Clustering of centromeres precedes bivalent chromosome pairing of polyploid wheats

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two cytosolic heat shock proteins (members of HSP70 and HSP90 families) were identified as SGT1-interactors in a yeast-two hybrid screen [18], and were recently shown using VIGS to be essential for non-host resistance in tobacco [19]. Heat shock proteins are abundant in all single and multicellular organisms, providing diverse ‘chaperon’ functions that enable folding and unfolding of other proteins, delivery of proteins for degradation, and assembly of multi-subunit complexes [20]. Interestingly, HSP70 and HSP90 proteins are key players in innate and adaptive immunity of mammals (involved in presenting proteins to the major histocompatibility complex), and are being used to develop immunotherapies for controlling cancers and infections in humans [20]. Progress since the pre-molecular description of induced accessibility from unorthodox combinations of host and parasitic microorganisms and recent advances from gene discovery is beginning to erode conceptual barriers in plant pathology. Are innovative controls of blight, mildew and rust diseases on the horizon for crops? Acknowledgements We are grateful to Al Ellingboe and Paul Williams for discussions that helped this article find its roots, and to BBSRC and the Gatsby Charitable Trust for funding our research on species level disease resistance.

References 1 Holub, E.B. (2001) The arms race is ancient history in Arabidopsis, the wild flower. Nat. Rev. Genet. 2, 516 – 527 2 Ouchi, S. and Oku, H. (1981) Susceptibility as a process induced by pathogens. In Plant Disease Control (Staples, R.C. and Toenniessen, G.H., eds), pp. 33 – 44, Wiley Press 3 Yarwood, C.E. (1977) Pseudoperonospora cubensis in rust-infected bean. Phytopathology 67, 1021 – 1022 4 Cooper, A. et al. (2002) Albugo candida (white rust) suppresses resistance to downy mildew pathogens in Arabidopsis thaliana. Plant Protection Sci. 38 (Special Issue 2), 474– 476 5 Vogel, J.P. et al. (2002) PMR6, a pectate lyase-like gene required for powdery mildew susceptibility in Arabidopsis. Plant Cell 14, 2095 – 2106 6 Nishimura, M.T. et al. (2003) Loss of a callose synthase results in salicylic acid-dependent disease resistance. Science 301, 969 – 972 7 Jacobs, A.K. et al. (2003) An Arabidopsis callose synthase, GSL5, is

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required for wound and papillary callose formation. Plant Cell 15, 2503– 2513 Mellersh, D.G. and Heath, M.C. (2003) An investigation into the involvement of defense signaling pathways in components of the nonhost resistance of Arabidopsis thaliana to rust fungi also reveals a model system for studying rust fungal compatibility. Mol. PlantMicrobe Interact. 16, 398 – 404 Takemoto, D. et al. (2003) GFP-tagging of cell components reveals the dynamics of subcellular re-organization in response to infection of Arabidopsis by oomycete pathogens. Plant J. 33, 775 – 792 Huitema, E. et al. (2003) Active defence responses associated with nonhost resistance of Arabidopsis thaliana to the oomycete pathogen Phytophthora infestans. Mol. Plant Pathol. 4, 487 – 500 Parker, J.E. et al. (1996) Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 8, 2033 – 2046 Jarosch, B. et al. (1999) The ambivalence of the barley Mlo locus: mutations conferring resistance against powdery mildew (Blumeria graminis f.sp. hordei) enhance susceptibility to the rice blast fungus Magnaporthe grisea. Mol. Plant-Microbe Interact. 12, 508 – 514 Collins, N.C. et al. (2003) SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425, 973 – 977 Yun, B-W. et al. (2003) Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew. Plant J. 34, 768 – 777 van Wees, S.C. and Glazebrook, J. (2003) Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv. phaseolicola is due to degradation products of salicylic acid. Plant J. 33, 733 – 742 To¨r, M. et al. (2003) The role of proteolysis in R gene mediated defence in plants. Mol. Plant Pathol. 4, 287 – 296 Peart, J.R. et al. (2002) Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants. Proc. Natl. Acad. Sci. U. S. A. 99, 10865 – 10869 Kanzaki, H. (2003) Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana. Mol. Plant Pathol. 4, 383 – 391 Takahashi, A. et al. (2003) HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 100, 11777– 11782 Srivastava, P. (2002) Roles of heat-shock proteins in innate and adaptive immunity. Nat. Rev. Immunol. 2, 185– 194 Alberts, B. et al. (2002) Molecular Biology of the Cell, 4th edn, Garland Science Publishing

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Clustering of centromeres precedes bivalent chromosome pairing of polyploid wheats Toma´s Naranjo and Eduardo Corredor Departamento de Gene´tica, Facultad de Biologı´a, Universidad Complutense, 28040 Madrid, Spain

Sexual reproduction of allopolyploid plants, with genomes from two or more related diploid ancestors, implies the formation of homologous bivalent pairing at the first meiotic division. In polyploid wheats, multivalent associations are corrected before recombination occurs. Recent analysis of chromosome arrangement at the onset of meiosis in tetraploid and hexaploid Corresponding author: Toma´s Naranjo ([email protected]). www.sciencedirect.com

wheats by Enrique Martinez-Perez and colleagues reveals that centromeres form into seven groups before the initiation of synapsis. These complex structures might be involved in the mechanism for sorting the chromosomes. Polyploidy has played a major role in the evolution of the Plant Kingdom. Most flowering plants, including important crops such as wheat, oat, cotton, coffee, sugar cane,

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potato or alfalfa, are polyploids [1]. Three or more chromosome sets, either from a given species (autopolyploids) or from related diploid species that sexually hybridized (allopolyploids), coexist in polyploids. At meiosis, more than two homologous or genetically related (homoeologous) chromosomes can compete for synapsis and recombination. If multivalent associations are formed at metaphase I, irregular chromosome segregation results at anaphase I and hence genetically unbalanced gametes. To circumvent this problem, many allopolyploids show a diploid-like meiotic behaviour. Tetraploid durum wheat, Triticum turgidum, (2n ¼ 4x ¼ 28, genomes AABB) and hexaploid bread wheat, Triticum aestivum (2n ¼ 6x ¼ 42, AABBDD) are representative examples. Although genomes A, B and D diverge from a common ancestor and retain similar genes sequences, polyploid wheats form only homologous bivalents at metaphase I. Homoeologous pairing is suppressed by the action of the Ph1 locus [2,3]. Enrique Martı´nez-Pe´rez and colleagues [4] have studied the initial stages of meiosis in polyploid wheats and report complex associations of centromeres that might be involved in the diploidizing mechanism of meiotic behaviour. Hexaploid wheat nuclei at premeiotic interphase retain the geometry of chromosomes at the previous anaphase, with centromeres at the nuclear periphery opposite the telomeres that spread on a hemisphere [5 –7]. The number of centromere sites approximates to half the chromosome number, indicating that centromeres are associated in pairs. The same happens in tetraploid wheat and wild related polyploids but centromeres are unassociated in the diploid progenitors [8]. Premeiotic centromere association caused by polyploidy is preferentially not-homologous (being either homoeologous or non-homologous). At the onset of meiosis, chromosomes adopt the bouquet configuration. Telomeres move and congregate at one site of the nuclear membrane while centromeres remain at the opposite pole, probably anchored to some cytoskeleton component [9]. Concomitant with the telomeres movement, chromatin decondenses and chromosomes elongate (Figure 1). After cognition and alignment of homologues, synapsis starts close to the telomeres and extends along the chromosome. The bouquet structure disorganizes before completion of synapsis. When a given chromosome undergoes synapsis with a different partner at each end, (a)

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a multivalent configuration results. In the course of prophase I, these multivalent associations are transformed into bivalents in the presence of the Ph1 locus but, in its absence, many of them persist until metaphase I [10 –12]. Seven clusters of centromeres at the onset of meiosis Using three-dimensional confocal fluorescence image stacks from anther sections labelled with the centromere (CCS1) and telomere (TTTAGGG repeat) DNA probes, Martı´nez-Pe´rez et al. examined the disposition of centromeres in meiotic cells at the bouquet stage. For their study they used the wild-type hexaploid wheat, the deletion mutant ( ph1b) lacking the Ph1 locus of hexaploid wheat, the wild-type tetraploid wheat, and the wild-type and ph1b mutant hexaploid wheat £ rye hybrids. These two types of hybrids with four homoeologous sets of chromosomes (three from wheat and one from rye) were studied because chromosome pairing is suppressed in the wild type but not in the mutant [13]. The telomere probe enabled them to select several meiocytes (7 to 12) before the formation of tight telomere clustering in each type of plant. Doublelabelling reveals the position and number of centromere signals in the three-dimensional reconstruction of these meiocytes. The results give a relatively simple and straightforward picture. Regardless of the number of chromosome sets and the presence or absence of homologues there is a reduction in the number of centromere signals to approximately seven diffuse groups that remain at the nuclear periphery on the pole opposite the telomeres. This suggests that super-clustering of centromeres does not occur at random but affects the chromosomes belonging to each one of the seven homoeologous groups. Martı´nez-Pe´rez et al. propose that centromere association, into pairs at premeiotic interphase and into seven clusters at the beginning of meiosis, provides a mechanism for sorting the chromosomes that operates before that derived from telomere congregation at the bouquet structure. At late bouquet stage, the seven centromere clusters form bipartite elongated structures in tetraploid wheat or tripartite elongated structures in hexaploid wheat, which resolve into two or three centromere pairs, respectively. Whereas the seven clusters become bipartite structures that are resolved into two centromere pairs in ph1b wheat £ rye hybrids, (c)

(d)

Figure 1. Germ cells at premeiotic interphase (a) and early meiotic stages (b-d) from a hexaploid wheat line with two chromosomes of rye. All images were obtained from spread cells of anthers after in situ hybridization with the following probes: rye genomic DNA (stained in green), the telomere tandem repeat pAt74 (stained in red) and the rye-specific centromere repeat pAWRC1 (stained in red and indicated by arrows). (a) Nucleus at premeiotic interphase with the homologous rye chromosomes occupying separated territories and centromeres and telomeres arranged as in the previous anaphase. (b) Nucleus at the onset of meiosis with telomeres clustering at the pole opposite the centromeres. (c) The bouquet is completely organized and chromatin is more decondensed than at previous stages. (d) Synapsis has been completed and the bouquet is disorganized. Scale bar ¼ 10 mm. www.sciencedirect.com

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Centromeres associate in seven groups

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Tripartite centromere structures

Centromeres in pairs

Telomeres Premeiotic interphase

Early bouquet

Bivalent and quadrivalent associations

Three homologous bivalents TRENDS in Plant Science

Figure 2. Pairing model of hexaploid wheat based on centromere clustering. Homologous and non-homologous centromere pairs (green structures) concentrate at one pole of the nucleus opposite the telomeres (orange structures) at premeiotic interphase. Three metacentric chromosome pairs from the same homoeologous group are shown in blue, brown and red. Concomitant with the formation of the bouquet structure, chromosomes of the same homoeologous group associate at the centromere site. While centromere clusters are resolved in tripartite structure, synapsis starts between homologous and between homoeologous chromosomes. Homoeologous synapsis is corrected in the presence of the Ph1 locus and centromere complexes are resolved in homologous pairs.

quadripartite structures are formed in the wild-type wheat £ rye hybrid, which are resolved in unpaired centromeres. This phenomenon allowed Martı´nez-Pe´rez et al. to assign the Ph1 gene with a role in the correction of interactions between homoeologous chromosomes. The presence of Ph1 enables discrimination and separation of non-homologous (homoeologous) centromeres, which pair in its absence. Hypothesis The occurrence of multiple interactions between chromosomes that congregate in a multimeric structure, which is different from the telomere bouquet, represents a feature of meiotic prophase I that is specific to polyploids. The formation of these complex centromere structures before the onset of synapsis, and their resolution before completion of synapsis, provides an opportunity to resort the chromosomes (Figure 2). Martı´nez-Pe´rez et al. postulate that pairing correction, which is under the control of the Ph1 locus, might be connected with different rates of condensation of homologous and homoeologous chromosomes. Pairing between homologues within the chromosome cluster can be more stable than pairing between homoeologues. Thus, homologous synapsis initiated at one chromosome end and confirmed at the multicentromere structure can proceed and be completed. By contrast, synapsis initiated between homoeologues and interpreted as wrong at the centromeres is immediately corrected. This appealing hypothesis is consistent with the formation of multivalent synaptonemal complexes and their transformation into bivalents before recombination occurs in polyploid wheats. A crucial test requires the development of probes that would allow researchers to visualize and discriminate by means of in situ hybridization the centromere sites of three homoeologous chromosome pairs in hexaploid wheat, or two in tetraploid wheat, respectively. This is a difficult task because of the huge size of the hexaploid wheat genome: 17 000 Mb compared to the www.sciencedirect.com

110 Mb genome of Arabidopsis thaliana or 430 Mb genome of rice. Current efforts on preparation of genomic resources for structural and functional characterization of the wheat genome should facilitate the isolation of appropriate chromosome-specific DNA probes. Likewise, isolation of Ph1 and its product should clarify the mode of action of this gene, which has been controversial since its discovery [14]. Unravelling the molecular nature of interactions that underlie centromere clustering is another challenge. Centromeres can associate in pairs during S phase in barley [15]. Germ cells of wheat might retain centromere association that occurred during the premeiotic S stage. Association of centromeres from chromosomes of the same homoeologous group in a more complex structure at the bouquet stage implies the existence of additional mechanisms for sorting such chromosomes and grouping them. Recognition of homologues and homoeologues among separated centromere pairs should be substantially different from the search for a partner at the chromosome ends, provided that all telomeres concentrate in one site. Polyploidy causes rapid genetic and epigenetic changes that can alter the transcriptome and phenotype [16]. Whether the expression of this innovative centromere function is a result of polyploidization or whether diploids have the potential of centromeres to associate before synapsis also needs to be answered. References 1 Masterson, J. (1994) Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperms. Science 264, 421 – 424 2 Riley, R. and Chapman, V. (1958) Genetic control of the cytologically diploid behaviour of hexaploid wheat. Nature 182, 713– 715 3 Sears, E.R. (1976) Genetic control of chromosome pairing in wheat. Annu. Rev. Genet. 10, 31 – 51 4 Martı´nez-Pe´rez, E. et al. (2003) Chromosomes form into seven groups in hexaploid and tetraploid wheat as a prelude of meiosis. Plant J. 36, 21 – 29 5 Arago´n-Alcaide, L. et al. (1997) Association of homologous chromosomes during floral development. Curr. Biol. 7, 905 – 908 6 Schwarzacher, T. (1997) Three stages of meiotic homologous chromosome

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pairing in wheat, cognition alignment and synapsis. Sex. Plant Reprod. 10, 324 – 331 Martı´nez-Pe´rez, E. et al. (1999) Homologous chromosome pairing in wheat. J. Cell Sci. 112, 1761– 1789 Martı´nez-Pe´rez, E. et al. (2000) Polyploidy induces centromere association. J. Cell Biol. 148, 233 – 238 Maestra, B. et al. (2002) Chromosome arrangement and behaviour of two rye homologous telosomes at the onset of meiosis in disomic wheat5RL addition lines with and without the Ph1 locus. Chromosome Res. 10, 655 – 667 Holm, P.B. and Wang, X. (1986) The effect of chromosome 5B on synapsis and chiasma formation in wheat, Triticum aestivum cv. Chinese Spring. Carlsberg Res. Commun. 53, 191 – 208 Martı´nez, M. et al. (2001) The synaptic behaviour of Triticum turgidum with variable doses of the Ph1 locus. Theor. Appl. Genet. 102, 751 – 758

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12 Martı´nez, M. et al. (2001) The Ph1 and Ph2 loci play different roles in the synaptic behaviour of hexaploid wheat Triticum aestivum. Theor. Appl. Genet. 103, 398– 405 13 Naranjo, T. et al. (1988) Chromosome pairing in hybrids of ph1b mutant wheat with rye. Genome 30, 639 – 646 14 Mikhailova, E.I. et al. (1998) The effect of the wheat Ph1 locus on chromatin organization and meiotic chromosome pairing analysed by genome painting. Chromosoma 107, 339 – 350 15 Jasencakova, Z. et al. (2001) Chromatin organization and its relation to replication and histone acetylation during the cell cycle in barley. Chromosoma 110, 83 – 92 16 Osborn, T.C. et al. (2003) Understanding mechanisms of novel gene expression in polyploids. Trends Genet. 19, 141 – 147 1360-1385/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2004.03.001

New signalling molecules regulating root hair tip growth Jozef Sˇamaj1,2, Frantisˇek Balusˇka1 and Diedrik Menzel1 1 2

Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-949 01 Nitra, Slovak Republic

Root hairs are tip-growing tubes that emerge from trichoblasts (hair-forming epidermal cells) along the length of the root. Signalling events involved in the formation of root hairs are largely unknown. However, two recent studies have revealed that signalling enzymes such as NADPH oxidase and phospholipase D are crucial for root hair growth and development. Reactive oxygen species (ROS) produced by NADPH oxidase activate calcium ion channels in the apical plasma membrane leading to the tip-focused calcium gradient, an inherent feature of growing root hairs. Root hairs facilitate water and nutrient uptake from the soil into the plant and help to anchor the plant body in the soil. They are exploratory tubes that grow exclusively at their apical domes (tip growth). Initiation and maintenance of polarity in growing root hairs is under genetic, hormonal and environmental control. During the past decade, root hairs have emerged as an exciting model system for uncovering general principles underlying cell polarity and driving polar growth in plants and other higher eukaryotes. Because root hairs are not essential for plant growth under laboratory conditions, mutant lines with root hair phenotypes can survive – a major advantage for genetic studies [1]. In contrast to root hairs, pollen tubes, another important cell model displaying tip growth, are more difficult to study because mutations of essential gametophytic genes are usually lethal. Here we highlight the recent discoveries about root hair tip growth and focus on emerging signalling pathways that regulate this process.

Corresponding author: Jozef Sˇamaj ([email protected]). www.sciencedirect.com

Cellular basis of root hair tip growth A tip-focused cytoplasmic calcium ion gradient, the actin cytoskeleton and polarly targeted vesicular traffic are crucial components of the tip-growth machinery in root hairs and pollen tubes [1– 3]. Continuous actin polymerization is required for their growth, as revealed by experiments with the potent actin filament-disrupting drug latrunculin B [4,5]. The phenotype of aberrant root hairs in the crooked mutant is caused by a mutation in the smallest subunit of the arp2/3 complex, resulting in impaired branching of actin filaments [6], supporting the view that the actin cytoskeleton is crucial for tip growth. Additionally, overexpression of two actin-binding proteins regulating the dynamic turnover of actin filaments, profilin and actin-depolymerizing factor ADF1, caused longer or shorter root hair phenotypes, respectively [7]. Moreover, recent genetic studies unambiguously demonstrated that ACTIN2 is essential for root hair initiation and growth [8,9]. Small Rho GTPases of plants called ROPs are believed to generate tip-focused F-actin and calcium ion gradients [10]. Lessons from root hair mutants: NADPH oxidase and phospholipase D are essential for tip growth Studies on root hair mutants have significantly improved our knowledge of the molecular components involved in root hair development [1] (Figure 1). Recently, Julia Foreman and colleagues [11] showed that root hair defective 2 (rhd2), a mutant forming root hair bulges but no elongated root hairs (Figure 1), has a mutation in a NADPH OXIDASE/RHD2 gene. NADPH oxidase/RHD2 is a key enzyme involved in intracellular signalling that produces reactive oxygen species (ROS) as second messengers. The authors localized ROS by fluorescent indicator dyes in the growing tips of root hairs of wild-type plants