Forum
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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228 Forum
Commentary
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
www.newphytologist.com © New Phytologist (2003) 158: 227 – 238
Commentary
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.
© New Phytologist (2003) 158: 227 – 238 www.newphytologist.com
Forum
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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Commentary
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.
www.newphytologist.com © New Phytologist (2003) 158: 227 – 238
Commentary
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
© New Phytologist (2003) 158: 227 – 238 www.newphytologist.com
Forum
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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Letters
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.
www.newphytologist.com © New Phytologist (2003) 158: 227 – 238
Letters
Letters
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).
© New Phytologist (2003) 158: 227 – 238 www.newphytologist.com
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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.
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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).
© New Phytologist (2003) 158: 227 – 238 www.newphytologist.com
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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).
www.newphytologist.com © New Phytologist (2003) 158: 227 – 238
Meetings
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
© New Phytologist (2003) 158: 227 – 238 www.newphytologist.com
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