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Charles University in Prague

Faculty of Science

Department of Plant Physiology

Academy of Sciences of the Czech Republic

Institute of Experimental Botany

Laboratory of Cell Biology

EXO70A1, SEC6 and SEC8 proteins in plant cell

morphogenesis

PhD Thesis

Mgr. Lukáš Synek

Prague 2007

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Supervisor: RNDr. Viktor Žárský, CSc.

Consultant: doc. RNDr. Fatima Cvrčková, Dr. rer. nat.

Following collaborators contributed to this work Mgr. Marek Eliáš – phylogenetic analysis

Michaël Quentin, PhD. – selection of the exo70B2 mutant

Nicole Schlager and Prof. Marie-Theres Hauser – ACC treatment, most experiments on phenotypic characterization of exo70A1 mutants were done in parallel in the laboratory of Prof. Hauser

Mgr. Edita Drdová – crossing of Arabidopsis plants, EXO70A1 protein purification and anti-EXO70A1 antibody testing

Acknowledgements

At this place I would like to thank all members of the Laboratory of cell biology (Institute of Experimental Botany) and the Laboratory of plant cell morphogenesis (Charles University) for their help and support at work on my thesis. Furthermore, I would like to thank Marek Eliáš for his fundamental contribution to this project and for helpful discussions. Last but not least, I would like to express my thanks to Prof. Marie-Theres Hauser (Laboratory of Plant Genetics, Institute of Applied Genetics and Cell Biology, BOKU - University of Natural Resources and Applied Life Sciences, Vienna) and to Rex Cole and Prof. John E. Fowler (Department of Botany and Plant Pathology, Oregon State University, Corvallis, USA) for a very nice and fruitful collaboration in this project.

This work was supported by the MŠMT ČR project LN00A081-SIDROS, the GAAV ČR grant

A6038410, and the EU RTN project TIPNET HPRN-CT-2002-00265.

List of Publications Synek, L., Schlager, N., Eliáš, M., Quentin, M., Hauser, M.T., Žárský, V. (2006). AtEXO70A1, a

member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. Plant Journal, 48: 54 - 72.

Cole, R.A., Synek, L., Žárský, V., Fowler, J.E. (2005). SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant Physiology 138: 2005-2018.

Žárský, V., Eliáš, M., Drdová, E., Synek, L., Quentin, M., Kakešová, H., Žiak, D., Hála, M., Soukupová, H. (2004). Do exocyst subunits in plants form a complex? Acta Physiol. Plant., 26: 146.

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Summary

The exocyst is a hetero-oligomeric protein complex that consists of eight subunits (Sec3, Sec5,

Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) and is involved in exocytosis. It has been extensively studied in yeast and animal cells, however evidence is now accumulating that the exocyst is also present in plants. Phylogenetic analysis of genes encoding plant homologs of the Exo70 exocyst subunit revealed that Arabidopsis and rice contain 23 and 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (Eliáš et al., 2003).

Expression analysis revealed that many genes of the EXO70 family exhibit tissue specific expression in Arabidopsis and that EXO70A1 is the most strongly expressed EXO70 isoform in the sporophyte. Based on the expression data we selected several Arabidopsis T-DNA insertional mutants in highly expressed EXO70 genes. Among them only two independent mutants in the EXO70A1 gene exhibited a mutant phenotype, which we characterized in detail. Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in the 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, exo70A1 homozygotes exhibit an array of phenotypic defects. The polarized cell growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 has a role in cell and organ morphogenesis that probably consists in regulation of exocytosis.

In addition, mutants in SEC6 and SEC8, single gene encoded exocyst subunits, were also obtained. Mutant alleles of both SEC6 and SEC8 genes exhibit pollen-specific transmission defect, so homozygous mutants are not produced (in collaboration with Rex Cole and John Fowler). We conclude that SEC6 and SEC8 genes are essential for pollen germination and pollen tube growth.

Abbreviations 3T3 cells mouse embryonic fibroblast cells ACC 1-aminocyclopropane-1-carboxylic acid ATH1 array Arabidopsis thaliana array (version 1) CLSM confocal laser-scanning microscopy DAG day after germination EST expressed sequence tag GEF GDP-GTP exchange factor GFP green fluorescent protein MDCK cells Madin–Darby canine kidney cells MS medium Murashige and Skoog medium MTOC microtubule-organizing center NAA naphtyl acetic acid PEG polyethylene glycol RT-PCR reverse transcriptase polymerase chain reaction T-DNA transferred DNA

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1 The Exocyst Complex

1.1 Introduction

Precise regulation of localized cell expansion and cell division is necessary for cell

morphogenesis. The executive morphogenetic process, exocytosis, is an essential vesicle traffic event mediating the secretion of hormones, neurotransmitters, enzymes, membrane proteins, lipids and in plants as well components of the cell wall to specific sites on the plasma membrane. Therefore, exocytosis is crucial for cell growth, cell-cell communication, and cell polarity establishment. Vesicle trafficking is intimately connected with cytoskeleton. Both are precisely regulated by a number of proteins such as small GTPases. Before the fusion of vesicles to the plasma membrane mediated by SNARE proteins, a multimeric complex, termed the exocyst, acts as a tethering complex. The exocyst specifies the vesicle docking site on the plasma membrane, resulting in spatially localized secretion.

The exocyst has been extensively studied in yeast and mammals (recently reviewed in Hsu et al., 2004). In both yeast and mammals, the exocyst complex consists of eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. It predominantly localizes at sites of extensive polarized exocytosis. In contrast to yeast and mammals, research on plants is just at the very beginning. However, the core components of the secretory pathway, e.g. SNARE proteins, coat complexes, or small GTPases appear to be highly conserved across eukaryotes (Dacks and Field, 2004; Sanderfoot and Raikhel, 2003), so it is likely that the exocyst complex is also present in plant cells and may play similar roles as in yeast and mammalian cells.

1.2 Functions of the exocyst complex

In yeast, the exocyst participates in bud-site establishment and has a role in growth of daughter

cell, especially during the stage of apical growth. Later in the cell cycle, the exocyst acts at cytokinesis in formation of the septum between mother and daughter cells (TerBush et al., 1996; Finger and Novick, 1997; Finger et al., 1998; Guo et al., 1999b). Importantly, both apical growth and septum formation involve extensive polarized exocytosis.

Most of the exocyst subunits were identified in the screen for yeast mutants defective in secretion (mutants in the late SEC genes) (Novick et al., 1980). Even for the later discovered subunits it was shown that their mutation or absence results in secretory defects (TerBush et al., 1996; Guo et al., 1999b). In general, null mutants in exocyst genes are inviable, whereas temperature-sensitive mutants block exocytosis and arrest growth of the daughter cell after the shift to restrictive temperature. Electron microscopic studies revealed the accumulation of 80- to 100-nm post-Golgi secretory vesicles in the mutant cells, probably due to defect in tethering vesicles to the plasma membrane (Novick et al., 1980; Finger and Novick, 1997; Finger et al., 1998; Guo et al., 1999a). The yeast mutants in exocyst genes clearly demonstrate the engagement of the exocyst complex in polarized exocytosis.

In animal cells, the exocyst seems to have a similar role in polarized exocytosis as in the yeast cells. In polarized epithelial cells, secretory vesicles are targeted by the exocyst complex to the basolateral membrane, but apparently not to the apical membrane (Grindstaff et al., 1998; Hsu et al., 1998). In neurons, the exocyst is required for neurite tip growth, neurite branching and synaptogenesis. Surprisingly, synaptic vesicle release at mature synapses was revealed to be independent of exocyst function (Hazuka et al., 1999; Vega and Hsu, 2001; Lalli and Hall, 2005; Murthy et al., 2003; Mehta et al., 2005). The exocyst has also been implicated in cytokinesis, reflecting its role in localized plasma-membrane expansion. Gromley et al. (2005) discovered that the exocyst is anchored by centriolin to the midbody and that is essential for the terminal step of cytokinesis, so called abscission. In addition, a screen for cytokinesis mutants in Drosophila identified the exocyst component Sec5 (Echard et al., 2004). Detailed molecular mechanism by which the exocyst functions in tethering is under investigation. Current observations suggest that under normal circumstances the exocyst holds vesicles in proximity to the plasma membrane long time enough for SNARE complexes to form and mediate fusion.

However, functions of the exocyst are not limited only to exocytosis. Recently, it has been shown that the exocyst probably promotes membrane recycling (Prigent et al., 2003; Sommer et al., 2005;

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Jafar-Nejad et al., 2005; Xu et al., 2005; Shen et al., 2006). Another data suggest a possibility that the exocyst may somehow link and coordinate the protein synthesis with protein targeting to the plasma membrane (Lipschutz et al., 2000 and 2003; Toikkanen et al., 2003; Wiederkehr et al., 2003). The exocyst can also interact with tubulin cytoskeleton, inhibit tubulin polymerization and modulate thus its dynamics underlying exocytosis (Vega and Hsu, 2001; Wang et al., 2004). Some other experiments revealed that the exocyst complex is also imvolved in actin organization (Aronov and Gerst, 2004; Zuo et al., 2006), filopodia formation ((Sugihara et al., 2002) and pre-mRNA splicing (Awasthi et al., 2001).

1.3 Structure of the exocyst complex

In both yeast and mammals, the exocyst complex consists of eight subunits: Sec3, Sec5, Sec6,

Sec8, Sec10, Sec15, Exo70, and Exo84 (Figure1A) (TerBush et al., 1996; Hsu et al., 1996; Guo et al., 1999b; Matern et al., 2001). Despite limited sequence homology between yeast and mammalian exocyst subunits (approx. 17% to 24% sequence identity) (Ting et al., 1995; Guo et al., 1997; Hazuka et al., 1997; Kee et al., 1997; Matern et al., 2001), the siblings have very similar molecular weights ranging from 70 to 140 kDa (TerBush et al., 1996; Hsu et al., 1996). More surprisingly, although overall sequence identity among different exocyst genes is less than 10%, all subunits are predicted to assume strikingly similar secondary and even tertiary structures.

Recent crystallographic studies of Exo70, Exo84, Sec6, and Sec15 structure indicate that all these subunits adopt rod-like shape and contain elongated domains formed from helical bundles (Dong et al., 2005; Wu et al., 2005; Hamburger et al., 2006; Sivaram et al., 2006). Based on the structures, on protein interaction studies, and on electron microscopic observations, current model of the exocyst complex was proposed. This model assumes that subunits align longitudinally together, keeping distant parts of their molecules flexible on one side of the complex, and resembling thus a ‘flower’ (Figure 1) (Munson and Novick, 2006).

Figure 1. Structure of the exocyst complex. A) Schematic model of the yeast exocyst complex, hypothesizing that each of the exocyst subunits has an elongated helical-bundle structure (Munson and Novick, 2006). B) Electron microscopic images of purified mammalian brain exocyst complexes, either unfixed (upper row) or fixed with glutaraldehyde (lower row) (Hsu et al., 1998). C) Tomographic slice images of vesicles connected by Y-shaped tethering complexes during cell plate formation in Arabidopsis (Seguí-Simaro et al., 2004). D) After the exocyst complex is fully assembled at a site specified by spatial landmarks, the arms of the complex may clamp together, tethering thus the vesicle to the membrane (Novick et al., 2006).

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1.4 Subcellular localization and the exocyst assemb ly Given the proposed role of the exocyst complex in vesicle tethering, it is crucial for the complex

to be properly positioned so that exocytosis will take place at the right site. The exocyst complex localizes to subdomains of the plasma membrane that represent sites of extensive polarized exocytosis (TerBush and Novick, 1995; Finger et al., 1998; Hsu et al., 1999; Vega and Hsu, 2001; Wang et al., 2002; Fielding et al., 2005). In contrast to t-SNARE proteins that are evenly distributed along the plasma membrane, the exocyst is precisely localized at specific sites (Haarer et al., 1996; Finger and Novick, 1997). Although the amino acid sequences of exocyst subunits predict soluble proteins, approx. 40% of the yeast exocyst complex and 70–90% of the mammalian exocyst complex was found associated with membranes (Bowser et al., 1992; Hsu et al., 1996; Grindstaff et al., 1998).

In budding yeast, the exocyst is localized at regions of polarized secretion at site of bud formation, at the apex of the growing bud (until the switch of apical growth to isotropic growth) and in the neck between the mother and daughter cell near the time of cytokinesis (Figure 2a). In fission yeast, the exocyst is required for the last step of cytokinesis (Wang et al., 2002). In mammalian cells, the exocyst localizes to the region of tight junctions and is responsible for targeting proteins to the basolateral membrane (Yeaman et al., 2001). In non-polarized MDCK cells, the exocyst resides mainly in cytoplasm or associated with trans-Golgi network and vesicles are targeted to random areas (Figure 2b). After cell polarization, the exocyst relocalizes to the sites of cell–cell interactions along the plasma membrane and eventually to the apex of the basolateral domain near the tight junctions. Vesicles are then targeted either to the apical membrane (Figure 2b, in green) or the basolateral membrane (in red). Upon depletion of calcium, the cells become non-polarized and the exocyst disperses to the cytosol (Grindstaff et al., 1998).

Exocyst function is also crucial for growth of neurite tips in neuronal cells (Hazuka et al., 1999) and for synaptogenesis (Murthy et al., 2003). In developing neurons, the exocyst is enriched in neurite growth tips. Later, one neurite becomes the axon, where the exocyst clusters at periodic intervals on the plasma membrane and implicates synaptogenesis (Figure 2c). Upon the formation of stable synapses (in blue), the exocyst is downregulated in these sites.

Figure 2. Subcellular localization of the exocyst complex (Hsu et al., 1999). a) yeast cells, b) Madin–Darby canine kidney cells, c) developing neurons. Description in the text. N – nucleus.

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Most of the exocyst subunits are delivered to exocytic sites on secretory vesicles, likely associated as a partial subcomplex on the vesicle surface (Finger et al., 1998; Boyd et al., 2004). However, Sec3 and partially Exo70 use a vesicle-independent transport mechanism and either one or both probably serve as spatial landmarks for recruitment of the rest of the exocyst complex to the plasma membrane (Finger et al., 1998; Boyd et al., 2004). A subset of exocyst subunits is probably preassembled on the vesicle surface, to which is anchored through Sec15p-Sec4p interaction or Sec15-Rab11 interaction in yeast or in mammals, respectively. This partial complex is delivered via cytoskeleton-based vesicle trafficking at sites of exocytosis marked by Sec3p and Exo70p subunits that had been localized in advance via interaction with Rho GTPases and other polarization factors (Boyd et al., 2004; Novick et al., 2006). Regulated exocyst assembly serves to both target and tether vesicles to sites of exocytosis.

1.5 Regulation of the exocyst by small GTPases

Although the central role of the exocyst complex in exocytosis is clearly evident, the knowledge of its control and regulation has been still fragmentary. Given the complexity of the exocyst, it is easy to imagine that the exocyst could integrate many different inputs. The overall picture that has emerged is that the exocyst serves as a focus point for a number of different signaling pathways, and by integrating these various signals, it determines where, when and how secretory vesicles land on the plasma membrane.

Indeed, research from both yeast and mammalian cells has identified several small GTPases that ‘talk’ to the exocyst. They include members of the Rab family (Sec4p, Rab11), the Rho family (Rho1p, Rho3, Cdc42p, TC10), the Ral family (RalA) and the Arf family (Arf6) that all belong to the Ras superfamily of small GTPases (reviewed in Lipschutz and Mostov, 2002; Novick and Guo, 2002; Camonis and White, 2005). Since the small GTPases have multiple effectors, exocyst function can be coordinated with other cellular processes. Very recently the first small GTPase was identified in plants interacting via an adaptor protein, ICR1, with the exocyst complex (Lavy et al., 2007).

1.6 The putative plant exocyst complex

Homologs to all eight exocyst subunits have been identified in silico in plant genomes, including

Arabidopsis thaliana (Cvrčková et al., 2001; Eliáš et al., 2003; Jurgens and Geldner, 2002). However, it remains to determine whether putative plant exocyst subunits assemble in a complex and share the same functions in plants as in yeast and animals. Sessile biology of the plant cell suggests that plant specific functions could be expected.

Exocyst subunits are typically encoded by single genes, eventually by two or three paralogous genes. For example, Arabidopsis has only SEC6 and SEC8 subunits represented as single-copy genes, while it has two SEC3, SEC5, SEC10 and SEC15 paralogs and three EXO84 paralogs. However, the EXO70 gene proliferated in to a large gene family specifically in land plants (Eliáš et al., 2003; Cannon et al., 2004). The Arabidopsis genome contains 23 EXO70 genes.

Recently, an electron tomographic analysis of cell plate formation during cytokinesis of somatic cells and pollen development in Arabidopsis uncovered the existence of 24-nm-long structures that tether membrane vesicles (Otegui and Staehelin, 2004; Seguí-Simarro et al., 2004), resembling the mammalian exocyst as observed in the electron microscope (Hsu et al., 1998) (Figure1B, C). In addition, a maize roothairless1 mutation, which is manifested by the failure of root hair primordia to elongate properly (a kind of polarized exocytosis), was shown to result from a transposon insertion into the homolog of the SEC3 gene (Wen et al., 2005). Arabidopsis exo70A1 mutants exhibit remarkable similar root hair phenotype to that of the roothairless1 mutant (Synek et al., 2006). Furthermore, phenotypic analysis of a series of mutants in the Arabidopsis SEC8 gene revealed that the putative SEC8 exocyst subunit is necessary for pollen tube germination and tip growth of pollen tubes (Cole et al., 2005).

These findings suggest that the exocyst is conserved in plants and facilitates the initiation and maintenance of polar growth. Therefore the putative plant exocyst may be involved in polarized exocytosis and cytokinesis.

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2 Aims of this Thesis

A. Analyze publicly available expression data of exocyst genes, including all genes of the EXO70

family, with respect to quantity and possible tissue specificity.

B. Inspect phenotypes of selected mutants in each cluster of EXO70 gene family and in putative exocyst subunits encoded by single genes (SEC6 and SEC8).

C. Determine subcellular localization of EXO70, SEC6 and SEC8 proteins in different plant tissues.

3 Results

3.1 Expression patterns of genes encoding exocyst s ubunits

To gain insight into the developmental and organ-specific regulation of the expression of

individual Arabidopsis genes encoding exocyst subunits we analyzed the publicly available Affymetrix ATH1 Arabidopsis genome array data in the Genevestigator database (Zimmermann et al., 2004). Except for EXO70A3, EXO70H4, and EXO70H6, all other 20 EXO70 genes are detectable in microarray experiments. According to their expression pattern, the expressed EXO70 genes can be divided roughly into four classes (Figure 3). EXO70A1, E2 and F1 are expressed in most organs and cells. Expression of EXO70B1, B2, D1, D2, D3, E1, G1, H7 is detectable in sporophytic tissues and organs but not during male gametophytic development. Complementary, EXO70A2, G2, H3, H5 are only active during pollen development. The last group of genes, EXO70H1, H2, H8, C1, C2, show a more restricted activity, being predominantly expressed in roots. In general, the most strongly expressed EXO70 gene in Arabidopsis is EXO70A1 (except for pollen development).

Transcripts of all genes encoding other exocyst subunits (SEC3, 5, 6, 8, 10, 15 and EXO84) are detectable in microarray experiments, except for SEC3b and EXO84a (Figure 3). Probes for both SEC3b and EXO84a are missing on the ATH1 array. In general, SEC and EXO84 genes are expressed in all tissues tested (Cole et al., 2005). Despite the fact that they are noticeably more abundant in root tips (lateral roots, elongation zone of roots), cell suspension and pollen, none of them shows strong tissue specific expression except for SEC15 genes. While the SEC15a gene is detectable exclusively in pollen, its closest relative, SEC15b, is complementary active predominantly in sporophytic tissues.

3.2 Phenotype of exo70 mutants 3.2.1 Insertional mutants in EXO70 genes

None of exo70B2, exo70D3, exo70F1, exo70G1, or exo70H7 homozygous insertional mutants

have showed any obvious difference from wild-type phenotype under standard laboratory conditions. However, exo70A1 homozygotes do exhibit a discernible semi-dwarf phenotype.

We obtained two different mutant lines, exo70A1-1 and exo70A1-2, with T-DNA insertions in the first intron and in the sixth exon, respectively. Heterozygotes EXO70A1/exo70A1-1 and EXO70A1/exo70A1-2 are indistinguishable from wild-type plants and their offspring segregates in the Mendelian 1:2:1 ratio. On the other hand, homozygous exo70A1-1 and exo70A1-2 plants exhibit developmental alterations. 3.2.2 EXO70A1 is required for hypocotyl elongation and tip growth of root hairs

Both exo70A1-1 and exo70A1-2 exhibit identical phenotypes. Mutant seedlings germinate

normally, and their cotyledons reach the same size and shape as wild-type ones (data not shown).

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Figure 3. Expression analysis of Arabidopsis exocyst genes. Expression data for Arabidopsis exocyst genes were retrieved from Genevestigator database. Sources of RNA for the expression analysis: seedlings (SD), lateral roots (LR), elongation zone of roots (EZ), aerial parts (AP), leaves (LV), flower buds (FB), uninucleate microspores (UM), bicellular pollen (BP), tricellular pollen (TP), mature pollen (MP), suspension (SP). However, both exo70A1 mutants develop significantly shorter etiolated hypocotyls than wild type as examined in 7-day-old seedlings (exo70A1-1: 11.1 +/- 1.8 mm, n = 16; exo70A1-2: 11.4 +/- 1.9 mm, n = 18; wild type: 16.3 +/- 1.6 mm, n = 35; t-test p-values < 0.01). To reveal whether this is due to shorter cell lengths or decreased number of cells we investigated files of epidermal cells. The average epidermal cell length in exo70A1-1 is shorter than in wild type (exo70A1-1: 415.3 +/- 149.6 µm, n = 56; wild type: 507.0 +/- 227.8 µm, n = 144; t-test p-value = 0.001), and a class of longest cells (> 800 µm) present normally in wild type is missing. Moreover, the number of cells per cell file is reduced in exo70A1-1 (exo70A1-1: 18.3 +/- 3.0, n = 9; wild type: 24.0 +/- 2.1, n = 9; t-test p-value = 0.015). These results show that EXO70A1 is involved in both cell division and cell elongation in etiolated hypocotyls.

Furthermore, root growth is slower after four days in exo70A1 mutants, resulting in shorter roots (exo70A1-1: 7.6 mm; exo70A1-2: 7.3 mm; wild type: 14.3 mm; at the day 7). However, the lengths of root epidermal cells indicate that epidermal cell expansion is not significantly affected (exo70A1-1: 121.4 +/- 38.3 µm; wild type: 116.7 +/- 42.8 µm; t-test p-value = 0.73). Lateral root initiation is retarded as well (done by N. Schlager and M.-T. Hauser).

We also observed a conditional tip growth defect of root hairs in exo70A1 mutants immediately after germination. Whereas on medium with 4.5% sucrose exo70A1-1 and exo70A1-2 initiate only bulges, which do not elongate, resulting in 'naked' roots (Figure 4), on medium without sucrose root hairs do elongate, but are significantly shorter than wild-type ones (Figure 4). Cultivation of exo70A1-1 mutants on medium supplemented with sorbitol or mannitol in concentration with comparable osmotic potential indicate that the inhibition of root hair elongation depends on the presence of sucrose and is independent of the surrounding osmotic potential (Figure 4).

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Figure 4. Root hair phenotype of exo70A1 mutants. Ten-day-old exo70A1-1 seedlings grown on solid MS medium supplemented with 4.5% sucrose initiate bulges, but do not elongate root hairs. On solid MS medium without sucrose or with 125 mM sorbitol exo70A1-1 seedlings produce short root hairs. Detailed images were taken at a position where root hairs do not further elongate. Bars = 500 µm (first two images) or 100 µm.

When seedlings were transferred from vertical agar plates to liquid medium and grown between

two slides for 24 h, higher percentage of branched root hairs was observed for exo70A1-1 mutants (56%, n = 101) than for wild types and heterozygotes (22%, n = 123). Along with the previous observation both facts are indicative for polar growth defects in exo70A1 mutants.

The sucrose-dependent inhibition of root hair growth was partly rescued by 5 µM ACC, but root hairs were still unable to reach completely the length of wild-type ones (done by N. Schlager and M.-T. Hauser). Root hairs of exo70A1-2 cultivated on a medium containing 10 nM or 100 nM naphtyl acetic acid (NAA) elongated similarly as wild-type ones However, the initiation of lateral roots was not enhanced by NAA. Therefore, we concluded that exo70A1-2 is partly insensitive to auxin.

3.2.3 The exo70A1 plants are smaller, exhibit reduced apical dominance and show an

indeterminate growth Upon the formation of true leaves, the shoot phenotype of exo70A1 mutants is becoming evident.

Plants are semi-dwarfish with much smaller rosette leaves (Figure 5a, b). Comparison of the size and number of epidermal cells of rosette leaves shows that the cell size is not changed, indicating that the smaller leaves are a result of lower cell number (exo70A1-1: 220 epidermal cells/mm2, 113 stomata/mm2; wild type: 217 cells/mm2, 90 stomata/mm2).

Although mutants develop secondary inflorescence earlier, the bolting time is similar to wild type (data not shown). However, the most dramatic phenotypical change occurs in the inflorescence architecture. Instead of initiating floral meristems at the flanks of the apical meristem, exo70A1-1 and exo70A1-2 develop also laterally secondary inflorescence meristems, resulting in an indeterminate highly branched inflorescence with cauline leaves (Figure 5d). This repetitive pattern of secondary inflorescences correlates with a significantly delayed senescence. The life span of exo70A1-1 mutants is about five months. Whereas wild-type plants reach maximum height of approx. 30 cm, exo70A1 mutants reach 12 cm at most (Figure 5e).

3.2.4 The exo70A1 mutants display impaired flower development and are nearly sterile

Although a large number of flowers develops during the indeterminate growth, the exo70A1

mutants are nearly sterile. Only four out of more than 100 propagated mutants produced in total 63 homozygous mutant seeds. Mutant flowers are smaller (Figure 5e) and have a variable number of stamens which was reduced to four or five in 33% of exo70A1-2 flowers. As the stamens reach the stigma in mutant flowers, heterostyly cannot be the reason for the infertility.

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Stigmatic papillae are not fully developed resembling the early stages of wild-type stigma development before pollination (Figure 5f). Siliques usually start but after a short phase of elongation they stop their development at length of only 2.1 +/- 0.2 mm (n = 40) and typically contain no seeds.

All anthers examined contained fewer pollen grains compared to the wild type. Microspores are released from tetrads, but pollen grains resemble wild-type mature pollen grains only very rarely (Figure 5g). They contain only one or two nuclei and their average diameter is distinctively smaller (exo70A1-2: 16.2 +/- 1.4 µm, n = 62; wild type: 19.8 +/- 1.3 µm, n = 37), suggesting a pollen development defect. The pollen of exo70A1-2 was unable to germinate in vitro and to pollinate wild-type flowers. Neither decrease in germination efficiency with respect to the wild type (maximum germination efficiency 62% for EXO70A1/exo70A1-2, 59% for wild type) nor dimorphic pollen was observed in EXO70A1/exo70A1-2, suggesting that the pollen defect depends on the sporophyte.

We conclude that the almost complete sterility of homozygous exo70A1 mutants is due to a combination of underdeveloped stigmatic papillae and disturbed anther/pollen development due to sporophytic dysfunction.

Figure 5. General phenotypes of exo70A1 mutants. a) Three-week-old plants cultivated in vitro. Bar = 1 cm. b) Rosette leaves of 4-week-old plants. Bar = 5 mm. c) Six-week-old plants in soil. Bar = 1 cm. d) Inflorescences with cauline leaves of exo70A1-1 and exo70A1-2 exhibit a repetitive pattern. Wild-type Col-0 inflorescence is displayed for comparison. Bars = 1 mm. e) Four-month-old exo70A1-1 exhibiting indeterminate highly branched inflorescence and delayed senescence. Bar = 1 cm. f) Stigmas after opening of flowers as seen in autofluorescence using CLSM. Bars = 0.1 mm. g) Pollen grains isolated from wild-type Col-0 and exo70A1-2 flowers after anthesis. Mutant pollen grains are smaller and mostly uninuclear as revealed by DAPI staining. Bars = 10 µm.

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3.3 Phenotype of sec6 and sec8 mutants

The lack of any discernible phenotype in several exo70 mutants (except for exo70A1 mutants)

was not surprising because we suppose functional redundancy of EXO70 genes within the EXO70 family. Therefore, we were interested how putative exocyst subunits encoded by a single gene in Arabidopsis (SEC6, SEC8) are affected by their disruption.

Two insertional mutants in the SEC6 and one in SEC8 were obtained. All three mutant lines showed identical characteristics. First, heterozygotes exhibited no difference from wild-type phenotype. Second, their offspring segragated in the 1:1 ratio (wild types : heterozygotes) and no homozygous mutants were found.

To distinguish whether the mutant allele transmission defect is male-specific or female-specific we crossed manually heterozygotes to wild type Col-0 (crossings performed by E. Drdová). Using heterozygotes as pollen recipients, we successfully harvested hybrid seeds. However, using heterozygotes as pollen donors, no mutant allele was identified by PCR genotyping within the crosses, suggesting an absolute pollen-specific transmission defect. To confirm this hypothesis we germinated pollen of heterozygous and wild-type plants in vitro. The maximum germination efficiency reached 30% for SEC6/sec6-2, 32% for SEC8/sec8 and 66% for wild type, indicating that the half of mutant pollen grains probably did not germinate. We conclude that both SEC6 and SEC8 are essential genes in Arabidopsis involved in pollen germination and/or pollen tube growth.

Detailed analysis of six different sec8 mutants was performed by our collaborators R. Cole and J. Fowler (Oregon State University) and included into the joint publication (Cole et al., 2005). 3.4 Subcellular localization of EXO70A1, SEC6 an SE C8

Overexpression of EXO70A1-GFP under the control of the 35S promoter in tobacco protoplasts, onion epidermal cells and Arabidopsis root cells caused ectopic localization of the fusion protein. Therefore the localization experiments were not very conclusive and expression under the native promoter will be necessary.

Figure 6. EXO70A1, SEC6 and SEC8 immunolocalization in tobacco pollen tubes. Projection of confocal sections (a) and medial single section (b) labeled by the anti-EXO70A1 antibody. A confocal section (c) and an image acquired using epifluorescence microscope (d) labeled by the anti-SEC6 antibody. Projection of confocal sections (e) and an image acquired using epifluorescence microscope (f) labeled by the anti-SEC8 antibody. Bar = 10 µm.

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To localize EXO70A1 by another approach we raised a mouse polyclonal antibody against the EXO70A1 protein (prepared with the help of E. Drdová). Indirect immunofluorescence was consequently performed on tobacco pollen, a model system for tip growth, because Arabidopsis pollen is difficult to harvest and germinate in a large amount. It is likely that the polyclonal anti-EXO70A1 antibody recognizes tobacco EXO70(s) as well due to high sequence homology among plant EXO70s. The immunolabeling revealed predominant tip localization (Figure 6). Brighter tiny dots could be discerned in the tip signal. Small spots, larger than the dots in the tip, were also present along the plasma membrane within the whole tube length and in the cytoplasm of the basal portion of the tube.

SEC6 was also localized to the tip of pollen tubes, but the signal within the rest of the tube was more diffuse, and its membrane association was less obvious (Figure 6). For indirect immunofluorescence we used a polyclonal mouse anti-SEC6 antibody (prepared by M. Eliáš and V. Žárský). Similarly, SEC8 was visualized using a polyclonal rabbit anti-SEC8 antibody (prepared by R. Cole and J. Fowler). SEC8 showed predominant localization at the tip of pollen tubes. However, bright spots distributed along the plasma membrane were observed similar to the anti-EXO70A1 labeling (Figure 6).

4 Discussion

In yeast and animal cells, Exo70, Sec6, and Sec8 proteins represent three of eight subunits of the

exocyst, a tethering complex, which is involved in final steps of exocytosis. The exocyst specifies the vesicle docking site on the plasma membrane and facilitates the fusion of vesicles to the plasma membrane mediated by SNARE proteins. Since exocytosis is an essential process for cell morphogenesis, cell-cell communication and cell polarity establishment, the requirement for proper functioning of the exocyst complex in the cell is obvious. This fact can be documented by the finding that mutants or knockouts in exocyst subunits are typically inviable (Novick et al., 1980; Novick et al., 1981; Friedrich and Soriano, 1991; EauClaire and Guo, 2003).

Homologs to all eight exocyst subunits have been identified in silico in plant genomes, including Arabidopsis thaliana (Cvrčková et al., 2001; Eliáš et al., 2003; Jurgens and Geldner, 2002). Putative plant exocyst subunits are often encoded by multiple genes in contrast to yeast and animals (Eliáš et al., 2003; Synek et al., 2006). Surprisingly, Arabidopsis contain in total 23 EXO70 genes. Several lines of genetic and functional evidence now indicate that the exocyst complex is also present in plants (Otegui and Staehelin, 2004; Seguí-Simarro et al., 2004; Cole et al., 2005; Wen et al., 2005; Synek et al., 2006). 4.1 Some EXO70 genes act probably redundantly in Arabidopsis

We started to characterize the function of the EXO70 family in Arabidopsis using a reverse

genetic approach. As described above, T-DNA insertions in EXO70B2, D3, F1, G1, and H7 genes did not result in a discernible mutant phenotype, despite the fact that most of the genes are highly expressed. Our preferable explanation is that paralogous EXO70 genes with redundant or overlapping function to each of the mutated EXO70B2, D3, F1, G1, or H7 genes are present in the cell. Similar expression patterns of related isoforms were revealed in Arabidopsis (Figure 3).

Why does the disruption of the EXO70A1 gene cause changes in the plant development and architecture? The EXO70A1 isoform seems to be the main Arabidopsis EXO70 based on the expression data (Figure 3) and equally its position in the phylogenetic tree suggests that it is most related to other eukaryotic EXO70 proteins (Eliáš et al., 2003; Synek et al., 2006). Therefore, we anticipate that EXO70A1 is a good candidate for the genuine exocyst subunit in plants. Unpublished biochemical data from our laboratory support this notion (M. Hála and V. Žárský).

The contradictory fact that homozygous exo70A1 mutants are viable unlike sec6 and sec8 mutants could be again explained by the functional redundancy among EXO70 isoforms. Nevertheless, the fact that the exo70A1 sporophyte is still so heavily phenotypically disabled indicates

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that other EXO70 gene/genes supply the EXO70A1 function only partially. Expression of the functionally redundant gene/genes could be either insufficient or limited only to certain tissues.

The sequence homology to the yeast and animal Exo70 exocyst subunit may not necessarily indicate a homologous cellular function. In the light of the balance hypothesis, saying that duplication of genes encoding subunits of complexes may lead to deleterious changes in stoichiometry and is therefore under negative selection (Papp et al., 2003), it is possible that multiplied EXO70 paralogs gained different functions. However, we cannot discount the possibility that more EXO70s in plants or even all of them can really incorporate into the exocyst complex. We can imagine that multiple EXO70 present in each tissue, as revealed by expression data, might compete about the incorporation into the exocyst complex, adjusting thus the exocyst function. The ongoing research aims to elucidate whether multiplied EXO70 genes possess redundant, overlapping or different functions.

4.2 The reduced organ size of exo70A1 mutants is mostly the result of organ-specific reduction of cell division In all organs from the exo70A1-1 and exo70A1-2 investigated, cell morphology generally does

not appear to be affected (omitting the special cases of polar growing cells; see next chapter). Epidermis of leaves and flowers consists of cells that have the same size as in wild type. Organ

size is, however, strongly diminished. The number of stamens is often reduced in mutant flowers, pointing to the possibility of reduced cell number in floral primordia. The number of hypocotyl epidermal cells, normally predetermined and constant (Gendreau et al., 1997), is significantly decreased in mutant etiolated seedlings. Thus, we suggest that in exo70A1 mutants cell division is affected, resulting in a decreased total number of cells and consequently in small plants. However, the short hypocotyls of exo70A1 mutants result partially from reduced cell length (see next chapter).

In eukaryotes, cell division depends on targeting of exocytic vesicles, e.g. to the cell plate in plants or cleavage furrow in animals (Guertin et al., 2002), and a direct role of the exocyst in this process has been demonstrated in S. cerevisiae (Dobbelaere and Barral 2004; VerPlank and Li, 2005), Sch. pombe (Wang et al., 2002 and 2003) and mammals (Fielding et al., 2005; Gromley et al., 2005). Interestingly, there is now indication that the cytokinetic role of the exocyst may be conserved also in plants, because exocyst-like particles were observed by electron tomography to tether vesicles during formation of cell plate in Arabidopsis meristematic cells (Seguí-Simarro et al., 2004) and developing pollen (Otegui and Staehelin, 2004). Therefore, we hypothesize that cell division is dependent on EXO70A1 and that the loss of its function leads to reduced cell division. 4.3 EXO70A1 is involved in polarized cell growth

While cells exhibiting less-polarized growth are unaffected by the mutation in EXO70A1, cells

featuring highly polarized growth show reduced length in exo70A1 mutants. It means that the dysfunction of the exocyst complex is strongly manifested in the situation of intensive polarized exocytosis.

Epidermal cells of etiolated hypocotyls of exo70A1 mutants show not only lower cell number but also reduced elongation compared to wild type. Elongation of hypocotyl cells in the dark well represents a kind of polar growth (Gendreau et al., 1997), indicating the importance of EXO70A1 for intensive polarized exocytosis. However, the most pronounced type of polar growth is tip growth, an extremely spatially focused cell expansion that has been characterized in root hairs and pollen tubes in plants (recently reviewed in Cole and Fowler, 2006). The mechanism that controls tip growth encompasses, beside a calcium gradient and the polarized actin cytoskeleton, also tip-directed vesicle trafficking. Possible roles of EXO70A1 in this process are discussed bellow.

Root hairs are short in exo70A1 mutants One of the most conspicuous phenotype of exo70A1 mutants is the lack of root hairs on medium

supplemented with 4.5% sucrose. The inhibitory effects of sucrose on the transition to tip growth in root hairs in exo70A1 mutants is not an osmotic effect of sucrose, and it is rescued by ACC treatment.

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Sucrose is known to influence ethylene signaling (Gazzarrini and McCourt, 2001; Gibson, 2004), which is required for root hair elongation (Pitts et al., 1998). It is possible that ethylene signaling pathway is more sensitive to sucrose in exo70A1 mutants than in the wild type. Nevertheless, the involvement of sucrose in plant signaling pathways (including auxin signaling) is incredibly complex (Gazzarrini and McCourt, 2001; Gibson, 2004).

The phenotype of exo70A1 mutants does not match any of the previously described mutants. However, the most similar among known root hair mutants are rhd3 and tip1 that resemble the exo70A1 mutants also in the stature and architecture of the plant (Schiefelbein and Somerville, 1990; Schiefelbein et al., 1993; Wang et al., 1997; Ryan et al., 1998). RHD3 (root hair defective) encodes a putative GTP-binding protein that controls anterograde vesicle trafficking between the endoplasmic reticulum and the Golgi compartments, thereby contributing to cell wall biogenesis and anisotropic cell expansion (Hu et al., 2003; Zheng et al., 2004). TIP1 (tip growth defective) codes for a protein with palmitoyl transferase activity that affects protein association with membranes, signal transduction, and vesicle trafficking but its exact cellular role has not been defined (Hemsley et al., 2005). Nevertheless, in contrast to exo70A1 mutants, where cell number is reduced and dimensions of non-polar growing cells are unaffected, cells of the rhd3 and tip1 mutants are typically smaller.

Furthermore, mutants in AXRl (Cernac et al., 1997), AXR2 (Wilson et al., 1990), AXR3 (Leyser et al., 1996) represent a group mutants that have short root hairs. Despite the fact that these mutants are auxin resistant in contrast to exo70A1 mutants, it might suggest a possible functional link between EXO70A1 and auxin transport and/or signaling. Indeed, we demonstrated that exogenous auxin analog (NAA) could promote root hair growth in exo70A1 mutants.

The higher frequency of branched root hairs in exo70A1 mutants observed in liquid medium without sucrose indicates that not only the tip growth itself is affected, but also the polarity is poorly established and/or maintained.

Importantly, a phenotype very similar to that of exo70A1 mutants has been recently reported for the maize roothairless1 (rth1) mutant bearing a transposon insertion in a gene coding for a homolog of the Sec3 exocyst subunit (Wen et al., 2005). Based on the Arabidopsis exo70A1 and maize rth1 phenotypes, we suggest that both the Arabidopsis EXO70A1 and maize RTH1 (SEC3) proteins function in vesicle delivery during tip growth of root hairs as subunits of the plant exocyst complex.

Stigmatic papillae are underdeveloped in exo70A1 mutants To our knowledge, the exact mechanism of stigmatic papillae extension has not been established

yet. However, similarity between the failure of stigmatic papillae elongation and the defect in root hair tip growth in exo70A1 mutants points to a possibility that papillae elongation might also proceed via tip growth.

Very recently, in a screen for floral phenotypes a novel mutant, termed hawaiian skirt (hws), was identified. The hws mutant fails to shed its reproductive organs, exhibits partial fusion of sepals and has extremely long stigmatic papillae. Contrary, overexpression of the HWS gene caused remarkably shorter stigmatic papillae than wild type. The HWS codes for an F-box protein, but the principle of its action in plant cell growth and development has not been clarified (Gonzalez-Carranza et al., 2007).

Studies on auxin distribution during flower development in Arabidopsis have revealed that auxin produced in growing flowers could be a major regulatory signal in flower morphogenesis (Aloni et al., 2006). Stigmatic papillae show high auxin concentration with a maximum shortly before pollination (Aloni et al., 2006), demonstrating a possible role of auxin in papillae development, which resembles the situation in root hairs as mentioned above.

Why is pollen tube growth unaffected in exo70A1 mutants? In contrast to root hairs and stigmatic papillae, tip-growth of pollen tubes harboring the exo70A1

mutation is unaffected. Mutant alleles do not exhibit reduced transmission, as indicated by the segregation ratio 3:1 in offspring of heterozygotes. Percentage of homozygous mutant seeds in the apical and basal half of siliques of EXO70A1/exo70A1-2 plants did not differ significantly, indicating that in vivo growth rate of mutant pollen tubes is not reduced compared to wild-type pollen tubes.

Recently, we clearly demonstrated together with our collaborators that the putative SEC8 exocyst subunit is involved in pollen germination and tube growth in Arabidopsis (Cole et al., 2005), which is well consistent with the expected role of the exocyst complex in polarized cell growth. Why do we not

16

observe a similar phenotype for the exo70A1 mutants? Although the EXO70A1 gene is expressed at least in some stages of pollen development and at low level, there is a closely related paralog, EXO70A2, whose expression is restricted to pollen and seems to be much stronger compared to that of EXO70A1 (Figure 3). Also other EXO70 isoforms such as C1, C2, H3, and H5 are highly and specifically expressed in pollen. We therefore suggest that pollen tube tip-growth is unaffected because EXO70A1 is not dominant in pollen, where pollen specific EXO70 isoforms mentioned above can obviously fully supply the EXO70 function.

The immunolabeling using polyclonal antibody raised against Arabidopsis EXO70A1 revealed predominant tip localization in tobacco pollen, where the exocytic machinery is located. The high sequence homology could allow other tobacco EXO70(s) isoforms to be recognized as well. The tip localization suggests an involvement of plant EXO70s in polarized exocytosis during tip growth.

The almost complete sterility of homozygous exo70A1 mutants probably results from a combination of underdeveloped stigmatic papillae and aberrant pollen development due to sporophytic defects in the anther function. A couple of mutants have been described where the defective tapetum, which is important for nutrition of developing pollen, results in the arrest of pollen development. In this respect, exo70A1 mutants resemble e.g. the ms1 (male sterility) Arabidopsis mutant, where pollen development is arrested at the uninuclear stage in (Wilson et al., 2001).

4.4 EXO70A1 affects meristem function The most dramatic phenotypic alterations in exo70A1 mutants occur in the inflorescence

architecture and lifespan. Mutants produce highly branched inflorescence with cauline leaves due to the recurrent initiation of new ectopic lateral inflorescences at the positions of wild-type flowers. Reversions of flower primordia to inflorescence primordia are very frequent in lfy-6 or ag-1 mutants due to weakening of the repression of inflorescence development and reprogramming of the original flower primordia (Okamuro et al., 1996, Tooke et al., 2005). Upon overexpression of AGL24, flower primordia are also reverted to inflorescence primordia (Yu et al., 2004). However, it is unclear how EXO70A1 might relate to function of these genes. Nevertheless, an involvement of the exocyst complex in cell fate determination was recently reported in Drosophila (Jafar-Nejad et al., 2005).

The longevity of exo70A1 mutants is certainly linked to the indeterminate growth based on reiterative initiation of new inflorescences along with the lack of seed production. A similar phenotype has been documented in different mutants including ms1 and mutants with floral meristem reversion (Wilson et al., 2001; Tooke et al., 2005). It is improbable that the longevity results from decreased sensitivity to ethylene because exo70A1 mutants exhibit normal triple response and their root hair development is sensitive to ethylene.

Another indication for affected meristem formation and function comes from roots. Short roots and retarded lateral root development in exo70A1 mutants could result from insufficient auxin supply, because it is known that auxin represents a key regulator of lateral root development. Laskowski et al. (1995) proposed that auxin is initially required to establish a population of rapidly dividing pericycle cells before their derivatives subsequently form hormone-autonomous meristems. Several auxin-related mutants have been described in Arabidopsis that affect this process at various stages (Celenza et al., 1995). For example, mutants in PLS (POLARIS), a gene that regulates auxin transport and root growth, initiate less lateral roots similar to exo70A1 mutants (Chilley et al., 2006).

Taken together, disturbances in meristem function in exo70A1 mutants might point to a possibility that EXO70A1 is involved in transport and distribution of auxin. Generally, auxin is known as a regulator of cell division and meristem function. Proper development of plant tissues requires polar auxin transport that depends on vesicle trafficking and distribution of PINs, auxin efflux carriers (Benková et al., 2003; Geldner et al., 2003; Xu and Scheres, 2005). Providing EXO70A1 function in exocytosis, we can speculate that EXO70A1 is involved in PIN recycling. Consequently, decreased auxin delivery could cause the defects in exo70A1 mutants described above. Thus, EXO70A1 might provide a link between exocytosis and auxin distribution. However, further treatment by exogenous auxin, visualization of auxin distribution and inspection of meristem anatomy are necessary.

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4.5 SEC6 and SEC8 are involved in polarized cell gr owth For both SEC6 and SEC8, as well as for EXO70A1, the localization to the tip of tobacco pollen

tubes and small patches along the plasma membrane was observed in our work. This is in concordance with previous results obtained from SEC6 and SEC8 mutant analysis that demonstrate the role of these proteins in pollen tube tip growth (Cole et al., 2005).

A series of six T-DNA insertional mutants in SEC8 was described by Cole et al. (2005). Three T-DNA insertions in SEC8 caused an absolute, pollen-specific transmission defect (null alleles). Therefore, homozygous mutants are not produced. Despite the sporophyte and pollen development showed no disturbances and all pollen grains responded to signals that initiate germination, mutant pollen grains were unable to germinate pollen tubes. The other three insertions, positioned in the last eighth of the SEC8 gene at the very 3’-end (most likely an individual domain), resulted only in a partial transmission defect of mutant alleles (hypomorphic alleles). In this case, detailed analysis revealed that pollen tube growth rate of mutant pollen grains is slower.

The additional sec8 mutant described here represents the seventh mutant allele and resembles the first group of mutants with the absolute transmission defect. The T-DNA insertion is positioned just between the two groups of insertions, so it helped to specify that at least 22 of 27 exons of the SEC8 gene are necessary for basal SEC8 function.

The phenotype of sec6 mutants seems to be identical to that of the three sec8 mutants associated with the absolute, pollen-specific transmission defect. Thus, we hypothesize that both SEC6 and SEC8 are involved in initiation and maintenance of the polarized growth of pollen tubes. Unfortunately, we cannot evaluate the root hair growth and hypocotyl cell elongation because homozygous mutants are not produced in the lines possessing the null alleles. In addition, homozygous sec8 mutants segregating out of lines with hypomorphic alleles are unaffected as well as all heterozygotes.

4.6 Do plants have the exocyst complex? This study on putative exocyst subunits EXO70A1, SEC6 and SEC8 along with the previously

published analyses of SEC3 (Wen et al., 2005; Lavy et al., 2007) and SEC8 (Cole et al., 2005) in plants establishes an essential base for further research on the putative plant exocyst complex. These observations are consistent with the hypothesis that EXO70A1, SEC6 and SEC8 proteins form the plant exocyst and are therefore required for polarized exocytosis in the plant cell.

Despite the accumulating genetic and functional evidences that support the presence of the exocyst complex in plants, its existence remains to be proved biochemically. However, we demonstrated here the involvement of three exocyst subunit homologs in plant morphogenesis driven by polarized exocytosis. Thus, it is most likely that plant cells do have an exocyst complex, which shares the structure and functions similar to yeast and animal cells.

5 Conclusions

I. Expression analysis showed that many genes of the EXO70 family exhibit tissue specific

expression in Arabidopsis and that EXO70A1 is the most strongly and ubiquitously expressed EXO70 isoform in the sporophyte.

II. Insertional mutants in six EXO70 genes were obtained. Only mutants in the EXO70A1 gene exhibit a discernible mutant phenotype. Mutations in EXO70B2, D3, F1, G1, and H7 are probably complemented by paralogous EXO70 genes with a redundant function.

III. While heterozygous EXO70A1/exo70A1 mutants have wild-type phenotype and segregate in the 1:2:1 ratio, homozygous exo70A1 mutants exhibit a semi-dwarf phenotype and are nearly sterile. Organs are generally smaller and plants show loss of apical dominance. They also display an indeterminate growth where instead of floral meristems new lateral inflorescences are initiated

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in a reiterative manner, suggesting a defect in meristem function. Taken together, EXO70A1 is crucial for cell and organ morphogenesis.

IV. Cells featuring strongly polarized growth such as hypocotyl cells, tip-growing root hairs and stigmatic papillae show reduced length in exo70A1 mutants, indicating that EXO70A1 may be involved in exocytosis. Pollen tube growth is unaffected because EXO70A1 is not dominant in pollen where other, pollen specific EXO70 paralogs are active.

V. Dramatically reduced fertility of exo70A1 mutants is caused by a combination of underdeveloped stigmatic papillae and disturbed anther/pollen development. The pollen defect is not cell autonomous but is caused by sporophytic dysfunction.

VI. Mutant alleles of both SEC6 and SEC8 exhibit pollen-specific transmission defect, so homozygous mutants are not produced. SEC6 and SEC8 genes seem to be essential for pollen germination and pollen tube growth. In the sporophyte, one functional allele of each, SEC6 and SEC8, is sufficient.

VII. EXO70A1, SEC6 and SEC8 are localized in the tip and small spots along the plasma membrane in tobacco pollen tubes, suggesting their possible association with vesicles and involvement in polarized exocytosis that occurs in the tip.

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Proteiny EXO70A1, SEC6 a SEC8 v morfogenezi rostlin né buňky

Charakteristickým rysem rostlinné buňky je morfogeneze založená na regulaci orientace buněčného dělení a polarizované buněčné expanzi. V těchto procesech hraje rozhodující úlohu dynamika cytoskeletu a endomembránového systému, především sekretorické dráhy. Na regulaci těchto procesů se podílejí malé GTPázy nadrodiny Ras. Jedním z efektorů těchto regulačních proteinů je komplex exocyst (zvaný též se6/sec8 komplex), který byl dosud studován pouze u kvasinek a živočichů.

Exocyst se v buňce účastní závěrečných kroků exocytózy, t.j. poslední fáze sekretorické dráhy. Ze studia exocystu u buněk kvasinek a živočichů vyplývá, že exocyst řídí směrování sekretorických váčků do oblastí při cytoplazmatické membráně (tzv. targeting), kde dochází k intenzivní a prostorově regulované exocytóze, a dále zajišťuje ukotvení těchto váčků k membráně (tzv. tethering). Tímto primárním přichycením usnadňuje následnou fúzi váčků s cytoplazmatickou membránou katalyzovanou proteiny SNARE. Funkce exocystu je tedy zásadní pro polarizovanou sekreci.

Kvasinkový i živočišný komplex exocyst se skládá z osmi podjednotek: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 a Exo84. Výsledky bioinformatické analýzy genomových sekvencí modelových rostlin prokázaly přítomnost genů homologních ke všem osmi podjednotkám (Eliáš et al., 2003; Jurgens and Geldner, 2002). Tato analýza také odhalila přítomnost více paralogních genů v genomu kódujících tutéž podjednotku. Např. u Arabidopsis jsou jediným genem kódovány pouze podjednotky Sec6 a Sec8, pro ostatní byly identifikovány nejméně dva příbuzné geny. Gen EXO70 však dokonce proliferoval do celé genové rodiny 23 paralogů (Eliáš et al., 2003; Cannon et al., 2004).

Výsledky naší laboratoře ukazují, že i v rostlinné buňce homologní proteiny pravděpodobně vytvářejí komplex exocyst (Hála a Žárský, nepublikováno). Další genetické a funkční důkazy poskytlo několik nedávných prací (Otegui and Staehelin, 2004; Cole et al., 2005; Wen et al., 2005; Synek et al., 2006; Lavy et al., 2007). Z analogie lze očekávat, že rostlinný exocyst bude pravděpodobně jedním z rozhodujících faktorů řídících lokalizovanou sekreci během buněčného dělení i polarizované buněčné expanze. Vzhledem ke specifickým rysům rostlinné buňky však můžeme u rostlin předpokládat i řadu odlišných funkcí.

Analýza údajů z databáze Genevestigator (Zimmermann et al., 2004) o expresi genů v

Arabidopsis ukázala, že řada genů rodiny EXO70 se exprimuje specificky, pouze v určitých pletivech. Přičemž v každém pletivu se exprimuje vždy více isoforem EXO70. Analýza dále odhalila, že dominantním genem celé rodiny z hlediska síly exprese je EXO70A1, který se intenzivně exprimuje ve všech pletivech sporofytu.

Získali jsme inzerční mutanty v šesti genech rodiny EXO70, z nichž pouze mutanti v genu EXO70A1 vykazují změnu fenotypu. Mutace v genech EXO70B2, D3, F1, G1 a H7 se neprojevují pravděpodobně kvůli přítomnosti jiných genů EXO70 s redundantní funkcí.

Heterozygotní mutanti EXO70A1/exo70A1 vypadají normálně a jejich potomstvo štěpí v poměru 1:2:1, což znamená, že funkce samčího ani samičího gametofytu není mutací ovlivněna. Homozygotní mutanti exo70A1 ovšem vykazují zakrslý vzrůst, mají menší nadzemní orgány i kratší kořeny a ztrácejí apikální dominanci. Protože epidermální buňky orgánů se neliší tvarem ani velikostí, předpokládáme, že menší velikost orgánů je dána sníženým počtem buněčných dělení. Role exocystu v cytokinezi byla dokumentována u kvasinek a živočichů (Guertin et al., 2002; Wang et al., 2002; Dobbelaere and Barral 2004; Fielding et al., 2005; Gromley et al., 2005). Zvláštní stavba květenství, kde se místo květních meristémů zakládají nová květenství, vede k neukončenému růstu. Tento projev ukazuje na poruchu meristému. Je tedy zřejmé, že protein EXO70A1 se významně podílí na morfogenezi rostliny.

Buňky vykazující výrazně polarizovaný růst, konkrétně kořenové vlásky, bliznové papily a buňky etiolovaných hypokotylů, mají porušenou elongaci. Z toho usuzujeme, že EXO70A1 se, stejně jako v buňkách kvasinek a živočichů, podílí na polarizované exocytóze, která je pro takový typ růstu nezbytná. Polarizovaný růst pylových láček naopak není mutací nijak ovlivněn, nejspíše proto, že v pylu se gen EXO70A1 exprimuje jen slabě a dominantní jsou jiné isoformy EXO70.

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Mutanti exo70A1 jsou obvykle sterilní, což je pravděpodobně způsobeno nedostatečně vyvinutými bliznovými papilami a poruchou ve vývoji pylu, který nedozrává. Tento defekt ve vývoji pylu je však podmíněn mutantním sporofytem, nikoliv pylem samotným.

Ve srovnání s EXO70, předpokládanou podjednotkou rostlinného exocystu, která je u Arabidopsis kódována 23 (pravděpodobně funkčně podobnými) geny, nás zajímal fenotyp mutantů v podjednotkách SEC6 a SEC8, z nichž každá je kódována pouze jedním genem. Zjistili jsme, že mutantní linie v genech SEC6 a SEC8 neprodukují homozygotní mutanty, protože nedochází k přenosu mutantní alely pylem. Defekt je způsoben neschopností pylu vytvořit pylovou láčku (Cole et al., 2005). Geny SEC6 a SEC8 jsou tedy pro Arabidopsis nezbytné.

Pomocí protilátek namířených proti proteinům EXO70A1, SEC6 and SEC8 jsme příslušné proteiny, respektive jejich homology, lokalizovali v pylových láčkách tabáku. Specifický signál jsme zjistili ve špičkách pylových láček, kde dochází k intenzivní exocytóze, což je v souladu se známou rolí exocystu v tomto procesu. EXO70A1 a SEC8 byly také lokalizovány v drobných partikulích distribuovaných podél cytoplazmatické membrány.

Tato práce o přepokládaných podjednotkách komplexu exocyst, EXO70A1, SEC6 a SEC8, ukazuje, že tyto proteiny hrají významnou roli v polarizované sekreci rostlinné buňky a podílejí se na procesech řízených exocystem. Společně s nedávno publikovanými pracemi o SEC3 (Wen et al., 2005; Lavy et al., 2007) a SEC8 (Cole et al., 2005) u rostlin tato práce naznačuje, že komplex exocyst existuje také u rostlin, a představuje tak základ pro jeho další studium. Citace: Cannon, S.B., Mitra, A., Baumgarten, A., Young, N.D., May, G. (2004). BMC Plant Biol. 4: 10.

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