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
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. Cellcell 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 be* Corresponding author; e-mail [email protected];
404 –727– 6586.
www.plantphysiol.org/cgi/doi/10.1104/pp.010753.
1508
fax
tween the distinct cell populations of two separate
organisms.
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.
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
Plant Physiology, December
Downloaded
2001, Vol.
from127,
on June
pp. 1508–1512,
16, 2017 - Published
www.plantphysiol.org
by www.plantphysiol.org
© 2001 American Society of Plant Biologists
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Update on Parasitic Plants
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 ␮m.
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).
WITHOUT EXUDED ENZYMES, HOW ARE THE
XENOGNOSINS RELEASED FROM HOST CELLS?
The answer initially came from studies with redoxsensitive 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
Plant Physiol. Vol. 127, 2001
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).
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
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1509
Keyes et al.
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.
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.
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
1510
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.
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.
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
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
Update on Parasitic Plants
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.
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.
SO HOW COULD REDOX ACTIVATION MEDIATE
SOCIAL CONTROLS?
All cells maintain a redox stationary state. The
plant cell in particular carries nuclear, mitochondrial,
Plant Physiol. Vol. 127, 2001
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
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1511
Keyes et al.
members of the plant kingdom to have both a basic
and practical impact throughout the 21st century.
ACKNOWLEDGMENTS
We gratefully acknowledge Howard Rees’ help with
confocal microscopy imaging and Patrick Liu’s assistance
in computer graphics.
Received August 16, 2001; returned for revision September
20, 2001; accepted September 20, 2001.
LITERATURE CITED
Boone L, Lynn DG (1995) Signal transduction in the initiation of germination. In MC Press, JD Graves, eds, Parasitic Flowering Plants. Chapman and Hall, London, pp
14–38
Chang M, Lynn DG (1986) The haustorium and the chemistry of host recognition in parasitic angiosperms. J Chem
Ecol 12: 561–579
Davies PJ (1995) Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers,
Dordrecht, The Netherlands
Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S,
Roberts K, Scheres B (1993) Cellular organization of the
Arabidopsis thaliana root. Development 119: 71–84
Fate GW, Lynn DG (1996) Xenognosin methylation is critical in defining the chemical potential gradient that regulates the spatial distribution in Striga pathogenesis.
J Am Chem Soc 118: 11369–11376
Fleming AJ, McQueen-Mason S, Mandel T, Kuhlemeier C
(1997) Induction of leaf primordia by the cell wall protein
expansin. Science 276: 1415–1418
Joo JH, Bae YU, Lee JS (2001) Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiol
126: 1055–1060
Jürgens G (2001) Apical-basal pattern formation in Arabidopsis embryogenesis. EMBO J 20: 3609–3616
Keyes WJ, O’Malley R, Kim D, Lynn DG (2000) Signaling
organogenesis in parasitic angiosperms: xenognosin generation, perception and response. Plant Growth Regul 19:
217–231
Kim D, Kocz R, Boone L, Keyes WJ, Lynn DG (1998) On
becoming a parasite: evaluating the role of wall oxidases
in parasitic plant development. Chem Biol 5: 103–117
Kuijt J (1969) The Biology of Parasitic Flowering Plants.
University of California Press, Berkeley
Lamb C, Dixon RA (1997) The oxidative burst in plant
disease resistance. Annu Rev Plant Physiol Plant Mol
Biol 48: 251–275
Lynn DG, Chang M (1990) Phenolic signals in cohabitation: implications for plant development. Annu Rev
Plant Physiol Mol Biol 41: 497–526
Lynn DG, Steffens JC, Kamat VS, Graden DW, Shabanowitz J, Riopel JL (1981) Isolation and characteriza-
1512
tion of the first host recognition substance for parasitic
angiosperms. J Am Chem Soc 103: 1868–1870
Mackenzie S, McIntosh L (1999) Higher plant mitochondria. Plant Cell 11: 571–585
Matvienko M, Wojtowicz A, Wrobel R, Jamison D, Goldwasser Y, Yoder JI (2001) Quinone oxidoreductase message levels are differentially regulated in parasitic and
non-parasitic plants exposed to allelopathic quinones.
Plant J 25: 1–15
O’Malley RC, Lynn DG (2000) Expansin message regulation in parasitic angiosperms: marking time in development. Plant Cell 12: 1455–1465
Ouedraogo JT, Maheshwan V, Berner DK, St-Pierre CA,
Belzile F, Timko MP (2001) Identification of AFLP markers linked to resistance of cowpea (Vigna unguiculata L.)
to parasitism by Striga gesnerioides. Theor Appl Genet
102: 1029–1036
Pei ZM, Murata Y, Benning G, Thomine S, Klusener B,
Allen GJ, Grill E, Schroeder JI (2000) Calcium channels
activated by hydrogen peroxide mediate abscisic acid
signaling in guard cells. Nature 406: 731–734
Press MC, Graves JD (1995) Parasitic Flowering Plants.
Chapman and Hall, London
Reinhardt D, Mandel T, Kuhlemeier C (2000) Auxin regulates the initiation and radial position of plant lateral
organs. Plant Cell 12: 507–518
Reinhardt D, Wittwer F, Mandel T, Kuhlemeier C (1998)
Localized upregulation of a new expansin gene predicts
the site of leaf formation in the tomato meristem. Plant
Cell 10: 1427–1437
Riopel JL (1979) Experimental studies in induction of haustoria in Agalinis purpurea. In LJ Musselman, AD Worsham, RE Eplee, eds, Proceedings of the Second International Symposium of Parasitic Weeds. North Carolina
State University, Raleigh, NC, pp 165–173
Rutter J, Reick M, Wu LC, McKnight SL (2001) Regulation
of clock and NPAS2 DNA binding by the redox state of
NAD cofactors. Science 293: 510–514
Shaykh CM, Soliday CL, Kolattukudy PE (1977) Proof of
the production of cutinase by Fusarium solini f. pisi during penetration into its host Pisum sativum. Plant Physiol
60: 170–172
Smith C, Dudley MD, Lynn DG (1990) Vegetative/parasitic transition: control and plasticity in Striga’s development. Plant Physiol 93: 208–215
Smith ER, Ruttledge T, Zeng Z, O’Malley RC, Lynn DG
(1996) A mechanism for inducing plant development: the
genesis of a specific inhibitor. Proc Natl Acad Sci USA 93:
6986–6991
Van den Berg C, Willemsen V, Hage W, Weisbeek P,
Scheres B (1995) Cell fate in the Arabidopsis root meristem determined by directional signaling. Nature 378:
62–65
Van den Berg C, Willemsen V, Hendriks G, Weisbeek P,
Scheres B (1997) Short-range control of cell differentiation in the Arabidopsis root meristem. Nature 390:
287–289
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001