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Plant Physiol, December 2001, Vol. 127, pp. 1508-1512
UPDATE ON PARASITIC PLANTS
Dancing Together. Social Controls in Parasitic Plant
Development
W. John
Keyes,
Jeannette V.
Taylor,
Robert P.
Apkarian, and
David
G.
Lynn*
Departments of Chemistry and Biology, Integrated Microscopy and
Microanalytical Facility, Emory University, Atlanta, Georgia
30322
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INTRODUCTION |
In contrast to animals, the basic
body plan of plants develops largely postembryonically and is directed
by two primary meristems located on opposite ends of a bipolar embryo
(Jürgens, 2001 ). The basal root meristem serves to extend the
primary root established in the embryo, whereas the apical shoot
meristem both maintains growth and provides a source of cells for new
organs such as leaves and flowers. In accordance, the shoot organ is
more complex, with new primordia forming in an established pattern,
whereas the root apex is streamlined and utilitarian, consisting of
long continuous files of developing cells radiating from the quiescent
center. In both organs, cell division is arrested and cell fate
established within the space of a few cell layers; cellular destiny is
precisely defined by geographical position. Cell-cell contacts,
"social controls," operating within the meristem appear to be most
critical for establishing the cellular pattern. Cell
ablation/regeneration experiments in the root have implicated direct
cell-cell surface contacts as critical for cell fate (Van den Berg et
al., 1995, 1997), and local auxin gradients appear necessary for the
differentiation of new organs in the shoot (Reinhardt et al.,
2000).
In the parasitic angiosperms, the root meristem can also give rise to
new organs (Kuijt, 1969). In Striga asiatica
(Scrophulariaceae), vegetative growth of the root meristem is aborted
on host contact and the tissue becomes committed to development of
the host attachment organ. This new organ, the haustorium, consists of
a host attachment structure, a heavily vascularized infection peg, and
thin filaments that infiltrate the host vascular bundles. The basal
meristems of these parasites therefore have acquired a capability
normally restricted to the shoot apex. Developmental checkpoints, which in this case exist at multiple stages of host/parasite integration, are
only traversed with specific host cell-derived cues. Successful attachment is achieved by the parasite's ability to read and respond to host cells, effectively allowing them into their developmental social circle. The expression "dancing together" then arises from the fact that the social controls on plant cellular development can be
experimentally interrogated between the distinct cell populations of
two separate organisms.
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WHAT ARE PARASITIC ANGIOSPERMS? |
It has been estimated that 1% of all flowering plants are
parasitic. In most cases, this parasitism is not obvious. For common eyebright, Indian paintbrush (Castilleja coccinea), gerardia
(Agalimis purpurea), and even witchweed (S. asiatica), the only distinguishing feature is the
subterranean attachment to host plants. As shown in Figure
1, the cellular anatomy of the
S. asiatica root meristem differs little from
that of other small seeds such as Arabidopsis (Dolan et al., 1993).
Longitudinal sections of the 1-d-old S. asiatica
seedling reveal an even more simplified architecture containing a
relatively small number of cylindrical layers (Fig. 1A). The most
remarkable feature is the apparent absence of a root cap, the meristem
surface being covered by the developing epidermal cells (Fig. 1B). The
cellular density of the inner stele also appears much reduced because
cell vacuoles emerge very early in the central zone. A direct
assignment of each individual developing file cannot be made with
certainty. In addition, a quiescent center is not readily apparent
without a thorough mitotic analysis.
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Figure 1.
The cellular structure of the S. asiatica seedling. A, Longitudinal section of a 12-h-old
seedling, stained with toluidine blue, showing the radicle and root
meristem. The shoot meristem does not emerge until contact with
the host is established; the shoot meristem and cotyledons are still
enclosed within the dark-staining seed coat. B, Enlargement of the root
meristem in A. The simple cellular structure is apparent in the small
number of files, each with a comparable level of vacuolarization. C,
Longitudinal section of the 20-h-old haustorium. The first 12 to 14 cells along each file swell radially. The arrows indicate the
haustorial hairs emerging from the epidermal layer. Bars = 50 µm.
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WHAT SIGNALS HOST COMMITMENT? |
The breakage of seed dormancy in S. asiatica
is dependent on small, diffusing, host recognition molecules, or
xenognosins, originating from the roots of its monocotyledonous hosts.
The agronomic impact of this parasite on grain production in
sub-Saharan Africa, as well as its spread to the southeastern United
States, has resulted in extensive efforts to identify these molecular signals. Several xenognostic germination signals have been discovered (Boone and Lynn, 1995) that in some cases are able to define precise chemical potential gradients around the host roots (Fate and Lynn, 1996). The breakage of dormancy is the most critical step in this commitment, starting a viability clock that runs only a few days without host attachment.
The second level of host integration, the development of the
host-parasite attachment organ (Fig. 1C), was naturally assumed to be
dependent on similar diffusible xenognosins (Riopel, 1979). Haustorial
development is not initiated when isolated seedlings are grown in
culture, and screens of commercially available plant exudates suggested
that small molecules were both necessary and sufficient for induction
of haustorial development. The identified xenognosins were unlike any
plant hormone known to mediate developmental commitments (Lynn et al.,
1981), and surprisingly were not found to be present in host root
exudates. Active compounds did appear after host wounding, leading to
the proposal that the parasite released oxidative enzymes, similar to
cutinase exudation by Fusarium solani
during haustorial penetration (Shaykh et al., 1977), to liberate
xenognosins from the host cell surface (Chang and Lynn, 1986). This
hypothesis was tested directly by screening for enzymatic activity.
Young S. asiatica seedlings were found, however,
to be particularly deficient in cell wall-localized oxidative enzyme activity at all points during xenognosis (Kim et al., 1998).
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WITHOUT EXUDED ENZYMES, HOW ARE THE XENOGNOSINS RELEASED FROM HOST
CELLS? |
The answer initially came from studies with redox-sensitive
probes, and later fluorescein diacetate, suggesting that the
S. asiatica root meristem constitutively produces
hydrogen peroxide (H2O2).
Exogenous applications of the enzyme catalase removes extracellular
H2O2, and blocks the
haustorial inducing activity of both isolated host cell wall
preparations and simple phenolic inducers (Kim et al., 1998).
Therefore, extracellular
H2O2 is necessary and can
be limiting in haustorial induction.
The epidermal cells of the S. asiatica meristem
constitutively produce
H2O2. As shown in Figure
2, oxidation of
CeCl3 occurs specifically within the apoplasmic
space of the cells along the surface of the meristem (Keyes et al.,
2000; W.J. Keyes, unpublished data). Conceptually analogous with radar
detection, a chemical potential gradient of
H2O2 would be maintained at
the root meristem and released from parasite epidermal cells that are
low in peroxidases and cell wall phenols to exploit host epidermal
cells rich in both (Fig. 3A). Upon
contact with host cell surfaces, the
H2O2 concentration serves
as a peroxidase cosubstrate, oxidatively releasing simple benzoquinone
xenognosins from host cell walls and creating a comparable benzoquinone
potential gradient radiating from the host cells (Fig. 3B). This
mechanism may be general; benzoquinone xenognosins are known to induce
haustorial development in many parasites (Lynn and Chang, 1990;
Matvienko et al., 2001). In Figure 3B, benzoquinone release is shown in
a second step downstream of the probing process, but these processes
occur simultaneously, together providing the continuous cross feeding
of benzoquinone over the extended time period necessary to ensure
commitment to haustoriogenesis (Smith et al., 1990).
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Figure 2.
Localization of
H2O2 accumulation in the
seedling tissue. Twelve-hour-old S. asiatica
seedlings were incubated in freshly prepared 5 mM
CeCl3 and 0.1 mM KCl for
2 h and fixed in 1.25% (v/v) glutaraldehyde/1.25% (v/v)
paraformaldehyde in 50 mM sodium cacodylate (CAB)
buffer for 1 h. After two 10-min washes in 50 mM
CAB buffer, the seedlings were dehydrated in a graded ethanol series
and exposed twice to propylene oxide. The fixed seedlings were embedded
in epoxy plastic, thin sectioned (70-80 nm), and observed with a
JEM-1210 transmission electron microscope (JEOL, Tokyo) at 80 kV. Electron-dense deposits of cerium perhydroxides accumulated at
interstitial locations between epidermal cells specifically in the
meristematic zone. The apoplastic junction between two cells along the
meristem surface are shown with cerium perhydroxides deposits (arrows).
Bar = 200 nm.
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Figure 3.
Proposed mechanism for the induction of haustorial
development. A, The growing S. asiatica seedling
produces H2O2 at the root
meristem, giving rise to a localized chemical potential gradient (red)
diffusing into the solution.
H2O2 is necessary and
generally the limiting substrate in peroxidase oxidation of cell
wall-localized phenolics, such as sinapic acid, into benzoquinones (Kim
et al., 1998). B, The accumulating quinones establish a similar
chemical gradient (blue), diffusing back to the parasite seedling and
signaling haustoriogenesis. Radial swelling and hair formation occur,
leading ultimately to the formation of the functional attachment organ
within 18 to 20 h.
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WILL THE CONSTITUTIVE RELEASE OF H2O2
IMPACT OTHER CELLULAR PROCESSES? |
Production of reactive oxygen species in plants is well documented
and a key component in plant defensive responses (Lamb and Dixon,
1997). H2O2 is a major
product of the oxidative burst, and its presence may be both directly
toxic to invading organisms and/or contribute to structural
reinforcement of the plant cell wall via cross-linking reactions
catalyzed by wall peroxidases. Here, curiously, the invading organism
exudes H2O2, certainly not
the best camouflage for a parasite, particularly considering that
reactive oxygen species generally activate systemic resistance in
plants. However, only low levels of
H2O2 accumulate alongside host cells. In normal defense responses,
H2O2 localized to the apoplast is consumed by wall peroxidases in both cross-linking reactions with pectic-associated phenols and in oxidative cleavage of
these phenols (Chang and Lynn, 1990; Kim et al., 1998). Under these
conditions, it might be expected that little
H2O2 would access the host
cell cytoplasm to activate counter resistance. What about the parasite
cells? Exogenously added fluorescein diacetate requires cytoplasmic
esterases to liberate the free phenol for oxidation, and allowed us to
stain the cytoplasm of the parasite meristem cells (Keyes et al.,
2000). This staining pattern is not altered by exogenous catalase
treatment. Therefore, at least part of the
H2O2 is produced
cytoplasmically in the parasite meristem, making it necessary to
consider how these cells cope with constitutive H2O2 production. It will be
equally important to consider how the host cells experience the
gradient of the benzoquinones.
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HOW ARE THE BENZOQUINONES PERCEIVED? |
At the host/parasite interface, which consists of a surface
contact between the epidermal cells of both organisms, the host cells
cross feed simple benzoquinones from their wall to the parasite cells
(Fig. 3B). Receptors for the benzoquinone xenognosins have not yet been
identified, but the available data suggest that they must function in a
series of one-electron benzoquinone oxidoreductions (Smith et al.,
1996). Terminal commitment to the host attachment organ occurs only
after several hours of xenognosin exposure, consistent with a
time-dependent accumulation of the necessary molecular components.
Without sufficient exposure time, the meristem reverts to vegetative
growth (Smith et al., 1990, 1996). It could be argued that this
exposure time requirement prior to terminal commitment improves the
probability of attaching to a viable host site, reporting on a minimal
threshold of benzoquinone precursor at the host cell surface.
As is evident in the longitudinal sections of Figure 1C, the inner
cells surrounding the forming vascular bundles swell radially, gradually forming an expanded surface area sufficient for host attachment. Somewhat later, the epidermal cells develop haustorial hairs that function as host attachment anchors. Consistent with the two
distinct cellular expansion events, at least two unique expansin
transcripts, saExp1 and saExp2, are induced by
the benzoquinone. Concomitant with that expression, the seedling
expansin, saExp3, is down-regulated (O'Malley and Lynn,
2000). As haustorial expansin message accumulation plateaus, the
meristem cells terminally commit to forming the new organ. At
intermediate xenognosin exposure times, haustorial expansin transcript
levels fall after benzoquinone removal. The time necessary for
re-induction correlates with the magnitude of expansin message
depletion. Benzoquinone perception appears to function as a
"molecular capacitor," allowing accumulation to a molecular charge
threshold necessary for commitment to haustorial development. Following
this analogy, the capacitor is both leaky and capable of being
recharged, ensuring developmental plasticity.
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IS EXPRESSION OF THE HAUSTORIAL EXPANSINS SUFFICIENT FOR ORGAN
DEVELOPMENT? |
Because the attachment organ can be viewed simply as the expansion
of preexisting cells, induction of saExp1 and
saExp2 may be sufficient to stage further haustorial
development. Micromanipulative addition of expansin induces out of
sequence development of leaf primordia in tomato
(Lycopersicon esculentum; Fleming et al., 1997);
however, these structures rarely developed past the initial stages, and
other factors appear to be involved. Auxin transport into the meristem
regulates expansin expression and induces complete leaf development
(Reinhardt et al., 2000), suggesting that hormonal signaling regulates
both initiation and radial position of organogenesis in the shoot
meristem. With this background, the haustorial expansin genes are
expected to be necessary for haustorial development and provide
valuable molecular markers for the commitment, but not to be sufficient
for complete haustoriogenesis.
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ARE PLANT HORMONES NECESSARY FOR HAUSTORIAL DEVELOPMENT? |
The answer to this question is not clear, but quite relevant.
Exogenous cytokinins will induce haustorial development in
S. asiatica, whereas auxins strongly inhibit both
cytokinin and benzoquinone induction (Keyes et al., 2000). Haustoria
originate as a recommitment of the root meristem pattern in
S. asiatica, in this case a meristem poised for
rapid transition to the parasitic mode, so it is not surprising that
plant hormones effect development. These hormones function in accord
with their site of synthesis, auxin in shoots and cytokinins in roots
(Davies, 1995), at the very least maintaining the poles of the plant
established in the embryo. However, exogenous addition of the
cytokinins does not establish physiological relevance to
haustoriogenesis, nor should it imply that the benzoquinones and
cytokinins necessarily share common or even overlapping induction pathways.
Attachment organs produced by cytokinin induction are morphologically
abnormal. This abnormality could arise from exposure to nonnatural
cytokinin structures, or from differences in temporal or spatial
application. For example, pleiotropic effects of the hormone on
spatially isolated cell types within the root meristem could be
expected to induce abnormal swelling events. In contrast, no such
abnormalities appear when the seedlings are incubated with the
benzoquinones. The benzoquinone xenognosins may be perceived only at
specific cell-cell contact surfaces and not across many cell types.
Contacts and cross feeding between the host and parasite epidermal
cells may establish local redox gradients, and only later, stage the
radial swelling of the internal cells and hair formation along the
epidermis through different mechanisms.
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SO HOW COULD REDOX ACTIVATION MEDIATE SOCIAL CONTROLS? |
All cells maintain a redox stationary state. The plant cell in
particular carries nuclear, mitochondrial, and plastid genomes, and the
intracellular communication between them depends on redox signaling
mechanisms (Mackenzie and McIntosh, 1999). Recent evidence connects
H2O2 in signaling cell
swelling in both stomatal control (Pei et al., 2000) and root
gravitropism (Joo et al., 2001). The mammalian circadian rhythm,
another system where events are clocked over a period of time, appears
to be entrained by the redox state of the NAD cofactors (Rutter et al.,
2001). Therefore, numerous possible mechanisms exist at the contact
surface where cross-feeding H2O2 and the benzoquinone
xenognosins could regulate both recognition and induction processes. As
additional genetic markers become available for different stages of the
developmental commitment (Matvienko et al., 2001; Ouedraogo et al.,
2001), it should become increasingly possible to define the signals and
molecular responses that allow epidermal cells of a parasite to dance
with its host.
The text on parasitic plants edited by Press and Graves (1995) points
out very clearly that cellular development is only one aspect of plant
physiology that has been impacted by an understanding of these
organisms. Parasitic members constitute a significant percentage of all
angiosperms and many elements of their emergence, evolution, and
ecology are noteworthy. Organelle genome evolution and biochemical
integration within the plant cell is altered dramatically when
assimilate comes from another plant. Overall source-sink relationships
are altered in both host and parasite with attachment. Organisms that
drain water, minerals, and even defensive chemistries from another
plant establish unique dependencies and advantages. Given the severe
economic impact these organisms still have on cereal production in
Africa, the Mediterranean Basin, and India, as well as timber
production in North America, we should expect continued investigations
of these unique members of the plant kingdom to have both a basic and
practical impact throughout the 21st century.
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ACKNOWLEDGMENTS |
We gratefully acknowledge Howard Rees' help with confocal
microscopy imaging and Patrick Liu's assistance in computer graphics.
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FOOTNOTES |
Received August 16, 2001; returned for revision September 20, 2001; accepted September 20, 2001.
*
Corresponding author; e-mail dlynn2{at}emory.edu; fax
404-727-6586.
www.plantphysiol.org/cgi/doi/10.1104/pp.010753.
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© 2001 American Society of Plant Physiologists
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TOP
INTRODUCTION
WHAT ARE PARASITIC ANGIOSPERMS?
