Plant Patogen Interactions

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Commentary Commentary ??? ? 0?? June 10.1111/j.1469-8137.2009.02931.x 2931 The Authors (2009). Journal compilation New Phytologist (2009) 1469-8137 0028-646X New Phytologist NPH2009 Oxford, UK Blackwell Publishing Ltd
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Plantpathogen interactions: a view from the evolutionary basement

The Vermin only teaze and pinch Their Foes superior by an Inch. So Natralists observe, a Flea Hath smaller Fleas that on him prey, And these have smaller Fleas to bite em, And so proceed ad infinitum Swift. On Poetry: a Rhapsody (1733)
Jonathan Swifts satiric verses warning aspiring poets of the adverse opinions of critics have much to recommend them as advice to biologists, struggling to persuade reviewers of the value of their work or committees of the impact of their research grants. But these lines, in particular, serve to remind us how all organisms (not just poets and biologists) engage in a struggle with pathogens, pests and parasites, and must needs deploy a variety of defensive strategies with which to repel them. In this issue of New Phytologist (pp. 432443), Mikko Lehtonen and his colleagues at the University of Helsinki describe how the war between plant and pathogen is waged by bryophytes, using the unique tools afforded by the model moss, Physcomitrella patens.
Using the highly efficient gene targeting technology available in Physcomitrella to create knockout mutant lines they have demonstrated that these peroxidase genes form an essential part of the moss defence mechanism.
It is estimated that 2040% of crop yield, worldwide, is lost to pathogen attack principally plant pathogenic fungi every year, and that this proportion would be even greater were it not for the intervention of crop-protection strategies. Clearly, plant pathogens pose a threat to agricultural monocultures. However, a glance out of the window reminds us that, left to their own devices, the plants which dominate our natural
environment ward off these depredations very effectively. Without a doubt, close observation of plants reveals incidences of pathogenesis, but it also reveals the frequent and characteristic fingerprints of effective defence against pathogens, usually in the form of the small and sporadic lesions that mark the activity of the hypersensitive response to infections. Flowering plants deploy highly effective countermeasures against microbial infection that rely on their ability to recognize a potential pathogen and neutralize it through the rapid release of a range of fungistatic substances, including reactive oxygen species (ROS), phytoalexins and specific antifungal enzymes such as chitinases. These are allied with programmed death of the infected cell and the modification of the surrounding cell walls to restrict the further passage of infection. The speed with which this response is mounted is the key to its effectiveness, and has been characterized, genetically, as a specific interaction between resistance (R) genes within the host plant and pathogenencoded avirulence (avr) loci. This gene-for-gene response is explained through a molecular recognition between an R-gene product essentially a plant receptor protein and a fungal avrspecified component to trigger an intracellular signal transduction pathway that triggers these events. Both R and avr loci are highly polymorphic, a consequence of the evolutionary arms race that develops as host and pathogen compete to survive (Dangl & Jones, 2001). Among the most prominent features of the plant response to infection is the oxidative burst: a rapid release of ROS in particular hydrogen peroxide (Bolwell et al., 2002). Hydrogen peroxide is multifunctional. It can serve directly as an antimicrobial agent, through its antiseptic properties, as well as participating in mechanical defence through the generation of papillae (Thordal-Christensen et al., 1997), the modification of cell walls (cross-linking and lignification through the incorporation of phenolic compounds) to restrict fungal spread, the synthesis of phytoalexins and as a potent intracellular and intercellular signalling compound to activate further responses (Torres et al., 2006; Almagro et al., 2009). By contrast, comparatively little is known about the pathogens of the lower plants, although pathogenic effects of fungal infection have been reported for a number of mosses (comprehensively reviewed by Davey & Currah, 2006). However, until recently, little was known of the mechanisms that the bryophytes deploy to resist infection. Because the dominant gametophyte stage of the moss life cycle lacks a protective cuticle, mosses should be particularly susceptible to infection. Add to this organs that are typically one cell thick, and the consequences of a successful infection are not difficult to envisage. However, it is clear that the mosses are a very successful taxon. They have flourished for the past 450 million years, and are the dominant
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form of vegetation in some ecosystems (e.g. polar regions). Comprising c. 10 000 species, the mosses are second only to the angiosperms in species diversity. It is not surprising therefore that this group of plants exhibits a robust response to infection. The wide range of molecular genetic tools, developed by the Physcomitrella research community, now provide researchers with the resources necessary to undertake a comprehensive molecular analysis of moss pathogen responses, and through comparative analysis, to place the existing information regarding the resistance of flowering plants within an evolutionary framework. Lehtonen et al. (2009) have isolated naturally occurring fungal bryo-pathogens of a moss species in the field (Racomitrium japonicum) and have shown that these are equally pathogenic to Physcomitrella. Having identified secretion of peroxidase as a rapid response to elicitation by the fungal cellwall-derived compound chitosan, they have identified the genes encoding this enzyme as a pair of duplicated Class III peroxidase genes clustering in a moss-specific phylogenetic clade. Using the highly efficient gene-targeting technology available in Physcomitrella to create knockout mutant lines, they have demonstrated that these peroxidase genes that either use or generate hydrogen peroxide form an essential part of the moss defence mechanism. The questions that arise from this are as follows. How does Physcomitrella and by analogy other mosses recognize the presence of the fungal elicitor? To what extent do the defence responses of mosses resemble those of higher plants? What can we learn from comparative studies about the evolution of the plant pathogen response? In recent years, considerable progress has been made in determining the molecular features of plantpathogen resistance mechanisms: an enterprise facilitated greatly through the use of the model angiosperm, Arabidopsis thaliana, and the extensive molecular genetic resources available for this species. Genomic analysis reveals that over 200 R genes are required for resistance, not only to fungal pathogens, but also to infection with bacteria and viruses, as well as to predation by invertebrate pests such as nematodes and insects. These genes display common features and are often clustered within the genome, suggesting that copy number expansion and diversification has been an important feature of their evolution (Sterck et al., 2007). In particular, the presence of leucine-rich repeat (LRR) sequences as a common feature of R-gene products indicates the basis for specific recognition of avr-dependent elicitors because these domains commonly participate in proteinprotein interactions and in specific ligand-binding (Kobe & Deisenhofer, 1995), and it is these regions of the R-gene products that display significantly high levels of polymorphism, indicative of the highly active selection operating on these loci (Dangl & Jones, 2001; Bakker et al., 2006). This combination of gene duplication and polymorphic variation provides plants with an adaptively flexible means of repelling pathogens.
It is now recognized that plant resistance to pathogens is multilayered: an initial response to contact with pathogenspecific elicitors (or pathogen-associated molecular patterns: PAMPs), backed up by a second phase of recognition of pathogen-derived effectors with which the pathogen seeks to suppress the response to an initial recognition event (Jones & Dangl, 2006; Boller & Felix, 2009). Notwithstanding this added complexity, the recognition of each of these pathogenic signals is based on Ravr interactions, and can lead to the activation of hypersensitive cell death and the release of antifungal microbial compounds. The R-gene-mediated pathogen response is clearly essential for the health of flowering plants, but how much is known about the evolution of this mechanism? Some features of the response are clearly ancient. R-gene products themselves share considerable similarity with proteins involved in the mammalian programmed cell death pathway (Dangl & Jones, 2001), suggesting that apoptosis is an ancient feature of multicellular organisms that has been recruited by evolution to provide a solution to similar problems in different organisms. Most R genes are distinctively modular, the most abundant class comprising sequences encoding nucleotide-binding (NB) domains, and a domain homologous with the Drosophila Toll and mammalian interleukin-2 receptors (TIR) that is thought to have an intracellular signalling role. To what extent are these activities widespread among the Plant Kingdom? The development of Physcomitrella for comparative functional genomic studies promises to shed light on this question. How then do mosses perceive and respond to pathogenic infection? Probably by using some of the same mechanisms that exist in flowering plants: previous studies have demonstrated that Physcomitrella will respond to infection with known angiosperm pathogens, such as Erwinia and Botrytis, in a manner analogous to angiosperms (Andersson et al., 2005; Ponce de Lon et al., 2007) and pathogen receptor type LRR-containing genes have been isolated from Physcomitrella (Akita & Valkonen, 2002) and also identified in other basal lineages, including liverworts and charophycean algae (Sasaki et al., 2007). With the publication of reference genomes for the mosses (Physcomitrella) and lycopods (Selaginella), we can now examine the extent of the gene families encoding LRR-domain genes. A rapid (and by no means exhaustive) search of these two genomes reveals over 150 (Physcomitrella) and 110 (Selaginella) LRR-containing genes, respectively. However, compared with Arabidopsis, only a handful have the characteristic motifs of the abundant flowering-plant TIRNBLRR family: in Physcomitrella, I found only 6 gene models containing TIR motifs, whilst 28 have NB domains; in Selaginella the corresponding numbers are 2 and 16, respectively. Therefore, it seems that whilst the expansion of LRR repeat genes is widespread among land plants, the evolutionary acquisition of the signalling domains may be a feature of the angiosperm radiation. This may underpin taxon-specific differences that have been recorded in the recognition of specific PAMPs (Qutob et al., 2006). It will
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be both practical and fruitful to apply the powerful reverse genetics tools afforded by Physcomitrella to investigate the functions of those receptors that do exhibit a high degree of similarity with their angiosperm counterparts. Nevertheless, responses elicited by microbial infection in Physcomitrella are similar in many ways to those in flowering plants, with the release of active oxygen species, mobilization of salicylic acid signalling (Andersson et al., 2005) and cell death (Ponce de Lon et al., 2007). It is tempting to speculate that necessary for the successful transition of green plants from an aquatic environment to colonize terrestrial habitats c. 470 million years ago was the evolution of an effective set of defences against airborne pathogens, and that the expansion and diversification of pathogen receptor recognition domains (the LRR domains) date from this time. This is clearly a question that can be addressed through comparative genomics, although at present no genome sequences exist for a taxon representative of the last common ancestor of the land plants. It is generally believed that the land plants are derived from the Charophycean algae (Karol et al., 2001), and whilst it is to be hoped that the genome sequence of either Chara or Coleochaete will be determined in the not-too-distant future, at present these taxa are currently only the subject of expressed sequence tag (EST) programmes, represented by fewer than 500 ESTs available in the NCBI Trace Archive. New sequencing technologies will undoubtedly play a part in rectifying this missing link in the genome databases, and it will clearly be of interest to determine the representation of the LRR gene set in either of these organisms. Until such times, we can only inspect the genome of the unicellular chlorophyte, Chlamydomonas reinhardtii, and that of its colonial relative Volvox carteri. Each of these organisms contains only a very restricted set of LRR genes (< 20), but these algae diverged from the green plant lineage approx. 1 billion years ago, and additionally are single-celled organisms. It is not unreasonable to suggest that a defence mechanism which operates primarily through the programmed death of host cells might not be the most evolutionary effective strategy for such organisms. Whatever the future holds, it is clear that there is much to be learnt about the acquisition of pathogen defence through the study of basal taxa. Andrew C. Cuming Centre for Plant Sciences, Leeds University, Leeds LS2 9JT, UK (tel +44 113 3433096; email a.c.cuming@leeds.ac.uk)
References
Akita M, Valkonen JPT. 2002. A novel gene family in moss (Physcomitrella patens) shows sequence homology and a phylogenetic relationship with the TIR-NBS class of plant disease resistance genes. Journal of Molecular Evolution 55: 595605. Almagro L, Gomez Ros LV, Belchi-Navarro SB, Bru R, Ros Barcelo A, Pedreno MA. 2009. Class III peroxidases in plant defence reactions. Journal of Experimental Botany 60: 377390. Andersson RA, Akita M, Pirhonen M, Gammelgard E, Valkonen JPT. 2005. Moss-Erwinia pathosystem reveals possible similarities in pathogenesis and pathogen defense in vascular and nonvascular plants. Journal of General Plant Pathology 71: 2328. Bakker EG, Toomajian C, Kreitman M, Bergelson J. 2006. A genome-wide survey of R gene polymorphisms. Plant Cell 18: 18031818. Boller T, Felix G. 2009. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology 60: 379406. Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F. 2002. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. Journal of Experimental Botany 53: 13671376. Dangl JL, Jones JDG. 2001. Plant pathogens and integrated defence responses to infection. Nature 411: 826833. Davey ML, Currah RS. 2006. Interactions between mosses (Bryophyta) and fungi. Canadian Journal of Botany 84: 15091519. Jones JDG, Dangl JL. 2006. The plant immune system. Nature 444: 323329. Karol KG, McCourt RM, Cimino MT, Delwiche CF. 2001. The closest living relatives of land plants. Science 295: 23512353. Kobe B, Deisenhofer J. 1995. Proteins with leucine rich repeats. Current Opinions in Structural Biology 5: 409416. Lehtonen MT, Akita M, Kalkkinen N, Ahola-Iivarinen E, Rholm G, Somervuo P, Thelander M, Valkonen JPT. 2009. Quickly released peroxidase of moss in defense against fungal invaders. New Phytologist 183: 432443. Ponce de Lon I, Oliver JP, Castro A, Gaggero C, Bentacor, Vidal S. 2007. Erwinia carotovora elicitors and Botrytis cinerea activate defense responses in Physcomitrella patens. BMC Plant Biology 7: 52. Qutob D, Kemmerling B, Brunner F, Kfner I, Engelhardt S, Gust AA, Luberacki B, Seitz HU, Stahl D, Rauhut T et al. 2006. Phytotoxicity and innate immune responses induced by Nep1-like proteins. Plant Cell 18: 37213744. Sasaki G, Katoh K, Hirose N, Suga H, Kuma K, Miyata T, Su Z-H. 2007. Multiple receptor-like kinase cDNAs from liverwort Marchantia polymorpha and two charophycean green algae, Closterium ehrenbergii and Nitella axillaris: extensive gene duplications and gene shufflings in the early evolution of streptophytes. Gene 401: 135144. Sterck L, Rombauts S, Vandepoele K, Rouz P, Vande Peer Y. 2007. How many genes are there in plants ( ... and why are they there)? Current Opinion in Plant Biology 10: 199203. Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB. 1997. Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant Journal 11: 11871194. Torres MA, Jones JDG, Dangl JL. 2006. Reactive oxtgen species signaling in response to pathogens. Plant Physiology 141: 373378. Key words: disease resistance, hypersensitive response, peroxidase, Physcomitrella patens, plant pathogen resistance, R genes.
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The hidden language of flowering plants: floral odours as a key for understanding angiosperm evolution?
We live in a world filled with flowering plants and insects, with both groups showing enormous species diversity. However, explaining this diversity has been a major challenge. One reason suggested for such species diversity is that angiosperms have established mutualistic relationships with different pollinator types, leading to co-evolution of both groups (Stebbins, 1970; Thien et al., 2000). Chemical communication via floral fragrances has been shown to play a central role in attracting pollinators (Knudsen et al., 2006), and it is reasonable to hypothesize that floral volatiles played a key role in early angiosperm evolution (Gottsberger, 1988; Thien et al., 2000). This is supported by observations that strong floral fragrances are a characteristic feature of many extant taxa from basal angiosperm lineages and that they play an important role for pollinator attraction in most species in these groups (Ervik & Knudsen, 2003; Silberbauer-Gottsberger et al., 2003). Unfortunately, compared with other plant groups (e.g. orchids) our knowledge on the odour chemistry of basal angiosperm lineages is still limited (Knudsen et al., 2006). Nevertheless, recent publications on the floral odours of basal groups have revealed interesting insights, for example, into the diversity of floral fragrances (Bernhardt et al., 2003; Goodrich et al., 2006; Teichert, 2008). The same is true for the paper on floral fragrances of Asimina (pawpaw) and Deeringothamnus species (Annonaceae) by Katherine Goodrich and Robert Raguso in this issue of New Phytologist (pp. 457469). First, the data on 10 species of the North American groups of Annonaceae fill an important gap in our knowledge, as Asimina and Deeringothamnus are the only temperate genera of this otherwise tropical family (Doyle & Le Thomas, 1997). Second, the fragrances they found, typical fermentation odours, are extremely unusual among angiosperm flowers. Four of the Asimina species, with wine red-coloured flowers (maroonphenotype), emit odours that resemble the scent of decaying organic matter. The scent blends comprise chemicals that are similar to the emissions of bakers yeast (e.g. acetic acid, ethyl acetate, ethanol, 3-methyl-1-butanol; see Goodrich et al., 2006) but they also contain compounds such as amino acid-derived aldoximes and nitriles. The scent composition suggests that these flowers might attract saprophilous flies and beetles, but more detailed investigations of the pollination biology of these species are needed. The four Asimina species and the two Deeringothamnus species with white flowers emit floral volatiles
that indicate a generalist pollination system. The floral scent of D. pulchellus was dominated by sweet-smelling benzenoid compounds normally found in flowers pollinated by moths. But there are two more striking findings in this paper: the high diversity of chemicals emitted from the flowers (Goodrich & Raguso detected 272 compounds in 11 species of only two genera), and the complex spatial and temporal odour patterns for female and male ontogenetic stages. Their study is also a fascinating example illustrating how complex and dynamic floral odours in basal angiosperms can be.
We are starting to understand that plants are quite flexible in

their odour chemistry and that this flexibility is linked with their evolutionary success.
Generalist vs specialist modes of pollination in basal angiosperm groups and the role of beetles
The hypothesis that beetles pollinated the early angiosperms dates back to Faegri & van der Pijl (1979, p. 51) and their mess and soil pollination mode concept in basal angiosperm taxa unspecialized (primitive) flowers utilize unspecialized (primitive) insects (beetles) as pollinators. Flowers with a mess and soil pollination mode often bear many anthers and deposit large amounts of pollen over the body surface of flower visitors while not offering nectar or lipids as a reward (Faegri & van der Pijl, 1979). It is true that beetles are a dominant pollinator group in many magnoliids, including Annonaceae, with up to nine beetle families involved in pollination (Bernhardt, 2000). However, beetles are interestingly not the dominant group of flower visitors in the first three branches of the angiosperm phylogenetic tree (ANITA grade, with 201 species) and it now seems that they represent a derived syndrome in the ANITA-grade plants (Thien et al., 2009). Furthermore, the magnoliids are dominated by beetle pollination and are regarded as specialized systems where flowers are specifically adapted to beetle pollinators (SilberbauerGottsberger et al., 2003). According to Thien et al. (2009), the affiliations of ANITA-grade species with their flower visitors suggest that Diptera are the strongest candidates as the first pollinators of early angiosperms. With more than 2500 species, Annonaceae are one of the most diverse families of the basal groups of flowering plants and the family has played an important role in discussions of the origin and evolution of angiosperms (Doyle & Le Thomas, 1997;
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Gottsberger, 1999). Annonaceae flowers usually contain no nectar but the flowers provide pollinators with nutritious floral tissues, pollen, and in some species, shelter inside a pollination chamber. In many, particularly night-active, Annonaceae species, thermogenesis enhances floral odour emission (Gottsberger, 1999). It has been estimated that c. 90% of the Annonaceae species studied so far seem to be adapted to beetle pollination (Teichert, 2008) with a differentiation into species pollinated by small beetles and large beetles (Silberbauer-Gottsberger et al., 2003). While many of the species in the ancestral genera of the family (e.g. Anaxagorea) are pollinated by small beetles, pollination by large beetles is a specialized condition within the family and was probably acquired late in its evolution (Gottsberger, 1999; Silberbauer-Gottsberger et al., 2003). However, other pollinator groups interacting with Annonaceae, such as thrips, flies, cockroaches and perfume-collecting euglossine bees (Gottsberger, 1999; Silberbauer-Gottsberger et al., 2003; Teichert, 2008 and references therein), also provide evidence for other specialized pollination systems within the Annonaceae.
Differentiation of Annonaceae pollination systems in odour space

Before the present study carried out by Goodrich & Raguso, floral fragrances of only a few, mostly Neotropical, Annonaceae had been analyzed (Jrgens et al., 2000; Teichert, 2008; Teichert et al., 2008). Based on the floral scent data of six Annonaceae species with small beetle pollination syndrome, which emit a wide range of different compounds in different compound classes, Jrgens et al. (2000) wondered whether the flower scent reflects the opportunistic behavior of the flower visitors. It is possible that Nitidulidae beetles, which normally live on, and eat, rotten bark and fruits, are attracted by a wide range of compounds that all might be indicative of fruits and/or rotten bark (Jrgens et al., 2000). Although there are only limited data available on the floral volatiles of Annonaceae, visualization of the combined data sets in odour space gives some evidence that different pollination types are represented by clearly separated odour clusters (Fig. 1). The analysis was based on nine different genera of the Annonaceae (21 species), including the data of Goodrich & Raguso (this issue of New Phytologist), Jrgens et al. (2000), Teichert et al. (2008), Teichert (2008) and unpublished data (A. Jrgens & G. Gottsberger). The analysis revealed that floral fragrances reflect both phylogenetic constraints/relationships and the pollination biology of the species (Fig. 1), also showing that unrelated species, associated with similar pollinator types, occupy the same odour space. For species with sweet fruity odour blends, a greenyellow colouration, and relatively small pollination chambers that are mainly pollinated by small beetles (Anaxagorea, Duguetia, Guatteria, Rollinia and Xylopia; Silberbauer-Gottsberger et al., 2003), the odour space is relatively large. These species probably represent different chemical strategies by using hydrocarbon
esters (Anaxagorea, Guatteria), naphthalene (Rollinia), benzenoid compounds (Xylopia), or monoterpenoids (Duguetia, Rollinia) to attract small beetles. Furthermore, the differences in odour chemistry might also reflect the taxonomic heterogeneity of the pollinating beetles. Although mainly pollinated by Nitidulidae, other beetles could play a role as additional pollinators (e.g. Staphylinidae, Chrysomelidae) and, in Xylopia aromatica, thrips also seem to play an important role. However, these are all subsumed under the small beetle pollination syndrome. Close to the small beetle pollination syndrome we find Annona glabra, a species pollinated by relatively small Chrysomelidae or Curculionidae beetles (Gottsberger, 1999) where Goodrich & Raguso found 3-pentanyl acetate and several monoterpenoids as major scent compounds. The spicy floral odour of Unonopsis stipitata shows similarities in the odour composition with that of A. glabra and emits many of the same monoterpenoids. However, U. stipitata is pollinated by perfume-collecting male euglossine bees and its scent is dominated by several monoterpenoids, particularly trans-carvone oxide, which was only present in this species (Teichert, 2008). Interestingly, trans-carvone oxide has been found several times in euglossine-pollinated plants of other unrelated plant families (e.g. orchids) and it has been suggested that this compound might be the key attractant for euglossine bees (Whitten et al., 1986). The Asimina and Deeringothamnus species analyzed by Goodrich & Raguso form a cluster separate from the other genera. Within this cluster we find a chemical differentiation of Asimina flowers with the maroon-phenotype, emitting fermented/decaying scents (suggesting a mimicry-based pollination strategy), from another cluster with flowers of the white-phenotype, emitting pleasant scents (suggesting honest signaling and a reward-based pollination strategy) (Fig. 1). It is known that fermenting odours elicit strong physiological and behavioral responses in fruit flies and some beetles (Stensmyr et al., 2003) and it seems likely that scent is mainly responsible for pollinator attraction in Asimina species with yeasty scent.
Floral fragrances as a key innovation promoting pollinator shifts and diversification in flowering plants
Stebbins (1970) wrote that more specialized vectors are attracted to flowers by a variety of stimuli, of which scent may be even more important than either shape or colour. Advances in our understanding of floral volatiles and their role in pollination have been enormous in the last 20 yr with the emergence of technological breakthroughs for floral volatile analysis. In 1993, Knudsen et al. compiled floral scent data on 441 species, and listed 700 compounds; in a recent update, Knudsen et al. (2006) listed 991 species and 1791 compounds. A rough calculation shows that the average number of new (identified) compounds per species has increased from 1.6 (in
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Fig. 1 Visualization of floral fragrance similarities and (hypothesized) pollinators of 21 Annonaceae species. Nonmetric multidimensional scaling was based on BrayCurtis similarities of 150 identified scent components. Asimina and Deeringothamnus species (triangles) and Annona glabra are from Goodrich & Raguso (2009) (open circle) (this issue of New Phytologist). The maroon-phenotype of Asimina flowers (closed triangles) and the white-phenotype (open triangles) are indicated. Unonopsis stipitata and Anaxagorea prinoides are from Teichert et al. (2008) and Teichert (2008); Anaxagorea brevipes, A. dolichocarpa, Xylopia aromatica, X. benthamii, Rollinia insignis and R. mucosa are from Jrgens et al. (2000); Guatteria foliosa and Duguetia asterotricha are from A. Jrgens & G. Gottsberger (unpublished data). The two-dimensional stress value is 0.15; ANOSIM Global R = 0.776; P < 0.01.
1993) to 1.8 (in 2006). Part of the increase can be attributed to the development of more sensitive technologies for sampling plant volatiles and better ways of compound identification (e.g. via database systems). Nevertheless, it seems that a plateau regarding the chemical diversity of angiosperm flowers has not yet been reached. Odour diversity of the Annonaceae species investigated so far seems to be relatively high compared with that of all angiosperms listed in Knudsen et al. (2006). The Annonaceae analyzed so far, representing 2.2% (22 species of 991) of the angiosperms, comprise 8.4% (150 compounds of 1791, not including the many unidentified compounds listed by Goodrich & Raguso) of the total odour diversity found in flowering plants. A key question for the coming decade will be whether angiosperm diversification is correlated with a potential for rapid changes in the floral scent composition. We are starting to understand that plants are quite flexible in their odour chemistry and that this flexibility is linked with their evolutionary success. The scent composition of flowers can be very complex, sometimes containing > 50 compounds per sample. In some cases, single key odour signals (like trans-carvone
oxide in Unonopsis stipitata) might be responsible for attracting specific pollinators (Teichert et al., 2008); however, the common situation in most flowering plants is that several compounds together attract a diverse array of different flower visitors. Not all are necessarily effective pollinators and other floral features often function as filters. In the Annonaceae, other factors, such as the size of the pollination chamber, floral colour and the reward system, need to be considered to understand the evolution of this group. In situations with multiple flower visitors and multiple volatiles emitted from a flower, compound X might be an attractant for pollinator A only, whereas compound Z might be an attractant for pollinator A plus B (or B only), etc. Thus, floral scent can be seen as a multichannel signal, and small evolutionary changes in the scent composition might open (or close) signal channels for flower visitors that might affect the plants fitness. Furthermore, insects are often opportunistic in their behavior, and the insect, as a signal receiver, may respond to a range of different chemicals. In such a scenario different chemical strategies may evolve, attracting the same type of pollinator but exploiting different aspects of its olfactory spectrum.
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Acknowledgements
I would like to thank Karl Duffy, Steve Johnson, Adam Shuttleworth and Taina Witt for valuable comments and discussion on the manuscript. Andreas Jrgens School of Biological and Conservation Sciences, University of KwaZulu-Natal, P. Bag X01 Scottsville, Pietermaritzburg 3209, South Africa (tel +27 (0)33 2605657; email juergensa@ukzn.ac.za)
References
Bernhardt P, Sage T, Weston P, Azuma H, Lam M, Thien LB, Bruhl J. 2003. The pollination of Trimenia moorei (Trimeniaceae): floral volatiles, insect/wind pollen vectors and stigmatic self-incompatibility in a basal angiosperm. Annals of Botany 92: 445458. Bernhardt P. 2000. Convergent evolution and adaptive radiation of beetlepollinated angiosperms. Plant Systematics and Evolution 222: 293320. Doyle JA, Le Thomas A. 1997. Phylogeny and geographic history of Annonaceae 1997. Gographie physique et Quaternaire 51: 353361. Ervik F, Knudsen JT. 2003. Water lilies and scarabs: faithful partners for 100 million years? Biological Journal of the Linnean Society 80: 539543. Faegri K, van der Pijl L. 1979. The principles of pollination ecology. Oxford, UK: Pergamon Press. Goodrich KR, Raguso RA. 2009. The olfactory component of floral display in Asimina and Deeringothamnus (Annonaceae). New Phytologist 183: 457469. Goodrich KR, Zjhra ML, Ley CA, Raguso RA. 2006. When flowers smell fermented: the chemistry and ontogeny of yeasty floral scent in pawpaw (Asimina triloba: Annonaceae). International Journal of Plant Sciences 167: 3346. Gottsberger G. 1988. The reproductive biology of primitive Angiosperms. Taxon 37: 630643.
Gottsberger G. 1999. Pollination and evolution in neotropical Annonaceae. Plant Species Biology 14: 143152. Jrgens A, Webber AC, Gottsberger G. 2000. Floral scent compounds of Amazonian Annonaceae species pollinated by small beetles and thrips. Phytochemistry 55: 551558. Knudsen JT, Eriksson R, Gershenzon J, Sthl B. 2006. Diversity and distribution of floral scent. The Botanical Review 72: 1120. Knudsen JT, Tollsten L, Bergstrm G. 1993. Floral scents a checklist of volatile compounds isolated by head-space techniques. Phytochemistry 33: 253280. Silberbauer-Gottsberger I, Gottsberger G, Webber AC. 2003. Morphological and functional flower characteristics of New and Old World Annonaceae with respect to their mode of pollination. Taxon 52: 701718. Stebbins GL. 1970. Adaptive radiation of reproductive characteristics in angiosperms. I: pollination mechanisms. Annual Review of Ecology and Systematics 1: 307326. Stensmyr MC, Giordano E, Balloi A, Angioy A-M, Hansson BS. 2003. Novel natural ligands for Drosophila olfactory receptor neurons. Journal of Experimental Biology 206: 715724. Teichert H. 2008. Pollination biology of cantharophilous and melittophilous Annonaceae and Cyclanthaceae in French Guiana. PhD thesis, University of Ulm, Ulm, Germany. Teichert H, Dtterl S, Zimma B, Ayasse M, Gottsberger G. 2008. Perfume-collecting male euglossine bees as pollinators of a basal angiosperm: the case of Unonopsis stipitata (Annonaceae). Plant Biology 11: 2937. Thien LB, Azuma H, Kawano S. 2000. New perspectives on the pollination biology of basal angiosperms. International Journal of Plant Sciences 161: 225235. Thien LB, Bernhardt P, Devall MS, Chen Z, Luo Y, Fan J-H, Yuan L-C, Williams JH. 2009. Pollination biology of basal angiosperms (ANITA Grade). American Journal of Botany 96: 166182. Whitten WM, Williams NH, Armbruster WS, Battiste MA, Strekowski L, Lindquist N. 1986. Carvone oxide: an example of convergent evolution in euglossine-pollinated plants. Systematic Botany 11: 222228. Key words: angiosperm, diversification, evolution, floral fragrances, pollination.
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Letters
Ussings conundrum and the search for transport mechanisms in plants
Plant transport physiologists have developed a range of models describing the movement of ions across cell membranes. However, while substantial progress has been made towards providing precise descriptions of the mechanisms underlying these fluxes, important instances remain in which the prevailing models cannot account for repeated observations, particularly in terms of energy transformations. As we shall show, disagreements with experimental findings may entail a revision of the proposed models, similarly to what has been required in animal transport studies (Ussing, 1994; see below). We present, as a key example, the futile cycling of sodium under toxic conditions (see Britto & Kronzucker, 2006, and discussed later), and show that unidirectional flux magnitudes measured by several groups, including our own, cannot be explained energetically by current models. We attempt to explain these observations by proposing alternative mechanisms of Na+ transport across the root-cell plasma membrane. The influx/efflux cycle of Na+ in plants has been attributed to the sophisticated activity of distinct transport proteins in
The Authors (2009) Journal compilation New Phytologist (2009)
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Table 1 Na+ fluxes from four studies, with predicted respiratory requirement Respiratory requirement (O2 consumption, mol g (root FW)1 h1) 120 60 48 21
Species Spergularia maritima Arabidopsis thaliana Oryza sativa (japonica rice) Oryza sativa (indica rice)
Na+ flux (mol g (root FW)1 h1) 600 300* 240* 107
Reference Lazof & Cheeseman (1986) Essah et al. (2003) Horie et al. (2007) Malagoli et al. (2008)
The asterisk (*) indicates influx measurements that are slightly higher than efflux; respiratory requirements are also therefore slightly higher than active efflux would require.
the plasma membrane (see Fig. 1 in Malagoli et al., 2008). According to this widely accepted view, the majority of Na+ influx into the plant cell occurs via nonselective cation channels (NSCCs; other possible Na+ influx transporters include partially specific potassium channels and HKT transporters), while Na+ efflux is thought to be mediated by a Na+/H+ exchanger, possibly SOS1 (Amtmann & Sanders, 1999; Munns & Tester, 2008). The consensus view of the thermodynamic conditions occuring during salinity stress is that: (1) root cytosolic Na+ activity ( aNa + ) is substantially lower than external aNa + (in one of the few studies that measured cytosolic aNa + directly, it was found to be as high as 28 mM, against an external aNa + of 150 mM (Carden et al., 2003)); and (2) the chemical potential difference for Na+ across the plasma membrane is amplified greatly by the electrical potential difference (approx. 80 mV in Carden et al., 2003), as per the Nernst equation. A steep electrochemical potential gradient results, favoring passive influx of Na+ into the cell, and an active efflux, powered by the electrochemical H+ gradient generated by plasma-membrane proton ATPases. To estimate the energy required to drive the Na+ fluxing proposed in this model, it is useful to consider that, in many cases, it appears to be almost purely cyclical; that is, relatively little Na+ accumulates in the plant compared with the large amount that initially enters, and ultimately exits, the system. Evidence for cyclic Na+ transport includes the loss, within 1 h, of 90% of 22Na+ absorbed in 10 min by corn roots (Cheeseman, 1982); the precipitous decline in apparent 24Na+ influx within 5 min of labeling intact Suaeda maritima roots (Lazof & Cheeseman, 1986; also see Britto & Kronzucker, 2006); the rapid (< 5 min) saturation of 22Na+ in roots of Arabidopsis (Essah et al., 2003); and efflux : influx ratios of 0.86 0.90 in rice (Malagoli et al., 2008), 0.920.95 in Puccinellia tenuiflora and wheat (Wang et al., 2009), and 0.95 in barley (Kronzucker et al., 2006). Thus, the active (efflux) component of this cycle is nearly equal to the passive (influx) component, and therefore influx measurements, which are more commonly found in the literature and more straightforward to interpret, can be used to approximate efflux, with only a slight overestimate (in fact,
the influx component may also entail an energy cost, despite being in the thermodynamically passive direction, because it involves an electrogenic uniport that must be counteracted by proton pumping; Na+ efflux cannot accomplish this, if it is indeed electroneutral, because of the exchange with H+). The required energy can then be predicted, in terms of O2 consumed per Na+ transported, on the basis of the following stoichiometric series that draws upon models of transport energization: a 1:1 stoichiometry for Na+/H+ antiport (Shi et al., 2000; Pardo et al., 2006; Yeo, 2007); a second 1:1 stoichiometry between H+ pumping and ATP hydrolysis (Briskin & ReynoldsNiesman, 1991; Palmgren, 2001); and a third stoichiometry, of 5:1, between ATP synthesized and O2 consumed in respiration (i.e. phosphorylation efficiency or P : O2 ratio; Poorter et al., 1991; Scheurwater, 1999; Kurimoto et al., 2004). To summarize, for every O2 consumed in respiration, five Na+ ions are extruded from the cell via Na+/H+ exchange. Energy demands will be even greater as a result of simultaneous Cl transport and other transport steps within the plant, such as across the tonoplast, or from root to shoot (see the analysis by Yeo, 1983). The problem with this analysis becomes evident upon examining actual Na+ flux magnitudes measured in plants and comparing them with O2 normally consumed in respiration. Table 1 predicts the O2-consumption rates theoretically associated with Na+ fluxes from a number of studies, based on the preceding analysis; in every case, these values are extremely high and almost certainly exceed the roots respiratory budget. For example, maximal root respiration was only approx. 30 mol of O2 g (root FW)1 h1 (assuming, conservatively, a fresh root weight : dry root weight ratio of 10) in a study of 24 wild plant species (Poorter et al., 1991), and was even less in a study of six crop species (Rao & Ito, 1998). These values fall far short of the predicted value in three of the four cases presented in Table 1. In the fourth case (Malagoli et al., 2008), measured respiration rates of only 11 mol g (root FW)1 h1 were compared directly with Na+ efflux; again, they were only half of what would be required to account for the flux. Clearly, because there are other demands on respiration (e.g. for growth, maintenance and the fluxes of other ions), these predicted values are in excess of what can be provided to power
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such large fluxes, according to the proposed mechanisms of transport and energy transduction. We have termed this lack of correspondence Ussings conundrum because it represents the same problem faced half a century ago by Hans Ussing, a pioneer of tracer-flux research (Ussing, 1947, 1994) and the co-originator of the UssingTeorell flux-ratio equation. Ussing had compared theoretically active chloride fluxes and respiration rates in frog epithelia, to find that the available energy was insufficient to account for the observed flux. He thus proposed an alternative transport mechanism, in which the flux of one ionic species in the uphill direction is energetically coupled to the flux of the same (or similar) ion in the downhill direction. This concept of exchange diffusion would later be developed into modern ideas of ionic symport and antiport (Maloney, 1994). The discrepancy that emerges in Table 1 is scarcely lessened if the UssingTeorell equation is applied to sodium fluxes. The energy calculations would then be based only upon the degree to which the total flux in the active (efflux) direction exceeds the maximum free-diffusion flux in that direction, which is a function of cytosolic electrochemical aNa + (Dainty, 1962; White, 2003). In practice, this diffusive flux can be calculated in relation to influx, and to the external activity, according to the UssingTeorell equation:
oc a o = co a c
Eqn 1
(a simplified version, where oc and co represent influx and efflux, and ac and ao represent cytosolic and external electrochemical activities, respectively; Teorell, 1949; Ussing, 1947; also see White, 2003, for critique). Applying values for membrane electrical potential ( 80 mV) and cytosolic aNa + ( 28 mM), both measured by Carden et al. (2003), at an external aNa + of 150 mM, we can calculate the electrochemical activity ratio (inside : outside) to be c. 28:3400 (derived first from the activity ratio 28:150, which is then increased by c. 22-fold as a result of the 80 mV electrical potential, according to the Nernst equation; see Nobel, 2005). This indicates that very little (< 1% of the magnitude of influx) of the efflux can be accounted for by passive diffusion. Moreover, this quantity is further reduced by the likelihood that the proposed channelmediated entry of Na+ is via a long pore, which exponentially reduces the back flux through the channel (Hodgkin & Keynes, 1955; Hille, 1992). Thus, like Ussing, we may be required to revise (if not abandon) one or more aspects of the proposed flux-energization model. In Ussings case, the concept of energetically coupled exchange diffusion was developed to address this concern (Ussing, 1994). Although few examples of ions exchanging with others of the same ionic species exist in the plant literature (Laties, 1959; cf. Horemans et al., 1998; Britto & Kronzucker, 2003), it is not impossible that the futile cycling of Na+ occurs
by this means, perhaps via an antiporter, evolved for another function, with low selectivity for monovalent cations. This possibility could be investigated by testing for trans-stimulation. Since the time of Ussings original conundrum, the broader concept of ionic symport and antiport has encompassed the exchange of similar, as well as dissimilar, ions (Maloney, 1994); in the case of sodium efflux, there may be many possibilities that have been overlooked (e.g. Na+/Cl symport; ColmeneroFlores et al., 2007). Indeed, the possibility that sodium exits the cell via processes other than Na+/H+ antiport, at least in some species, was suggested in a study of 16 crop plant species (Mennen et al., 1990). Interestingly, much of the ground work that established Na+/H+ antiport as a leading model for Na+ efflux was conducted under low-Na+ conditions (Colombo et al., 1979; Jacoby & Teomy, 1988; Mennen et al., 1990), where energetic considerations are, by comparison to the present discussion, trivial. In addition, the stoichiometries of ATP hydrolysis and proton pumping may not be uniform (Baunsgaard et al., 1996; Low & Rausch, 1996; Morsomme et al., 1996). More radical solutions may also have to be considered. For instance, it may be that Na+ is not extruded via the plasma membrane by transport proteins, but is secreted via membrane vesicles, as has been suggested for Na+ transport (Lazof & Cheeseman, 1986; Flowers & Colmer, 2008, and references therein), and more recently shown for photoassimilate (Etxeberria et al., 2007) and Mn2+ (Peiter et al., 2007) transport. A still more unconventional proposal is that the majority of apparent ion fluxes exceeding the cells respiratory capacity are not across the plasma membrane at all, but result from the misinterpretation of tracer accumulating in extracellular spaces, particularly in leaves, as has been demonstrated to some degree in rice plants (Flowers et al., 1991; Gong et al., 2006).
Acknowledgements
We wish to thank Eduardo Blumwald for helpful discussions, and the Canada Research Chair Program and the Natural Sciences and Engineering Research Council of Canada for funding this work. Dev T. Britto and Herbert J. Kronzucker* University of Toronto, 1265 Military Trail, Toronto, Ontario, Canada, M1C 1A4 (*Author for correspondence: tel +1 416 287 7436; email herbertk@utsc.utoronto.ca)
References
Amtmann A, Sanders D. 1999. Mechanisms of Na+ uptake by plant cells. Advances in Botanical Research 29: 75112. Baunsgaard L, Venema K, Axelsen KB, Villalba JM, Welling A, Wollenweber B, Palmgren MG. 1996. Modified plant plasma membrane
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stimulates H+-transport byt not ATP hydrolysis of the V-ATPase. Journal of Experimental Botany 47: 17251732. Malagoli P, Britto DT, Schulze LM, Kronzucker HJ. 2008. Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, an relationship to salinity tolerance. Journal of Experimental Botany 59: 41094117. Maloney PC. 1994. Bacterial and plant antiporters. Journal of Experimental Biology 196: 439442. Mennen H, Jacoby B, Marschner H. 1990. Is sodium-proton antiport ubiquitous in plant cells? Journal of Plant Physiology 137: 180183. Morsomme P, de Kerchove dExaerde A, De Meester S, Thines D, Goffeau A, Boutry M. 1996. Single point mutations in various domains of a plant plasma membrane H+-ATPase expressed in Saccharomyces cerevisiae increase H+-pumping and permit yeast growth at low pH. EMBO J. 15: 551326. Munns R, Tester M. 2008. Salinity tolerance in higher plants. Annual Review of Plant Biology 59: 651681. Nobel PS. 2005. Physicochemical and environmental plant physiology, 3rd edn. Amsterdam, the Netherlands: Elsevier Academic Press, 118148. Palmgren MG. 2001. Plant plasma membrane H+-ATPases: powerhouses for nutrient uptake. Annual Review of Plant Biology 52: 817845. Pardo JM, Cubero B, Leidi EO, Quintero FJ. 2006. Alkali cation exchangers: roles in cellular homeostasis and stress tolerance. Journal of Experimental Botany 57: 11811199. Peiter E, Montanini B, Gobert A, Pedas P, Husted S, Maathuis FJM, Blaudez D, Chalot M, Sanders D. 2007. A secretory pathway-localised cation diffusion facilitator confers plant manganese tolerance. Proceedings of the National Academy of Science, USA 104: 85328537. Poorter H, Van der Werf A, Atkin O, Lambers H. 1991. Respiratory energy requirements depend on the potential growth rate of a plant species. Physiologia Plantarum 83: 469475. Rao TP, Ito O. 1998. Differences in root system morphology and root respiration in relation to nitrogen uptake among six crop species. Japan Agricultural Research Quarterly 32: 97103. Scheurwater I, Clarkson DT, Purves J, Van Rijt G, Saker L, Welschen R, Lambers H. 1999. Relatively large nitrate efflux can account for the high specific respiratory costs for nitrate transport in slow-growing grass species. Plant and Soil 215: 123134. Shi HZ, Ishitani M, Kim CS, Zhu JK. 2000. The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proceedings of the National Academy of Sciences, USA 97: 68966901. Teorell T. 1949. Membrane electrophoresis in relation to bioelectrical polarization effects. Archives des Sciences Physiologiques 3: 205219. Ussing HH. 1947. Interpretation of the exchange of radio-sodium in isolated muscle. Nature 160: 262263. Ussing HH. 1994. Does active transport exist? Journal of Membrane Biology 137: 9198. Wang C-M, Zhang J-L, Liu X-S, Li Z, Wu G-Q, Cai J-Y, Flowers TJ, Wang S-M. 2009. Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+. Plant, Cell & Environment (in press; DOI: 10.1111/j.13653040.2009.01942.x) White PJ. 2003. Ion transport. In: Thomas B, Murphy DJ, Murray BG, eds. Encyclopedia of Applied Plant Sciences. Oxford, UK: Elsevier, 625634. Yeo A. 1983. Salinity resistance: physiologies and prices. Physiologia Plantarum 58: 214222. Yeo A. 2007. Salinity. In: Yeo A, Flowers TJ, eds. Plant solute transport. Oxford, UK: Blackwell, 340370. Key words: exchange diffusion, flux coupling, low-affinity transport, sodium transport, transport energetics, UssingTeorell equation.
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H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast. Plant Journal 10: 45158. Briskin DP, Reynolds-Niesman I. 1991. Determination of H+/ATP stoichiometry for the plasma membrane H+-ATPase from red beet (Beta vulgaris L.) storage tissue. Plant Physiology 95: 242250. + Britto DT, Kronzucker HJ. 2003. Trans-stimulation of 13 NH4 efflux provides evidence for the cytosolic origin of tracer in the compartmental analysis of barley roots. Functional Plant Biology 30: 12331238. Britto DT, Kronzucker HJ. 2006. Futile cycling at the plasma membrane: A hallmark of low-affinity nutrient transport. Trends in Plant Science 11: 529534. Carden DE, Walker DJ, Flowers TJ, Miller AJ. 2003. Single-cell measurements of the contributions. Plant Physiology 131: 676683. Cheeseman JM. 1982. Pump-leak sodium fluxes in low-salt corn roots. Journal of Membrane Biology 70: 157164. Colmenero-Flores JM, Martinez G, Gamba G, Vazquez N, Iglesias DJ, Brumos J, Talon M. 2007. Identification and functional characterization of cation-chloride cotransporters in plants. Plant Journal 50: 278292. Colombo R, Bonetti A, Lado P. 1979. Promoting effect of fusicoccin on Na+ efflux in barley roots: evidence for a Na+-H+ antiport. Plant, Cell & Environment 2: 281285. Dainty J. 1962. Ion transport and electrical potentials in plant cells. Annual Review of Plant Biology 13: 379402. Essah PA, Davenport RJ, Tester M. 2003. Sodium influx and accumulation in Arabidopsis thaliana. Plant Physiology 133: 307318. Etxeberria E, Gonzalez P, Pozueta J. 2007. Mannitol enhanced fluid-phase endocytosis in storage parenchyma cells of celery (Apium graveolens) petioles. American Journal of Botany 96: 10411045. Flowers TJ, Colmer TD. 2008. Salinity tolerance in halophytes. New Phytologist 179: 945963. Flowers TJ, Hajibagheri MA, Yeo AR. 1991. Ion accumulation in the cell walls of rice plants growing under saline conditions evidence for the Oertli hypothesis. Plant, Cell & Environment 14: 319325. Gong HJ, Randall DP, Flowers TJ. 2006. Silicon deposition in the root reduces sodium uptake in rice (Oryza sativa L.) seedlings by reducing bypass flow. Plant, Cell & Environment 29: 19701979. Hille B. 1992. Ionic channels of excitable membranes, 2nd edn. Sunderland, MA, USA: Sinauer. Hodgkin AL, Keynes RD. 1955. The potassium permeability of a giant nerve fibre. Journal of Physiology 128: 6188. Horemans N, Asard H, Van Gestelen P, Caubergs RJ. 1998. Facilitated diffusion drives transport of oxidised ascorbate molecules into purified plasma membrane vesicles of Phaseolus vulgaris. Physiologia Plantarum 104: 783789. Horie T, Costa A, Kim TH, Han MJ, Horie R, Leung HY, Miyao A, Hirochika H, An G, Schroeder JI. 2007. Rice OSHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth. EMBO Journal 26: 30033014. Jacoby B, Teomy S. 1988. Assessment of Na+/H+ antiport in ATP-depleted red beet slices and barley roots. Plant Science 55: 103106. Kronzucker HJ, Szczerba MW, Moazami-Goudarzi M, Britto DT. 2006. The cytosolic Na+:K+ ratio does not explain salinity-induced growth impairment in barley : a dual-tracer study using 42K+ and 24Na+. Plant, Cell, & Environment 29: 22282237. Kurimoto K, Day DA, Lambers H, Noguchi K. 2004. Effect of respiratory homeostasis on plant growth in cultivars of wheat and rice. Plant, Cell & Environment 27: 853862. Laties GG. 1959. Active transport of salt into plant tissue. Annual Review of Plant Physiology 10: 87112. Lazof D, Cheeseman JM. 1986. Sodium transport and compartmentation in Spergularia marina partial characterization of a functional symplasm. Plant Physiology 81: 742747. Low R, Rausch T. 1996. In suspension-cultured Daucus carota cells salt stress
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Questions about floral (dis)integration

Successful pollen dispersal requires complex interactions for pollen to be loaded from flowers onto a pollen vector and then be deposited on stigmas as the vector subsequently encounters other flowers. These interactions involve many floral and inflorescence traits, so that coordination of their functions should promote pollen dispersal, leading to the hypothesis that angiosperm flowers are functionally integrated organs (reviewed by Armbruster et al., 2004). Several specialized pollination systems, such as heterostyly, secondary pollen presentation, and the fusion of anther(s) and stigma(s) into the orchid column and asclepiad gynostegium, provide obvious examples of floral integration, but what is the evidence for general integration? This question has been examined for almost 50 years (Berg, 1960), but with inconsistent results (reviewed by Armbruster et al., 2004; see Ashman & Majetic, 2006, for results based on genetic correlations), and variation in the extent of floral integration within angiosperms remains poorly understood. In this context, the recent article by Ordano et al. (2008) in New Phytologist, reporting a survey of 55 studies of floral integration, is of particular interest. Based on this survey, they concluded that flowering plants have lower floral integration ... than expected by a randomly generated distribution (page 1189), a finding clearly at odds with the integration hypothesis. However, certain aspects of Ordano et al.s analysis seem inappropriate and I demonstrate here that correction of this problem leads instead to strong evidence that flowers are usually integrated. The studies surveyed by Ordano et al. (2008) used the population variance of eigenvalues, V(), from principalcomponents analyses of floral traits as an index of floral integration. In such an analysis, all eigenvalues will equal one when the m traits are uncorrelated (not integrated), resulting in a minimum variance of zero; whereas, if all traits are perfectly, positively correlated (highly integrated), the first eigenvalue would equal m, the number of variables, and the remaining eigenvalues would equal zero, resulting in the maximum possible variance (which coincidentally equals m numerically). To generate their random null distribution, Ordano et al. calculated the integration index for simulated samples based on trait correlations randomly chosen from a uniform probability distribution (page 1186) and found that the average expected integration index was 32.7% (SD = 8.5%) of the maximum possible. Use of a uniform distribution proposes that when traits are uncorrelated strong correlations occur as often as weak correlations, owing solely to sampling error. For productmoment correlations, which are used in most studies of floral integration, such a uniform distribution is appropriate only for samples of four observations (Stuart & Ord, 1987). By contrast, the null distribution of productmoment corre-
Fig. 1 Frequency distributions of (a) 1000 simulated integration indices based on trait correlations drawn randomly from a uniform distribution (white bars), as modeled by Ordano et al. (2008), or from the distribution of productmoment correlation coefficients (grey bars), and (b) observed integration indices for 36 species (summarized by Ordano et al., 2008). The vertical dashed lines indicate the mean for each distribution. For (a) each simulated sample represented 30 observations of five variables and the true population correlation for both parent distributions was 0: variation resulted solely from sampling error.
lations approaches a normal distribution as the sample size increases (Stuart & Ord, 1987), so strong correlations should occur rarely for studies with reasonable samples when the null hypothesis is true. As a result of their use of a uniform distribution of correlations, Ordano et al.s simulations should generally have overestimated the expected integration index for randomly associated traits. I illustrate this problem with the results from two sets of 1000 simulations for n = 30 observations of m = 5 traits: one using a uniform distribution of correlation coefficients; and the other based on the null distribution of productmoment correlation coefficients. The mean integration index for simulations based on uniform correlations is 26.7% of the
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maximum possible (Fig. 1a, white histogram) and of the magnitude of the results of Ordano et al., which were based on varying numbers of traits and observations. By contrast, the mean integration index based on the null distribution of productmoment correlations was an order of magnitude smaller, at 2.76% of the maximum (Fig. 1a, grey histogram). This outcome is consistent with the prediction of 100(m 1)/ mn = 2.67%, based on the expected variance of eigenvalues for productmoment correlations when the null hypothesis is true (Wagner, 1984; also see Chevrud et al., 1989: note that this expectation approaches an asymptotic maximum of 100/ n as the number of traits considered increases). In light of these results, the average integration index of 21.5% (SD = 15.4%), observed for 36 plant species in 16 families in the survey of Ordano et al. (Fig. 1b), indicates, in contrast to their conclusion, that flowers are much more highly integrated than expected based on random trait correlations. Indeed, over 80% of the observed integration indices exceed the maximum null simulation based on productmoment correlations (compare grey histograms in Fig. 1a,b), indicating that floral integration is the rule, rather than the exception. Thus, rather than questioning whether flowers are integrated, attention should now focus on the causes of the extensive variation in integration (Fig. 1b) and its reproductive consequences. Lawrence D. Harder Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 (Author for correspondence: tel +1 403 2206489; email harder@ucalgary.ca)
Phenotypic integration: between zero and how much is too much

Letter Letter
References
Armbruster WS, Plabon C, Hansen TF, Mulder CPH. 2004. Floral integration, modularity, and precision: distinguishing complex adaptations from genetic constraints. In: Pigliucci M, Preston K, eds. Phenotypic integration: studying the ecology and evolution of complex phenotypes. New York, NY, USA: Oxford University Press, 2349. Ashman T-L, Majetic CJ. 2006. Genetic constraints on floral evolution: a review and evaluation of patterns. Heredity 96: 343352. Berg RL. 1960. The ecological significance of correlation pleiades. Evolution 14: 171180. Chevrud JM, Wagner GP, Dow MM. 1989. Methods for the comparative analysis of variation patterns. Systematic Zoology 38: 201213. Ordano M, Fornoni J, Boege K, Domnguez CA. 2008. The adaptive value of phenotypic floral integration. New Phytologist 179: 11831192. Stuart A, Ord JK. 1987. Kendalls advanced theory of statistics, Volume 1. Distribution theory. New York, NY, USA: Oxford University Press. Wagner GP. 1984. On the eigenvalue distribution of genetic and phenotypic dispersion matrices: evidence for a nonrandom organization of quantitative character variation. Journal of Mathematical Biology 21: 7795. Key words: eigenvalue variance, floral integration, floral morphology, null distribution, pollination.
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The hypothesis that flowers constitute mechanical devices designed by natural selection to ensure pollen donation/ reception through the concerted action of a suite of correlated (integrated) traits is an appealing and broadly accepted idea (Bell, 1985). Two questions derive from this hypothesis. The first is whether flowers are, in fact, integrated modules. Most studies evaluating this question have found significant levels of floral integration and a marked heterogeneity among species (Armbruster et al., 1999, 2004; Prez et al., 2007; Prez-Barrales et al., 2007; Ordano et al., 2008). Thus, the available evidence already indicates that flowers are indeed integrated (Ordano et al., 2008). A different, but equally relevant, question is whether or not flowers exhibit relatively high levels of phenotypic integration. Because in most plant species flower functioning depends on the interaction between floral and pollinator morphologies, it has been suggested that flowers should have relatively high levels of phenotypic integration (Stebbins, 1950, 1970; Faegri & van der Pijl, 1966). Obviously, responses to these two questions require different approaches and rely on distinct biological reasons. In his letter to New Phytologist, Harder (2009; this issue, pp. 247248) argues that our conclusion that flowering plants have lower floral integration than expected by a randomly generated distribution (Ordano et al., 2008, p. 1189) is flawed because of the inappropriateness of our analysis. He further concluded that: floral integration is the rule, rather than the exception. While we agree with his general conclusion regarding the ubiquity of floral integration, we believe that his criticism is based on confusing the two questions presented above. Accordingly, the disagreement presented by Harder (2009) merits a thorough discussion of these two questions. In doing so, we attempt to clarify possible sources of misinterpretation in the paper by Ordano et al. (2008).
Question 1. Are flowers integrated?

Since the seminal work of Berg (1960), evidence has accumulated supporting the expectation that flowers are integrated modules (Armbruster et al., 1999, 2004; Prez et al., 2007; Prez-Barrales et al., 2007; Ordano et al., 2008), a pattern observed for other functional modules in animal taxa (Wagner et al., 2007; Pavlicev et al., 2009). By using the observed distribution of integration values reported by Ordano et al. (2008), and the statistical approach developed by Wagner (1984) and Cheverud et al. (1989), Harders analyses confirmed that flowers are significantly integrated. The null hypothesis used by Harder (2009) is that of no correlation among
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characters, and thus any integration level above the expected value, given by the sampling error E(V()) = (M 1)/N, would indicate that a correlation matrix is significantly integrated (Cheverud et al., 1989). Because the average integration value observed among flowering plants was 21.5% (Ordano et al., 2008), and the expected value obtained by Harder (2009) was only 2.76%, the null hypothesis is readily rejected (sampling error obtained from an approximated normal distribution of productmoment correlation coefficients). Accordingly, Harders exercise (2009) just confirmed previous results supporting the expectation that flowers express significant levels of phenotypic integration. Moreover, we agree that the use of a uniform distribution of productmoment correlation coefficients to determine whether a given level of integration is significantly different from zero is not a valid procedure. So far, these results add to the mounting evidence showing that flowers, and many other functional modules in several organisms, express significant levels of integration.
Question 2. Do flowers have high levels of floral integration?

Harders (2009) test evaluates whether a group of traits are indeed integrated, but it does not tell us if the magnitude of integration is low or high. In addition to the presence/absence question, one could also ask whether the special case of plant pollinator interactions really favoured the evolution of high levels of phenotypic integration. The natural history of plants and pollinators is full of examples showing that efficient pollen transfer depends on the interaction between flower and pollinator morphologies (Faegri & van der Pijl, 1966; Fenster et al., 2004). Accordingly, answering if flowers are highly integrated modules requires a different null hypothesis from that used for testing question 1. In this sense, Ordano et al. (2008) looked for the appropriate null distribution that should be used to determine whether an observed level of integration was relatively low or high. To this end, Ordano et al. (2008) built an expected distribution of integration values based on randomly generated correlation matrices. These matrices, in turn, were obtained from randomly sampling productmoment correlation coefficients (ranging from 1 to 1). This procedure ensures that low, intermediate and high values have the same probability of being selected. Integration values were then calculated for each matrix (Wagner, 1984) and were used to build the expected distribution of integration. Such a distribution is centred on the average expected (significant) value of integration for a random association of independent correlation coefficients. Consequently, testing question 2 would determine whether a given integration value corresponds to a lower level or a higher level than that expected by chance. In fact, the expected distribution of integration values obtained by Ordano et al. (2008) can be used for any other functional module to determine what level of integration can be considered as low or high. A couple of examples suggests that there is a huge
variation in the magnitude of integration. For instance, integration levels as high as 77.16% have been found for the wing in the northern goshawk (Accipiter gentilis; Pavlicev et al., 2009), while that of the fruit of Prunus persica is 21.79% (Badenes et al., 1998). Other modules, like the facial skull of papionins, can range from 33.33% in Papio cynocephalus to 18.33% in Macaca nemestrina (Cheverud, 1989). Thus, the low level of integration found among flowering plants may not be an exception. Overall, we agree with Harder (2009) in that floral integration is the rule. Having taken this position, we must also take another. Demonstrating that flowers are integrated organs does not mean that they are highly integrated. The analyses in Ordano et al. (2008) were intended to test question 2 and concluded that flowers have relatively low levels of floral integration, rejecting the expectation that flowers are highly integrated organs. Thus, besides understanding the causes of variation in the levels of floral integration, floral evolutionary biologists could go forward in exploring why flowers are apparently little integrated (Fornoni et al., 2008). Juan Fornoni1, Mariano Ordano2, Karina Boege1 and Csar A. Domnguez1* Departamento de Ecologa Evolutiva, Instituto de Ecologa, Universidad Nacional Autnoma de Mxico, Apartado Postal 70-275, C.P. 04510, Mxico Distrito Federal, Mxico; 2Centro de Investigaciones sobre Regulacin de Poblaciones de Organismos Nocivos (CIRPON), Fundacin Miguel Lillo, Pasaje Caseros 1050, T4001MVD, San Miguel de Tucumn, Tucumn, Argentina (*Author for correspondence: tel +52 55 5622 9039; email: tejada@servidor.unam.mx)
1
References
Armbruster WS, Di Stilio VS, Tuxill JD, Flores TC, Velsquez-Runk JL. 1999. Covariance and decoupling of floral and vegetative traits in nine neotropical plants: a re-evaluation of Bergs correlation pleiades concept. American Journal of Botany 86: 3955. Armbruster WS, Plabon C, Hansen TF, Mulder CPH. 2004. Floral integration, modularity, and accuracy: distinguishing complex adaptations from genetic constraints. In: Pigliucci M, Preston K, eds. Phenotypic integration: studying the ecology and evolution of complex phenotypes. New York, NY, USA: Oxford University Press, 2349. Badenes ML, Martnez-Calvo J, Llacer G. 1998. Estudio comparativo de la calidad de los frutos de 26 cultivares de melocotonero de origen norteamericano y dos variedades poblacin de origen espaol. Investigacin Agraria, Produccin y Proteccin Vegetales (INIA Espaa) 13: 5770. Bell GB. 1985. On the function of flowers. Proceedings of the Royal Society of London B 224: 223265. Berg RL. 1960. The ecological significance of correlation pleiades. Evolution 14: 171180. Cheverud JM. 1989. A comparative analysis of morphological variation patterns in the Papionins. Evolution 43: 17371747. Cheverud JM, Wagner GP, Dow MM. 1989. Methods for the comparative analysis of variation patterns. Systematics Zoology 38: 201213.
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Prez-Barrales R, Arroyo J, Armbruster WS. 2007. Diffferences in pollinator faunas may generate geographic differences in floral morphology and integration in Narcissus papyraceus (Amaryllidaceae). Oikos 116: 19041918. Stebbins GL. 1950. Variation and evolution in plants. New York, NY, USA: Columbia University Press. Stebbins GL. 1970. Adaptive radiation of reproductive characteristics in angiosperms, I: pollination mechanisms. Annual Review of Ecology and Systematics 1: 307326. Wagner GP. 1984. On the eigenvalue distribution of genetic and phenotypic dispersion matrices: evidence for a nonrandom organization of quantitative character variation. Journal of Mathematical Biology 21: 7795. Wagner GP, Pavlicev M, Cheverud JM. 2007. The road to modularity. Nature Review Genetics 8: 921931. Key words: correlation matrix, floral morphology, modularity, phenotyic evolution, phenotypic integration.
Meetings Meeting Report ??? ? 0?? May 10.1111/j.1469-8137.2009.02906.x 2906 2009 Meetings
Faegri K, van der Pijl L. 1966. The principles of pollination ecology. Oxford, UK: Pergamon Press. Fenster CB, Armbruster WS, Wilson P, Thomson JD, Dudash MR. 2004. Pollination syndromes and floral specialization. Annual Review of Ecology, Evolution and Systematics 35: 375403. Fornoni J, Boege K, Domnguez CA, Ordano M. 2008. How little is too little? The adaptive value of floral integration. Communicative & Integrative Biology 1: 5658. Harder LD. 2009. Questions about floral (dis)integration. New Phytologist 183: 247248. Ordano M, Fornoni J, Boege K, Domnguez CA. 2008. The adaptive value of phenotypic floral integration. New Phytologist 179: 11831192. Pavlicev M, Cheverud JM, Wagner GP. 2009. Measuring morphological integration using eigenvalue variance. Evolutionary Biology 36: 157170. Prez F, Arroyo MTK, Medel R. 2007. Phylogenetic analysis of floral integration in Schizanthus (Solanaceae): does pollination truly integrate corolla traits? Journal of Evolutionary Biology 20: 17301738.
Meetings
Gearing up for comparative genomics: analyses of the fungal class Dothideomycetes
Dothideomycetes Comparative Genomics session: 25th Fungal Genetics Conference, Pacific Grove, CA, USA, March 2009
The Fungal Genetics Conferences, held biannually on the grounds of the Asilomar Conference Center in Pacific Grove, CA, USA, are the premier gatherings for fungal geneticists world-wide. This years conference, the 25th (http://www. fgsc.net/25thFGC/FGC25.htm), provided four very full days of talks, workshops and posters for almost 1000 participants. Fungi exceed every other kingdom of Eukaryotes in the number of genomic sequences completed or in progress. With this rich resource of available sequences, the focus is now switching from analyses of single genomes to comparative analyses across multiple genomes as a way to understand fungal biology and the interactions of fungi with plants. Fungi in the class Dothideomycetes are leading the way, and a full concurrent session was devoted to Dothideomycetes Comparative Genomics. This subject was chosen because the Dothideomycetes is one of the largest and most important groups of fungi that collectively infect almost every major monocot and dicot crop, whether for food, feed, fiber or fuel. In addition to plant pathogens, the class includes fungi with an unparalleled ecological, life history and metabolic diversity. Dothideomycetes are present on every continent, including Antarctica (Selbmann et al., 2005), and are important to ecosystem health and global carbon cycling as saprophytes and degraders of plant biomass. Many are lichenized (Del Prado et al., 2006) or are otherwise tolerant of environmental extremes including heat, cold and humidity (Ewaze et al., 2007). Some produce enzymes that help degrade rocks while others can capture and metabolize ethanol vapors (Tribe et al., 2006). A few are pathogens of humans or livestock. Cladosporium herbarum and Alternaria alternata are ubiquitous colonizers of dead plant biomass that play an important role in global carbon cycling, but in addition they are the two most commonly detected human allergens and a leading cause of asthma (Gioulekas et al., 2004). Thus, Dothideomycetes are extremely important to human as well as plant health. With seven Dothideomycetes genomes sequenced from the orders Capnodiales and Pleosporales (Table 1) and more on the way, a critical mass for comparative analysis has been achieved. The purpose of the Dothideomycetes Comparative Genomics session was to highlight recent progress on comparative analyses of this group. Talks were selected from submitted abstracts because they presented new tools for highthroughput functional analysis that could be applied to other sequenced genomes, or used a comparative genomics approach to reveal new insights into the biology of these fungi. Most Dothideomycetes are plant pathogens, and understanding how they interact with their plant hosts was a major focus of the session.
No claim to original US government works Journal compilation New Phytologist (2009)
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Genetic analysis of Cochliobolus sativus

Shaobin Zhong (North Dakota State University, Fargo, ND, USA) began the session with a talk on new tools for genomic analyses of Cochliobolus sativus (asexual stage: Bipolaris sorokiniana), a pathogen that causes kernel blight, root rot and seedling blight of barley (Hordeum vulgare), wheat (Triticum aestivum) and other grasses. It also causes spot blotch, a very important foliar disease in the northern USA and in tropical environments of Southeast Asia. Genetic and genomic tools are being developed for unraveling the pathogenicity of this fungus, which is highly specialized into several races on barley. Genetic mapping and pulsed-field gel electrophoresis identified the genomic locations for single factors controlling the virulence of the fungus in barley and wheat, which are now being pursued through map-based cloning strategies. Techniques for transformation and RNA-mediated gene silencing were developed for functional analyses that will help to pin down the virulence factors once a genomic sequence becomes available. This is an up-and-coming system with great potential for elucidating hostpathogen interactions in the Dothideomycetes.
Tools for functional analysis in the Pleosporales

Salim Bourras (INRA-Bioger, Versailes, France) moved the focus to high-throughput tools for functional analysis of Leptosphaeria maculans, the black leg fungus of canola (Brassica napus) and other Brassicaceous crops. This fungus alternates necrotrophic and biotrophic growth within a field season. The genome sequence has approximately 12 000 genes but their density varies with position between gene-poor, AT-rich regions (with 1 gene per 29 kb) and GC-equilibrated isochores containing one gene approximately every 2.4 kb. To assign function to these genes, Agrobacterium tumefaciens-mediated transformation (ATMT) was developed to generate a large
mutant library. The number of targeted genes was 279, of which 169 have an assigned function. Forty-three genes were recovered that potentially were involved in pathogenicity. The program will be extended and validated with similar large insertional-mutant libraries that are available for the rice blast pathogen, Magnaporthe oryzae, and should help to elucidate the mechanisms by which L. maculans interacts with its Brassica hosts. High-throughput mutant generation by ATMT was further elaborated by Carrie Smith (Oklahoma State University, Stillwater, OK, USA) working on Phoma medicaginis, a pathogen that causes a defoliating leaf spot of alfalfa (Medicago sativa) in large areas of the USA and also infects the model legume Medicago truncatula. This fungus is asexual and readily forms conidia in pycnidia on artificial media as well as on host plants. ATMT was used to generate an insertional-mutant library and identify genes involved in pathogenicity. Over 1000 mutants were generated (0.03% efficiency) and 10 were selected for an in-depth analysis. Several mutants with insertions in hypothetical proteins had obvious, visible phenotypes such as cracked pycnidia or lack of melanization. One mutant had fluffy, white hyphae that produced no pycnidia or conidia, while one with an insertion in a poly-A RNA polymerase gene produced small and few pycnidia and had reduced pathogenicity. This gene was similar to caffeine-induced death (Cid) genes in Schizosaccharomyces pombe. Homologs were identified in the Dothideomycetes Stagonospora nodorum and Pyrenophora tritici-repentis. With a sequenced host genome and an efficient T-DNA tagging system, P medicaginis is poised to become an important organism . for the analysis of Dothideomyceteshost interactions.
Tools for comparative genomics analysis

The development of tools for comparative analysis has lagged behind the accumulation of genomic sequence data. To address this problem, The Joint Genome Institute ( JGI) of the US Department of Energy (DOE) has pioneered a unique approach
Table 1 Sequence resources available currently for comparative analyses of Dothideomycetes genomes Genome size (Mb) 30.3
Species Alternaria brassicicola
Order Pleosporales
Coverage 6.4
Resources Washington University (http://genome.wustl.edu/ genome.cgi?GENOME=Alternaria%20brassicicola) JGI (jgi.doe.gov/Abrassicicola) JGI (http://jgi.doe.gov/Cochliobolus) JGI (http://jgi.doe.gov/Mfijiensis) JGI (http://jgi.doe.gov/Mgraminicola) Broad (http://www.broad.mit.edu/annotation/genome/ pyrenophora_tritici_repentis.3/Info.html) JGI (http://jgi.doe.gov/Ptritici_repentis) Broad (http://www.broad.mit.edu/annotation/genome/ stagonospora_nodorum.2/Home.html) JGI (http://jgi.doe.gov/Snodorum)
Cochliobolus heterostrophus Mycosphaerella fijiensis Mycosphaerella graminicola Pyrenophora tritici-repentis
Pleosporales Capnodiales Capnodiales Pleosporales
10.0 7.1 Finished 6.9
34.9 73.4 39.7 37.8
Stagonospora nodorum
Pleosporales
> 10
37.2
JGI, The Joint Genome Institute.
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to software development by asking communities of users to request new tools that are directly needed to further their research. Andrea Aerts (DOE-JGI, Walnut Creek, CA, USA) presented the JGI annotation pipeline for Dothideomycetes genomes. To support the JGI mission of community annotation, six Dothideomycetes genomes (Table 1) are currently available on their web portal, including three that were sequenced outside JGI and, in addition, they have organized on-site annotation jamborees to facilitate whole-genome annotation. The annotation pipeline comprises repeat masking, data mapping, gene prediction, annotation and validation, and culminates in public release of the data. Functional categories according to GO (Gene Ontology), KOG (euKaryotic Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) assignments can be browsed for each genome, which provides an excellent tool for comparative genomics analyses. Side-by-side analysis of all six genomes facilitates rapid and efficient comparisons, and it is simple to switch from one species to another. An overview of all browsers, synteny, VISTA (http://genome.lbl.gov/vista/index.shtml) conservation and protein cluster viewers was provided, demonstrating the excellent support that the JGI provides to the Dothideomycetes community. VISTA conservation tracks for all Dothideomycetes genomes and the synteny viewer allow syntenic relationships to be identified and analyzed efficiently. The Dot Plot viewer readily showed the differences between essential and dispensable chromosomes of the Mycosphaerella graminicola genome. These new tools provide an excellent resource for the Dothideomycetes research community and also should be useful for comparative genomics analyses in other organisms, including plants.
at least three of the C. fulvum effectors, two of which are also present in M. graminicola and other Dothideomycetes. Based on these findings, the current hypothesis is that closely related pathogens share some effectors that are most probably inherited from a common ancestor and are adapted to specific host plants. This is the first report on effectors shared among pathogens of such widely divergent hosts, suggesting that similar strategies might have been employed by related plant pathogens that infect unrelated host plants. These results have led to a new model for effectoromics, in which related Dothideomycetes fungi share a set of common effectors that facilitate colonization of many different host species, but have diverged for other effectors that enable host specialization.
Mycosphaerella graminicola to the fore

Sarrah Ben MBarek (Plant Research International, Wageningen, The Netherlands) presented the current status of the M. graminicola sequence analysis, the first finished genome of a filamentous fungus. High-density linkage mapping with over 2000 sequenced markers enabled a perfect alignment of the genetic map with the genome sequence over all 21 chromosomes. Genome plasticity in this fungus was evident from polymorphisms in chromosome length and number. Meiosis, in contrast to mitosis, frequently generates chromosome number polymorphisms (CNPs), particularly for chromosomes 14 21. These chromosomes are missing frequently in progeny with no visible effect on viability or virulence and appear to be dispensable (Wittenberg et al., in press). Comparative genomic hybridizations using a NimbleGen (Roche NimbleGen Inc., Madison, WI, USA) whole-genome array confirmed the origin of CNPs. The dispensable chromosomes are smaller, have lower gene densities, have a higher density of transposons and contain many unclassified genes that could code for novel proteins. They also contain a high number of redundant copies of genes that are unique on the essential chromosomes. For example, extra copies of tubulin genes on the dispensable chromosomes appeared to be pseudogenes compared with those on the essential chromosomes. The JGI browsers (described by Andrea Aerts) enabled high-resolution selfsynteny analyses showing that the dispensable chromosomes are a mosaic of redundant blocks of virtually all other chromosomes. However, the synteny with other Dothideomycetes such as Stagonospora nodorum and M. fijiensis is extremely low. The presence of eight dispensable chromosomes is unique for fungi. However, their function is unknown and will be the subject of future analyses. Braham Dhillon (Purdue University, West Lafayette, IN, USA) continued analyses of the M. graminicola genome with research on the amplification and apparent inactivation of a gene for cytosine methylation. Analysis of the repetitive fraction of the genome identified a family of 28 repeats, 23 of which contained a region similar to a DNA methyltransferase
Effectors shared among species

Several talks were related to the finished genome of the septoria tritici blotch fungus of wheat, Mycosphaerella graminicola, or the draft sequence of its close relative, the banana black Sigatoka (or leaf streak) pathogen, Mycosphaerella fijiensis. Ioannis Stergiopoulos (Wageningen University, The Netherlands) reported on a comparative study between M. fijiensis and Cladosporium fulvum (syn.: Passalora fulva), a nonobligate, biotrophic pathogen of tomato (Solanum lycopersicum) and reference organism for hostpathogen interactions. To date, 10 effectors have been identified from C. fulvum. All are small, cysteine-rich proteins that are secreted into the apoplast and whose recognition in tomato is mediated by cognate Cf (for C. fulvum) resistance proteins. Although demonstrated for only a few, all of the effector proteins are assumed to be virulence factors (Stergiopoulos & De Wit, 2009). So far no homologs of the C. fulvum effectors had been identified in other fungi, and thus they were considered to be species specific. However, a careful search in the genome sequence of M. fijiensis, based on a combination of comparative genomics and structural analysis, identified putative homologs for
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(DNMT) gene. All copies except for one were located near the telomeres, with the remaining copy on chromosome 6. The DNMT gene was single copy in nine other fungal genomes and, based on syntenic relationships identified by a comparative analysis with the genome sequence of M. fijiensis, the copy on chromosome 6 probably was the original before amplification. Interestingly, all copies of the DNMT gene, including the putative original on chromosome 6, showed evidence of repeat-induced point mutation (RIP), a mechanism in fungi for inactivating transposons by introducing stop codons into reading frames. Tests for methylation showed that M. graminicola lacks cytosine methylation, even though it was present in close relatives. Accidental amplification followed by RIP appears to be a novel mechanism for inactivation of single-copy genes in fungi. In the final talk of the session, Eva Stukenbrock (University of Aarhus, Aarhus, Denmark) reported on the putative genetic basis of speciation in Mycosphaerella based on comparative genomics. A previous analysis of sequence data had shown very recent divergence times among M. graminicola, two undescribed new species from wild grasses in Iran and the somewhat more distantly related barley pathogen, Septoria passerinii (Stukenbrock et al., 2007). Therefore, genomic comparisons with M. graminicola could reveal evolutionary changes that occurred during speciation. Resequencing one of these related species revealed two haplotypes in the mitochondrial sequences, indicating the first possible report of heteroplasmy in the Dothideomycetes. The nuclear genome sequence was very similar and showed a high degree of synteny with the essential chromosomes 113 of the finished genome of M. graminicola. In contrast, synteny with the dispensable chromosomes 1421 was considerably lower. Regions of repetitive DNA correlated well with nonaligned DNA except for the eight dispensable chromosomes of M. graminicola, probably as a result of their higher content of transposons. A browser developed for comparative genomics showed a significant difference in the ratio of substitutions in both coding and noncoding DNA of the essential and dispensable chromosomes, suggesting that they have different evolutionary patterns. In particular, the ratio of nonsynonymous to synonymous substitutions (dN/dS) was markedly higher on the dispensable chromosomes, giving a strong indication of possible directional selection.
these analyses to illuminate hostpathogen interactions will increase greatly once genome sequences also are available for the host plants. The future of comparative genomics is increasingly bright. The recent session on Dothideomycetes Comparative Genomics provided a first small hint of what is to come as more fungal and plant genomes are sequenced.
Acknowledgements
We thank Francine Govers and Jay Dunlap for programming the Dothideomycetes Comparative Genomics session of the 25th Fungal Genetics Conference, and Andrea Aerts, Sarrah Ben MBarek, Salim Bourras, Braham Dhillon, Carrie Smith, Ioannis Stergiopoulos, Eva Stukenbrock, and Shaobin Zhong for speaking at the session and for verifying a previous draft of this report. Stephen B. Goodwin1* and Gerrit H. J. Kema2 USDA-ARS, Crop Production and Pest Control Research Unit, Department of Botany and Plant Pathology, 915 West State Street, Purdue University, West Lafayette, IN 479072054, USA; 2Plant Research International B.V., Wageningen University and Research Centre, P.O. Box 16, 6700 AA Wageningen, The Netherlands (*Author for correspondence: tel +1 (765) 494-4635; email steve.goodwin@ars.usda.gov or sgoodwin@purdue.edu)
1
References
Del Prado R, Schmitt I, Kautz S, Palice Z, Lcking R, Lumbsch HT. 2006. Molecular data place Trypetheliaceae in Dothideomycetes. Mycological Research 110: 511520. Ewaze JO, Summerbell RC, Scott JA. 2007. Physiological studies of the warehouse staining fungus, Baudoinia compniacensis. Mycological Research 111: 14221430. Gioulekas D, Damialis A, Papakosta D, Spieksma F, Giouleka P, Patakas D. 2004. Allergenic fungi spore records (15 years) and sensitization in patients with respiratory allergy in Thessaloniki-Greece. Journal of Investigational Allergology & Clinical Immunology 14: 225231. Selbmann L, de Hoog GS, Mazzaglia A, Friedmann EI, Onofri S. 2005. Fungi at the edge of life: cryptoendolithic black fungi from Antarctic desert. Studies in Mycology 51: 132. Stergiopoulos I, De Wit PJGM. 2009. Fungal effector proteins. Annual Review of Phytopathology 47: 233263. Stukenbrock EH, Banke S, Javan-Nikkhah M, McDonald BA. 2007. Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation. Molecular Biology and Evolution 24: 398411. Tribe HT, Thines E, Weber RWS. 2006. Moulds that should be better known: the wine cellar mould, Racodium cellare Persoon. Mycologist 20: 171175. Wittenberg AHJ, van der Lee TAJ, Ben MBarek S, Ware SB, Goodwin SB, Kilian A, Visser RGF, Kema GHJ, Schouten HJ. (in press). Meiosis drives extraordinary genome plasticity in the haploid fungal plant pathogen Mycosphaerella graminicola. PLoS One. Keywords: comparative, Dothideomycetes, fungi, genetics, genomics, host pathogen interactions, tools.
Perspectives
As more genomes are sequenced, the potential power of comparative analyses increases. Being able to search complete genomes for homologs of interesting genes has revealed new insights into the evolution of effectors and the genes on dispensable chromosomes, and has also revealed a potentially new mode of inactivation of an otherwise single-copy gene. Estimates of selection on all genes in an organism will identify those that are under directional selection and might be involved in hostpathogen interactions and speciation. The power of
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New Phytologist is owned by a non-profit-making charitable trust dedicated to the promotion of plant science, facilitating projects from symposia to open access for our Tansley reviews. Complete information is available at www.newphytologist.org. Regular papers, Letters, Research reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication as-ready via Early View our average submission to decision time is just 29 days. Online-only colour is free, and essential print colour costs will be met if necessary. We also provide 25 offprints as well as a PDF for each article. For online summaries and ToC alerts, go to the website and click on Journal online. You can take out a personal subscription to the journal for a fraction of the institutional price. Rates start at 139 in Europe/$259 in the USA & Canada for the online edition (click on Subscribe at the website). If you have any questions, do get in touch with Central Office (newphytol@lancaster.ac.uk; tel +44 1524 594691) or, for a local contact in North America, the US Office (newphytol@ornl.gov; tel +1 865 576 5261).

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Plantpathogen interactions: a view from the evolutionary basement

The Author (2009) Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 237239 237 www.newphytologist.org 237

New Phytologist (2009) 183: 237239 www.newphytologist.org

The Author (2009) Journal compilation New Phytologist (2009)

The Author (2009) Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 237239 www.newphytologist.org

We are starting to understand that plants are quite flexible in

New Phytologist (2009) 183: 240243 www.newphytologist.org

The Author (2009) Journal compilation New Phytologist (2009)

Differentiation of Annonaceae pollination systems in odour space

The Author (2009) Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 240243 www.newphytologist.org

New Phytologist (2009) 183: 240243 www.newphytologist.org

The Author (2009) Journal compilation New Phytologist (2009)

The Authors (2009) Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 243246 www.newphytologist.org

New Phytologist (2009) 183: 243246 www.newphytologist.org

The Authors (2009) Journal compilation New Phytologist (2009)

The Authors (2009) Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 243246 www.newphytologist.org

New Phytologist (2009) 183: 243246 www.newphytologist.org

The Authors (2009) Journal compilation New Phytologist (2009)

Questions about floral (dis)integration

The Author (2009) Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 247248 www.newphytologist.org

Phenotypic integration: between zero and how much is too much

Question 1. Are flowers integrated?

New Phytologist (2009) 183: 248250 www.newphytologist.org

The Authors (2009) Journal compilation New Phytologist (2009)

Question 2. Do flowers have high levels of floral integration?

The Authors (2009) Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 248250 www.newphytologist.org

New Phytologist (2009) 183: 250254 www.newphytologist.org

No claim to original US government works Journal compilation New Phytologist (2009)

Genetic analysis of Cochliobolus sativus

Tools for functional analysis in the Pleosporales

Tools for comparative genomics analysis

Species Alternaria brassicicola

Cochliobolus heterostrophus Mycosphaerella fijiensis Mycosphaerella graminicola Pyrenophora tritici-repentis

Pleosporales Capnodiales Capnodiales Pleosporales

10.0 7.1 Finished 6.9

34.9 73.4 39.7 37.8

JGI, The Joint Genome Institute.

No claim to original US government works Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 250254 www.newphytologist.org

Mycosphaerella graminicola to the fore

Effectors shared among species

New Phytologist (2009) 183: 250254 www.newphytologist.org

No claim to original US government works Journal compilation New Phytologist (2009)

No claim to original US government works Journal compilation New Phytologist (2009)

New Phytologist (2009) 183: 250254 www.newphytologist.org

About New Phytologist

New Phytologist (2009) 183: 250254 www.newphytologist.org

No claim to original US government works Journal compilation New Phytologist (2009)

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