in tobacco, all NtEILs were found to be expressed in all organs. In addition, using two ethylene-response marker genes, tissue- specific ethylene responses were identified within the ovary. However, mRNA of all NtEILs was detected in all cells of the ovary. Together, these data suggest that developmental cues that cause the tissue-specific ethylene responses are integrated downstream of the primary ethylene-signaling pathway.

MATERIALS AND METHODS

Plant material and treatments Seeds of Nicotiana tabacum L. cv. Petit Havana SR1 were derived from the Solanaceae germplasm collection of the Botanical garden of the University of Nijmegen, The Netherlands (http://www-bgard.sci.kun.nl). Plants were grown under standard greenhouse conditions with additional lighting. Plant tissues were harvested, frozen in liquid nitrogen and stored at -80oC. For experiments with ethylene treatments, flowers at stage 9 (according to Koltunow et al., 1990) were harvested, placed in a desiccator with the petioles submerged in water and incubated for 24 h with air or with air containing 1 ^l/l ethylene. The ethylene concentration was confirmed by gas chromatography at the beginning and at the end of the treatment.

Isolation of NtEIL cDNAs A tobacco ovary cDNA library was constructed from poly(A)+ RNA purified from ovaries at stage 1 to stage 6. cDNA synthesis and cloning was preformed by using the ZAP Express cDNA synthesis and cloning kit from Stratagene (La Jolla, CA, USA) according to the manufacturers instructions. A partial cDNA was amplified from this cDNA library by PCR, using the following degenerated primers: GTMYARRGCHCAAGAYGG and GMHGTCATYTTVTCCTGY. The resulting fragment was used to screen the same cDNA library under low stringency conditions according to the manufacturers instructions. The cDNA clones were sequenced using vector and gene-specific synthetic primers. The 5’-end of the NtEIL1 cDNA was extended with 254 bp by PCR amplification of a 648 bp fragment from the cDNA library, using an upstream primer based on the highly homologous NtEIL2 sequence (GGGAAGTCATCAATTTGAG) and a downstream primer specific for NtEIL1 (GAAGCTCCTGCAAAGTG). To extend the 5’-end of the NtEIL4 cDNA with 110 bp, a 1038 bp fragment was amplified with a primer based on the highly homologous NtEIL3 sequence (GAGGATTTT GGGT ATT ATCT GG) and an NtEIL4-specific primer (ACCAGCTGAGGACAAGGC). The large overlapping part of the resulting fragments was used to ensure that the fragments belonged to the desired cDNA clone. All five NtEILs were sequenced completely at least two times. The presence of the stop codon in NtEIL3 was confirmed by sequencing a clone obtained independently by RT-PCR. Genbank

39 Chapter 3 accession numbers are AY248903 (NtEIL1), AY248904 (NtEIL2), AY248905 (NtEIL3), AY248906 (NtEIL4), AY248907 (NtEIL5).

Phylogenetic analysis Protein sequences were aligned using the ClustalW program (version 1.82 at http://www.ebi.ac.uk/clustalw/index.html; Thompson et al., 1994). The Phylip package (version 3.5c at http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html; Felsenstein, 1989) was used to calculate a distance matrix and to generate a tree using the bioneighbour-joining method (Gascuel, 1997). An unrooted tree was plotted using the drawtree program of the package.

RNA analysis Total RNAs were extracted using the method of Van Eldik et al. (1995). 10^g of total RNA was separated on a formaldehyde agarose gel and blotted on Hybond-N membrane (Amersham Biosciences, Buckinghamshire, UK). Northern hybridization was carried out according to the instructions for Hybond-N. Washing was done with a 1xSSC buffer at 57oC. NtEIL probes were prepared from restriction fragments corresponding to bp 2004-2392 of NtEIL1 (EcoRV/ScaI), bp 1987-2277 of NtEIL3 (BglII/MscI) and bp 1907­ 2241 of NtEIL5 (EcoRI/HindIII). In-situ hybridization studies were performed as described by Cox and Goldberg (1988) with minor modifications. Immediately after ethylene-treatment the ovaries were excised and fixed overnight at 4°C in 1% glutaraldehyde, 4% paraformaldehyde solution prepared in 0.1 M phosphate buffer (pH 7.0), dehydrated, cleared, and embedded in paraffin. Eight-micrometer-thick sections were hybridized to 33P-labeled antisense RNA probes at a specific activity of 4 to 5 x 108 dpm/^g. Probes were transcribed from a tobacco ACC-oxidase cDNA clone (Weterings et al., 2002), a cloned PCR fragment corresponding to bp 1753-2068 of the tobacco ERS homolog (Terajima et al., 2001) and from clones of the NtEIL fragments described above. Hybridization with sense probes was used to determine the background signal. After hybridization and emulsion development, digital photographs were taken with a CoolSNAP CCD camera (Roper Scientific, USA) using bright-field illumination on a Leitz Orthoplan (LSM, Wetzlar, Germany). The images were enhanced using Adobe Photoshop 5.0 (San Jose, CA).

RESULTS

The tobacco EIN3 family To identify tobacco EIN3 family members, a PCR based approach was taken using degenerate oligonucleotides based on the sequence of the four Arabidopsis EIN3 family proteins (Chao et al., 1997). A 570-bp fragment was amplified from a cDNA library made from tobacco ovaries. This fragment was 97% homologous to TEIL, a recently identified tobacco EIN3 family member (Kosugi and Ohashi, 2000) and was used to screen the same

40 The tobacco EIN3 family

Figure 1. Analysis of the tobacco EIN3 family. A. Schematic structural diagram of the deduced NtEIL proteins and the Arabidopsis EIN3 protein (Chao et al., 1997), indicating the location of the acidic domain, the basic domains (BD-I to BD-V) and the proline-rich region. The number of amino acids is indicated between brackets. B. Phylogenetic analysis of EIN3 homologs from tobacco (with exception of the short NtEIL3), tomato (Tieman et al., 2001) and Arabidopsis (Chao et al., 1997). Accession numbers At5g10120 and At5g65100 refer to putative proteins from Arabidopsis identified by the Arabidopsis Genome Initiative (2000).

tobacco cDNA library under low stringency. The seventeen positive clones that resulted from this screening corresponded to five different EIN3-like cDNAs, which were named NtEIL1 (Nicotiana tabacum E[N3-LIKE1; previously TEIL) to NtEIL5. The cDNAs of NtEIL1 and NtEIL4 were extended to obtain full-length clones (see materials and methods). The sequence identity between the NtEIL cDNAs ranges from 48% to 96%, with NtEIL1/2 and NtEIL3/4 being highly homologous pairs. Analysis of the NtEIL cDNAs revealed that they share predicted amino acid sequence similarity to the Arabidopsis EIN3 protein, with the exception that NtEIL3 has a premature stop codon. Figure 1A shows that the NtEIL proteins possess many of the structural features found in the Arabidopsis EIN3 family. All proteins have an acidic domain at the N-terminus and, with the exception of the shorter NtEIL3 protein, contain five basic domains, a proline-rich region and a C-terminus rich in asparagine. The mono-amino acid repeats of asparagine and glutamine present in the C- terminus of Arabidopsis EIN3 and EIL1, respectively, are absent in the NtEILs. As in AtEIN3, the amino acids between the acidic N-terminus and the basic domain II are predicted to form a coil (Lupas, 1996). To examine the relation between the EIN3-like proteins from tobacco, tomato and Arabidopsis, a phylogenetic analysis was performed. Figure 1B shows that each branch containing a tomato EIL also contained one or two of the tobacco EILs. Furthermore, all tobacco and tomato proteins proved to be more related to Arabidopsis EIN3 and EIL1 than to the other EILs.

Accumulation of NtEIL mRNA in cells with different ethylene responses Accumulation of NtEIL mRNA in plant organs was determined by northern analysis. Because of the high homology between the cDNAs of NtEIL1 and NtEIL2 (96%) and between the cDNAs of NtEIL3 and NtEIL4 (96%) the probes used in this experiment did not

41 Chapter 3

Figure 2. Expression analysis of the NtEIL genes and ACO and ERS genes. A. Northern blots, loaded with 10 |j.g total RNA from different organs, were hybridized with the probes as indicated in the left panel. Equal loading of the gels was confirmed by ethidium bromide (EtBr) staining. B. In-situ expression analysis of the ERS and ACO genes in ovaries of tobacco flowers incubated in air or ethylene. C. In-situ expression analysis of the NtEIL genes in ovaries of tobacco flowers incubated in air. Sections of ovaries in B and C were hybridized with sense and antisense probes, exposed for 5 days (ERS and ACO probes) or 27 days (NtEIL probes) and examined using bright-field illumination. Hybridization signal appears as black grains. ep, epidermal layer of placenta; ov, ovule; vb, vascular bundles; cw, carpel wall. Bar = 100 ^m. Color figure, see appendix.

discriminate between these related NtEILs, but they did discriminate between the other NtEILs as confirmed by dot-blot analysis (data not shown). The probes recognized a single fragment of about 2.6 kb. Figure 2A shows that mRNA of the NtEILs was present at almost similar levels in all plant organs examined, although the NtEIL1/2 signal was somewhat higher in root and the NtEIL3/4 and NtEIL5 signals somewhat lower in seeds. To be able to compare the expression of the NtEILs between cells that have different ethylene responses, in-situ hybridization analysis of two genes that were known to be ethylene-inducible, the tobacco ERS gene and ACC-oxidase (ACO) gene (Terajima et

42 The tobacco EIN3 family al., 2001; Rieu et al., 2003), was performed first. Figure 2B shows that after treatment with ethylene, the cells of the vascular bundles and placental epidermis gave a strongly increased ERS and ACO hybridization signal. By contrast, the cells of the ovules showed a strong increase in ERS hybridization signal and only a weak increase in ACO signal and the cells of the carpel wall did not show an effect of ethylene on the hybridization signal for any of the two marker genes. To see if these different responses could be related to expression patterns of specific NtEILs, sections from the same ovaries were hybridized with probes against NtEIL1/2, NtEIL3/4 and NtEIL5. As shown in figure 2C, all NtEILs were expressed throughout the ovary, albeit at a low level. The level and pattern of NtEIL expression were not influenced by ethylene-treatment (data not shown). Taken together, these results show that, although different cells within the ovary respond differently to ethylene, the NtEILs do not show differential expression.

DISCUSSION

Five different NtEIL cDNAs from tobacco that encode deduced proteins with high similarity to the Arabidopsis EIN3 protein have been isolated (Fig. 1A). Highest homology was found between the N-termini, which contain the DNA-binding domain and the dimerization domain (Kosugi and Ohashi, 2000; Solano et al., 1998). When compared to tomato, a closely related solanaceous species, one or two close homologs for each of the three LeEILs were found (Fig. 1B), which is probably due to the amphidiploid nature of tobacco. The fact that the LeEILs are functionally similar to Arabidopsis EIN3 (Tieman et al., 2001) suggests that the NtEILs fulfill the EIN3 function in tobacco. Indeed, it has been shown that over-expression of NtEIL1 in Arabidopsis causes a constitutive triple response phenotype (Kosugi and Ohashi, 2000). The introduction of an early stop codon in the NtEIL3 gene seems to be a very recent event, as the remainder of the open reading frame on the cDNA is still intact and would generate a protein with 96% identity to NtEIL4. So, although partial transcription factors can have a function as negative regulators (e.g. CAPRICE; Lee and Schiefelbein, 2002), this is probably not the case for NtEIL3. From genetic studies in Arabidopsis it is not clear whether the EIN3 family members are functionally redundant or have distinct functions. Recently, analysis of ein3/eil1 double mutants showed that EIN3 and EIL1 are fully responsible for ethylene-signal transmission during the triple-response of seedlings and act redundantly during this process (Alonso et al., 2003). The inability of the remaining family members to compensate for EIN3/EIL1 deficiency may reflect functions in other tissues or during other stages of development or functions other than in ethylene signaling. In tomato, silencing experiments pointed towards functional redundancy within the EIN3 family of this species (Tieman et al., 2001). If tissue- specificity of ethylene responses is regulated at the level of the EIN3 component, the different EIN3 family members need to be expressed or activated in a tissue-specific manner. It has been observed before that different plant organs react differently to ethylene. In addition, it was demonstrated that even within an organ, different tissues can

43 Chapter 3

react differently to ethylene, using the ERS and ACO genes as markers for the ethylene response in the tobacco ovary (Fig. 2B). However, it is shown that all NtEILs are expressed in all organs and in all ovary cells (Fig. 2A, C). Therefore, these results imply that the differences in ethylene response are not due to differences in composition of the primary ethylene-signaling pathway at the level of EIN3. Although it should be noted that almost nothing is known about the accumulation and activity of the EIN3 proteins, all data collected so far on ethylene signaling and responses fit in a model in which the primary signaling pathway is similar in all cells, and in which the unique developmental signals and the ethylene signal are integrated downstream of this primary signaling pathway. Combinatorial control of the expression of ethylene-responsive genes such as those from the Ethylene Response Factor (ERF) family (Solano et al., 1998; Fujimoto et al., 2000) may well be the actual point of regulation.

ACKNOWLEDGEMENTS

The authors wish to thank Jelle Eygensteyn for assistance with sequencing, the greenhouse personnel for excellent plant material, and Liesbeth Pierson for help with microscopy. This research was funded by grant 805-22-681 of the Netherlands Organization for Scientific Research.

REFERENCES

Abeles FB, Morgan PW, Salveit MEJ (1992) Ethylene in Plant Biology, Ed Second. Academic Press, San Diego, USA Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284: 2148-2152 Alonso JM, Stepanova AN, Solano R, Wisman E, Ferrari S, Ausubel FM, Ecker JR (2003) Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 100: 2992-2997 Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796-815 Bleecker AB, Estelle MA, Somerville C, Kende H (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 241: 1086-1089 Chang C, Kwok SF, Bleecker AB, Meyerowitz EM (1993) Arabidopsis ethylene-response gene E TR 1: Similarity of product to two-component regulators. Science 262: 539-544 Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell 89: 1133-1144 Cox KH, Goldberg RB (1988) Analysis of plant gene expression. In CH Shaw, ed, Plant molecular biology: a practical approach. IRL Press, Oxford, pp 1-34 Felsenstein J (1989) PHYLIP - phylogeny inference package (version 3.2). Cladistics 5: 164-166 Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis ethylene- responsive element binding factors act as transcriptional activators or repressors of GCC box­ mediated gene expression. The Plant Cell 12: 393-404 Gascuel O (1997) BIONJ: an improved version of the NJ algorithm based on a simple model of

44 The tobacco EIN3 family sequence data. Molecular Biology and Evolution 14: 685-695 Hua J, Chang C, Sun Q, Meyerowitz EM (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 269: 1712-1714 Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR, Meyerowitz EM (1998) EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. The Plant Cell 10: 1321-1332 Hua J, Meyerowitz EM (1998) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94: 261-271 Johnson PR, Ecker JR (1998) The ethylene gas signal transduction pathway: a molecular perspective. Annual Review Of Genetics 32: 227-254 Kende H (1993) Ethylene biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 44: 283-307 Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72: 427-441 Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. The Plant Cell 2: 1201-1224 Kosugi S, Ohashi Y (2000) Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3 (EIN3) homolog. Nucleic Acids Research 28: 960-967 Lee MM, Schiefelbein J (2002) Cell pattern in the Arabidopsis root epidermis determined by lateral inhibition with feedback. The Plant Cell 14: 611-618 Lincoln JE, Fischer RL (1988) Diverse mechanisms for the regulation of ethylene-inducible gene expression. Molecular and General Genetics 212: 71-75 Lupas A (1996) Prediction and analysis of coiled-coil structures. Methods in Enzymology 266: 513­ 525 Ouaked F, Rozhon W, Lecourieux D, Hirt H (2003) A MAPK pathway mediates ethylene signaling in plants. EMBO Journal 22: 1282-1288 Raghothama KG, Maggio A, Narasimhan ML, Kononowicz AK, Wang G, D'Urzo MP, Hasegawa PM, Bressan RA (1997) Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region. Plant Molecular Biology 34: 393-402 Rieu I, Wolters Arts M, Derksen J, Mariani C, Weterings K (2003) Ethylene regulates the timing of anther dehiscence in tobacco. Planta 217: 131-137 Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR (1995) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway. Genetics 139: 1393-1409 Ruperti B, Cattivelli L, Pagni S, Ramina A (2002) Ethylene-responsive genes are differentially regulated during abscission, organ senescence and wounding in peach (Prunus persica). Journal of Experimental Botany 53: 429-437 Sakai H, Hua J, Chen QG, Chang C, Medrano LJ, Bleecker AB, Meyerowitz EM (1998) ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 95: 5812-5817 Solano R, Stepanova A, Chao QM, Ecker JR (1998) Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE- FACTOR1. Genes and Development 12: 3703-3714 Terajima Y, Nukui H, Kobayashi A, Fujimoto S, Hase S, Yoshioka T, Hashiba T, Satoh S (2001) Molecular cloning and characterization of a cDNA for a novel ethylene receptor, NT-ERS1, of tobacco (Nicotiana tabacum L.). Plant Cell Physiology 42: 308-313 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680 Tieman DM, Ciardi JA, Taylor MG, Klee HJ (2001) Members of the tomato LeEIL (EIN3-like) gene family are functionally redundant and regulate ethylene responses throughout plant development. The Plant Journal 26: 47-58 Van Eldik GJ, Vriezen WH, Wingens M, Ruiter RK, Van Herpen MMA, Schrauwen JAM, Wullems GJ (1995) A pistil-specific gene of Solanum tuberosum is predominantly expressed in the

45 Chapter 3 stylar cortex. Sexual Plant Reproduction 8: 173-179 Verlinden S, Boatright J, Woodson WR (2002) Changes in ethylene responsiveness of senescence-related genes during carnation flower development. Physiologia Plantarum 116: 503­ 511 Weterings K, Pezzotti M, Cornelissen M, Mariani C (2002) Dynamic 1-aminocyclopropane-1- carboxylate-synthase and -oxidase transcript accumulation patterns during pollen tube growth in tobacco styles. Plant Physiology 130: 1190-1200 Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 88: 553-558

46 Chapter 4

Ethylene regulates the timing of anther dehiscence in tobacco

Ivo Rieu, Mieke Wolters-Arts, Jan Derksen, Titti Mariani and Koen Weterings

Planta (2003) 217: 131-137

ABSTRACT

We investigated the involvement of ethylene signaling in the development of the reproductive structures in tobacco (Nicotiana tabacum L.) by studying flowers that were insensitive to ethylene. Ethylene-insensitivity was generated either by expression of the mutant etr1-1 ethylene-receptor allele from Arabidopsis thaliana or by treatment with the ethylene-perception inhibitor 1-methylcyclopropene (MCP). Development of ovaries and ovules was unaffected by ethylene-insensitivity. Anther development was also unaffected, but the final event of dehiscence was delayed and was no longer synchronous with flower opening. We showed that in these anthers degeneration of the stomium cells and dehydration were delayed. In addition, we found that MCP-treatment of detached flowers and isolated, almost mature anthers delayed dehiscence whereas ethylene-treatment accelerated dehiscence. This indicated that ethylene has a direct effect on a process that takes place in the anthers just before dehiscence. Because a similar function has been described for jasmonic acid in Arabidopsis, we suggest that ethylene acts similarly to or perhaps even in concurrence with jasmonic acid as a signaling molecule controlling the processes that lead to anther dehiscence in tobacco.

INTRODUCTION

Genetic studies of Arabidopsis, Petunia and other plant species have revealed a regulatory network that controls the formation of the floral meristem and identity of the floral organ primordia (Smyth, 2001). Recently, data on the further differentiation and development of the individual floral organs have accumulated (e.g. Bowman et al., 1999; Sanders et al., 1999; Hase et al., 2000). Hormones produced by the floral organs appear to have a role mainly in the synchronization of the development of the floral organs. In Petunia, it has been shown that gibberellin produced in developing anthers is transported

47 Chapter 4 to the petals where it acts as a signal that initiates the second phase of petal development including petal expansion and pigmentation (Weiss 2000). In Arabidopsis, there is evidence that jasmonic acid produced in the stamens synchronizes the later stages of flower development, namely pollen maturation, anther dehiscence and flower opening (Sanders et al., 2000; Ishiguro et al., 2001). In many species, ethylene and its precursor 1- aminocyclopropane-1-carboxylate (ACC) are produced in the pistil upon pollination and translocated to the other floral organs to induce senescence (Woltering et al., 1995; O’Neill, 1997; Jones and Woodson, 1999). Studies in orchids have identified ethylene as a signaling molecule between stigma and ovary that promotes ovary development upon pollination (Zhang and O’Neill, 1993; Bui and O’Neill, 1998). In tobacco, ethylene may also function earlier in flower development as suggested by studies on ovule and in vitro pollen development (De Martinis and Mariani, 1999; Chibi and Matilla, 1994) and expression studies of ethylene receptor genes in developing flowers (Zhang et al., 2001a; Zhang et al., 2001b). To further study the effect of ethylene signaling on development of the reproductive organs in the tobacco flower, we analyzed plants that were rendered ethylene-insensitive either genetically or chemically. We found that these plants produce normally developing flowers, in which the process of anther dehiscence is delayed with respect to flower opening. Furthermore, we showed that anther dehiscence in detached flowers and in isolated anthers could be accelerated by treatment with ethylene and delayed by treatment with an ethylene-perception inhibitor. This indicates that ethylene acts directly on anthers to promote dehiscence and in that way synchronizes anther dehiscence with flower opening in tobacco.

MATERIALS AND METHODS

Plant material Seeds of Nicotiana tabacum L. cv. Samsun NN and N. tabacum L. cv. Petit Havana SR1 were derived from the Solanaceae germplasm collection of the Botanical garden of the University of Nijmegen, The Netherlands (http://www-bgard.sci.kun.nl). Seeds of the ethylene-insensitive Tetr mutant line 18 of N. tabacum L. cv. Samsun NN (Knoester et al., 1998) were kindly provided by dr. H.J.M. Linthorst, University of Leiden, The Netherlands. Because of the enhanced pathogen-sensitivity of this mutant, the Tetr plants and the wild­ type Samsun NN plants were raised on sand supplemented with fertilizer. All plants were grown in a standard growth chamber with a long-day light regime. Because the wild-type cultivars Petit Havana and Samsun NN behaved in an identical way in all experiments, results for Petit Havana are presented as wild-type. Flower stages were based on flower length and corolla appearance, as described by Koltunow et al. (1990).

Plant treatments To generate ethylene-insensitive flowers an attached branch of wild-type N. tabacum cv. Petit Havana was treated with 1-methylcyclopropene (MCP; Biotechnologies

48 Ethylene and anther dehiscence for Horticulture, Walterboro, USA; sold as EthylBloc containing 0.43% 1-MCP; Sisler and Serek, 1997). The branch was sealed airtight in a 1.5-l glass container that was flushed with air containing 5 ^l/l MCP at a rate of 3 l/h. All flowers that were used in this study were treated for at least 2 weeks. For the in situ localization experiment, flowers at stage 9 were cut from the plants, placed in a desiccator with the petioles submerged in water and incubated for 24 h with air or with air containing 1 ^l/l ethylene. The ethylene concentration was confirmed by gas chromatography at the beginning and at the end of the treatment. Anther-dehiscence experiments were carried out with detached flowers at stage 9 (n = 13) and stage 10 (n = 16) and isolated anthers at stage 10 (n = 17, from 17 flowers). Flowers, with the petioles submerged in water, were incubated in a desiccator with air, air containing 1 ^l/l ethylene or air containing 5 ^l/l MCP. To determine anther dehiscence, the desiccators were opened for 15 min at 4 h intervals during the day and the treatment gas was replaced after examination. Likewise, dehiscence of isolated anthers was studied. Again, the ethylene concentration was confirmed at each incubation.

In situ localization experiments In-situ hybridization studies were performed as described by Cox and Goldberg (1988) with minor modifications. Immediately after ethylene-treatment the ovaries were excised and fixed overnight at 4°C in 1% glutaraldehyde, 4% paraformaldehyde solution prepared in 0.1 M phosphate buffer (pH 7.0), dehydrated, cleared, and embedded in paraffin. Eight-micrometer-thick sections were hybridized to 33P-labeled antisense RNA probes transcribed from tobacco 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA clone pMPZ2 (Weterings et al., 2002) at a specific activity of 4 x108 to 5x108 dpm/^g. Hybridization with a sense ACO probe was used to determine the background signal. After hybridization and emulsion development, digital photographs were taken with a CoolSNAP CCD camera (Roper Scientific) using bright-field illumination on a Leitz Orthoplan (LSM, Wetzlar, Germany). The images were enhanced using Adobe Photoshop 5.0 (San Jose, CA, USA).

Light-microscopy Preparations for light-microscopy were made according to Wolters-Arts et al. (1996). Anthers and ovaries were fixed for 2 h in 2% glutaraldehyde, 0.1 M phosphate buffer (pH 7.2), at room temperature followed by post-fixation in 1% OsO4 in the same buffer. After dehydration, samples were embedded in ERL (Polysciences, Warrington, PA, USA). One-micrometer-thick sections were stained with 0.1% toluidine blue in 1% borax and examined with a Leitz Dialux 20EB microscope (LSM). Bright-field photographs were taken with Kodak Gold 100 film (ISO 100/21°) and were enhanced using Adobe Photoshop 5.0 after digitizing.

49 Chapter 4

Figure 1. In-situ expression analysis of the ACO gene in ovaries of non-treated and MCP-treated tobacco (Nicotians tabacum) flowers incubated in air or ethylene. Sections of ovaries were hybridized with an antisense ACO probe and examined using bright-field illumination. Hybridization signal appears as black grains. ep Epidermal layer of placenta, ov ovule, pl placenta, vb vascular bundles, cw carpel wall. Bar = 500 ^m.

RESULTS

Flowers of the Tetr transformant and MCP-treated tobacco plants are ethylene­ insensitive To characterize the phenotype of tobacco flowers developing in the absence of ethylene signaling, we used flowers in which ethylene-insensitivity was generated either genetically or chemically. The first type of flower was derived from Tetr tobacco plants (Knoester et al., 1998) that had been transformed with the mutant etr1-1 allele of the ethylene receptor gene ETR1 from Arabidopsis thaliana. Ethylene-insensitivity of Tetr seedlings and flowers has been shown by Knoester et al. (1998). The second type was generated by treating wild-type tobacco flowers with the ethylene perception inhibitor 1- methylcyclopropene (MCP) while still attached to the plant. In order to confirm that both types of flower were ethylene-insensitive, we monitored pollination-induced corolla senescence, which is a well-characterized ethylene-response (Abeles et al., 1992). We found that in wild-type flowers the corolla senesced within 3 days after pollination, whereas in Tetr and MCP-treated flowers senescence was delayed by over a week (data not shown). This indicated that the corollas of the Tetr plant and the MCP-treated plant have a strongly decreased response to ethylene. To ascertain that the exogenously applied MCP had also reached the tissues deeper in the flower, we analyzed the expression of a gene that is normally induced by ethylene. To this purpose we investigated the mRNA accumulation of the ACO gene (De Martinis and Mariani, 1999) in the ovary of non-treated and MCP-treated flowers by in-situ hybridization. Figure 1 shows that non-treated ovaries kept in air have a low ACO hybridization signal in the vascular bundles and in the epidermal layer of the placenta. Treatment with ethylene caused an increase of this

50 Ethylene and anther dehiscence

Figure 2. Stomium degeneration in wild-type and ethylene-insensitive tobacco anthers. Sections of anthers from wild-type, Tetr and MCP-treated plants at stage 9, 11 and 12 were stained with toluidine blue and examined using bright-field illumination. co, connective; en, endothecium; ep, epidermis; ms, microspores; st, stomium; vb, vascular bundle. Bars = 100 ^m.

hybridization signal while a new hybridization signal was found in the rest of the placenta and in the ovules. In MCP-treated ovaries kept in air, a hybridization signal above background level was visible in vascular bundles, but the signal was much weaker than in non-treated ovaries. Moreover, in MCP-treated ovaries this signal did not increase when they were treated with ethylene. Similar results were obtained with Tetr ovaries (data not shown). This demonstrates that MCP penetrated well into the flower tissues and caused ethylene-insensitivity even in the deeper cell layers. Taken together, these results showed that both types of flower were insensitive to ethylene and could thus be used for further studies.

51 Chapter 4

wild-type wild-type Tetr MCP-treated

Figure 3. Shrinkage of cells in the vascular bundle in wild-type and ethylene-insensitive tobacco anthers. Sections of anthers at stage 12 from wild-type, Tetr and MCP-treated plants were stained with toluidine blue and examined using bright-field illumination. aw, anther wall; pa, parenchyma cells; vb, vascular bundle. Bar = 100 ^m.

Ovary and anther development are normal in ethylene-insensitive tobacco, but anther dehiscence is delayed In order to determine whether ethylene-insensitivity had an effect on development of the reproductive organs, we performed a detailed morphological study of anthers and ovaries at all 12 developmental stages described by Koltunow et al. (1990). To this end, anthers and ovaries from wild-type and ethylene-insensitive flowers were embedded in resin and transverse sections were examined using light microscopy. In wild-type ovaries at stage 1, ovule primordia protruded from the placenta and at stage 5 they consisted of a funiculus and a nucellus covered by one integument. These ovules had enlarged by stage 9 and at flower stage 12 an embryo sac was present inside the mature ovules (data not shown; De Martinis and Mariani, 1999). The morphology of the ovaries and ovules of the MCP-treated and Tetr plants was identical to that of the wild-type (data not shown). In wild-type anthers at stage 1, the epidermis, endothecium, vascular bundle, connective, circular cell cluster, stomium, tapetum and microspore tetrads were all present. By stage 5, the cells of the circular cell cluster had degenerated, secondary wall thickenings (fibrous bands) had appeared in the endothecium, and microspores had formed. At stage 9, the tapetum was fully degenerated and the anther had become bilocular. Figure 2 shows that the stomium cells were still fully turgid at stage 9. At stage 11 however, the stomium cells had collapsed and the connective was completely degenerated (not shown). Ultimately, at stage 12 the stomium cells separated causing anther dehiscence and pollen release. In the two types of ethylene-insensitive tobacco flower, all of the above-mentioned morphological markers of anther development behaved identically to wild-type markers with the exception of stomium degeneration and anther dehiscence. At flower stage 11, the stomium of the anthers of both types of ethylene-insensitive flower was still intact and as a result the anthers had not dehisced at stage 12 (Fig. 2). Further examination showed that the shrinkage of the cells in the vascular bundle that was visible in wild-type anthers at stage 12 was absent in anthers from ethylene-insensitive flowers

52 Ethylene and anther dehiscence

Figure 4a-c. The effect of MCP, air and ethylene on anther dehiscence in tobacco. Detached flowers or isolated anthers were placed in a desiccator and incubated in air, MCP or ethylene. Anthers were analyzed at regular intervals and a flower was scored as dehisced when all five anthers had dehisced. (a) Flowers incubated from stage 9. (b) Flowers incubated from stage 10. (c) Anthers incubated from stage 10.

(Fig. 3). Although in ethylene-insensitive flowers dehiscence of the anthers was delayed by one to a few days (data not shown), the pollen was functional as self-pollination resulted in viable seeds. Taken together, these data indicate 1) that cellular differentiation and development of the reproductive tissues and gametophytes is not affected by ethylene-insensitivity, 2) that stomium degeneration and anther dehiscence are delayed in ethylene-insensitive tobacco flowers, and 3) that shrinkage of the cells in the vascular bundle is absent in ethylene-insensitive anthers at stage 12.

53 Chapter 4

Ethylene directly influences the last stage of anther dehiscence Because the delay in dehiscence of the anthers did not seem to be caused by a general delay in anther development, but rather by a specific delay of stomium degeneration and breakage, we hypothesized that only the very last stage of dehiscence was influenced by ethylene-insensitivity. To test this, we treated almost mature, detached flowers with MCP, air or ethylene and measured the time to anther dehiscence. Figure 4a shows that anther dehiscence was accelerated when flowers at stage 9 were treated with ethylene. By contrast, treatment with MCP delayed anther dehiscence compared to the control with air. In flowers of stage 10 (Fig. 4b), dehiscence could be delayed by MCP- treatment, but no appreciable acceleration effect of ethylene was detected anymore. Similar results were obtained with detached anthers at stage 10 (Fig. 4c), which demonstrates that ethylene has a direct effect on anthers. Furthermore, we noticed that treatment of stage-9 flowers with MCP also caused a slight delay in pigmentation and expansion of the corolla (data not shown). Taken together, these data indicate 1) that ethylene produced by the flower promotes anther dehiscence, and 2) that it does so via a direct effect on anther tissue.

DISCUSSION

Although genes regulated by ethylene are expressed in the ovary (Fig. 1), we did not find an appreciable difference in ovary and ovule development by blocking ethylene perception. This is in contrast to what was observed previously by De Martinis and Mariani (1999) in plants in which ethylene biosynthesis was inhibited and may suggest that the down regulation of a specific ACC-oxidase gene caused additional physiological differences. Similarly, we did not find any effect on pollen development, in contrast to previous evidence that ethylene is required for pollen maturation in vitro (Chibi and Matilla, 1994). However, a striking effect of the inhibition of ethylene perception was observed in anther dehiscence (Fig. 2). In tobacco, the anthers dehisce at flower opening at stage 12. Dehiscence occurs via decay and breakage of a ridge of stomium cells in the anther wall (Beals and Goldberg, 1997). We found that in ethylene-insensitive plants and detached flowers treated with MCP, anther dehiscence was delayed and we obtained similar results when we blocked ethylene perception in isolated anthers at stage 10 (Fig. 4). This indicates that ethylene acts directly on anthers to promote dehiscence. Although the results in figure 4c would suggest that the anther itself produces this ethylene, we cannot say this with certainty as we may have induced wound-ethylene by detaching the anthers. Between stage 10 and dehiscence at stage 12, the two main processes that take place in the anther are dehydration (Koltunow et al., 1990; Bonner and Dickinson, 1989) and stomium cell degeneration (Beals and Goldberg, 1997). We found that in anthers from ethylene-insensitive flowers the stomium differentiation and degeneration events were not detectably different from those of the wild-type. However, the stomium cells degenerated at

54 Ethylene and anther dehiscence a later time in development (Fig. 2). Thus, it is tempting to speculate that ethylene accelerates degeneration of the stomium cells, for example by promoting programmed cell death (PCD). Stomium cell degeneration has some characteristics of PCD, like positive TUNEL staining (M. Aarts, University of Wageningen, The Netherlands, personal communication; Balk and Leaver, 2001) and the expression of a thiol endopeptidase (Koltunow et al., 1990) and ethylene has been shown to promote PCD in other cases (Orzaez and Granell, 1997; Gunawardena et al., 2001; De Jong et al., 2002). In addition, ethylene could accelerate stomium cell degeneration by enhancing the expression or activity of cell wall-degrading enzymes, which have been shown to be involved in stomium cell separation (Jenkins et al., 1999; Roberts et al., 2002). Notably, cell wall-degrading enzymes that are active during anther dehiscence in Brassica napus and Arabidopsis are also active at seed pod dehiscence, and this process can be delayed by application of an ethylene-synthesis inhibitor (Jenkins et al., 1999; Petersen et al., 1996; Child et al., 1998; Roberts et al., 2000). In the last stage before dehiscence, the anthers also dehydrate and there is evidence that this causes the collapse of the cells in the vascular bundle in the tip of the filament (Keijzer et al., 1987a, b) We show that this did not occur in ethylene-insensitive anthers at stage 12, suggesting that these anthers are less dehydrated (Fig. 3). Interestingly, a class of aquaporins that is up-regulated in tobacco anthers during the last stages of development was identified recently (M. Bots and C. Mariani, University of Nijmegen, The Netherlands, personal communication), making the genes that encode these proteins a possible target for the ethylene signal. The delayed stomium cell degeneration could then be an indirect effect of the defective dehydration. Remarkably, the Arabidopsis mutant ddel that has delayed anther dehiscence also shows a delay in stomium cell degeneration and a defect in anther dehydration (Sanders et al., 2000; Ishiguro et al., 2001). This mutant is impaired in the production of jasmonic acid, which is functionally related to ethylene in the induction of some plant defense responses (Penninckx et al., 1998; Schenk et al., 2000; Sasaki et al., 2001). By contrast, delayed anther dehiscence has never been reported for ethylene-insensitive Arabidopsis mutants. In conclusion, we have shown that ethylene promotes anther dehiscence in tobacco by a direct effect on anther tissue and in that way synchronizes anther dehiscence with flower opening. The fact that ethylene, like jasmonic acid, promotes the final steps of stamen and corolla development at the same time (Ishiguro et al., 2001) suggests that it acts as an inter-organ signaling molecule during the last stages of flower development, but this remains to be established more firmly by studies on the site of hormone synthesis and translocation. Determination of the targets of the ethylene and jasmonic acid signals in the anther will show whether ethylene in tobacco and jasmonic acid in Arabidopsis perform homologous functions or whether they act partially redundantly in both species. The latter would explain why the tobacco Tetr and Arabidopsis ddel plants show delayed rather than inhibited anther dehiscence.

55 Chapter 4

ACKNOWLEDGEMENTS

The authors thank Mark Aarts for critical reading of the manuscript and for sharing data prior to publication. We are grateful to Huub Linthorst for Tetr transformant seeds and to Gerard Bogemann and Liesbeth Pierson for technical assistance. All plants used in this study were in perfect condition due to excellent greenhouse personnel. This research was funded by grant 805-22-681 of the Netherlands Organization for Scientific Research.

REFERENCES

Abeles FB, Morgan PW, Salveit MEJ (1992) Ethylene in Plant Biology, Ed Second. Academic Press, San Diego Balk J, Leaver CJ (2001) The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. The Plant Cell 13: 1803-1818 Beals TP, Goldberg RB (1997) A novel cell ablation strategy blocks tobacco anther dehiscence. The Plant Cell 9: 1527-1545 Bonner LJ, Dickinson HG (1989) Anther dehiscence in Lycopersicon esculentum Mill. I. Structural aspects. New Phytologist 113: 97-115 Bowman JL, Baum SF, Eshed Y, Putterill J, Alvarez J (1999) Molecular genetics of gynoecium development in Arabidopsis. Current Topics in Developmental Biology 45: 155-205 Bui AQ, O'Neill SD (1998) Three 1-aminocyclopropane-1-carboxylate synthase genes regulated by primary and secondary pollination signals in orchid flowers. Plant Physiology 116: 419-428 Chibi F, Matilla AJ (1994) The involvement of ethylene in in vitro maturation of mid-binucleate pollen of Nicotiana tabacum. Journal of Experimental Botany 45: 529-532 Child RD, Chauvaux N, John K, Ulvskov P, Van Onckelen HA (1998) Ethylene biosynthesis in oilseed rape pods in relation to pod shatter. Journal of Experimental Botany 49: 829-838 Cox KH, Goldberg RB (1988) Analysis of plant gene expression. In CH Shaw, ed, Plant molecular biology: a practical approach. IRL Press, Oxford, pp 1-34 De Jong AJ, Yakimova ET, Kapchina VM, Woltering EJ (2002) A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells. Planta 214: 537-545 De Martinis D, Mariani C (1999) Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants. The Plant Cell 11: 1061­ 1071 Gunawardena A, Pearce DM, Jackson MB, Hawes CR, Evans DE (2001) Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize (Zea mays L.). Planta 212: 205-214 Hase Y, Tanaka A, Baba T, Watanabe H (2000) FRL1 is required for petal and sepal development in Arabidopsis. The Plant Journal 24: 21-32 Ishiguro S, Kawai Oda A, Ueda K, Nishida I, Okada K (2001) The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. The Plant Cell 13: 2191-2209 Jenkins ES, Paul W, Craze M, Whitelaw CA, Weigand A, Roberts JA (1999) Dehiscence-related expression of an Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus. Plant Cell and Environment 22: 159-167 Jones ML, Woodson WR (1999) Interorgan signaling following pollination in carnations. Journal of the American Society for Horticultural Science 124: 598-604 Keijzer CJ, Hoek IHS, Willemse MTM (1987a) The processes of anther dehiscence and pollen dispersal. III. The dehydration of the filament tip and the anther in three monocotyledonous species.

56 Ethylene and anther dehiscence

New Phytologist 106: 281-287 Keijzer CJ, Hoek IHS, Willemse MTM (1987b) The development of the staminal filament of Gasteria verrucosa. Acta Botanica Neerlandica 36: 271-282 Knoester M, Van Loon LC, Van Den Heuvel J, Hennig J, Bol JF, Linthorst HJM (1998) Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi. Proceedings of the National Academy of Sciences of the United States of America 95: 1933-1937 Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. The Plant Cell 2: 1201-1224 O'Neill SD (1997) Pollination regulation of flower development. Annual Review of Plant Physiology and Plant Molecular Biology 48: 547-574 Orzaez D, Granell A (1997) DNA fragmentation is regulated by ethylene during carpel senescence in Pisum sativum. The Plant Journal 11: 137-144 Penninckx I, Thomma B, Buchala A, Metraux JP, Broekaert WF (1998) Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. The Plant Cell 10: 2103-2113 Petersen M, Sander L, Child R, Van Onckelen H, Ulvskov P, Borkhardt B (1996) Isolation and characterisation of a pod dehiscence zone-specific polygalacturonase from Brassica napus. Plant Molecular Biology 31: 517-527 Roberts JA, Elliott KA, Gonzalez-Carranza ZH (2002) Abscission, dehiscence, and other cell separation processes. Annual Review Of Plant Biology 53: 131-158 Roberts JA, Whitelaw CA, Gonzalez-Carranza ZH, McManus MT (2000) Cell separation processes in plants - models, mechanisms and manipulation. Annals of Botany 86: 223-235 Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu YC, Lee PY, Truong MT, Beals TP, Goldberg RB (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sexual Plant Reproduction 11: 297-322 Sanders PM, Lee PY, Biesgen C, Boone JD, Beals TP, Weiler EW, Goldberg RB (2000) The Arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway. The Plant Cell 12: 1041-1061 Sasaki Y, Asamizu E, Shi bat a D, Nakamura Y, Kaneko T, Awai K, Amagai M, Kuwata C, Tsugane T, Masuda T, Shimada H, Takamiya X, Ohta H, Tabata S (2001) Monitoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways. DNA Research 8: 153-161 Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proceedings of the National Academy of Sciences of the United States of America 97: 11655-11660 Sisler EC, Serek M (1997) Inhibitors of ethylene responses in plants at the receptor level: recent developments. Physiologia Plantarum 100: 577-582 Smyth DR (2001) Flower development. Current Biology 10: R82-R84 Weiss D (2000) Regulation of flower pigmentation and growth: multiple signaling pathways control anthocyanin synthesis in expanding petals. Physiologia Plantarum 110: 152-157 Weterings K, Pezzotti M, Cornelissen M, Mariani C (2002) Dynamic 1-aminocyclopropane-1- carboxylate-synthase and -oxidase transcript accumulation patterns during pollen tube growth in tobacco styles. Plant Physiology 130: 1190-1200 Woltering EJ, Somhorst D, Van Der Veer P (1995) The role of ethylene in interorgan signaling during flower senescence. Plant Physiology 109: 1219-1225 Wolters-Arts M, Derksen J, Kooijman JW, Mariani C (1996) Stigma development in Nicotiana tabacum. Cell death in transgenic plants as a marker to follow cell fate at high resolution. Sexual Plant Reproduction 9: 243-254 Zhang JS, Xie C, Shen YG, Chen SY (2001a) A two-component gene (NTHK1) encoding a putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco. Theoretical and Applied Genetics 102: 815-824 Zhang JS, Xie C, Wu XL, Du BX, Chen SY (2001b) Tobacco two-component gene NTHK2. Chinese Science Bulletin 46: 574-577 Zhang XS, O'Neill SD (1993) Ovary and gametophyte development are coordinately regulated by auxin and ethylene following pollination. The Plant Cell 5: 403-418

57

Chapter 5

Evolution of unitegmy in the euasterids

Ivo Rieu, Richard Feron, Ank Jansen, Tamara van Mölken, Titti Mariani and Koen Weterings

ABSTRACT

The integument is a sporophytic structure that surrounds the embryo sac in the mature ovule and develops into the seed coat after fertilization. The number of ovular integuments has varied during seed plant evolution. Within the angiosperms, the number has been reduced from two to one several times, as in the ancestor of the euasterids. Four different evolutionary pathways have been described for this reduction, but morphological studies have not revealed which of these accounts for the unitegmy of the euasterids. To obtain information on the homology between the euasterid single integument and the two Arabidopsis integuments, the genetic programs of these structures were compared. Integument specific expression in Arabidopsis has been reported only for the AP3 and INO genes, both expressed in the outer integument. The finding that a tobacco AP3 ortholog shows enhanced expression in the integument suggests similarity with the outer integument of Arabidopsis. However, as we could not isolate a tobacco INO homolog and the promoter of the INO gene was silent in the tobacco integument, this suggestion could not be supported further.

INTRODUCTION

The integument is a sporophytic structure that surrounds the sporangiophore, or nucellus, in seed plants. The nucellus, in turn, bears the megasporangium. Together with the funiculus these structures define the ovule. After fertilization, the integuments develop into the seed coat. Based on fossil evidence, several theories for the origin of the integument have been proposed (reviewed in Herr, 1995). The most widely accepted theory states that the integument has evolved through fusion of sterile telomes or stem axes that surrounded a central megasporangium (De Haan, 1920). This theory was supported by a recent analysis of Paleozoic and extant ovules, although this study suggested that at least some of the integumentary telomes must have been fertile themselves (Herr, 1995). During further evolution of the seed plants, the number of integuments has varied. The fact that all of the putatively basal angiosperms are bitegmic

59 Chapter 5

(i.e. have two integuments) indicates that this was the morphology of the ancestral angiosperm ovule (Bouman, 1984; Endress and Igersheim, 2000). Although this bitegmic state is preserved in most of today’s angiosperms, many unrelated families include unitegmic species. This means that the evolutionary changeover from bitegmy to unitegmy must have taken place multiple times during angiosperm radiation (Philipson, 1974; Corner 1976; Bouman 1984), which is reflected in the existence of several non-homologous evolutionary pathways to unitegmization. Examples of four different pathways have been described (Bouman and Callis, 1977; Boesewinkel and Bouman, 1991): 1) the retardation or complete suppression of the development of one of the two integuments, 2) the fusion of the primordia of the two integuments, 3) the shifting of the inner integument along the outer integument and 4) the shifting of the outer integument along the inner integument. A strong correlation between sympetaly and unitegmy was noticed already long ago. According to a recent reclassification of the angiosperms, most of these sympetalous families belong to the asterids (Angiosperm Phylogeny Group, 1998). A detailed evaluation of the evolutionary patterns of unitegmy in the asterids indicated that this trait was present in the ancestor of the clade of euasterids, and may have been present already in the ancestor of the whole asterid clade (Albach et al., 2001a, b). This means that at least in the euasterids the unitegmic state is homologous. However, it is not clear through which pathway the shift towards unitegmy took place in the euasterid ancestor. From studies of some euasterid species, it was concluded that the integument was of full dermal origin (Satina, 1945; Bhandari et al., 1976). Because this character is usually associated with the inner integument, it was interpreted as an indication for homology between the euasterid integument and the ancestral inner integument (Corner, 1976). A detailed ontogenetical study of the ovules of Gentiana cruciata suggested that the single integument in this euasterid species was of dermal plus subdermal derivation (Bouman and Schrier, 1979). Because there has only been one report of a family in which subdermal cells contribute to the inner integument (i.e. Euphorbiaceae; Bor and Bouman, 1974), this seemed to be an indication for homology to the outer integument. Furthermore, the position of the dermal and subdermal initials suggested that the single integument of G. cruciata has been derived from a dermal inner and a subdermal outer integument by means of integumentary shifting (Bouman and Schrier, 1979). The dermal integuments in other euasterids may than have evolved later, as dermalization of the outer integument has also occurred multiple times during angiosperm evolution (Bouman, 1984). But the observations of Bouman and Schrier are contradicted by Shamrov (1991, 2000a, b), who reported that the integument in G. cruciata and Swertia iberica (both Gentianaceae) is fully dermal, because the deeper initials, which start dividing later, are themselves of dermal origin. Therefore, there is still no consensus about the pathway that led to unitegmy in the euasterids. All studies performed so far, have been based on comparison of the integuments in different plant species using morphological data only. Due to the recent advances in molecular biology, it has now become feasible to directly compare the genetic programs underlying the development of the integuments. To this end, we compared the single integument of tobacco (Nicotiana tabacum), a euasterid, with the two integuments of

60 Evolution of unitegmy

Arabidopsis thaliana, as a reference species from the rosids. The rosids are closely related to the asterids, but still possess both ancestral integuments. We followed two strategies: 1) search in literature for genes that are known to be expressed in the integuments of euasterids or Arabidopsis and compare the obtained data sets. The overlap between the two data sets could be extended through the study of the expression patterns of genes specific for one of the two Arabidopsis integuments in tobacco, and 2) study the activity of promoter regions of the same Arabidopsis genes when placed in tobacco. The main problem that we encountered was that a very limited number of genes are known to be integument-expressed. Only two Arabidopsis genes are known to be expressed specifically in one of the two integuments: the AP3 gene and the INO gene, both are specific to outer integument (Modrusan et al., 1994; Villanueva et al., 1999). We show that the tobacco AP3 homolog NtDEF has a broad expression pattern in the ovule, but is expressed markedly more in the integument. This suggests similarities between the euasterid integument and the ancestral outer integument. However, as we could not isolate a tobacco INO homolog and the promoter of the INO gene was silent in the tobacco integument, this suggestion could not be supported further. In conclusion, the data obtained are not sufficient to reveal the evolutionary pathway to unitegmy in the euasterids.

MATERIALS AND METHODS

Plant material Seeds of Nicotiana tabacum L. cv. Petit Havana SR1 were derived from the Solanaceae germplasm collection of the Botanical garden of the University of Nijmegen, The Netherlands (http://www-bgard.sci.kun.nl). The tobacco plants were grown in soil under standard greenhouse conditions with additional artificial lighting to obtain a 16:8 hours light:dark regime. Seeds of Arabidopsis thaliana L. ecotype Col-0 were obtained from the Nottingham Arabidopsis Stock Center. Arabidopsis plants were grown in a 3:1 mixture of soil and vermiculite and kept in a growth chamber under a 16:8 hours light:dark regime at 22°C and ambient relative humidity. Flower stages are according to Koltunow et al. (1990) for tobacco and Schneitz et al. (1995) for Arabidopsis. cDNA library screening A 200 bp fragment of the INO transcript was amplified by PCR using primers GGCTTCCTTCATTCCTCTC and TATTGACAACTTGGTAAACACG on cDNA made from Arabidopsis inflorescence mRNA. The fragment corresponds to the part of the transcript located between the regions coding for the zinc finger and YABBY domains. Using this INO fragment as a probe, approximately 106 plaques were screened as described before (Rieu et al., 2003). Hybridization and washing of the filters was done under low stringency conditions [hybridization at 50oC and washing with 1xSSC (0.15 M NaCl, 0.015 M sodium citrate) at 50o C]. Eleven weakly hybridizing clones were isolated and sequenced. Screening for a tobacco ANT homolog was done with a probe made from a 382 bp

61 Chapter 5 fragment of the ANT transcript that was amplified from a cDNA library of tobacco ovaries at flower stage 1 to 6 (Rieu et al., 2003) using primers aactttggtgtttgctatggat and attccccatatacccctacat. This fragment corresponds to a part of the transcript located at the 5’ side of the region coding for the AP2 domain. Screening of approximately 106 plaques with the ANT probe under the same conditions as used for INO resulted in three clones corresponding to one transcript. The longest cDNA clone was sequenced twice using vector and gene-specific primers and named NtANT-like (Genbank AY461432).

RT-PCR Total RNAs were extracted from isolated tobacco ovules at flower stage 5 using the method of Van Eldik et al. (1995). First strand cDNA was synthesized according to the protocol of the ZAP Express cDNA synthesis and cloning kit (Stratagene, La Jolla, CA, USA). Using the Codehopper program (Rose et al., 1998), degenerate primers for the zinc finger and YABBY domains were designed based on the amino acid sequence of the published Arabidopsis YABBY proteins, with preference for INO and taking into account the tobacco codon usage. The resulting zinc finger primer was ccatatacttctctgttaatgactgttactgtnm gntgygg and the YABBY domain primer was tgagccttaattctttgaatttcttcyttdatraa. PCR was carried out under low stringency conditions with annealing at 48oC.

RNA analysis Total RNAs were extracted using the method of Van Eldik et al. (1995). 10^g of total RNA was separated on a formaldehyde agarose gel and blotted on Hybond-N membrane (Amersham Biosciences, Buckinghamshire, UK). A probe against NtDEF (Davies et al., 1996) was made from a fragment obtained by PCR with primers catatggattggttgagcaag and atacgcacttcccttatcca on cDNA made from tobacco anther mRNA. This fragment corresponds to a small part of the 3’ end of the coding region and the 3’ UTR. Northern hybridization was carried out under high stringency conditions according to the instructions for Hybond-N. In-situ hybridization studies were performed as described by Cox and Goldberg (1988) with minor modifications. Tobacco ovaries were excised and fixed overnight at 4°C in 1% glutaraldehyde, 4% paraformaldehyde solution prepared in 0.1 M phosphate buffer (pH 7.0), dehydrated, cleared, and embedded in paraffin. Eight-micrometer-thick sections were hybridized to 33P-labeled antisense RNA probes at a specific activity of 4 to 5 x 108 dpm/^g. The probe against NtDEF was transcribed from the cloned fragment used for northern hybridization. A probe against NtANT-like was transcribed from a fragment obtained by digestion of the cloned NtANT-like cDNA with SacI, which also digested the vector at a site just 5’ of the start of the cDNA. Hybridization with sense probes was used to determine the background signal. After hybridization and emulsion development, digital photographs were taken with a CoolSNAP CCD camera (Roper Scientific, USA) using bright-field illumination on a Leitz Orthoplan (LSM, Wetzlar, Germany). The images were enhanced using Adobe Photoshop 5.0 (San Jose, CA, USA).

62 Evolution of unitegmy

Construction of P-INO::GUS fusion and plant transformation A 1.8 kb fragment of the 5’ flanking region of the INO gene was obtained using PCR with the primers gtcgacttgtggctgtagaattaggacgttgtgttgg and ggagctttgccatggagagtgtgtgtgtacg. The fragment was cloned upstream of the GUS coding region in the vector pCAMBIA1305.1 resulting in construct P-INO::GUS. The transformation vector was transferred to Agrobacterium tumefaciens strain LBA4404 using freeze thaw transformation (Chen et al., 1994). Arabidopsis plants were transformed by the floral dip method, as described by Clough and Bent (1998). T1 seeds were grown on agar plates containing 20 ^g/mL hygromycin. Resistant seedlings were transplanted to soil. Tobacco plants were transformed using the standard leaf disk transformation and regeneration method (Tavazza et al., 1988). After shoot and root induction on medium containing 50 ^g/mL hygromycin, resistant plants were tranferred to soil and grown in the greenhouse.

^-Glucuronidase (GUS) activity analysis Histochemical staining for GUS activity was performed as described by Jefferson et al. (1987). To prevent oxidation, all solutions used for tobacco material were complemented with 0.05% Na-metabisulfide, or 0.3% during the acetone treatment. For staining of tobacco ovules, ovaries were excised, the carpel wall was removed and 100 |im thick sections were made in 0.1 M phosphate buffer (pH7.2) using a vibratom. For staining of Arabidopsis ovules, sepals, petals, anthers and stigma were removed from the flowers. Prior to staining, all material was fixed for 15 minutes in 90% acetone at 0°C, followed by three washes in 1 M phosphate buffer (pH 7.2) for 15 minutes at room temperature. Tissues were stained in 0.1 M phosphate buffer (pH 7.2), 10 mM EDTA, 0.2% triton X-100, 2 mM KiFe(CN)6, 2 mM K4Fe(CN)6, 1 or 2 mM 5-bromo-4-chloro-3-indolyl p-D-glucuronide cyclohexylamine salt (X-gluc) at 37°C, and stored in 70% ethanol at 4°C. Whole flowers were visualized using a dissecting microscope (Zeiss, Oberkochen, Germany) and photographed with a mounted CCD camera. Tobacco vibratom sections and dissected Arabidopsis ovules were mounted in a clearing solution containing 50% w/w chloral hydrate, 25% w/w lactic acid, 25% phenol (CLP; Beeckman and Engler, 1994) and visualized with a Zeiss Axiovert 135 TV using DIC optics. Digital photographs were taken with a CCD camera and the images were enhanced using Adobe Photoshop 5.0.

RESULTS

Few genes from euasterids and Arabidopsis are known to be expressed preferentially in the integument To determine whether Arabidopsis thaliana could function as a reference species in this comparative study, we combined data on the number of integuments in the angiosperm families (Watson and Dallwitz, 1992; Stevens, 2001) with the most recent phylogenetic tree published by the APG (Angiosperm Phylogeny Group, 2003; fig. 1).

63 Chapter 5

— 2 — gunnerales

2 _ core _ 2 — berberidopsidales eudicots __dilleniales/ caryophyllales

------— 1/2— santalales

_ — saxifragales/ vitales/rosids

— 1/2— asterids

Figure 1. A phylogenetic tree indicating the relationship between various core eudicot clades. The numbers indicate the deduced number of integuments in the ancestor of the respective clade.

Because the exact relation between the eudicots other than the gunnerales is not resolved yet, no statistical analysis could be performed to predict the points of character change. The simplest solution, however, seemed to be that the state in the core eudicots remained bitegmic and that the shifts to unitegmy took place at or near the base of the asterid branch and the santanales branch. This implies that the two integuments in plants belonging to the other sister clades of the asterids are homologous to the two integuments that were present in the last bitegmic ancestor of the asterids. As Arabidopsis is a member of the rosids, it probably represents a good reference species in this study. As a first approach to compare the genetic programs of the euasterid single integument with the two integuments of Arabidopsis, we performed a thorough search in literature for genes expressed in the integuments. In order to limit the result to genes that are of interest for our study, we used three additional selection criteria. (1) To be sure that a gene was integument-expressed, only those were included for which a clear in-situ localization of the expression was known. (2) Genes expressed only after fertilization, when the integuments have started to differentiate further into a seed coat, were excluded. (3) Also, genes that are known to be expressed ubiquitously were excluded. Although many genes are reported to be ovule-expressed, table 1 shows that only few of them met the selection criteria, mainly due to a lack of in-situ expression data. In the light of the comparison, the genes that are expressed in both Arabidopsis integuments are not informative, but they can reveal similarity between a general integument program in Arabidopsis and the program in the euasterid integument. The highly homologous MADS-box genes AGL11 and FBP7/11 exemplify this. AGL11 is expressed in the whole ovule, including both integuments in Arabidopsis, although at later stages there might be some preference for the outer integument (Rounsley et al., 1995; M. Yanofsky, University of California San Diego, USA, personal communication). Apart from expression in the ovule primordium, FBP7/11 transcript is found in the whole developing Petunia integument (Angenent et al., 1995).

64 Evolution of unitegmy

Table 1. Overview of genes shown to be expressed in the integument(s) of euasterid species or of Arabidopsis by in-situ localization studies. Genes marked with * and genes marked with ** are putative orthologs.

Euasterid Species Expression in Reference gene integument

NpCal1 N. plumbaginifolia Endothelium Borisjuk et al. (1998) FBP2 P. hybrida Endothelium Immink et al. (2002) DEFH21 A. majus Inner layer Becker et al. (2002) LeT6 L. esculentum Inner layers Janssen et al. (1998) SmCP S. melongena Inner layers Xu and Chye (1999) PhADF1 P. hybrida Outer layers Mun et al (2002) HPDEF* Hieracium Whole Guerin et al.(2000) FBP7/11** P. hybrida Whole Angenent et al.(1995) AOC L. esculentum Whole Hause et al. (2000) LapA L. esculentum Whole Chao et al. (1999)

Arabidopsis gene

SKP1 A. thaliana Endothelium Porat et al. (1998) AP3* A. thaliana Outer Modrusan et al. (1994) INO A. thaliana Outer Villanueva et al. (1999) AGL1 A. thaliana Both Flanagan et al. (1996) AGL11** A. thaliana Both Rounsley et al. (1995) AGL13 A. thaliana Both Rounsley et al. (1995) ANT A. thaliana Both Elliot et al. (1996) NZZ A. thaliana Both Schiefthaler et al. (1999) SAP A.thaliana Both Byzova et al. (1999) SIN1 A. thaliana Both Golden et al. (2002) TSO1 A. thaliana Both Song et al. (2000)

Most interesting are the genes that are expressed specifically in one of the two Arabidopsis integuments. Expression of their euasterid orthologs may provide information on the location of homologous cells in the euasterid integument. The endothelium, formed by the inner integument of mature Arabidopsis ovules, specifically expresses SKP1. It encodes a cell cycle regulators otherwise expressed in meristems and cells undergoing division (Porat et al., 1998). SKP1 homologs from the euasterid Antirrhinum, FAB genes, are also active in ovule tissue, although the precise location has not been determined (Ingram et al., 1997). The AP3 gene in Arabidopsis has been shown to be expressed specifically in the outer integument of mature ovules (Modrusan et al., 1994). Of all the euasterid AP3 orthologs that have been identified, only for the Hieracium HPDEF gene in- situ data on ovules are available, and they show that HPDEF is expressed in the whole developing integument (Guerin et al., 2000). Villanueva et al. (1999) reported that the INO gene is also expressed specifically in the outer integument of Arabidopsis, early in its development. No INO orthologs from other species have been described so far. A third class of genes, only known to be expressed in the euasterid integument, are not informative at this moment, but might become interesting if in-situ data for their Arabidopsis orthologs become available. The DEF21 MADS-box gene is expressed

65 Chapter 5 specifically in the inner layer of the developing Antirrhinum integument (Becker et al., 2002). Expression of its Arabidopsis homolog ABS in the inner layer of the inner integument has not yet been examined, but it is expected because functional ABS is necessary for the differentiation of the endothelium from this cell layer (Nesi et al., 2002). For all other genes from euasterid species that met our criteria, expression was only analyzed at ovule maturity, when the integuments were already fully developed. Another MADS-box gene from Petunia, FBP2, and a calreticulin gene from Nicotiana are expressed in the differentiated endothelium (Immink et al., 2002; Borisjuk et al., 1998), whereas two other genes, LeT6 and SmCP, seem to have a somewhat broader expression pattern, encompassing multiple inner cell layers of the integument. LeT6 is a tomato KNOX gene, otherwise associated with meristem activity (Janssen et al., 1998). Its overall expression pattern is largely overlapping with that of the most homologous gene from Arabidopsis, STM (Long et al., 1996). However, STM expression has not been observed in ovules (Jeff Long, Caltech, USA, personal communication). SmCP encodes a cysteine proteinase from brinjal (Solanum melongena) and is also expressed in the inner layers of the integument of mature ovules (Xu and Chye, 1999). By contrast, an actin-depolymerizing factor, PhADF1, is expressed specifically in the outer layers of the mature Petunia integument (Mun et al., 2002). So far, no such a pattern has been described for the few studied ADF genes from Arabidopsis (Dong et al., 2001). Finally, two genes whose expression is normally related to defense responses are expressed in the tomato integument at ovule maturity: AOC, encoding an enzyme involved in jasmonic-acid biosynthesis, and LapA (Hause et al., 2000; Chao et al., 1999). Nothing is known about the expression of the Arabidopsis AOC homologs and the only studied Arabidopsis LapA homolog is expressed constitutively (Bartling and Nosek, 1994). Taken together, the survey indicates similarities between the genetic programs of the euasterid integument and the two integuments of Arabidopsis, as indicated by the expression of FBP7/11 and AGL11 genes. More specifically, it shows a similarity between the euasterid integument and the Arabidopsis outer integument, in the form of HPDEF and AP3 expression. The expression of DEF21 and the function of ABS indicate a similarity between the inner layer of the integument of the euasterids and that of the inner integument of Arabidopsis.

A homolog of AP3 is expressed in the whole ovule, but INO homologous transcripts were not detected in tobacco To extend the knowledge on the gene expression program in the euasterid integument, we started with the isolation of a homolog for the ANT gene, which has a function in ovule primordium growth and integument growth (Elliot et al., 1996). We chose tobacco as a representative for the euasterids. Screening of a cDNA library of tobacco ovaries with the N-terminal part of ANT (without the conserved AP2 domains), yielded a cDNA clone that encoded an AP2 domain protein. When compared to all (putative) proteins identified by the Arabidopsis Genome Initiative (2000), the deduced protein clearly showed highest homology to ANT, both inside and outside the AP2 domains, and therefore we

66 Evolution of unitegmy

A

ANT (1) ------MKSFCDNDDNNHSN----- TTNLLGFSLSSNMMKMGGRGG-REAIYSSSTSSAATSSSSVPPQLVVGDNTSNFGVCYGSNPNG-GI NtANT-like (1) MKMKSMNDDNNNCSSHRSSNNNNNNNSNSHNDASSNWLGFSLSPHMKMEVTSSEPQHQHHQFNNQSSALAQSFYLSSSPMNVTSNTSALCYGVGENNPFG

ANT (80) HMSVMPLRSDGSLCLMEALNRSSHSNHHQDSS-PKVEDFFG-THHNNTSHKEAMDLSLDSLFYl -- TTHEPNTTTNFQEFFSFPQTRNHEEET NtANT-like (101) iSLSVMPLKSDGSLCIMEALSRSHADAMVQSSSSPKLEDFLGGASQYGNHEREAMALSLDSLYY!YHQNEGYYSPMHCHGMYQESLLDETKPTQISHCDAQ

ANT (171) RNYGNDPSLTHGGSFNVGVYGEFQQSLSLSMSPGSQSSCITGSHHHQQNQNQNHQSQNHQQISEALVETSVGFETTTMAAAKKKRGQEDWWGQKQIV NtANT-like (201) MTAVSGNELKSWGHYAIDQHINETCSSMVGAGGGGGTSGSGGGTVGGNDLQSLSLSMNPGSQSSCVTPRQISPNELECVAIETKKRASGKVVQ--KQPV

ANT (271) RKSIDTFGQRTSQYRGVTRHRWTGRYEAHLWDNSFKKEGHSRKGRQVYLGGYDMEEKAARAYDLAALKYWGPSTHTNFSAENYQKEIEEDMKNMTRQEY NtANT-like (299) RKSIDTFGQRTSQYRGVTRHRWTGRYEAHLWDNSCKKEGQTRKGRQVYLGGYDMEEKAARAYDLAALKYWGPSTHINFPLENYQKELEDMKNMTRQEY

ANT (371) HLRRKSSGFSRGASIYRGVTRHHQHGRWQARIGRVAGNKDLYLGTFGTQEEAAEAYDVAAIKFRGTNAVTNFDITRYDVDRIMSSNTLLSG NtANT-like (399) HLRRKSSGFSRGASMYRGVTRHHQHGRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDVAAIKFRGVNAVTNFDISRYDVEKIMASNTLPAGELARRNKER

ANT (470) SIVV] tedqtalnavveggsn:KE ------VSTPERLLSFPAIFALPQVN---- -QKMFGSNMGGNMSPWTS1N ------NtANT-like (499) EPIE ILSDHKNEEACVQNDNN]'NNNGNNVTDWKMVSYQSSNPSLGNYRNPTFSMALQDLIGIDLMNSSNHPILDD

ANT (532) ------PNAEL KTVALTLPQMPVFAAWADS NtANT-like (599) SPDKSAASLVFAKPTNVNACIPSAQLRPIPVSISHLPVFAALNDA

B

CRC HCGNLSFLTT FVVKPPEKKQRLPSAYNR NtYAB1 s v s m TPIRPPEKRQRVPSAYNR YAB3 SVTV EANRPPEKRQRVPSAYNR YAB2 MHCTNLLSLNI PPIRPPEKRQRVPSAYNR NtYAB2 HCTNMWTVNM IVNRPPEKRQRVPSAYNQ NtYAB3 HCTNMWSVNM IVNRPPEKRQRVPSAYNQ YAB5 HCTNLWSVNM IVNRPPEKRQRVPSAYNQ FIL CCTNLLSVNM PVNRPPEKRQRVPSAYNR INO HCTSLLSVNL VVNKPPEKRQRAPSAYNC NtYAB4 HCTNLLPGLL VINRPPEKRQRVPSAYNR

zf YABBY

Figure 2. Comparison of the identified tobacco ANT-like and YABBY cDNAs with homologous Arabidopsis sequences. A. Alignment of the deduced NtANT-like protein with the ANT protein from Arabidopsis. The two AP2 domains are underlined. Identical amino acids (43%) are shown in dark gray, highly similar amino acids (9%) in light gray. B. Alignment of the partial zinc finger (zf) and YABBY domains (both underlined) of the deduced tobacco YABBY proteins (NtYAB1-4) with the six Arabidopsis YABBY proteins. Adjacent amino acids that showed conservation were included in the alignment. The phylogenetic tree was calculated from the alignment using the neighbor-joining algorithm.

considered the gene a tobacco ANT ortholog (NtANT-like; fig. 2A). Sections of tobacco ovaries at various stages of flower development were hybridized with a probe against a non-conserved part of NtANT-like. Figure 3 shows that NtANT-like was expressed at a low level in all tissues of the ovary. In the ovule, the signal was distributed evenly at all stages of development. Information on the homology between the integuments may come from expression patterns of orthologs of the INO and AP3 genes (table 1). The INO gene belongs to the YABBY gene family, whose members are characterized by the presence of a C2C2 zinc finger and a YABBY domain, separated by a variable linker domain (Bowman, 2000). Because no YABBY genes from other species had been described, it was unknown whether only the zinc finger and YABBY domains or also the remaining parts of the proteins are conserved in the orthologs. We first tested whether transcripts with homology

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Figure 3. In-situ expression analysis of the NtANT-like gene and the NtDEF gene in tobacco ovules. Sections of ovaries at different stages of flower development were hybridized with a sense and antisense probe, exposed for 5 days and examined using bright-field illumination. Hybridization signal appears as black grains. f, funiculus; i, integument; m, developing megaspore; op, ovule primordium. Bar = 0.5 mm. Color figure, see appendix.

to the linker domain of INO were present in the tobacco ovary. Screening of the cDNA library of tobacco ovaries with this domain of the INO cDNA did not yield any homologous clone. Therefore, a PCR approach was taken. The degenerate oligonucleotides were based on the zinc finger and YABBY domains of all YABBY proteins, with preference for INO. Because the Arabidopsis YABBY genes contain introns inside the linker domain and inside the YABBY domains, these primers were used for analysis of the mRNAs and not of genomic DNA. A control RT-PCR on mRNA from Arabidopsis inflorescences, in which five out of six YABBY genes are expressed (Bowman, 2000), yielded only fragments belonging to INO (six out of six clones), confirming that our primers had a preference for INO.

68 Evolution of unitegmy

Subsequently, we performed a PCR on the tobacco ovary cDNA library and an RT-PCR on mRNA isolated from tobacco ovules at stage 5 of flower development. Because ovule development is not synchronized in tobacco (De Martinis and Mariani, 1999; data not shown), this sample contained ovules in which the integuments had just initiated and ovules in which the integuments had reached the top of the nucellus. Sequencing of eleven of the resulting clones showed that four different fragments had been amplified, and that all belonged to tobacco YABBY family members. After removal of the primers, the putative translation of the fragments consisted of two amino acids belonging to the zinc finger domain, the complete linker domain and fourteen amino acids belonging to the YABBY domain. Based on this translation, two fragments (NtYAB2, NtYAB3; four PCR clones) seemed to be most homologous to YAB5. As the linker domains of the other clones did not show homology to any of the Arabidopsis YABBY proteins, we used only the partial conserved domains for comparison with the Arabidopsis YABBY family members (fig. 2B). One fragment (NtYABI; one PCR clone) showed highest homology to YAB3, and one fragment (NtYAB4; six PCR clones) could not be aligned specifically well with any of the Arabidopsis YABBY proteins. Thus, using the above approaches, we did not identify a tobacco ortholog of INO. Although the expression pattern of HPDEF in the Hieracium integument had already been described, we considered it useful to extend this observation with the tobacco AP3 homolog NtDEF (Davies et al., 1996). Northern analysis with a probe against a non­ conserved region of the NtDEF cDNA confirmed that in the flower, NtDEF was expressed in the petals and anthers and, much weaker, in the pistil (data not shown). The same probe was used to hybridize sections of tobacco ovaries at various stages of development. Figure 3 shows that NtDEF was expressed in the ovule primordia at stage 1 and 2. At subsequent stages, the strongest signal was visible in the developing integument. Expression could also be detected in the developing megaspore. At flower maturity (stage 12), the hybridization signal had decreased and was distributed equally over the funiculus and integument. Together, we have shown that a putative tobacco ANT ortholog is expressed in the tobacco ovary and in the whole ovule. NtDEF, an ortholog of AP3, is also expressed in the whole ovule, but at the stages at which the integument grows around the nucellus, it is expressed markedly higher in the integument. This confirms the similarity between the the single euasterid integument and the Arabidopsis outer integument. We could not corroborate this with the identification of a cDNA of a tobacco INO ortholog, despite the fact that the primers used for RT-PCR showed a preference for INO in Arabidopsis.

A GUS fusion with the promoter of INO marks the outer integument in Arabidopsis, but is silent in the tobacco integument If there is enough similarity in the trans-acting factors, cis-acting elements from one species may be functional in a different species. Because we did not succeed in isolating a tobacco INO homolog, we decided to study the activity of the promoter of the Arabidopsis INO gene in tobacco. We made a transcriptional fusion of the putative INO promoter with

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Arabdiopsis stage 3-11 2-111 2 -V 3-11 4-1

Tobacco stage 2 3 5

Figure 4. Expression of the P-INO::GUS reporter construct in transgenic Arabidopsis and tobacco plants, analyzed with a dissecting microscope and a light microscope with DIC optics. Arabidopsis inflorescence and ovules were stained for GUS activity for 24 hours, using 1 mM X-gluc. GUS staining is observed in the whole flower, and in the outer integument of the ovules. Tobacco flower and ovule were stained for 24 hours, using 2 mM X-gluc. No GUS staining was found in ovules of the transgenic tobacco plants. Bar = 2.0 mm in the photograph of the tobacco flower, 0.5 mm in the photograph of the Arabidopsis flower and 25 ^m in the photographs of ovules. Color figure, see appendix.

the coding sequence of E. coli p-glucuronidase (GUS). To confirm the correct activity of the putative promoter, the construct was introduced in Arabidopsis. GUS activity was detectable in three out of five independent Arabidopsis P-INO::GUS lines. Figure 4 shows that GUS activity was observed in the whole inflorescence, which is not in agreement with the endogenous INO expression pattern in Arabidopsis (Villanueva et al., 1999). However, in the ovule the GUS expression was comparable to the endogenous INO RNA accumulation pattern, as the GUS staining was specifically found in cells giving rise to the outer integument at stage 2-III and in the proximal cells of the developing outer integument at stage 2-V. Weak GUS staining was found in the outer integument until ovule maturity (stage 4-I). Transformation of tobacco yielded 39 independent transformants. GUS staining was found in all flower organs of tobacco, but detailed analysis of the five plants with strongest GUS expression showed that P-INO was not active in the ovules.

DISCUSSION

The question about the evolutionary pathway to unitegmy in the euasterids is already long standing, and it was mainly investigated by morphological studies. By comparing the gene expression programs in the developing integuments of euasterids and a reference species we tried to obtain information regarding their homologies. That the

70 Evolution of unitegmy inner and outer integuments of Arabidopsis are distinct structures with different genetic programs is illustrated by the existence of Arabidopsis mutants with an inner integument- or outer integument-specific phenotype (Schneitz et al., 1997). However, a large set of integument marker-genes is still lacking (table 1).

Similarities in the basal ovule development program The expression patterns of the MADS-box genes AGL11 and FBP7/11, likely orthologs, suggest a similarity between the basal programs found in the Arabidopsis and Petunia ovules, including the integuments (Rounsley et al., 1995; Angenent et al., 1995; Angenent and Colombo, 1996). FBP7/11 have been shown to be involved in determination of the identity of the ovule primordia (Angenent et al., 1995), but based on their expression patterns, FBP7/11 and AGL11 may have an extended function in the developing integument. Multiple loci from Arabidopsis have been described to be necessary at later stages of ovule development, like primordium outgrowth, patterning of the proximal-distal axis and morphogenesis of the various ovular tissues (reviewed in Gasser et al., 1998). A number of the corresponding genes have been identified, among which the ANT gene. This gene seems to be required repeatedly for cell proliferation during ovule development (Elliot et al., 1996; Schneitz et al., 1998). That an orthologous gene may be involved in ovule primordium and integument growth in the euasterids is indicated by our findings that the NtANT-like gene is expressed in the tobacco ovule (fig. 3).

Similarities in induced secondary differentiation programs Our survey identified a number of genes specific to one of the two integuments of Arabidopsis (table 1), some of which are presumably involved in endothelium formation or function. Similarities between endothelium differentiation in the euasterids and Arabidopsis are suggested in particular by the involvement of orthologous B-sister MADS-box genes ABS and DEF21 (Becker et al., 2002; Nesi et al., 2002). However, it is possible that the differentiation of an endothelium is not part of the autonomous developmental program of the integuments, but instead, is induced by signals from the nucellar region. If so, the identity of the innermost layer of integument cells might not be critical for the development of an endothelium and consequently, its presence cannot be regarded as characteristic for the inner integument. At the moment no Arabidopsis mutants with a reduced inner integument are available to study this relation. The presence of an endothelium in the Arabidopsis spo mutant, which is defective in megasporogenesis (Yang et al., 1999), indicates that the putative signal can not come from the developing megagametophyte. However, as nucellar cells are still present at the start of endothelium differentiation (Schneitz et al., 1995), the inducing signals could be coming from these sporophytic cells. Similarly, some aspects of seed coat development could rely on induced deveopmental programs. Genes involved in seed coat development may have been incorporated into our data set, namely if these genes are already expressed before fertilization. This might be the case for the cysteine proteinase SmCP. Its overall expression pattern in brinjal suggests that it marks cells undergoing programmed cell death

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(PCD), and its expression in the mature brinjal ovule is therefore likely to be linked to the PCD of the inner layers of the developing seed coat (Xu and Chye, 1999). The inner integument of Brassica napus also undergoes PCD after fertilization and this is accompanied by the inner integument-specific expression of BnCysP1, which belongs to the same papain family of cysteine proteases (Wan et al., 2002). Although this shows that similar processes take place in the inner integument of B. napus and the inner layers of the brinjal integument it does not constitute strong evidence for evolutionary homology between the cell layers.

Similarities in integument initiation and development programs Following the above considerations, the only genes that may be used as markers for one of the two integuments are the AP3 and INO genes, both expressed in the outer integument of Arabidopsis. As reported for Hieracium, the tobacco AP3 ortholog is expressed in the whole ovule. However, we found it to be expressed significantly higher in the integument compared to other ovular tissues. This shows a similarity between the genetic programs of the tobacco integument and the Arabidopsis outer integument. Because a clear relation between the INO expression pattern and function has been established in Arabidopsis (Villanueva et al., 1999; Meister et al., 2002), knowledge about expression of a putative tobacco INO ortholog would be even more informative. However, we could not identify an INO homologous transcript in the tobacco integument and the promoter of INO was silent in the integument of tobacco. This suggests that there are no cells with outer integument identity in the tobacco ovule. Alternatively, it could also be that the similarity between the INO genes of Arabidopsis and tobacco is too low for detection. Likewise, the inactivity of the promoter might mean that the trans-acting factors regulating INO expression in the Arabidopsis outer integument are not present in the tobacco ovule, but could also mean that they are present but not conserved well enough to be able to interact with the cis-elements of the Arabidopsis INO promoter. Notably, a putative AP3 promoter that mimicked endogenous AP3 expression in Arabidopsis, seemed to have lost its specificity in tobacco, where it was active in the whole flower, including the whole ovules, thus masking any possible specific activity in the integuments (data not shown).

Perspective In this paper we presented two approaches to study integument evolution in the euasterids: 1) comparison of the transcripts present in the integuments of the euasterids and Arabidopsis, and 2) investigation of the activity of promoters of integument-specific Arabidopsis genes in a euasterid. The data obtained from two such heterologous- expression experiments were not informative, because in both cases no specific activity was observed in the heterologous environment. With this approach the activity of a whole set of transacting factors is tested at the same time, and inactivity of one of the factors can be decisive for the outcome. The phylogenetic distance between the species compared here may be too large for this approach.

72 Evolution of unitegmy

The first strategy seemed to be more promising, although a reliable comparison could not be made on basis of the currently available gene expression data. The isolation of more markers for each integument is a prerequisite for the success of this strategy. Most markers can probably be found at the initial stages of integument development when the identities are established. Such early markers are also less likely to be associated with induced genetic programs. Because of the difficulties that can be expected with separating the two integuments at early stages, expansion of the set of marker genes is probably better achievable by continuing the forward genetic screenings, than by using direct transcript profiling of isolated integument cells. Knowledge about gene functions obtained from the forward genetic studies, could also be used to complement the descriptive study with a functional study. Expression of the SUP gene in the Arabidopsis ovule, for example, could not be correlated specifically with development of one of the two integuments (Sakai e t al., 1995). A functional study, however, showed it to be essential for development of the outer integument only (Meister e t al., 2002). Information on the function of a euasterid SUP ortholog could thus be useful.

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76 Chapter 6

Summary and concluding remarks

Plants are one of the most important components of life on Earth. They convert energy radiating from the sun into organic compounds, which are used by a variety of consumers, including the fungi and animals. Plant shape is determined by the number, place and plane of cell division and subsequent cell growth (Meyerowitz, 1997). In addition to form, cells also differ in function. Both aspects of cellular development are determined by the genetic program of a cell. Whereas in animals a cell’s lineage is a major factor in determining the genetic program, external and endogenous signals appear more important in plants (Howell, 1998). As a result, plants are more flexible in their development. Plant hormones play a crucial role in this endogenous, or intercellular, signaling. They are compounds that are released by a cell to the apoplast and translocated to other cells, where low concentrations are perceived by a receptor protein. The signal is transduced further to the nucleus, where it changes the genetic program. Ethylene is one of the five classic plant hormones and very different responses to enhanced ethylene concentration have been described. In light grown seedlings, hypocotyl cells become elongated in response to ethylene (Smalle e t al., 1997), whereas in dark grown seedlings, ethylene treatment results in shorter cells (Guzman and Ecker, 1990). Roots respond to ethylene with the formation of extra root hairs from the epidemal cells (Tanimoto e t al., 1995), and in many fruits the cells respond with degradation of their cell walls, at least, if the fruit is at the correct stage of development (Abeles e t al., 1992). Although some of the genes modulated by ethylene may be similar, there are clearly differences between the genetic responses as well. The reason for this is that the response of a cell to a certain hormone is determined by the interaction between the hormone signal and other signals that the cell perceives or has perceived before. Not much is known about the level at which interactions between the ethylene signal and other signals take place (Gazzarrini and McCourt, 2001; Genoud and Metraux, 1999). It is possible that the interactions occur downstream of the primary signal transduction pathways, via combinatorial control of transcription of response genes. Alternatively, signals may directly influence eachothers primary signal transduction pathway, so that the output from the pathways is different in the various plant cells. In the ethylene signal transduction pathway, several components are encoded for by a gene family. If the family members differ in their activities, differential expression of these members may be one of the causes of differential responses to ethylene. An example of such a component is the ethylene receptor. It has been shown in several plant species that the receptor family members are differentially expressed (Hua et al., 1998;

77 Chapter 6

Tieman and Klee, 1999; Lashbrook e t al., 1998), but little is known about the activity of the various proteins. A major way in which the activity of receptors is adjusted in animals is via heterodimerization or multimerization (Heldin 1995; Bray e t al., 1998). Because some ethylene receptors have been shown to form homodimers (Schaller e t al., 1995; Hall e t al., 2000), it has been suggested that they may also form heterodimers. To investigate if such interactions are possible, we studied whether the ethylene receptors are present at the same location in the cells (chapter 2). By fusing the Arabidopsis ethylene receptor ERS1 with the fluorescent EYFP protein, it was possible to locate the protein in vivo. In isolated protoplasts, the fusion protein was associated with the endoplasmic reticulum (ER). However, high expression of the fusion protein resulted in disturbance of the secretory pathway and formation of aggregates. When expressed in Arabidopsis plants, these problems were not observed. In these plants, the receptor was associated with an intracellular structure that resembled the ER. ETR1, the ethylene receptor most closely related to ERS1 and with which it constitutes subfamily I, has also been shown to reside in the ER membrane (Chen e t al., 2002). Therefore, it is conceivable that these two proteins form an ethylene receptor together. Such direct interactions could not be shown, however, because the expression of the EYFP tagged receptors was too low for fluorescence-based interaction studies. Interestingly, an ethylene receptor from subfamily II appeared to be localized at the plasma membrane (Xie e t al., 2003), revealing a potentially important difference between the two subfamilies. That these two subfamilies differ in their activities is confirmed by the finding that the mutant phenotype of a double null-mutant of the two subfamily I members could not be rescued by overexpression of subfamily II members (Wang e t al., 2003). A second component of the ethylene signal transduction pathway that is encoded for by a gene family is the EIN3 transcription factor. In chapter 3, the isolation and characterization of the tobacco EIN3 family is described. It is shown that all members are expressed constitutively. Similar results have been obtained with the tomato EIN3 family, whose members are expressed almost equally in all major tomato plant organs (Tieman et al., 2001). Moreover, they seem to act functionally redundant at different stages of tomato plant development. Together, these data indicate that there is no regulation of ethylene responses at the level of this trancscription factor. Recently, more components of the ethylene response pathway have been revealed, including proteins that function in a MAPkinase pathway (Ouaked e t al., 2003). Because MAPkinase pathways are involved in multiple signaling pathways (Tena e t al., 2001), interactions with other signaling pathways may occur at this level. Furthermore, an example of combinatorial control of transcription has been identified. The ERF1 transcription factor is an EIN3 response gene involved in ethylene responses, but its expression is also regulated by another hormone, jasmonic acid (Lorenzo e t al., 2003). Because ERF1 is a member of a large family of proteins with different activities (Fujimoto e t al., 2000), combinatorial control of these proteins could well be involved in differential ethylene responses. In chapter 4, a novel role for ethylene is described. In ethylene-insensitive flowers, anther dehiscence was delayed and was no longer synchronous with flower opening. We

78 Summary and concluding remarks showed that in these anthers degeneration of the stomium cells and dehydration were delayed. Because anther dehiscence could be delayed by inhibition of ethylene-perception and accelerated by ethylene-treatment in isolated, almost mature anthers, ethylene must have a direct effect on a process that takes place in the anthers just before dehiscence. Interestingly, a similar role has been found for the plant hormone jasmonic acid (JA) in Arabidopsis, by use of the JA deficient dde1 mutant (Sanders et al., 2000). Because of the mild phenotype of the JA deficient Arabidopsis plants and the ethylene-insensitive tobacco plants (i.e. delayed dehiscence, instead of inhibited dehiscence), it was suggested that ethylene and jasmonic acid may act partially redundantly. However, generation of ethylene- insensitivity in the Arabidopsis dde1 mutant, by introduction of the etr1-1 mutation, did not enhance the delayed anther dehiscence phenotype (data not shown).

A link between integument development and ethylene was suggested by the phenotype of ethylene-deficient tobacco flowers (De Martinis and Mariani, 1999) and the homology between the ANT gene, involved in integument growth, and the ERF genes, involved in ethylene signaling (Reichmann and Meyerowitz, 1998). However, due to the absence of a mutant integument phenotype in ethylene-insensitive tobacco plants (chapter 4), the function of ethylene in integument development could not be investigated further. Chapter 5 describes a study aimed to reveal the homology between the tobacco integument and the two integuments of Arabidopsis. Two integuments is the ancestral state in the angiosperms (Herr, 1995; Endress and Igersheim, 2000), and information on homology between the different integuments could be useful to determine the evolutionary pathway that has led to unitegmy in the euasterids. By comparing gene expression programs in the integuments, we revealed a similarity between the single tobacco integument and the outer Arabidopsis integument. However, similarities between gene expression programs or gene functions do not indicate evolutionary homoly between cells or tissues in all cases, as genes can be recruited to different structures in a process of parallel or convergent evolution (Abouheif, 1997). Therefore, data from multiple, unrelated marker genes are necessary and should best be combined with data from detailed morphological and cell lineage analyses.

REFERENCES

Abeles FB, Morgan PW, Salveit MEJ (1992) Ethylene in Plant Biology, Ed Second. Academic Press, San Diego, USA Abouheif E (1997) Developmental genetics and homology: a hierarchical approach. Trends in Ecology and Evolution 12: 405-408 Bray D, Levin MD, Morton-Firth CJ (1998) Receptor clustering as a cellular mechanism to control sensitivity. Nature 393: 85-88 Chen YF, Randlett MD, Findell JL, Schaller GE (2002) Localization of the ethylene receptor ETR1 to the endoplasmic reticulum of Arabidopsis. The Journal of Biological Chemistry 277: 19861-19866 De Martinis D, Mariani C (1999) Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants. The Plant Cell 11: 1061­ 1071

79 Chapter 6

Endress PK, Igersheim A (2000) Gynoecium structure and evolution in basal angiosperms. International Journal of Plant Sciences 161 Suppl: S211-S223 Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis ethylene- responsive element binding factors act as transcriptional activators or repressors of GCC box­ mediated gene expression. The Plant Cell 12: 393-404 Gazzarrini S, McCourt P (2001) Genetic interactions between ABA, ethylene and sugar signaling pathways. Current Opinion in Plant Biology 4: 387-391 Genoud T, Metraux J-P (1999) Crosstalk in plant cell signaling: structure and function of the genetic network. Trends in Plant Science 4: 503-507 Guzman P, Ecker JR (1990) Exploiting the triple response of Arabidopsis to identify ethylene- related mutants. The Plant Cell 2: 513-523 Hall AE, Findell JL, Schaller GE, Sisler EC, Bleecker AB (2000) Ethylene perception by the ERS1 protein in Arabidopsis. Plant Physiology 123: 1449-1457 Heldin CH (1995) Dimerization of cell surface receptors in signal transduction. Cell 80: 213-223 Herr JM (1995) The origin of the ovule. American Journal of Botany 82: 547-564 Howell SH (1998) Molecular genetics of plant development. Cambridge University Press, Cambridge Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR, Meyerowitz EM (1998) EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. The Plant Cell 10: 1321-1332 Lashbrook CC, Tieman DM, Klee HJ (1998) Differential regulation of the tomato ETR gene family throughout plant development. The Plant Journal 15: 243-252 Lorenzo O, Piqueras R, Sanchez Serrano JJ, Solano R (2003) ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. The Plant Cell 15: 165-178 Meyerowitz EM (1997) Genetic control of cell division patterns in developing plants. Cell 88: 299­ 308 Ouaked F, Rozhon W, Lecourieux D, Hirt H (2003) A MAPK pathway mediates ethylene signaling in plants. EMBO Journal 22: 1282-1288 Riechmann JL, Meyerowitz EM (1998) The AP2/EREBP family of plant transcription factors. Biological Chemistry 379: 633-646 Sanders PM, Lee PY, Biesgen C, Boone JD, Beals TP, Weiler EW, Goldberg RB (2000) The Arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway. The Plant Cell 12: 1041-1061 Schaller GE, Ladd AN, Lanahan MB, Spanbauer JM, Bleecker AB (1995) The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer. The Journal of Biological Chemistry 270: 12526-12530 Smalle J, Haegman M, Kurepa J, Van Montagu M, Van Der Straeten D (1997) Ethylene can stimulate Arabidopsis hypocotyl elongation in the light. Proceedings of the National Academy of Sciences of the United States of America 94: 2756-2761 Tanimoto M, Roberts K, Dolan L (1995) Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. The Plant Journal 8: 948 Tena G, Asai T, Chiu WL, Sheen J (2001) Plant mitogen-activated protein kinase signaling cascades. Current Opinion in Plant Biology 4: 392-400 Tieman DM, Klee HJ (1999) Differential expression of two novel members of the tomato ethylene- receptor family. Plant Physiology 120: 165-172 Tieman DM, Ciardi JA, Taylor MG, Klee HJ (2001) Members of the tomato LeEIL (EIN3-like) gene family are functionally redundant and regulate ethylene responses throughout plant development. The Plant Journal 26: 47-58 Wang WY, Hall AE, R OM, Bleecker AB (2003) Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission. Proceedings of the National Academy of Sciences of the United States of America 100: 352-357 Xie C, Zhang JS, Zhou HL, Li J, Zhang ZG, Wang DW, Chen SY (2003) Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco. The Plant Journal 33: 385-393

80 Nederlandse samenvatting

Planten behoren tot de belangrijkste levensvormen op aarde. Ze zetten de energie die uitgezonden wordt door de zon om in organische verbindingen, die vervolgens gebruikt worden door vele andere aardse levensvormen. De vorm van een plant wordt bepaald door het aantal, de plaats en het vlak van de celdelingen in de plant en door de uiteindelijke vorm van de cellen. Behalve in vorm, verschillen plantencellen ook van elkaar in functie. Deze beide aspecten van een plantencel, vorm en functie, worden bepaald door het genetische programma van de cel. Terwijl dit genetische programma in dierlijke organismen vooral bepaald wordt door de afkomst van een cel (zijn zogenaamde cellijn), lijken bij planten de interne en externe signalen die een cel continu opvangt veel belangrijker te zijn. Daardoor zijn planten flexibeler zijn in hun ontwikkeling. Plantenhormonen spelen een belangrijke rol bij de interne signalering, oftewel de communicatie tussen de plantencellen. Het zijn stoffen die door een cel worden uitgescheiden naar de ruimtes tussen de cellen, zich verplaatsen naar andere cellen, en daar, al in zeer lage concentraties, worden waargenomen met behulp van receptoreiwitten. In de betreffende cellen wordt er vervolgens, via een cellulaire signaaltransductieweg, een signaal gestuurd naar de celkern, alwaar het genetische programma wordt veranderd. Ethyleen is een van de vijf klassieke plantenhormonen en verhoging van de concentratie van dit hormoon kan tot verschillende reakties van de cellen leiden. Cellen van zaailingen die in het licht groeien, bijvoorbeeld, worden langer in aanwezigheid van extra ethyleen, terwijl cellen van zaailingen die in het donker groeien juist korter worden in aanwezigheid van extra ethyleen. Cellen in de buitenste cellaag van de wortels reageren op ethyleen door zich vaker om te vormen tot wortelhaarcellen, en in veel vruchten reageren de cellen met het afbreken van hun celwanden, tenminste, als de vrucht zich in de juiste ontwikkelingsfase bevindt. In deze voorbeelden zal een aantal van de genen die gemoduleerd worden door ethyleen identiek zijn, maar er zullen ook zeker verschillen zijn in de genetische reakties. De oorzaak is dat de reaktie van een cel op het waarnemen van een bepaald hormoon, in dit geval ethyleen, bepaald wordt door interacties tussen het hormoonsignaal en andere signalen die de plantencel opvangt of al heeft opgevangen. Het is echter niet bekend waar de interactie tussen het plantenhormoon ethyleen en andere signalen plaatvindt. Mogelijk gebeurt dit pas nadat het ethyleensignaal via de cellulaire signaaltransductieweg is doorgegeven naar de celkern, middels gezamenlijke controle over de genen die kunnen worden aan- en uitgeschakeld. Een andere mogelijkheid is dat de signalen elkaars cellulaire signaaltransductiewegen beïnvloeden, zodat de uitkomst van deze wegen niet gelijk is in de verschillende plantencellen. Enkele bekende componenten van de cellulaire signaaltransductieweg voor ethyleen worden gecodeerd door genfamilies. Mits de verschillende familieleden ook verschillende activiteiten hebben, kan differentiële expressie van de leden van deze genfamilies een oorzaak zijn voor het verschil in de reaktie op ethyleen tussen

81 Samenvatting verschillende plantencellen. Een mogelijk voorbeeld van zo een component is de ethyleenreceptor. De verschillende ethyleenreceptoreiwitten komen inderdaad differentieel tot expressie. Over de activiteiten van deze verschillende eiwitten is echter weinig bekend. Uit onderzoek aan dierlijke modelsystemen is gebleken dat de activiteit van hormoonreceptoreiwitten sterk beïnvloed kan worden door de vorming van heterodimeren tussen verschillende leden van receptoreiwitfamilies. Omdat van enkele ethyleenreceptoreiwitten bekend is dat ze homodimeren vormen, is vaak gesuggereerd dat ze misschien ook heterodimeren kunnen vormen. Om dit te onderzoeken, hebben we allereerst bekeken of verschillende ethyleenreceptoreiwitten zich op dezelfde plek in de plantencellen bevinden. Dit is beschreven in hoofdstuk 2. Door het ethyleenreceptoreiwit ERS1 van de zandraket (Arabidopsis thaliana) te fuseren aan het fluorescerende eiwit EYFP hebben we kunnen vaststellen dat het ERS1 eiwit zich in het endoplasmatisch reticulum van de cellen bevond. Aangezien uit onderzoek verricht door anderen al bekend was dat een ander ethyleenreceptoreiwit uit dezelfde plant zich ook op deze plek in de cellen bevindt, is het mogelijk dat deze twee eiwitten heterodimeren met elkaar vormen. Dat dit ook echt gebeurt konden we niet onderzoeken, vanwege een te lage hoeveelheid ethyleenreceptoreiwitten in de plantencellen. Een tweede component van de cellulaire signaaltransductieweg voor ethyleen die door een familie van genen wordt gecodeerd, is de EIN3 transcriptiefactor. In hoofdstuk 3 wordt de isolatie en karakterisering van de EIN3 familie van tabak beschreven. Er wordt aangetoond dat alle familieleden in alle onderzochte organen en celtypen tot expressie komen. Een vergelijkbaar onderzoek in tomaat liet al zien dat de verschillende EIN3 familieleden gelijke functies hebben in de tomatenplant. Deze studies suggereren dat de reaktie van een cel op ethyleen niet gereguleerd wordt door regulatie van de expressie van de EIN3 familieleden. Waar de interactie tussen het ethyleensignaal en andere signalen plaatsvindt, hebben we niet kunnen vaststellen. Wellicht vindt dit al plaats op het nivo van de ethyleenreceptor. Recentelijk zijn nog enkele andere componenten uit de cellulaire ethyleensignaaltransductieweg beschreven die mogelijk beïnvloed kunnen worden door andere signalen. Daarnaast is er een transcriptiefactor beschreven, waarvan de expressie gereguleerd wordt door zowel ethyleen als door een andere plantenhormoon. In hoofdstuk 4 wordt een nieuwe rol voor ethyleen beschreven. In ethyleenongevoelige tabaksbloemen was het openen van de helmknoppen vertraagd en daardoor niet langer synchroon met het openen van de bloemen. We konden laten zien dat de degeneratie van een groepje cellen, de stomiumcellen, in deze helmknoppen vertraagd was en dat de helmknoppen minder uitgedroogd waren. Omdat het openen ook in losse, bijna volwassen helmknoppen geremd kon worden door de waarneming van ethyleen te blokkeren en versneld kon worden door extra ethyleen te geven, moet ethyleen een direkt effect hebben op een proces dat plaatsvindt in de helmknoppen, vlak voor het opengaan. Een eerdere studie aan tabaksbloemen met een verlaagde ethyleenproductie suggereerde dat ethyleen ook een rol speelde bij de ontwikkeling van de zaadknop. De vervolgstudie naar deze rol van ethyleen werd niet voortgezet, omdat de zaadknoppen

82 Samenvatting normaal ontwikkelden in tabaksbloemen die ongevoelig waren voor ethyleen (hoofdstuk 4). Hoofdstuk 5 beschrijft een studie naar de evolutionaire homologie tussen het enkele vlies rond de zaadknoppen van tabak (behorend tot de asteriden) en de twee vliezen rond de zaadknoppen van de zandraket. Omdat de aanwezigheid van twee zaadknopvliezen de oersituatie is in bloeiende planten, zou informatie over de homologie tussen de genoemde vliezen inzicht kunnen verschaffen in de evolutie van het enkele zaadknopvlies in de asteriden. Overeenkomsten tussen het genetische programma van het enkele zaadknopvlies van tabak en het buitenste zaadknopvlies van de zandraket konden worden aangetoond. Interpretatie van dergelijke data is echter moeilijk, en daarom zijn meer gegevens noodzakelijk voor het eenduidig vaststellen van de evolutionaire homologie.

83

Dankwoord

Bij aanvang van mijn studie biologie stond mij duidelijk voor ogen wat ik wilde: onderzoek doen aan de natuur, het milieu, aan zoogdieren en ecosystemen. Biologie "in het groot”, dus. Een lange reis is echter moeilijk te plannen, en zo belandde ik al tijdens mijn studie bij Titti op de afdeling Celbiologie van de Plant. En wat bleek: een laboratorium was helemaal niet zo’n nare plek als ik me had voorgesteld! Vooral omdat ik door jou, Titti, ontdekte dat achter het "kleine” werk in een lab ook grotere biologische vragen schuilgaan. Vanaf de eerste tot en met de laatste dag ben jij een uitstekende leermeester geweest ("denk aan de controles!”; "niet afdwalen!”; "wat wil je hiermee zeggen?”). Het was bijzonder om te voelen dat jij ons, je AlO’s, altijd op de eerste plek had staan. Behalve van de wetenschap, hoop ik dat ik ook iets heb opgestoken van jouw vele andere kwaliteiten, die de afgelopen vijf jaar niet alleen tot een heel leerzame, maar ook onvergetelijk mooie tijd hebben gemaakt: jouw kwaliteiten om mensen te begeleiden, te motiveren en zelfvertrouwen te geven en om de sfeer in een groep zo goed te houden. Ik kwam elke dag met erg veel plezier naar de afdeling. Titti, bedankt voor alle steun als begeleider en voor alle liefde als mens. Ik ben blij en trots een van jouw "jongens” te zijn! Als tweede wil ik Koen bedanken. Vanaf het moment dat je bij ons op de afdeling kwam heb je mij (en anderen) zonder eigenbelang geholpen bij de projecten; daar heb ik veel bewondering voor! De uren dat we samen hebben gezeten aan een buro, een (isotopen) labtafel en jouw tuintafel voelden nooit als een plicht, maar als een voorrecht. Ik heb veel opgestoken van de aandachtige wijze waarop jij werkt, zowel bij het opzetten als bij het uitvoeren en rapporteren van onderzoek. Maar bovenal heeft je enthousiasme en je brede kennis van -, en interesse in de ontwikkelingsbiologie mij enorm gemotiveerd! Een belangrijke redenen waarom het werken op het lab mij zo beviel, was de sterke interactie met collega’s. Ik vind dat ik ongelofelijk geluk heb gehad dat ik kon samenwerken met Barend, Wim en Richard, Maurice, Ana en Chiara, Jeroen, Marc en Karin, Raymond, Flora, Manoko en Tamara. Echt een supergroep! Met jullie heb ik veel gelachen, flink gediscussieerd, af en toe geruzied, lekker gegeten en gedronken, ver gefietst, mooie en lelijke muziek geluisterd, goed gepraat, gezellig gebiljard en vriendschap gesloten. Bedankt allemaal! Else, je hebt het al vaker gehoord, en dat is omdat het waar is: jij bent er altijd, voor alles en voor iedereen. Knap vind ik dat. Jouw kamer voelde voor mij als een veilige, warme en menselijke plek op de afdeling. Bedankt! Jan, Mieke, Peter, Huub en Bart, zonder jullie was het werk onmogelijk en de afdeling minder veelzijdig en aangenaam geweest. Hopelijk word ik ooit zo wijs als jullie. Door Walter, Yvette, Harry, Jeroen, Theo en Gerard werd mij telkens weer duidelijk: we werken aan echte, levende, mooie planten! Al zaten we ver uitelkaar, ik voelde me

85 Dankwoord

meer met jullie verbonden dan jullie misschien denken. Bedankt voor de gezelligheid en de goede zorgen! Liesbeth, Jelle en Rien, jullie grote bijdrage aan mijn proefschrift bestaat uit (bijna) al de foto’s en DNAsequenties. Bedankt dat ik telkens weer langs kon komen voor zulke goede hulp. Tom, Janny, Filip, Stefan, Wim, Theo en José, George, Rinus en Ton, bedankt voor jullie steun en leuk dat ik jullie heb leren kennen! De afgelopen jaren werden verlicht door vier getalenteerde stagestudenten: Michiel, Marijn, Tamara en Ank, hartstikke bedankt voor jullie inzet in soms zware tijden! Dan zijn er nog de "verre” mensen met wie ik heb samengewerkt, al moet ik zeggen dat het meestal eenrichtingsverkeer (richting mij) was: Joop en Dorus, bedankt voor de schemerige uren achter de CLSM; Gerco en Stefan, bedankt voor jullie hulp bij het Arabidopsis werk; Dominique en Ernst, bedankt dat jullie je beschikbaar stelden als externe begeleiders; Peter en Casper, bedankt voor jullie hulp bij het onderzoek naar de subcellulaire localizatie van ERS1. Tenslotte Paul, Lia en Ruben. Ik vertelde jullie misschien weinig over mijn werk, maar dat kwam omdat ik het zo lekker vond om bij jullie in een hele andere wereld te komen. Ik hou van jullie!

Andrea, ich liebe dich!

86 Curriculum vitae

Ivo Rieu werd geboren op 25 oktober 1975 te Breda. Hij begon in 1993 aan de Universiteit Utrecht met de studie biologie, welke hij vanaf 1994 voortzette aan de Katholieke Universiteit Nijmegen. De hoofdvakstage verrichtte hij op de afdeling Celbiologie van de Plant bij Prof. Dr. C. Mariani, w aar hij onderzoek deed naar het effect van overstroming op de activiteit van ethyleenbiosynthesegenen in moeraszuring. Zijn bijvakstage verrichte hij op de vakgroep Bosbouw aan de Universiteit Wageningen onder leiding van Dr. F.J.J.M. Bongers. Hiervoor deed hij veldonderzoek in Frans Guyana naar de invloed van omgevingsfactoren op de overleving van zaailingen. Tijdens zijn studie werkte hij als studentassistent bij verscheidene cursussen. In 1998 behaalde hij zijn doctoraal examen biologie cum laude. In hetzelfde jaar begon hij aan een promotieonderzoek bij de afdeling Celbiologie van de Plant onder leiding van Prof. Dr. C. Mariani. De resultaten hiervan staan beschreven in dit proefschrift. Naast zijn promotieonderzoek begeleidde hij een aantal stagestudenten en was hij lid van de AIO- raad van de onderzoeksschool Experimentele Plantenwetenschappen. Vanaf januari 2004 is hij werkzaam als postdoctoraal onderzoeker bij Rothamsted Research in Harpenden, Engeland, onder leiding van Dr. P. Hedden, waar hij onderzoek doet naar de afbraak van het plantenhormoon gibberelline.

87

Appendix

Color figures chapter 2

89 Appendix

Chapter 2, figure 2:

Fluorescence analysis FDA viability staining EYFP ERS1-EYFP EYFP ERS1-EYFP

Chapter 2, figure 3:

CFP CFP-ER ST-tmdCFP GFP-Zm7hvr

ERS1-EYFP

+ #

90 Appendix

Chapter 2, figure 4:

YFP channel CFP channel Overlay

ERS1-EYFP + CFP

ERS1-EYFP + A CFP-ER i (

ERS1-EYFP + ST-tmdCFP

Chapter 2, figure 6:

91

Appendix

Color figures chapters 3 and 5

93 Appendix

Chapter 3, figure 2:

94 Appendix

Chapter 5, figure 3:

Chapter 5, figure 4:

95