Litter decomposition, ectomycorrhizal roots and the 'Gadgil'effect

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Editorial Blackwell Science, Ltd

Welcome to new editors Mark Rausher and Chris Cobbett have recently joined the Editorial Board of New Phytologist – we are delighted to welcome them to our expanding journal. New Phytologist appoints new Editors in order to widen the collective knowledge base of the Board and to provide clear indications of specific areas of plant science research in which we would like to encourage the submission of manuscripts. Mark is based at the Department of Biology, Duke University, USA where his research encompasses the evolution and ecology of mating systems and plant interactions with ‘enemies’ (Rausher, 2001). The diverse range of research topics in Mark’s lab can be seen on his web page (http:// www.biology.duke.edu/research_by_area/eeob/rausher.html) – current efforts are directed at quantifying genetic variation in insect resistance and identifying those processes that maintain genetic variation in natural populations of plants. The addition of Mark to the Board will enhance New Phytologist’’s expertise in evolutionary biology, an area already reinvigorated in the journal through the appointment of Loren Rieseberg (Ayres, 2001; Rieseberg, 2001; Rieseberg et al., 2002). This is an area in which we are focussing attention this year in the 11th New Phytologist Symposium (see http://www.newphytologist.org/plantspeciation) and is one

References Armbruster WS. 2001. Evolution of floral form: electrostatic forces, pollination, and adaptive compromise. New Phytologist 152: 181–183. Ayres P. 2001. Welcome to new editors. New Phytologist 149: 153. Bleeker PM, Schat H, Vooijs R, Verkleij JAC, Ernst WHO. 2003. Mechanisms of arsenate tolerance in Cytisus striatus. New Phytologist 157: 33 – 38. Brouat C, McKey D. 2001. Leaf-stem allometry, hollow stems, and the evolution of caulinary domatia in myrmecophytes. New Phytologist 151: 391– 406. Cobbett CS. 2000. Phytochelatins and heavy metal tolerance in plants. Current Opinion in Plant Biology 3: 211– 216. Gatehouse JA. 2002. Plant resistance towards insect herbivores: a dynamic interaction. New Phytologist 156: 145 –169. Ha S-B, Smith AP, Howden R, Dietrich WM, Bugg S, O’Connell MJ, Goldsbrough PB, Cobbett CS. 1999. Phytochelatin synthase genes from Arabidopsis and the yeast, Schizosaccharomyces pombe. Plant Cell 11: 1153 –1164. Hartley-Whitaker J, Woods C, Meharg AA. 2002. Is differential phytochelatin production related to decreased arsenate influx in arsenate tolerant Holcus lanatus? New Phytologist 155: 291– 225.

in which the journal is now establishing a very strong reputation (Armbruster, 2001; Brouat & McKey, 2001; Shaw, 2001; Gatehouse, 2002; Tewksbury, 2002). Chris is a double first for New Phytologist. He is our first editorial appointment from Australia (Department of Genetics, University of Melbourne) and is also the first Editor in a rapid growth area for New Phytologist – heavy metal tolerance and phytoremediation (Ha et al., 1999; Cobbett, 2000). Research in Chris’ lab can be viewed at http://www.genetics.unimelb. edu.au/Cobbett/CC.html – the emphasis is on identifying the genetic mechanisms by which plants cope with different toxic metals. His research group was the first to show that a particular class of metal-binding peptides, the phytochelatins, is essential for cadmium tolerance. Research in the area of heavy metal tolerance has expanded steadily for the past few years to become a regular feature of New Phytologist, and one in which the Trust focussed at the 9th Symposium last year (Kraemer, 2003). Recent highlights in the journal in this area have included studies of phytochelatin production (Hartley-Whitaker et al., 2002; Pawlik-Skowronska et al., 2002) and the surge of interest in arsenic tolerance (Lombi et al., 2002; Meharg, 2002; Zhao et al., 2002; Bleeker et al., 2003). Ian Woodward Editor-in-Chief

Kraemer U. 2003. Phytoremediation to phytochelatin – plant trace metal homeostasis. New Phytologist 158: 4 – 6. Lombi E, Zhao F-J, Fuhrmann M, Ma LQ, McGrath SP. 2002. Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata. New Phytologist 157: 33 – 38. Meharg AA. 2002. Arsenic and old plants. New Phytologist 156: 1– 8. Pawlik-Skowronska B, di Toppi LS, Favali MA, Fossati F, Pirszel J, Skowronski T. 2002. Lichens respond to heavy metals by phytochelatin synthesis. New Phytologist 156: 95 –102. Rausher MD. 2001. Coevolution and plant resistance to natural enemies. Nature 411: 857– 864. Rieseberg LH. 2001. Chromosomal rearrangements and Speciation. Trends in Ecology and Evolution 16: 351– 358. Rieseberg LH, Widmer A, Arntz MA, Burke JM. 2002. Directional selection is the primary cause of phenotypic diversification. Proceedings of the National Academy of Sciences, USA 99: 12242 –12245. Shaw J. 2001. Antagonistic pleiotrophy and the evolution of alternate generations. New Phytologist 152: 365 – 374. Tewksbury JJ. 2002. Fruits, frugivores and the evolutionary arms race. New Phytologist 156: 137–144. Zhao FJ, Dunham SJ, Mcgrath SP. 2002. Arsenic hyperaccumulation by different fern species. New Phytologist 156: 27– 31. Commentary 158

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Commentary Litter decomposition, ectomycorrhizal roots and the ‘Gadgil’ effect What is the Gadgil effect? In field and laboratory experiments in New Zealand, Gadgil & Gadgil (1971, 1975) found that when ectomycorrhizal roots were excluded from Pinus radiata litter, the rate of litter decomposition increased dramatically over a 12-month period. This ‘Gadgil effect’ was attributed to stimulated colonization and exploitation of litter by ectomycorrhizal fungi at the expense of litter decomposing saprotrophs, and was used to explain the accumulation of the slowly degraded organic ‘mor’ humus horizons which characteristically form in forests of ectomycorrhizal tree species. However, attempts to corroborate the ‘Gadgil effect’ in subsequent studies have met with a variety of results, and the area has become a contentious area of soil microbiology. The study of Koide & Wu reported in this issue (pp. 401– 407) provides a simple additional explanation for the ‘Gadgil effect’ and goes some way to account for inconsistencies in the literature dealing with this subject.

Explanations The simplest explanation for the ‘Gadgil effect’ has been the direct inhibition of saprotrophic organisms by ectomycorrhizal fungi. Although some ectomycorrhizal fungi have the capacity to break down organic components of litter these are small relative to saprotrophic fungi (Colpaert & van Tichelen, 1996). Exploitation of litter resources by ectomycorrhizal fungi in preference to saprotrophic fungi would therefore result in reduced rates of litter decomposition. Some ectomycorrhizal fungi have indeed been shown to outcompete saprotrophic fungi for territory (Lindahl et al., 2001), although the outcome of interactions between these fungal groups appears to depend on species and carbon availability to each of the combatants. Ectomycorrhizal mycelium can also cause substantial inhibition of the activities of soil bacteria, although again, the size of the effect varies between different species of ectomycorrhizal fungus (Olsson et al., 1996). The production of antibiotic compounds by ectomycorrhizal fungi, particularly organic acids, has been implicated in the direct inhibition of saprotrophic organisms by ectomycorrhizal mycelium (Rasanayagam & Jeffries, 1992). Direct competition between ectomycorrhizal fungi and saprotrophs for nitrogen has also been implicated in the ‘Gadgil

effect’. Nitrogen availability is a key factor determining rates of cellulose degradation by saprotrophic organisms (Park, 1976). Abuzinadah et al. (1986) suggested that the selective exploitation and translocation of N from litter to the host plant could result in N limitation to saprotrophic organisms, leading to reduced rates of litter decomposition. Further studies have clearly demonstrated that colonization of litter by ectomycorrhizal fungi reduces the quality of litter remaining, by increasing the C : N ratio, and depleting N, P and K contents (Bending & Read, 1995). The magnitude of the ‘Gadgil effect’ could therefore be expected to depend on the availability of N and other nutrients in the particular soil being studied.

A new view Koide & Wu have provided an alternative explanation for the ‘Gadgil effect’. Litter decomposition, ectomycorrhiza density and moisture content were followed over a 12month period in a Pinus resinosa plantation. It was found that litter decomposition was reduced as ectomycorrhiza density increased, confirming the findings of Gadgil & Gadgil (1971, 1975). Significantly, it was shown that litter moisture content was also reduced as ectomycorrhiza density increased. Moisture content is a key determinant of forest litter decomposition, affecting the size, composition and activities of saprotrophic communities (Robinson, 2002). The drying induced by ectomycorrhizal roots in the study of Koide & Wu was of the order of magnitude known to be sufficient to cause substantial reduction in litter decomposition rates. Similar evidence for changes in litter moisture content following colonization by ectomycorrhizal roots and mycelium have been reported in many studies (Griffiths et al., 1990; Parmalee et al., 1993; Zhu & Ehrenfeld, 1996). The possibility that water removal contributes to the ‘Gadgil effect’ would provide some explanation for discrepancies in the literature dealing with this subject. The significance of changes to soil moisture content would depend on the prevailing weather conditions, and would therefore vary according to season and between years. In dry weather, when water could become a factor limiting the growth and activities of saprotrophic organisms, the uptake and translocation of water by ectomycorrhizal roots could result in the inhibition of saprotrophic organisms, reducing litter decomposition. During wet weather, changes to litter moisture content caused by ectomycorrhizal roots may have little effect on saprotrophic organisms. However, it is likely that any ectomycorrhizal root induced changes to litter moisture content would need to be maintained over a prolonged

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period to have long-term impacts on decomposition (O’Neil et al., 2003). Ectomycorrhiza induced change to soil moisture content is evidently only one of several factors which may interact to control the impacts of ectomycorrhizal roots on litter decomposition rates. Zhu & Ehrenfeld (1996) found stimulated decomposition of litter following colonization by ectomycorrhizal roots in the mineral soil of a Pinus resinosa plantation, despite consistently lower moisture contents in ectomycorrhiza colonized litter relative to uncolonized litter. Parmalee et al. (1993) showed that in organic forest soil horizons moisture content, microbial biomass, microbial growth rate and extractable N declined with increasing root density, although the sizes of faunal groups were not affected. However in the soil’s mineral horizon, increasing root density stimulated microbial growth and faunal communities. Similarly, Priha et al. (1999) showed that the presence of ectomycorrhizal roots reduced rates of soil respiration in the organic horizon of a mor forest soil, but stimulated decomposition in the underlying mineral horizon.

Perspectives The suppression of litter decomposition induced by mycorrhizal roots thus appears to be limited to organic soil horizons which have low N availability and are probably more susceptible to fluctuations in moisture content. In mineral soils the presence of ectomycorrhizal roots evidently stimulates the activities of carbon-limited saprotrophic organisms following the rhizodeposition of labile carbon. These interactions must be further complicated by the considerable functional diversity that exists between different species of ectomycorrhizal fungus with respect to water uptake and translocation, nitrogen mobilization and translocation, and antagonistic interactions. Gary D. Bending Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK (email [email protected])

References Bending GD, Read DJ. 1995. The structure and function of the vegetative mycelium of ectomycorrhizal plants. V. Foraging behaviour and translocation of nutrients from exploited litter. New Phytologist 130: 401– 409. Colpaert JV, van Tichelen KK. 1996. Decomposition, nitrogen and phosphorus mineralization from beech leaf litter colonized by ectomycorrhizal or litter-decomposing basidiomycetes. New Phytologist 134: 123 –132. Gadgil RL, Gadgil PD. 1971. Mycorrhiza and litter decomposition. Nature 233: 133.


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Gadgil RL, Gadgil PD. 1975. Suppression of litter decomposition by mycorrhizal roots of Pinus radiata. New Zealand Journal of Forest Science 5: 33 – 41. Griffiths RP, Caldwell BA, Cromack K Jr, Morita R. 1990. Douglas-fir forest soils colonized by ectomycorrhizal mats. I. Seasonal variation in nitrogen chemistry and nitrogen cycle transformation rates. Canadian Journal of Forest Research 20: 211–218. Koide RT, Wu T. 2003. Ectomycorrhizas and retarded decomposition in a Pinus resinosa plantation. New Phytologist 158: 401– 407. Lindahl B, Stenlid J, Finlay RD. 2001. Effects of resource availability on mycelial interactions and 32P transfer between a saprotrophic and an ectomycorrhizal fungus in soil microcosms. FEMS Microbiology Ecology 38: 43 –52. O’Neil EG, Johnson DW, Ledford J, Todd DE. 2003. Acute seasonal drought does not permanently alter mass loss and nitrogen dynamics during decomposition of red maple (Acer rubrum L.) litter. Global Change Biology 9: 117–123. Olsson PA, Chalot M, Baath E, Finlay RD, Soderstrom B. 1996. Ectomycorrhizal mycelia reduce bacterial activity in a sandy soil. FEMS Microbiology Ecology 21: 77–96. Park D. 1976. Carbon and nitrogen levels as factors influencing fungal decomposers. In: Anderson JM, Macfaydon A, eds. The role of terrestrial and aquatic organisms in decomposition processes. Oxford, UK: Blackwell Scientific Publications, 41–46. Parmalee RW, Ehrenfeld JG, Tate RL. 1993. Effects of pine roots on microorganisms, fauna and nitrogen availability in 2 soil horizons of a coniferous forest spodosol. Biology and Fertility of Soils 15: 113 –119. Priha O, Grayston SJ, pennanen T, Smolander A. 1999. Microbial activities related to C and N cycling and microbial community structure in the rhizosphere of Pinus sylvestris, Picea abies and Betula pendula seedlings in an organic and mineral soil. FEMS Microbiology Ecology 30: 187–199. Rasanayagam S, Jeffries P. 1992. Production of acid is responsible for antibiosis by some ectomycorrhizal fungi. Mycological Research 11: 971–976. Robinson CH. 2002. Controls on decomposition and soil nitrogen availability at high latitudes. Plant and Soil 242: 65 – 81. Zhu W, Ehrenfeld JG. 1996. The effects of mycorrhizal roots on litter decomposition, soil biota, and nutrients in a spodsolic soil. Plant and Soil 179: 109 –118. Key words: Gadgil effect, mycorrhizas, roots, nutrition, decomposition. Commentary 158

Breaking physical dormancy in seeds – focussing on the lens Why do seeds germinate in spring? Surprisingly, the answer to this apparently simple question is still not fully understood, even in a plant family as extensive and as agronomically important as the legumes. The seeds of the legumes, in

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common with those of many other species (Box 1), have a mechanism of physical dormancy, with water-impermeable seed (or fruit) coats. Intriguingly, however, there is a water gap in the impermeable layer and it is this structure which formed the focus of research into dormancy-break in the work of Van Asshe et al. described in this issue (pp. 315– 323). With water gaps also present in so many other species, this has very wide implications.

Water-impermeable coats Species with water-impermeable seed (or fruit) coats – physical dormancy – occur in some 15 plant families (sensu APG, 1998), including the Fabaceae (subfamilies Caesalpinioideae, Mimosoideae, and Papilionoideae). This impermeability of the coat is caused by the presence of one or more palisade layers of lignified malphigian cells (macrosclereids) tightly packed together and impregnated with water-repellant chemicals (Rolston, 1978; Werker, 1980–81). An anatomical structure in the impermeable layer(s) functions as the ‘water gap’, seven types of which have been described (Baskin et al., 2000; water gaps have so far not been described in three families with physical dormancy – the Curcurbitaceae, Rhamnaceae, and Sapindaceae). In legumes the water-gap is the lens (Fig. 1).

when germination will occur. Thus, understanding how timing of germination of seeds with physical dormancy is controlled in nature means determining the environmental conditions required for the water gap to open. As seeds with other kinds of dormancy have very specific temperature requirements for dormancy-break (Baskin & Baskin, 1998), it is logical that temperature should also be an important factor in breaking physical dormancy, and this certainly appears to be the case. For example, high and highly fluctuating temperatures promote dormancy break in impermeable seeds of Stylosanthes humilis and S. hamata (Fabaceae) during the hot, dry season in northern Australia (McKeon & Mott, 1982). Similarly a 15°C difference in amplitude of daily temperature fluctuations in an opening (gap) in a tropical rain forest in Vera Cruz, Mexico, promoted germination (67%) of seeds of Heliocarpus donnell-smithii (Malvaceae). Only 25% of the seeds germinated under the forest canopy, where the daily temperature fluctuation was 5°C (VazquezYanes & Orozco-Segovia, 1982). Furthermore, many species whose seeds have physical dormancy appear after fire in the habitat (e.g. Iliamna spp. (Malvaceae)), and exposure of their seeds to temperatures of ≥ 70°C result in high germination percentages (Baskin & Baskin, 1997).

How to open the water gap Water gaps are closed at seed maturity (Fig. 1b), and then open in response to an appropriate environmental signal. The water gap is dislodged, or in the case of the lens the macrosclereids pull apart (Fig. 1c), thereby creating an entry point for water into the seed. Once open, water gaps cannot close. Since opening of the water gap is necessary for seeds with physical dormancy to germinate, this event indirectly controls Box 1 Physical dormancy Species with water-impermeable seed (or fruit) coats – physical dormancy – occur in the following plant families (sensu APG, 1998; Baskin et al., 2000): • Anacardiaceae • Bixaceae • Cannaceae • Cistaceae • Cochlospermaceae • Convolvulaceae (including Cuscutaceae) • Curcurbitaceae • Dipterocarpaceae (subfamilies Montoideae and Pakaraimoideae, but not Dipterocarpoideae) • Fabaceae (subfamilies Caesalpinioideae, Mimosoideae and Papilionoideae) • Geraniaceae • Malvaceae (including Bombacacaceae, Sterculiaceae, and Tiliaceae) • Nelumbonaceae • Rhamnaceae • Sapindaceae • Sarcolaenaceae

Fig. 1 Sagittal sections of a stylized seed of a Papilionoid legume. (a) Whole seed. (b) Portion of the seed coat showing lens closed. (c) Portion of the seed coat showing lens open. Cl, cleft; Cu, cuticle; E, embryo; H, hilum; L, lens; M, micropyle; P, impermeable palisade layer of seed coat; RL, radicle lobe.


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New insights into breaking physical dormancy While temperature appears so important for breaking dormancy, the responses of seeds with physical dormancy to natural temperature regimes are not well understood. For example, how/why do seeds of some legumes in the temperate zone germinate only in spring? In a 2-yr germination phenology study on 14 herbaceous species of legumes, Van Assche et al. identified six species that germinated mainly in spring, and determined the temperatures required to break physical dormancy in their seeds. Fresh seeds of five of the six legumes mostly did not germinate at constant (5, 10, 23, 30°C) or at alternating (20/10°C) temperatures. Furthermore, few seeds of five of the species germinated when they were kept on moist filter paper at 5°C for 2 months (simulating winter) and were then transferred to 23°C. In the sixth species, Trifolium pratense, germination ranged from 16 to 28%, regardless of treatment or test condition. In an experiment on five of the spring-germinating species, seeds of four of them chilled at 5°C for 2 months germinated to higher percentages at alternating (15/6, 20/10°C) than at constant (10, 23°C) temperatures, and in three species more seeds germinated at 15/6 than at 20/10°C. Seeds of Trifolium pratense germinated equally well at 10, 15/6, and 20/10°C. Significantly, Van Assche et al. showed that most seeds remained impermeable at 5°C and became permeable only after being subjected to 15/6 and/or 20/10°C, or to 10°C for T. pratense. In another experiment, buried seeds of the 14 species were exposed to natural temperature regimes in Belgium, and at regular intervals for up to 28 months samples of each species were exhumed and tested for germination at constant (23°C) and at alternating (15/6, 20/10, 30/20°C) temperature regimes. The six spring-germinating species exhibited a peak of germination when exhumed in spring, but little or no germination occurred at other times of the year. The temperature regime simulating early spring (15/ 6°C) was optimal for germination for five of the six species, with 15/6 and 20/10°C being equally suitable for germination of the sixth. Depending on the species, little, or no, germination occurred at the constant temperature, regardless of the time of year seeds were tested.

Water gaps as environmental signal detectors Why was seed germination of the six species restricted to spring? In general, germination of exhumed seeds in spring decreased with an increase in the alternating temperature regime; thus, germination in the field in summer is prevented by high temperatures. However, this does not explain why seeds exhumed in summer and tested at 15/6 and 20/10°C failed to germinate. In detailed studies on Melilotus albus seeds, Van Assche et al. showed that seeds


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failed to germinate if they were chilled at 5°C and then held at 20°C for 1 month before being transferred to 15/6°C. However, seeds of M. albus chilled at 5°C for 2 months and then moved directly to 15/6°C germinated to high percentages. Thus, germination in spring requires that seeds be subjected to a sequence of two temperature regimes: 1. Chilling 2. Low alternating temperatures. If the two temperature regimes are separated by a period of relatively high constant temperature, seeds lose the ability to respond to low alternating spring temperatures. If seeds of these legumes are buried in soil and not exposed to alternating spring temperatures at the end of winter, they lose their ability to respond to low alternating temperatures. Therefore, although temperatures in late autumn are approx. 15/6°C, seeds would not be capable of responding to them and thus do not become permeable in autumn. Any seeds that fail to germinate in spring would be prevented from doing so until some following spring, after they are chilled and subsequently exposed to low alternating temperatures. Van Assche et al. found that 26–92% of the seeds of the six species were impermeable after 2.5 years of burial under natural temperature regimes.

Perspectives Under natural temperature regimes, buried seeds of the six legumes did not cycle between physical dormancy and nondormancy, but there was cycling with regard to ability of seeds to respond to the second phase (i.e. low alternating temperatures) of the dormancy-breaking requirement. These discoveries help explain how timing of germination of seeds with physical dormancy is controlled in nature in temperate zones. As in other species whose seeds have physical dormancy, the conditions required for opening of the water gap ‘fine-tune’ germination of the species to the habitat. In the spring-germinating legumes, the two-step temperature requirements for opening of the water gap allow this special anatomical structure to act as a signal detector not only for the arrival of spring, but also for the depth of seeds in the soil. Carol C. Baskin Department of Biology and Department of Agronomy, University of Kentucky, Lexington, KY 40506–0225, USA (tel +1859 2573996; fax +1859 2571717; email [email protected])

References Angiosperm Phylogeny Group (APG). 1998. An ordinal classification for families of flowering plants. Annals of the Missouri Botanical Garden 85: 531–553.

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Baskin JM, Baskin CC. 1997. Methods of breaking seed dormancy in the endangered species Iliamna corei (Sherff ) Sherff (Malvaceae), with special attention to heating. Natural Areas Journal 17: 313–323. Baskin CC, Baskin JM. 1998. Seeds. Ecology, biogeography, and evolution of dormancy and germination. San Diego, CA, USA: Academic Press. Baskin JM, Baskin CC, Li X. 2000. Taxonomy, anatomy and evolution of physical dormancy in seeds. Plant Species Biology 15: 139 –152. McKeon GM, Mott JJ. 1982. The effect of temperature on the field softening of hard seed of Stylosanthes humilis and S. hamata in a dry monsoonal climate. Australian Journal of Agricultural Research 33: 75 – 85. Rolston MP. 1978. Water impermeable seed dormancy. The Botanical Review 44: 365 – 396.

Van Assche JA, Debucquoy KLA, Rommens WAF. 2003. Seasonal cycles in the germination capacity of buried seeds of some Leguminosae (Fabaceae). New Phytologist 158: 315 – 323. Vazquez-Yanes C, Orozco-Segovia A. 1982. Seed germination of a tropical rain forest pioneer tree (Heliocarpus donnell-smithii ) in response to diurnal fluctuation of temperature. Physiologia Plantarum 56: 295 – 298. Werker E. 1980– 81. Seed dormancy as explained by the anatomy of embryo envelops. Israel Journal of Botany 29: 22 – 44. Key words: dormancy, seed germination, water gap, legumes, physical dormancy.

Letters Reproductive success by unusual growth of pollen tubes to ovules Sexual reproduction in flowering plants depends on successful transfer of pollen that encloses male gametes from anthers to pistils. The pollen tube that carries male gametes must find its way to the ovule, which houses the female gametes, to achieve fertilization. While much attention has been paid to the pollination process in attempts to understand flower diversity and plant–pollinator interactions, a recent finding in arrowhead Sagittaria (Alismataceae) – a mechanism of reallocation of pollen tubes described by Wang et al. (2002) – highlights just how little we know about the fate of pollen in postpollination events. Nevertheless, it does appear that unusual, diverse growth pathways of pollen tubes may, potentially, provide a supplementary means of reproductive assurance under conditions of unpredictable pollination.

2001; Higashiyama et al., 2001; Wheeler et al., 2001). Once an ovule has been fertilized, the other pollen tubes usually stop growing towards it (Cheung & Wu, 2001). This pathway of pollen tube growth characterizes almost all observed plant species, but exceptions do exist. Some redundant pollen tubes reaching an ovary in the apocarpous gynoecium of Sagittaria potamogetifolia could grow through the base of the ovary and the receptacle tissue into adjacent unfertilized ovules (Fig. 1). Wang et al. (2002) harvested more than 10 achenes after applying approx. 30 pollen grains to one stigma of this aquatic

Postpollination events in Sagittaria When a pollen grain lands on a compatible stigmatic surface, it germinates and extrudes a pollen tube. In general, the pollen tube elongates within the transmitting tissue in the style, eventually reaching the ovary, where it enters an ovule and penetrates the embryo sac, and then releases the sperm cells for fertilization (Cheung, 1995). Pollen tube growth has been used as a model system to study signal transduction in plants (Cheung & Wu, 2001; Herrero,

Fig. 1 Unusual pollen tube growth in the apocarpous gynoecium of Sagittaria potamogetifolia (Alismataceae). The longitudinal section through the pistillate flower of this aquatic plant shows redundant pollen tubes from one pistil entering adjacent unfertilized ovules. The diagram is courtesy of Xiao-Fan Wang.


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monoecious herb in which one pistil has one ovule, confirming that pollen tubes enter neighbouring ovules when pollen is deposited on a single stigma. The first report of such intercarpellary growth of pollen tubes was in Illicium (Illiciaceae) by Williams et al. (1993), which was ignored by Wang et al. (2002). Both independent observations found that pollen tube growth between ovules in apocarpous angiosperms enhanced reproduction. This strategy of unusual pollen tube growth may provide supplementary reproductive assurance in plants that experience inadequate pollination. Seed production is often limited by pollen because of the stochastic nature of pollinator services (Burd, 1994). Plants have developed diverse strategies to enhance pollination, such as prolongation of floral longevity, or increased attractiveness to pollinators by floral advertisements and rewards. Recent studies have demonstrated that diverse floral designs function principally to facilitate effective pollen transfer (Barrett, 2002). However, pollen is usually deposited unequally on the multiple stigmas of a gynoecium (Carr & Carr, 1961). For example, the pollen load ranged from 0 to 60 grains per stigma in apocarpous Liriodendron chinense (Magnoliaceae) in which some unpollinated pistils do not set seeds (Huang & Guo, 2002). The development of syncarpy with associated stigmas is a major innovation in angiosperms (Mulcahy, 1979; Endress, 1982), which may permit pollen tubes to cross between carpels and increase pollen competition during pollen tube growth in the pistils (Carr & Carr, 1961; Mulcahy, 1979; Endress, 1982; Williams et al., 1993; Armbruster et al., 2002). An early observation in Daucus carota (Apiaceae) showed that pollen tubes growing through either style could cross over and eventually fertilize either ovule (Borthwick, 1931). This phenomenon was observed in another genus, Lomatium (Apiaceae), and seems common in syncarpous flowers with separate styles (M. Schlessman, unpublished). The main advantage of fused carpels relates to offspring quality, increasing the intensity of pollen competition (Mulcahy, 1979; Endress, 1982; Armbruster et al., 2002). Pollen tube growth involving a long pathway has been observed in Dalechampia (Euphorbiaceae), in which species have expanded stigmatic surfaces. When pollen grains land on the lateral stigmatic surfaces, pollen tubes grow first to the stylar lip, bend 180 degrees, and then grow through the style to the ovules (Armbruster et al., 1995). Despite these potential advantages and the prevalence of syncarpy, reverse transitions from syncarpy to apocarpy do occur in angiosperms (Stebbins, 1974; Armbruster et al., 2002). Theoretical analyses suggest that the repeated evolution of fused carpels is influenced by pollination dynamics (Armbruster et al., 2002). In addition, a comparative analysis suggests that polycarpic plants seem no more likely to be pollen limited than monocarpic plants (Larson & Barrett, 2000).


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Evolutionary strategies It will be interesting to know how many angiosperms show such intercarpellary growth of pollen tubes, and how this phenomenon has evolved. Given that this pollen tube behaviour promotes fertility under conditions of unpredictable pollination, why do many plants seem not to employ this mechanism when pollen limits plant fecundity in various taxa? An alternative solution to the problem of inadequate pollination in plants is the shift to selfpollination (Baker, 1955; Wyatt, 1988; Schoen et al., 1996), which may have occurred in the genus Sagittaria, which is basically monoecious. The retention of functional stamens within perfect flowers of S. guyanensis, an andromonoecious species, could be selected for to allow self-fertilization in cases of inadequate pollination (Huang, 2003). This poses an interesting evolutionary question: how have the different strategies to diminish pollen limitation evolved in Sagittaria? In cleistogamous flowers of the Malpighiaceae pollen tubes reach ovules by growing through the filament into the receptacle from the indehiscent anther (Anderson, 1980). Many chasmogamous flowers experiencing loss of pollinators in unfavorable habitats may adopt this means to achieve fertilization, although this has been little studied. For example, in the underwater pollen tubes of the insect-pollinated Ranalisma rostratum (Alismataceae) achieve fertilizations by growing from indehiscent anthers to reach stigma surfaces (Wang et al., 1993). Another unusual pattern of pollen tube growth was observed in the monoecious Callitriche (Callitrichaceae), an aquatic genus. In underwater conditions, pollen grains germinate in the indehiscent staminate flower. Pollen tubes grow down the filament, and through vegetative tissue across to the pistillate flower, and enter the ovary from the base (Philbrick, 1984). These observations of unusual pollen tube growth in both dicotyledons and monocotyledons, although largely unexplored, suggest diverse means of achieving reproductive assurance during pollination. Pollen tubes growing through various different tissues to target ovules also provide natural cases to support the recent experimental observation that pollen tube growth is guided by a signal derived from the synergid cell of unfertilized ovules (Cheung & Wu, 2001; Higashiyama et al., 2001).

Acknowledgements The author wishes to thank Mark Schlessman for providing unpublished data and, as well as Amots Dafni, for discussion; Spencer Barrett, Scott Armbruster and Lynda Delph for their valuable comments; Sarah Corbet for correcting English and providing helpful suggestions on an earlier draft of the manuscript; and three anonymous reviewers for their improvements to the manuscript. The

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research was supported by the National Science Foundation of China (Grant no. 30070054). Shuang-Quan Huang College of Life Sciences, Wuhan University, Wuhan 430072, China (email [email protected])

References Anderson WR. 1980. Cryptic self-fertilization in the Malpighiaceae. Science 207: 892–893. Armbruster WS, Debevec EM, Willson MF. 2002. Evolution of syncarpy in angiosperms: theoretical and phylogenetic analyses of the effects of carpel fusion on offspring quantity and quality. Journal of Evolutionary Biology 15: 657– 672. Armbruster WS, Martin P, Kidd J, Stafford R, Gogers DG. 1995. Reproductive significance of indirect pollen-tube growth in Dalechampia (Euphorbiaceae). American Journal of Botany 82: 51–56. Baker HG. 1955. Self-compatibility and establishment after ‘long distance’ dispersal. Evolution 9: 347–348. Barrett SCH. 2002. The evolution of plant sexual diversity. Nature Reviews Genetics 3: 274 –284. Borthwick HA. 1931. Development of the macrogametophyte and embryo of Daucus carota. Botanical Gazette 92: 23 – 44. Burd M. 1994. Bateman’s principle and reproduction: the role of pollen limitation in fruit and seed set. Botanical Review 60: 83 –139. Carr SGM, Carr DJ. 1961. The functional significance of syncarpy. Phytomorphology 11: 249–256. Cheung AY. 1995. Pollen–pistil interactions in compatible pollination. Proceedings of the National Academy of Sciences, USA 92: 3077–3080. Cheung AY, Wu H. 2001. Pollen tube guidance: right on target. Science 293: 1441–1442. Endress PK. 1982. Syncarpy and alternative modes of escaping disadvantages of apocarpy in primitive angiosperms. Taxon 31: 48 –52. Herrero M. 2001. Ovary signals for directional pollen tube growth. Sexual Plant Reproduction 14: 3 –7. Higashiyama T, Yabe S, Sasaki N, Nishimura Y, Miyagishima S,

Kuroiwa H, Kuroiwa T. 2001. Pollen tube attraction by the synergid cell. Science 293: 1480 –1483. Huang S-Q. 2003. Flower dimorphism and the maintenance of andromonoecy in Sagittaria guyanensis subsp. lappula (Alismataceae). New Phytologist 157: 357–364. Huang S-Q, Guo Y-H. 2002. Variation of pollination and resource limitation in a low seed-set tree, Liriodendron chinense (Magnoliaceae). Botanical Journal of the Linnean Society 140: 31–38. Larson BMH, Barrett SCH. 2000. A comparative analysis of pollen limitation in flowering plants. Biological Journal of the Linnean Society 69: 503–520. Mulcahy DL. 1979. The rise of the angiosperms, a genecological factor. Science 206: 20 –23. Philbrick CT. 1984. Pollen tube growth within vegetative tissues of Callitriche (Callitrichaceae). American Journal of Botany 71: 882–886. Schoen DJ, Morgan MT, Bataillon T. 1996. How does self-pollination evolve? Inferences from floral ecology and molecular genetic variation. Philosophical Transactions of the Royal Society of London, Series B 351: 1281–1290. Stebbins GL. 1974. Flowering plants. Evolution above the species level. Cambridge, MA, USA: The Belknap Press of Harvard University Press. Wang X-F, Tao Y-B, Lu Y-T. 2002. Pollen tubes enter neighbouring ovules by way of receptacle tissue, resulting in increased fruit-set in Sagittaria potamogetifolia Merr. Annals of Botany 89: 791–796. Wang J-B, Wang X-F, Chen J-K, Wang H-Q, Li Y-Q. 1993. A preliminary study of reproductive traits in Ranalisma rostratum (Alismataceae). Journal of Wuhan University (Natural Science Edition) 39: 130 –132. Wheeler MJ, Franklin-Tong VE, Franklin FCH. 2001. The molecular and genetic basis of pollen–pistil interactions. New Phytologist 151: 565–584. Williams EG, Sage TL, Thien LB. 1993. Functional syncarpy by intercarpellary growth of pollen tubes in a primitive apocarpous angiosperm, Illicium floridanum (Illiciaceae). American Journal of Botany 80: 137–142. Wyatt R. 1988. Phylogenetic aspects of the evolution of self-pollination. In: Gottlieb LD, Jain SK, eds. Plant evolutionary biology. London, UK: Chapman & Hall, 109–131. Key words: apocarpy, pollen tube growth, postpollination events, reproductive assurance, syncarpy. Meetings 158


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Meetings Exploring plant–microbe interactions using DNA microarrays Functional genomics of plant–microbe interactions – the 10th New Phytologist Symposium, Nancy, France, October 2002 Functional genomics, facilitated by DNA microarray technology, has vast potential for our understanding of plant– microbe systems. But how useful are the data when there is limited genomic information and the organisms cannot yet be genetically manipulated? During the 10th New Phytologist Symposium in Nancy, France, the potential and the problems associated with using DNA microarray technology for studying the molecular background of plant–microbe interactions were discussed.

‘There is a danger that microarray experiments will lead to a vast accumulation of data that cannot be meaningfully interpreted’

The main application of the DNA microarray technique has so far been in the analysis of gene expression in model organisms such as Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Caenorhabiditis elegans, simply because their complete genome sequences are available. However, DNA microarray technology is now rapidly being applied to studies of other organisms, including plant pathogens and symbionts (Martin, 2001). Complete genome sequences are available for a number of important bacterial pathogens and symbionts and genome sequencing of several fungi is under way. In the absence of fully sequenced genomes, information from large sets of expressed sequence tags (ESTs) is well suited for constructing cDNA arrays. Genome sequences are also becoming available for several plant hosts including rice, legumes and poplar. There is, clearly, great potential in applying DNA microarray technology to examining plant–microbe systems, as this will substantially increase our knowledge of the genetic background behind these interactions. However, concerns have been raised about the usefulness of the data obtained from microarray experiments in organisms for which there is limited genomic information and that cannot be genetically manipulated. To what extent can the complex and large data sets generated from microarray experiments in nonmodel organisms be meaningfully interpreted and validated? How can microarray data from different experiments and labs be compared?

Expression profiles The power of DNA microarray technology Since their introduction in the mid-1990s, DNA microarrays (Box 1) have become one of the major tools in functional genomics for exploring the genome-wide patterns of gene expression in an organism (Colebatch et al., 2002a).

Box 1

The first demonstrations of the applicability of the DNA array technique for monitoring the gene expression of plant– microbe interactions originate from studies on defence reactions in Arabidopsis. Schenk et al. (2000) analysed the expression of genes in Arabidopsis either infected by the incompatible fungal pathogen Alternaria brassicicola or treated with the defence-related

The DNA array technique

The DNA array technique is in principle very simple. Thousands of DNA sequences (typically presynthesized oligonucleotides or inserts from cDNA libraries) are printed onto glass slides or nylon sheets using a robotic arrayer. To compare the abundance of these genes in a sample, RNA or DNA is extracted (the ‘target’), labelled and hybridized to the arrayed DNA (the ‘probe’). After washing, the probe is detected by fluorescence scanning or phosphor imaging. The primary data in microarray experiments consist of scans of the array (images). The spots on the images are quantified and the intensities are normalized. The final step is to identify genes that are significantly up- or down-regulated and to identify clusters of coregulated genes (regulons). The rationale behind the approach is that genes displaying similarity in expression pattern might be functionally related and governed by the same genetic control mechanism (Brown & Botstein, 1999).


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signalling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene. Analysis of the expression data obtained indicated the existence of a considerable network of regulatory interactions and coordination of signalling pathways, which had not been observed previously when analysing a few genes at a time. Maleck et al. (2000) monitored changes in gene expression induced during the systemic acquired resistance response (SAR) in Arabidopsis. Various levels of the SAR response were observed following chemical treatment and when analysing mutants with a constitutive or repressed SAR phenotype. Groups of genes with common regulatory patterns were identified. In addition, a common promotor element in one of the regulons was identified by searching for binding motifs in the upstream regions of the predicted translation start sites. During the meeting in Nancy, Nikolaus Schlaich (RWTH-BioIII Pflanzenphysiologie, Aachen, Germany) reported on a study in which cDNA arrays were being used to examine changes in the metabolism of Arabidopsis during infection with the bacterial plant pathogen Pseudomonas syringae pv. tomato. Based on the patterns of genes expressed during infection, it was possible to identify major shifts in the metabolism of the host (Scheideler et al., 2002). In addition, Laurent Zimmerli (Department of Plant Biology, Stanford University, CA, USA) presented data from recent experiments comparing the expression of genes in Arabidopsis leaves infected by compatible and incompatible powdery mildew species. As a result of the ever-increasing rate at which genomes and ESTs are being sequenced (Tunlid & Talbot, 2002), the DNA array technique is now rapidly being applied to studies of a number of parasitic and symbiotic microorganisms and their corresponding host plants. For example, arrays have been constructed to examine the interactions between arbuscular mycorrhizal (AM) fungi and legumes (Martin Parniske, John Innes Centre, Norwich, UK; Philipp Franken, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany; Maria Harrison, The Samuel Roberts Noble Foundation, Ardmore, OK, USA; Franken & Requena (2001)), nitrogen-fixing bacteria and legumes (Michael Udvardi, Max Planck Institute of Molecular Plant Physiology, Golm, Germany; Colebatch et al. (2002b); Sprent (2002)), ectomycorrhizal fungi and trees (Sébastien Duplessis, INRA, Nancy, France; Voiblet et al. (2002); Anders Tunlid, Department of Microbial Ecology, Lund, Sweden), parasitic nematodes and plants (Pierre Abad, INRA, Antibes Cedex, France), and between pathogenic fungi and host plants (Regine Kahmann, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany). Notably, sequences (probes) have been obtained in several of these projects from both the microbe and the host, which will allow direct examination of the interaction between the pathogen/symbiont and the host at the transcript level. The application of microarray technology to these plants and microorganisms will provide a considerable amount of

new data for the scientific community. Concerns were expressed that the characterization of gene expression patterns in these organisms will be much less valuable than those of model organisms such as Arabidopsis and S. cerevisiae. The interpretation of the data obtained from DNA array studies in these organisms is, to a large extent, dependent on our knowledge of the many genes that have been characterized by the classical methods of genetics, molecular biology and biochemistry. Corresponding data are lacking for most parasitic and symbiotic microorganisms and their host plants. However, the rate of evolution of many genes with respect to both sequence and function has been so slow that characterization in one organism can suffice for many or all. For example, even when sequences from such evolutionarily distant organisms as S. cerevisiae and C. elegans are compared, the function of 20% of the genes encoded by the nematode could be indicated by knowing the function of the yeast orthologue (Chervitz et al., 1998). This suggests that clusters of coregulated genes identified in DNA array experiments of plant–microbe interactions will, in many cases, contain at least some genes that encode proteins with orthologues that have been functionally characterized in other organisms including one or more of the models (Brown & Botstein, 1999). In many cases this information can serve as a starting point for generating new hypotheses for the mechanisms of pathogenesis and symbiosis. There are several other ways in which investigators applying the DNA microarray technique to plant–microbe systems can benefit from the efforts of those working with model organisms.

Design of microarray experiments DNA microarray experiments are costly in terms of equipment, consumables and time. Therefore, it is important that the experiments be carefully planned and executed. Well-designed array experiments improve the quality and reliability of the data (Yang & Speed, 2002). The community of scientists working on functional genomics in Arabidopsis have devoted substantial efforts to determining the best practice for DNA array experiments. Experiments have shown that careful probe selection, physical design of the array, and experimental design, including the number of biological replicates, can have a considerable impact on the quality of the microarray data obtained. However, the best methods of scanning, extraction, normalization and data analysis have not yet been determined. Until this is done, it is recommended that each microarray experiment be run through a series of quality tests (Finkelstein et al., 2002). Further information can be found on the homepages of the Arabidopsis Information Resource (TAIR, http:// www.arabidopsis.org) and the Genomic Arabidopsis Resource Network (GARNET, http://www2.york.ac.uk/res/ garnet/garnet.htm).


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Validation of results Validation of DNA array results using other techniques is critical for establishing the biological significance of the data. Expression levels of key genes identified in array analysis should be confirmed using RT-PCR or Northern blots. In many cases the function of these genes cannot be inferred by sequence similarities to well-characterized genes, and their function must be examined by genetic and molecular methods. Although there are many plant symbionts and pathogens that cannot be genetically manipulated, there are a few that can be transformed to generate knockout and conditional mutants. Analysis of such mutants can provide important information on gene function. In addition, DNA array analysis in organisms exhibiting a loss of function mutation or over-expression of a transcription factor or genes involved in signalling pathways may result in the identification of downstream genes. Such analyses are now under way in the corn smut fungus Ustilago maydis (Regine Kahmann, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany). Furthermore, it should be remembered that expression profiling using DNA arrays only assays functionality in an indirect way. mRNA molecules are just transmitters of the instructions for synthesizing proteins, while it is proteins and metabolites that are the functional entities in the cell. Extensive efforts have been devoted to developing methods for analysing the proteome and metabolome in model organisms, but there are still major technical and conceptual difficulties in analysing these ‘omes’ (Oliver, 2002). Michael Udvardi (Max Planck Institute of Molecular Plant Physiology, Golm, Germany) presented data showing how metabolome analysis using GC-MS has been used to profile the metabolites in nitrogen-fixing nodules in legumes and to follow leads on potentially new aspects of metabolism uncovered by DNA array analysis. In addition, this group is using proteome analysis to identify nutrient transporters in the symbiosome membrane which separates the rhizobia from the plant cell cytoplasm.

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transcriptome data, so that they can apply the normalization procedure that is most appropriate for the biological problem that they are studying (Oliver, 2002). Therefore, current work towards defining international standards for depositing microarray results in public databases is very welcome (Brazma et al., 2001) (http://www.mged.org).

Conclusion The DNA microarray technique was developed for gene expression profiling in model organisms with complete genome sequences, but is now being widely applied in studies of other organisms including important plant pathogens, symbionts and their hosts. Exciting new information will be gained from these studies, including broadened knowledge on the molecular background of plant–microbe interactions, and the gap in information on gene function between these organisms and the currently favoured model organisms will narrow. However, there is a danger that microarray experiments may lead to a vast accumulation of data that cannot be meaningfully interpreted. For this reason, DNA microarray experiments should be carefully designed, array data must be validated using other techniques, and the expression data should be made available in databases with public access.

Acknowledgements The 10th New Phytologist Symposium, ‘Functional genomics of plant–microbe interactions’ in Nancy, 23–25 October 2002 was sponsored by the New Phytologist Trust, INRA and The Noble Foundation. The workshop participants are grateful to the organizers Francis Martin, Maria Harrison, Nicholas Talbot and Jonathan Ingram. Anders Tunlid Department of Microbial Ecology, Lund University, Ecology Building, SE-223 62 Lund, Sweden (email [email protected])

Sharing microarray data There will soon be a need to share DNA microarray data between research groups working on plant–microbe interactions. There are several reasons for this. One is that most array experiments will identify dozens (if not hundreds) of genes that are differentially regulated and only a few of them can be studied in detail by one laboratory. Another reason is that a common database on transcript profiles from a number of different experiments and plant– microbe systems can function as a ‘compendium’ for comparative studies on gene expression in different species, tissues, treatments and growth conditions. For such analysis it is essential that researchers have access to each other’s raw


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Botstein D. 1998. Comparison of the complete protein sets of worm and yeast: orthology and divergence. Science 282: 2022–2028. Colebatch G, Kloska S, Trevaskis B, Freund S, Altmann T, Udvardi MK. 2002. Novel aspects of symbiotic nitrogen fixation uncovered by transcript profiling with cDNA arrays. Molecular Plant–Microbe Interactions 15: 411– 420. Colebatch G, Trevaskis B, Poyser SJ. 2002a. Functional genomics: tools of the trade. New Phytologist 153: 27– 36. Colebatch G, Trevaskis B, Poyser SJ. 2002b. Symbiotic nitrogen fixation in the postgenomics era. New Phytologist 153: 37– 42. Finkelstein D, Ewing R, Gollub J, Sterky F, Cherry JM, Somerville S. 2002. Microarray data quality analysis: lessons from the AFGC project. Plant Molecular Biology 48: 119 – 131. Franken P, Requena N. 2001. Analysis of gene expression in arbuscular mycorrhizas: new approaches and challenges. New Phytologist 150: 517–523. Maleck K, Levine A, Eulgem T, Morgan A, Schmid J, Lawton KA, Dangl JL, Dietrich RA. 2000. The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nature Genetics 26: 403 – 410. Martin F. 2001. Frontiers in molecular mycorrhizal research – genes, loci, dots and spins. New Phytologist 150: 499 –507.

Oliver SG. 2002. Functional genomics: lessons from yeast. Philosophical Transactions of the Royal Society of London B 357: 17–23. Scheideler M, Schlaich NL, Fellenberg K, Beissbarth T, Hauser NC, Vingron M, Slusarenko AJ, Hoheisel JD. 2002. Monitoring the switch from housekeeping to pathogen defense metabolism in Arabidopsis thaliana using cDNA arrays. Journal of Biological Chemistry 277: 10555 –10561. Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM. 2000. Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proceedings of the National Academy of Sciences, USA 97: 11655 –11660. Sprent J. 2002. Knobs, knots and nodules – the renaissance in legume symbiosis research. New Phytologist 153: 2 – 6. Tunlid A, Talbot NJ. 2002. Genomics of parasitic and symbiotic fungi. Current Opinion in Microbiology 5: 513 –519. Voiblet C, Duplessis S, Encelot N, Martin F. 2001. Identification of symbiosis-regulated genes in Eucalyptus globulus – Pisolithus tinctorius ectomycorrhiza by differential hybridisation of arrayed cDNAs. Plant Journal 25: 181–191. Yang YH, Speed T. 2002. Design issues for cDNA microarray experiments. Nature Reviews Genetics 3: 579 –588. Key words: Microarrays, DNA arrays, functional genomics, plant–microbe interactions, mycorrhizas.
