Download Roles of root border cells in plant defense and regulation of

Plant Soil (2012) 355:1–16
DOI 10.1007/s11104-012-1218-3
MARSCHNER REVIEW
Roles of root border cells in plant defense and regulation
of rhizosphere microbial populations by extracellular
DNA ‘trapping’
Martha C. Hawes & Gilberto Curlango-Rivera &
Zhongguo Xiong & John O. Kessler
Received: 2 November 2011 / Accepted: 11 March 2012 / Published online: 27 March 2012
# Springer Science+Business Media B.V. 2012
Abstract
Background As roots penetrate soil, specialized cells
called ‘border cells’ separate from root caps and contribute a large proportion of exudates forming the rhizosphere. Their function has been unclear. Recent findings
suggest that border cells act in a manner similar to that of
white blood cells functioning in defense. Histone-linked
extracellular DNA (exDNA) and proteins operate as
‘neutrophil extracellular traps’ to attract and immobilize
animal pathogens. DNase treatment reverses trapping
and impairs defense, and mutation of pathogen DNase
results in loss of virulence.
Scope Histones are among a group of proteins secreted
from living border cells. This observation led to the discovery that exDNA also functions in defense of root caps.
Experiments revealed that exDNA is synthesized and
exported into the surrounding mucilage which attracts,
traps and immobilizes pathogens in a host-microbe specific manner. When this plant exDNA is degraded, the normal resistance of the root cap to infection is abolished.
Conclusions Research to define how exDNA may
operate in plant immunity is needed. In the meantime,
the specificity and stability of exDNA and its association with distinct microbial species may provide an
important new tool to monitor when, where, and how
soil microbial populations become established as
rhizosphere communities.
Responsible Editor: Philippe Hinsinger.
M. C. Hawes (*) : G. Curlango-Rivera
Department of Soil, Water and Environmental Sciences,
University of Arizona,
429 Shantz Building, #38, 1177 E. Fourth St., POB 210038,
Tucson, AZ 85721-0038, USA
e-mail: [email protected]
Z. Xiong
Division of Plant Pathology and Microbiology,
School of Plant Sciences, University of Arizona,
Tucson, AZ 85721, USA
J. O. Kessler
Physics Department, University of Arizona,
Building 81,
Tucson, AZ 85721, USA
Keywords Root border cells . Mucilage . Root cap .
Extracellular DNA (exDNA) . Root exudates .
Rhizosphere colonization
Abbreviations
exDNA Extracellular DNA
DAPI
4′,6-diamidino-2-phenylindole
One of the take-home messages is that spatial and
temporal variability act to confound root research
(Zobel and Wright 2005). There is an urgent need
to develop new approaches and methods for probing rhizodeposition (Jones et al. 2009).
2
Background
Critical needs for sustainable practices in agriculture
have been considered in many excellent articles (e.g.
Brady and Weil 2010; Compant et al. 2005; Donato et
al. 2010; Pinton et al. 2007; Sylvia et al. 1998; Zobel
and Wright 2005). The root-soil interface is a target
where positive changes can yield stable improvement
in fertility, water use, and disease control leading to
increased crop productivity with reduced damage to
the environment (Bruehl 1987; Gilbert et al. 1996;
Marschner et al. 2011; Rovira 1991; Schroth and
Snyder 1961; Uren 2001). Efforts to apply biological
control to root systems have been a focus of interest
for decades with promising results and progress in
understanding mechanisms (Handelsman and Stabb
1996; Hirsch 2004; Loh et al. 2002; Morris and
Monier 2003; Pierson and Pierson 2007; Weller
1988; Zentmyer 1963). Of special interest are
carbon allocation to the root and its delivery to
the soil environment (Curl and Truelove 1986;
Kuzyakov 2001; Lynch and Whipps 1990). If exudates control microbial growth, then controlling
the composition, timing, and localization of root
exudation would seem to be a reasonable approach
to stimulate the growth of beneficial microorganisms
at the expense of pathogens (Bednarek et al. 2010;
Broeckling et al. 2008; Liu et al. 2005).
Unfortunately, despite ever-increasing precision in
measuring carbon deposition and microbial colonization in the rhizosphere, the goal of developing predictive models, let alone controlling the process for crop
improvement, has eluded researchers (Bowen and
Rovira 1976; Cooper and Rao 2006; Darrah and
Roose 2001; Handelsman 2004; Hinsinger 2001;
Hinsinger et al. 2011; Luster et al. 2009). Apart from
the extremes of environment and composition encountered in soils, the process of root exudation per se, as
detailed below, is an intrinsically dynamic process
that can be difficult to predict even under controlled
conditions (Brady and Weil 2010; Lynch and
Whipps 1990; Watt et al. 2006). Here we describe
challenges and opportunities presented by the recent
discovery that extracellular DNA (exDNA) is a
component of exudates whose delivery into the
rhizosphere is controlled by metabolically active
cells at the root apex.
Plant Soil (2012) 355:1–16
Lots of exudates at the root tip, not much microbial
colonization: why?
Microbial growth in the rhizosphere, by definition, is
increased relative to that in bulk soil (Rovira 1969).
This phenomenon is attributed to the plant’s release of
nutrient-rich exudates that can support the growth of
diverse microbiota. Therefore, regions of the root that
release more exudates might be predicted to support a
corresponding increase in microbial growth relative to
that in other regions. The root cap has been reported to
be a primary source of exudate in experiments using
diverse species and conditions (Dennis et al. 2010;
Jones et al. 2009; Lundegardh and Stenlid 1944;
Lynch and Whipps 1990; McDougall and Rovira
1970; Odell et al. 2008; VanEgeraat 1975; Wood
1967). In direct measurements from whole roots of
young legume seedlings grown in hydroponic or plate
culture under aseptic conditions, for example, more
than 90 % of the total fresh or dry weight derives from
the root cap (Griffin et al. 1975; Gunawardena et al.
2005). Therefore it would seem reasonable to predict
that root exudate-stimulated microbial populations
would predominate at the root cap under more complex conditions.
Instead, root caps of cereals, legumes, and other
agronomically important species repeatedly have been
found to be free of infection and colonization. In fieldgrown wheat Foster et al. (1983) reported that, ‘Unlike
the rest of the root surface, the root cap as seen in
scanning electron micrographs is generally quite devoid
of microbial colonies.’ On tomato roots inoculated with
Fusarium, ‘the root cap is not an important site of
colonization’ (Lagopodi et al. 2002). On tomato inoculated with Pseudomonas fluorescens, ‘the root cap was
always devoid of bacteria’ (Gamalero et al. 2005).
Similar results occurred on maize root caps inoculated
with P. fluorescens, but upon removal of root caps
colonization of the apex developed (Humphris et al.
2005). On pea roots inoculated with spores of pathogenic fungi, then incubated in warm, moist conditions,
the root cap remains sterile despite being ensheathed
within a mantle of fungal hyphae (Gunawardena and
Hawes 2002). Newly synthesized plant cells like
those in the region of elongation are more susceptible to infection than older tissue with lignified
cell walls (Hawes et al. 2000). Because root caps
Plant Soil (2012) 355:1–16
also are comprised of newly synthesized cells generated by meristems in the root apex, this was an
especially surprising observation (Curlango-Rivera
and Hawes 2011). New insight into the nature and
function of root cap defense systems may yield an
answer to this long-standing mystery: Sometimes,
the carbon-based ‘exudates’ may act to trap, immobilize and inhibit microbial growth rather than
serving as a passive nutrient base.
Extracellular DNA (exDNA) and protein in root tip
defense
The recognition that exDNA is a key component of
root exudates involved in border cell ‘extracellular
trapping’ (Hawes et al. 2011) followed a long history
of clues whose significance was overlooked until
Brinkmann et al. (2004) documented the importance
of exDNA in mammalian defense. VanEgeraat (1975)
documented that the primary source of root exudates
from young healthy seedlings under laboratory conditions is the root apex. Seedlings were placed onto
damp filter paper for 24 h, then removed and the paper
was dried and sprayed with ninhydrin (2,2-dihydroxyindane-1,3-dione) which reacts with lysine present in
peptides and proteins. Positive reactions were limited
to sites where root caps had been in contact with the
filter paper. In older seedlings, an additional source is
the site of lateral root emergence from the pericycle.
However, chromatographic profiles of the material
released from these natural wound sites are similar to
those of root extracts, while profiles of material released from the root cap are distinct. As VanEgeraat
(1975) recognized, ‘The process by which compounds are exuded from the root tip region is
completely different from the release following
damage of the root....Exudation by the root tip
might be more selective so that certain specific
compounds would be liberated.’
This prediction proved correct, despite the longstanding presumption that apart from a high molecular
weight ‘slime’ or mucilage secreted from root caps,
exudates from root tips primarily are the product of
cytoplasmic contents leaking from dead ‘sloughed’
cells (Esau 1967; Levy-Booth et al. 2007; Voeller et
al. 1964). Synonyms for ‘sloughed’ are ‘putrid’ and
3
‘gangrenous.’ Border cells, once termed ‘sloughed
root cap cells,’ instead are metabolically active cells
which exhibit host specific susceptibility and resistance to infection (Goldberg et al. 1989; Sherwood
1987). The border cell gene expression profile is distinct from that of progenitor cells in the root cap but
parallel across diverse species (Brigham et al. 1998;
Wen et al. 2008). Two-dimensional gel electrophoresis
of proteins synthesized by the root cap during a 1h test period (Fig. 1a) also yielded a profile markedly
distinct from that of border cells (Fig. 1b) (Brigham et
al. 1995). Most surprising was that the profile of
proteins extracted from intact border cells (Fig. 1b)
was markedly similar to that of a secretome with >100
proteins synthesized and exported during the same
experiment (Fig. 1c). Extracellular proteins were
found to play a key role in defense of the root tip:
when treated with protease at the time of inoculation
with spores of a pathogenic fungus, the normal resistance to root tip infection was abolished (Wen et al.
2007b). Among the proteins were antimicrobial
enzymes long known to be associated with plant and
mammalian defense (De-la-Pena and Vivanco 2010;
Kwon et al. 2008). Therefore, it was perhaps not
surprising that their destruction altered the normal
root defense processes. Treatment with protease
also resulted in disintegration of a surrounding
mucilage layer and release of bacteria within the
layer (Wen et al. 2007a). These data support the
suggestion by Matsuyama et al. (1999) that proteins may play a role in the structural integrity of
the matrix, even though protein comprises only a
small fraction of the matrix composition (Bacic et
al. 1986; Chaboud and Rougier 1990; Moody et
al. 1988).
The discovery that histone H4 was among the proteins synthesized and exported into the extracellular
matrix was a surprise, given the long-established role
of histones in assembly of genetic material inside the
cell (Wen et al. 2007a). However, emerging research
provided insights into alternative functions of histones, including potent antimicrobial activity in the extracellular environment (Bergsson et al. 2005;
Kawasaki and Iwamuro 2008; Patat et al. 2004; Wang
et al. 2009; Xu et al. 2009). Of special interest were
reports of a role for histones in extracellular chemotaxis and ‘trapping’ of pathogens by neutrophils in the
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Plant Soil (2012) 355:1–16
Root cap
Border cells
Supernatant
b
c
a
Fig. 1 The root cap secretome. After a 1-h period of labelling,
large differences in protein profiles from (a) root caps and (b)
border cells of Pisum sativum L. are evident using twodimensional gel electrophoresis. c More than 100 proteins are
synthesized and exported from living cells during the test period
(from Brigham et al. 1995). Examples of proteins common to
root caps and border cells (black arrows), specific to root caps
(open triangles), or specific to border cells (closed triangles) are
denoted. Plant Physiol 143:773–783 (www.plantphysiol.org)
“Copyright American Society of Plant Biologists”
mammalian immune response, because a very similar
process occurs in border cells in response to plant
pathogens (Gochnauer et al. 1990; Goldberg et al.
1989; Gunawardena et al. 2005; Hawes and Pueppke
1987; Hawes et al. 1988; Zhu et al. 1997). ‘Neutrophil
extracellular traps’ (NETs) were first described by
Zychlinsky and coworkers (Brinkmann et al. 2004),
and now have been implicated in defense against
diverse pathogens and other aspects of immune
responses in mammals (Abdallah et al. 2012; Amulic
and Hayes 2011; Brinkmann and Zychlinsky 2007;
Harding and Kubes 2012; Medina 2009; Mitroulis et
al. 2011; Park et al. 2012; Urban et al. 2006; Wardini
et al. 2010; Wen et al. 2012; Yost et al. 2009; Young et
al. 2011). As with the border cell slime layer (Fig. 2),
NET formation can occur rapidly in response to specific signals, in the absence of cell death (Pilszik et al.
2010). Experiments therefore were carried out to determine (1) whether the presence of extracellular histone surrounding border cells, like neutrophils, is
associated with exDNA; and if so, (2) to determine if
enzymatic degradation of border cell exDNA, like
NET exDNA, interferes with resistance to infection
(Gunawardena and Hawes 2002). The results of these
experiments revealed that, like the plant proteins
exported from the root cap and border cells (Brigham
et al. 1995), plant DNA is synthesized and exported
into the root cap extracellular matrix during a 1-h
period when no cell death occurs (Wen et al. 2009).
Initial sequence analysis revealed that the exDNA
structure is related to nuclear DNA, but is enriched
in repetitive sequences. When this exDNA was degraded by addition of DNase I concomitant with the
inoculation by a pathogenic fungus, the frequency of
root cap infection increased from a mild local necrosis in <5 % of inoculated roots to 100 % infection,
with rotting of each root tip and proliferation of
fungal hyphae (Wen et al. 2009). As in exDNAbased extracellular trapping in mammals, the root
tip resistance to fungal infection is associated with
aggregation of the fungus and inhibition of its
growth (Gunawardena et al. 2005; Medina 2009).
The extracellular trapping phenomenon is hostmicrobe specific, with no aggregation or growth
inhibition of nonpathogenic fungi (Gunawardena
and Hawes 2002; Jaroszuk-Scisel et al. 2009).
Host specific chemotaxis and extracellular trapping
of pathogens by border cells was described previously,
but was presumed to involve aspects of pathogenesis,
not defense (Goldberg et al. 1989; Hawes and
Pueppke 1987; Hawes and Smith 1989). Agrobacterium tumefaciens chemotaxis toward border cells of a
host species was measured using swarm agar assays
(Fig. 2a) (Hawes et al. 1988) or direct microscopic
observation (Fig. 2b,c). Within hours, strings and
strands of immobilized bacteria develop (Fig. 2b).
Bacteria adhere to the surface in a layer that is impervious to removal by washing in water (Fig. 2c).
Plant Soil (2012) 355:1–16
5
a
b
c
d
e
f
Fig. 2 Host specific binding of bacteria within an inducible extracellular ‘trap’ produced by border cells. a Within seconds of adding
the pathogen Agrobacterium tumefaciens to border cells of a host
species (P. sativum L.), chemotaxis toward the cells is evident. The
large arrow denotes the leading edge of the bacterial swarm; the
small arrow denotes border cell sample. b Within 1–2 h, interconnecting strands of bacteria (white arrow) develop between border
cells (black arrow). c Trapping of bacteria is evident within the
surrounding mucilage of individual border cells of the host species,
pea (arrow); d No chemotaxis occurs in response to cells from a
nonhost species (Avena sativa L.), and bacteria are excluded from
rather than trapped within the surrounding mucilage layer (visualized using India ink, which does not penetrate the mucilage)
(arrow). No mucilage layer is induced when a nonpathogenic strain
of E. coli is added to border cells of pea (e) or oats (f), and no
trapping occurs at the cell surface. Scale bar: 15 μm
Adding the plant pathogen to border cells of a nonhost
species triggers no chemotaxis or attachment within
the surrounding mucilage (Fig. 2d). The human pathogen E. coli added to border cells (Fig. 2e, f) was not
associated with chemotaxis, attachment, or production
of a mucilage layer in either plant species. Minimal
growth can be measured in remaining unattached bacteria or in bacteria growing on mucilage as a sole
carbon source, but whether the trapped pathogenic
bacteria are viable is unclear (Knee et al. 2001; Zhu
et al. 1997). Similar patterns of specificity were
reported in association between maize border cells
and bacterial species including Rhizobium, E. coli,
Pseudomonas, Bacillus, Streptomyces and Cytophaga
(Gochnauer et al. 1990). It will be of interest to examine the role of exDNA in this phenomenon, and to
explore the possibility that clusters and strings of
viable but not culturable (VBNC) colonies found in
the rhizosphere might be related to exDNA based
trapping (Gamalero et al. 2004).
The high molecular weight polysaccharide-based
mucilage exported from root caps has been studied
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Plant Soil (2012) 355:1–16
in various species but DNA has not been included in
analyses to date (Bacic et al. 1986; Chaboud and
Rougier 1990; Foster 1981a, b, 1982; Jones and Morre
1973; Knee et al. 2001; Lynch and Staehelin 1995; Miki
et al. 1980; Newcomb 1967; Oades 1978; Read et al.
1999; Sealey et al. 1995; Watt et al. 1993). Therefore
details of how exDNA is synthesized, exported, and
integrated into the extracellular matrix remain to be
established (Hawes et al. 2011). However, the presence
of nucleic acids among exudates of healthy roots was
reported (Curl and Truelove 1986; Fries and Forsman
1951; Lundegarth and Stenlid 1944; Stenlid 1944), and
its active synthesis and export into the extracellular
matrix of the root cap periphery also were documented
(Phillips and Torrey 1971). Using the fluorescent stain
DAPI, which binds to A-T rich strands of DNA and can
pass through intact cell membranes to reveal DNA
within cells or outside the cell boundaries (Kubista et
al. 1987), exDNA is readily detected within border cell
mucilage (Wen et al. 2009). In the presence of stimulating bacteria, DAPI staining occurs within border cells,
throughout the surrounding expanded mucilage layer
(Fig. 3a) and within trapped bacteria (Fig. 3a, arrow).
Fig. 3 exDNA from the
root tip of pea. a DAPI
staining of a pea border cell
with bacteria trapped within
the surrounding mucilage
layer. Scale bar: 15 μm. b
SYTOX green staining of
border cell exDNA strands
and c other structures. Scale
bar: 10 μm. 3A, 3 C, photos
by Fushi Wen. 3B, photo by
Sarah O’Connor
Staining border cell populations and associated
mucilage with SYTOX green, a high-affinity nucleic
acid stain which is not taken into living cells, reveals
extracellular material ranging from strands (Fig. 3b) to
distinctive structures (Fig. 3c). These structures are similar in appearance to those produced by neutrophils
(Patel et al. 2010; Pilszik et al. 2010).
Questions of particular interest are the nature of the
exDNA structure(s) involved in trapping and how they
might interface with other polymers within the root cap
mucilage (Bacic et al. 1986; Knee et al. 2001). One
possibility is that the ‘stickiness’ of DNA alone might
be sufficient to trap added microorganisms. If so, then
addition of DNA alone would be predicted to result in
trapping. No such result occurred upon addition of
salmon sperm DNA or pea genomic DNA to microbes
(Wen et al. 2009). An alternative hypothesis is that
distinct sequences organized in specific structures are
required. In support of this model are observations by
Van’t Hof and colleagues (Van’t Hopf and Bjerknes
1982; Kraszewska et al. 1985), who described a distinct
class of DNA produced by P. sativum root caps during
the G2-M transition, the point in the root cap meristem
a
b
c
Plant Soil (2012) 355:1–16
7
The presence of DNA from plants and other organisms
in the soil is well established (Izano et al. 2008;
Vlassov et al. 2007; Whitchurch et al. 2002). Plant
exDNA has been presumed to be derived by leakage
from dead cells (Levy-Booth et al. 2007). The discovery that secretion of exDNA from root caps instead is a
component of a complex, inducible, and carbonexpensive defense mechanism may be useful in tracking as well as modelling rhizosphere community structure. The programmed separation of cells from the root
cap was long presumed to be a product of continuous
cell cycle activity within the root cap meristem in
parallel with such activity in the apical meristem
(e.g. Clowes 1971; Whipps and Lynch 1983). If correct, then a continuous detection of exudates at the tip
would be a predicted result. Direct observations of
rhizosphere structure even under controlled conditions
do not support this paradigm (Iijima et al. 2003). The
viability and number of border cells that a root cap can
release daily are conserved within families and can
range from 0 to 10,000 cells a day (Hawes et al.
2003; Hawes and Pueppke 1986). For a given root,
the process of root cap turnover is not continuous but
instead is induced or repressed in a species- and
genotype- specific manner in response to endogenous
Fig. 4 Variation in border cell delivery from root caps under
controlled conditions. The length of the root at time zero is
denoted with white arrows. As roots elongate side by side in
the same plate of water agar, the presence of border cells
detectable by direct microscopic observation ranges from none
(inset photo, left) to intermittent clumps (inset photo, center) to a
continuous robust sheath (center). (Plant Physiol 119:417–428,
(www.plantphysiol.org) Copyright American Society of Plant
Biologists). Inset, center: Border cells released from tips of
elongating root of Lithospermum erythrorhizon. The border
cells from this species express a red pigment, shikonin, which
facilitates detection of their presence at intervals along the root
surface (arrows). (Plant Physiol 119:417–428, (www.plantphysiol.org) Copyright American Society of Plant Biologists)
cell cycle when border cell separation occurs (Brigham
et al. 1998). Like root cap exDNA (Wen et al. 2009), this
‘extrachromosomal DNA’ is related to nuclear DNA but
is distinguishable based on the prevalence of repetitive
sequences (Kraszewska et al. 1985). The programmed
delivery of characteristic exDNA patterns as an integral
component of the matrix could provide a tool to examine underlying patterns of rhizosphere carbon deposition
and microbial colonization and allow progress toward exploiting the system for crop improvement.
Factors known to influence border cell delivery are
summarized below.
Factors controlling delivery of exDNA-based traps
from root caps
Border cell populations
8
Plant Soil (2012) 355:1–16
and environmental signals (Brigham et al. 1998;
Ponce et al. 2005). Therefore, when seedlings are
grown under identical conditions side by side in petri
dishes, the delivery of mucilage and border cells can
vary from nothing to intermittent clumps to a continuous sheath surrounding the root from base to tip
(Fig. 4). The variation is illustrated schematically because even with direct microscopic observation on
sterile plates the differences can be difficult to detect
(Fig. 4, inset photos). Some species exhibit border
cell specific expression of pigmented metabolites
which provide a convenient marker for cell dispersal
(Brigham et al. 1999). Thus, Saccharum officinarum,
Sorghum vulgare and Lithospermum erythrorhizon
have pink, purple, and red border cells, respectively.
This pigmentation facilitates recognition of rhizosphere distribution patterns that otherwise would
be obscure (Fig. 4, inset center). Variation in root
exudation and rhizosphere colonization has been
proposed to be a major obstacle to agronomic application of promising discoveries like biological control
(Cooper and Rao 2006; Sylvia et al. 1998). Understanding factors controlling carbon delivery via border
cells may be key to monitoring and controlling rhizosphere community structure (Lee and Hirsch 2006;
Smucker and Erickson 1987).
a
b
Fig. 5 Instantaneous swelling and dispersal of P. sativum border cells in response to immersion in free water. a At 99+%
humidity, the root tip is smooth, and the presence of border cells
is undetectable except with scanning electron microscopy (inset). Scale bar: 0.5 mm. b Addition of a droplet of water results
in swelling of border cells away from the tip within 10–15 s.
For roots of any given plant, one major factor
controlling border cell release is the availability of free
water (Fig. 5) (Odell et al. 2008). Roots of legumes,
cereals, cucurbits and most other crop species are
programmed to produce a species-specific number of
cells (Hawes and Pueppke 1986). When that number
has accumulated on the cap periphery the same set
may remain on the root for an extended period without
any new cells being produced. This appears to result
from the accumulation of an extracellular signal within
the surrounding mucilage to a level that suppresses
cap turnover except when diluted in water (Brigham et
al. 1998). The properties of the mucilage are such that
it can hold 1000X its weight in water (Guinel and
McCully 1986). Yet even at 99 % humidity, in the
absence of free water, the mucilage remains ‘dry’
like a sponge without moisture (Fig. 5a) and highresolution microscopy is necessary to detect the
ensheathed border cells (Fig. 5a, inset). Upon addition
of water–including, for example, a drop resulting from
condensation on the inside of a petri plate falling onto
the root–the mucilage immediately expands (Fig. 5b)
and border cells are dispersed into suspension
(Fig. 5c). It is important to note that the drop of water
not only causes the dissociation of the existing group
of ca 4,000 cells from the cap periphery, but also
c
Scale bar: 0.5 mm. c Gentle agitation of the plate by tapping one
side results in immediate dispersal of border cells into the
suspension, in the direction of the applied force. Scale bar:
0.5 mm. Staining with the vital stain fluorescein diacetate,
which only accumulates inside living cells, reveals border cell
viability of 95–100 % (inset). Scale bar: 10 μm
Plant Soil (2012) 355:1–16
9
triggers renewed cell cycle instantaneously (Brigham
et al. 1998). Within 5 min, mitosis increases within the
root cap meristem, and dozens of new cells emerge
from the periphery. Activation of the quiescent center
also occurs, and cell production proceeds until a new
set has accumulated within 24 h (Ponce et al. 2005). It
seems obvious that within the soil environment, where
a continuous film of free water at the root tip would be
intermittent for most crops in most conditions, this
factor alone could account for much of the variability
in root tip carbon deposition that occurs.
Other factors that can vary the number of border
cells and associated products released into the rhizosphere, include soil type, physical abrasion, day
length, root age and growth rate (Iijima et al. 2000,
2003; Odell et al. 2008; Somasundaram et al. 2008;
Wuyts et al. 2006). Sodium fluoride added to wheat
roots can stimulate changes in number of border cells
and in level of protein secretion (Bozhkov et al. 2007).
Carbon dioxide, aluminum, boron, and plant pathogens stimulate changes in border cell production in a
plant species- and genotype-specific manner with
distinct responses at different developmental stages
(Cannesan et al. 2011; Chen et al. 2008; Liu et al.
2007; Miyasaka and Hawes 2001; Pan et al. 2004;
Tamas et al. 2005; Zhao et al. 2000; Zhu et al. 2003).
For example, increased carbon dioxide inhibits border
cell production in P. sativum during germination, but
results in increased cell production in seedlings (Fig. 6).
Border cell production in Medicago sativa seedlings, in
contrast, is impervious to similar changes in carbon
dioxide (Zhao et al. 2000). Continuous culture of roots
in high concentrations of certain sugars and secondary
metabolites results in marked increases in mucilage
production by maize roots (Jones and Morre 1973;
Knudson 1917). Transient exposure of roots to metabolites including rhamnose, caffeine, and flavonoids for
several minutes, a condition more likely to occur under
natural conditions, can specifically induce or repress
border cell production without affecting rate of root
growth (Curlango-Rivera et al. 2010). Altered expression of genes controlling cell cycle or cell wall solubilization at the cap periphery results in altered border cell
production, and transient changes in their expression
due to diverse environmental signals could influence
the process as well (Wen et al. 1999; Woo et al. 2004).
A new study reporting an ‘extraordinary sheath’ of
material triggered on roots of Acacia magnum grown
in hydroponic culture, highlights the importance of understanding factors controlling this avenue of carbon
deposition and their impact on rhizosphere structure
(Endo et al. 2011).
Fig. 6 Effects of carbon dioxide on border cell separation from
root tips (arrows) of P. sativum seedlings. a Cells from a single
root tip observed with a dissecting microscope after 3 days in
(A) ambient (0.03 % CO2 vs 21 % O2); or (b) 6 % CO2 vs 15 %
O2). Increased O2 alone had no effect on cell production. From
Plant Physiology 122:181–188, used with permission. (www.
plantphysiol.org) Copyright American Society of Plant
Biologists
Single cells
Morphology of border cell detachment from the cap
periphery can range from a population of single cells
in suspension to finger-like strands of cells to an entire
root cap (Endo et al. 2011; Hamamoto et al. 2006;
Vicre et al. 2005; Wen et al. 2008). The significance of
these variations with respect to exDNA-based trapping
is unknown, but the variation in amount and composition of carbon-based material can be substantial even
on a single-cell basis. For many years, border cells
were called ‘sloughed root cap cells’ to reflect the
presumption that delivery of the cell populations must
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Plant Soil (2012) 355:1–16
reflect a process of falling away from the root as a
consequence of cell death (e.g. Uren 2001). This notion prevailed, despite repeated documentation that the
cells from most species are metabolically active as
they detach from the root cap and can survive for
extended periods in liquid culture (Caporali 1983;
Gautheret 1933; Hawes and Wheeler 1982; Stubbs et
al. 2004). Knudson (1919) reported that border cells
released from Zea mays or P. sativum grown in hydroponic culture, with or without glucose, remained
100 % viable for more than one month. Even more
surprising was the observation that the cells export
enzymes and other proteins (Rogers et al. 1942) and
can remain metabolically active after detachment into
the soil environment (Vermeer and McCully 1982).
Continued secretion of mucilage from border cells
can occur for days after detachment from roots
grown in soil (Hawes and Brigham 1992; Hawes et
al. 1998). Like white blood cell ‘granules’, border
cells contain abundant storage particles which may
provide energy for survival and response to signals
in the extracellular environment (Feldman 1985;
Newcomb 1967).
The mucilage produced by individual border cells
after separation from the root cap also is a dynamic
process. An increase in the diameter of the mucilage
layer is induced almost instantaneously in a speciesand genotype-specific manner in response to exposure
to bacteria (Figs. 2, 3), fungi (Wen et al. 2009), and
aluminum (Miyasaka et al. 2000). Border cells from
pea, for example, can form aggregates containing
hundreds of cells and associated mucilage (Fig. 7a),
or exist as isolated cells with variable layers of surrounding mucilage (Fig. 7b) (Wen et al. 2007a). Given
that such variation can occur in controlled environments and that each cell can trap thousands of bacterial cells, the potential for creating variable islands
that confound efforts to measure carbon deposition
and its impact on rhizosphere colonization in the
soil, is obvious. With recognition of the ‘trapping’
function of border cells, on the other hand, these
seemingly inexplicable phenomena may be easier
to understand. The observation by Guinel and
McCully (1987) that border cells can continue to
expand after detachment from the root as single
cells, also is less surprising in the context of their
proposed functions in ‘border patrol.’ If border
cells trap heavy metals and pathogens and control
the growth of deleterious microorganisms in the
vicinity of plant roots, then a capacity to achieve
an increased surface area would be a predictable
benefit to the plant rather than an egregious waste
of fixed carbon (Fig. 8).
In addition to proteins, DNA and polysaccharides,
the root cap and border cell exudates include primary
and secondary metabolites that function in signalling
and recognition of beneficial as well as pathogenic
microbes (e.g. Baluska et al. 1996; Graham 1991;
Maxwell and Phillips 1990; Peters and Long 1988).
The mixture also contains feedback signals that may
influence rate and direction of root growth and development (Baluska et al. 1996; Caffaro et al. 2011;
Moore and Fondren 1986). The potential for creating
changes in the composition of border cell products has
been demonstrated by studies of cotton engineered to
resist insect damage by expression of crystal (CRY)
proteins from Bacillus thuriengensis. BT toxin is delivered through exudates of engineered plants into the
Fig. 7 Variation in aggregation of detached border cell
populations ranges from (a)
a cohesive mass containing
hundreds of border cells to
(b) individual cells. Mucilage layers, detected by
staining with India ink
which is excluded, are present on viable cells but disintegrate rapidly after cell
death (block arrow). Scale
bar: 20 μm
a
b
Plant Soil (2012) 355:1–16
11
Fig. 8 Border cell expansion of the volume of a single cell
(black arrows denote each end of the cell) by >10-fold 7 days
after detachment from the root cap. The nucleus (white arrow)
and cytoplasmic strands are evident within the living cell. Inset:
The original size of border cells within this sample is illustrated
by showing for comparison a cell within the same population
which died before any growth occurred. Scale bar: 30 μm
soil where it can exhibit a half-life of up to 234 days
(Saxena and Stotzky 2001; Tapp and Stotzky 1997).
Direct measurements of Cry proteins revealed that
roots of all genetically modified lines tested synthesize
and export BT toxin, and that root caps, border cells
and root mucilage are sources of this material (Knox
and Vadakattu 2005; Knox et al. 2007). The environmental impact is not clear at this time, but the results
suggest that reproducible changes in the soil environment already have been accomplished via changes in
root cap delivery systems of genetically modified
crops.
dynamics of the rhizosphere and its components
in the interest of fostering sustainable methods for
agriculture (Ceccherini et al. 2009; Levy-Booth et
al. 2007; Pietramellara et al. 2009). If used in
conjunction with holistic tracking methods that
combine laboratory and field assessment (e.g.
Knox et al. 2009), a goal of harnessing the plant’s
ability to control root exudation and rhizosphere
community structure may not be unrealistic
(Atkinson et al. 1975; Knox et al. 2009; Liu et
al. 2005).
Conclusions
The discovery that exDNA plays a role in plant
defense raises more questions than it answers, and
additional research is needed before conclusions
can be drawn regarding a general role in plant
immunity. The new data do reinforce the premise
that a simple model of nutrient rich material leaking from roots and feeding microbial growth in
general is inadequate (De-La-Pena et al. 2010).
The important role of metabolites secreted into
the ‘apoplast’ has long been recognized (Brisson
et al. 1994; Kwon et al. 2008). Understanding the
nature and function of the ‘exudates’ delivered by
the root cap may offer insights into how the
natural immunity of the root cap might be extended to more vulnerable sites including the region of
elongation, where most soilborne pathogens initiate infections (Hawes et al. 2000). The controlled
delivery of exDNA may complement new tools
available to define the structural and functional
Acknowledgements We gratefully acknowledge support for
our research in this area from the National Science Foundation
(NSF# 1032339 to MCH and ZX) and the Department of Energy (DOE DEAC02-06CH11357 to JOK). We thank Dr. Virginia
Rich for critical reading of the manuscript.
We dedicate this review to the memory of W. D. ‘Dietz’
Bauer.
References
Abdallah DS, Lin C, Ball CJ et al (2012) Toxoplasma gondii
triggers release of human and mouse neutrophil extracellular traps. Infection Immun 80:768–777
Amulic B, Hayes G (2011) Neutrophil extracellular traps. Curr
Biol 21:R297–R298
Atkinson TG, Neal JL, Larson RI (1975) Genetical control of
the rhizosphere of wheat. In: Bruehl GW (ed) Biology and
control of soil-borne plant pathogens. American Phytopathological Society, St. Paul
Bacic A, Moody SF, Clarke AE (1986) Structural analysis of
secreted root slime from maize. Plant Physiol 80:771–777
Baluska F, Volkmann D, Hauskrecht M, Barlow PW (1996)
Root cap mucilage and extracellular calcium as modulators
of cellular growth in postmitotic growth zones of the maize
root apex. Bot Acta 109:25–34
12
Bednarek P, Kwon C, Schulze-Lefert P (2010) Not a peripheral
issue: secretion in plant-microbe interactions. Curr Opin
Plant Biol 13:378–385
Bergsson G, Agerberth B, Jornvall H, Gudmundsson GH (2005)
Isolation and identification of antimicrobial components
from the epidermal mucus of Atlantic cod (Gadus
morhua). FEBS J 272:4960–4969
Bowen GD, Rovira AD (1976) Microbial colonization of plant
roots. Ann Rev Phytopathol 114:121–144
Bozhkov AI, Kuznetsova YA, Menzyanova NG (2007) Interrelationship between the growth rate of wheat roots, their
excretory activity and the number of border cells. Russian J
Plant Physiol 54:97–103
Brady NC, Weil RR (2010) Elements of the nature and properties of soils. Prentice Hall
Brigham LA, Woo HH, Nicoll SM, Hawes MC (1995) Differential expression of proteins and messenger-RNAs from
border cells and root tips of pea. Plant Physiol 109:457–
463
Brigham LA, Woo HH, Wen F, Hawes MC (1998) Meristemspecific suppression of mitosis and a global switch in gene
expression in the root cap of pea by endogenous signals.
Plant Physiol 118:1223–1231
Brigham LA, Michaels PJ, Flores HE (1999) Cell-specific production and antimicrobial activity of naphthoquinones in
roots of Lithospermum erythrorhizon. Plant Physiol
119:417–428
Brinkmann V, Zychlinsky A (2007) Beneficial suicide: why
neutrophils die to make NETs. Nat Rev Microbiol 5:577–
582
Brinkmann V, Brichard U, Goosmann C et al (2004) Neutrophil
extracellular traps kill bacteria. Science 303:1532–1535
Brisson RF, Tenhaken R, Lamb C (1994) Function of oxidative
cross linking of cell wall structural proteins in plant disease
resistance. Plant Cell 6:1703–1712
Broeckling CD, Broz AK, Bergelson J, Manter DK, Vivanco J
(2008) Root exudates regulate soil fungal community composition and diversity. Appl Environ Microbiol 74:738–
744
Bruehl GW (1987) Soilborne plant pathogens. Macmillan Publishing Company, NY, USA
Caffaro MM, Vivanco JM, Gutierrez BFH et al (2011) The
effect of root exudates on root architecture in Arabidopsis
thaliana. Plant Growth Regul 64:241–249
Cannesan MA, Gangneux C, Lanoue A et al (2011) Association
between border cell responses and localized root infection
by pathogenic Aphanomyces euteiches. Ann Bot 108:459–
469
Caporali L (1983) Cytological study of cultured cells from
maize root cap. Plant Sci Lett 31:231–236
Ceccherini MT, Ascher J, Agnelli A et al (2009) Experimental
discrimination and molecular characterization of the extracellular soil DNA fraction. Antonie Van Leeuwenhoek
96:653–657
Chaboud A, Rougier M (1990) Comparison of maize root mucilages isolated from root exudates and root surface extracts
by complementary cytological and biochemical investigations. Protoplasma 156:163–173
Chen W, Liu P, Xu G et al (2008) Effects of aluminum on the
biological characteristics of cowpea root border cells. Acta
Physiol Plant 30:303–308
Plant Soil (2012) 355:1–16
Clowes FAL (1971) The proportion of cells that divide in root
meristems of Zea mays L. Ann Bot 35:249–261
Compant S, Duffy B, Nowak J et al (2005) Use of plant growth
promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action and future prospects. Appl
Environ Microbiol 71:4951–4959
Cooper JE, Rao JR eds (2006) Molecular approaches to soil,
rhizosphere and plant microorganism analysis. CABI,
Oxfordshire, UK, Cambridge MA, USA
Curl EA, Truelove B (1986) The rhizosphere. Advanced series
in agricultural sciences, vol. 15, Springer-Verlag, BerlinHeidelberg-New York-Tokyo
Curlango-Rivera G, Hawes MC (2011) Root tips moving
through soil: an intrinsic vulnerability. Plant Signal Behavior 6:1–2
Curlango-Rivera G, Duclos DV, Ebolo JJ, Hawes MC (2010)
Transient exposure of root tips to primary and secondary
metabolites: impact on root growth and production of
border cells. Plant Soil 332:267–275
Darrah PR, Roose T (2001) Modeling the rhizosphere. In:
Pinton R, Varanini Z, Nannipieri P (eds) The rhizosphere:
biochemistry and organic substances at the soil-plant interface. Marcel Dekker, Inc., New York, pp 327–372
De-la-Pena C, Vivanco JM (2010) Root-microbe interactions:
the importance of protein secretion. Curr Proteonomics
7:265–274
De-la-Pena C, Badri DV, Lei Z et al (2010) Root secretion of
defense-related proteins is development-dependent and
correlated with flowering time. J Biol Chem 285:30654–
30666
Dennis PG, Miller AJ, Hirsch PR (2010) Are root exudates more
important than other sources of rhizodeposits in structuring
rhizosphere bacterial communities? FEMS Microbiol Ecol
72:313–327
Donato JJ, Moe LA, Converse BJ et al (2010) Metagenomic
analysis of apple orchard soil reveals antibiotic resistance
genes encoding predicted bifunctional proteins. Appl Environ Microbiol 76:4396–4401
Endo I, Tange T, Osawa H (2011) A cell-type-specific defect in
border cell formation in the Acacia mangium root cap
developing an extraordinary sheath of sloughed-off cells.
Ann Bot 108:279–290
Esau K (1967) Plant anatomy. Wiley, New York
Feldman LJ (1985) Root gravitropism. Physiol Plant 65:341–344
Foster RC (1981a) Polysacharrides in soil fabrics. Science
214:665–667
Foster RC (1981b) The ultrastructure and histochemistry of the
rhizosphere. New Phytol 89:263–273
Foster RC (1982) The fine structure of epidermal cell mucilages
of roots. New Phytol 91:727–740
Foster RC, Rovira AD, Cock TW (1983) Ultrastructure of the
root-soil interface. American Phytopathological Society,
St. Paul
Fries N, Forsman B (1951) Quantitative determination of certain
nucleic acid derivatives in pea root exudate. Physiol Plant
4:410–420
Gamalero E, Lingua G, Capri FG et al (2004) Colonization
pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron
microscopy. FEMS Microbiol Ecol 48:79–87
Plant Soil (2012) 355:1–16
Gamalero E, Lingua G, Tombolini R et al (2005) Colonization
of tomato root seedling by Pseudomonas fluorescens
92rkG5: spatio-temporal dynamics, localization, organization, viability and culturability. Microbial Ecol 50:289–297
Gautheret MR (1933) Cultures of cells isolated from the root
cap. CR Acad Sci 186:638–640
Gilbert GS, Clayton MK, Handelsman J, Parke JL (1996) Use of
cluster and discriminant analysis to compare rhizosphere
bacterial populations following biological perturbation.
Microbial Ecol 32:123–147
Gochnauer MB, Sealey LJ, McCully ME (1990) Do detached
root cap cells influence bacteria associated with maize
roots? Plant Cell Environ 13:793–801
Goldberg NP, Hawes MC, Stanghellini ME (1989) Specific attraction to and infection of cotton root cap cells by zoospores of
Pythium dissotocum. Can J Bot 67:1760–1767
Graham TC (1991) Flavonoid and isoflavonoid distribution in
developing soybean seedling tissues and in seed and root
exudates. Plant Physiol 95:594–603
Griffin GJ, Hale MG, Shay FJ (1975) Nature and quantity of
sloughed organic matter produced by roots of axenic peanut plants. Soil Biol Biochem 8:29–32
Guinel FC, McCully ME (1986) Some water-related physical
properties of maize root cap mucilage. Plant Cell Environ
9:657–666
Guinel FC, McCully ME (1987) The cells shed by the root cap
of Zea: their origin and some structural and physiological
properties. Plant Cell Environ 10:565–578
Gunawardena U, Hawes MC (2002) Tissue specific localization
of root infection by fungal pathogens: role of root border
cells. Mol Plant Microbe Int 15:1128–1136
Gunawardena U, Rodriguez M, Straney D et al (2005) Tissue
specific localization of root infection by Nectria haematococca: mechanisms and consequences. Plant Physiol
137:1363–1374
Hamamoto L, Hawes MC, Rost TL (2006) The production and
release of living root cap border cells is a function of root
apical meristem type in dicotyledonous angiosperm plants.
Ann Bot 97:917–923
Handelsman J (2004) Metagenomics: application of genomics to
uncultured microorganisms. Microbiol Mol Biol Rev
68:669–685
Handelsman J, Stabb EV (1996) Biocontrol of soilborne plant
pathogens. Plant Cell 8:1855–1869
Harding M, Kubes P (2012) Innate immunity in the vasculature:
interactions with pathogenic bacteria. Curr Opin Microbiol
15:85–91
Hawes MC, Wheeler H (1982) Factors affecting victorininduced cell death: temperature and plasmolysis. Physiol
Plant Pathol 20:137–144
Hawes MC, Pueppke SG (1986) Sloughed peripheral root cap
cells: yield from different species and callus formation
from single cells. Am J Bot 73:1466–1473
Hawes MC, Pueppke SG (1987) Correlation between binding of Agrobacterium tumefaciens by root cap cells
and susceptibility of plants to crown gall. Plant Cell
Rep 6:287–290
Hawes MC, Smith LY (1989) Requirement for chemotaxis
in pathogenicity of Agrobacterium tumefaciens on
roots of soil-grown pea plants. J Bacteriol 171:5668–
5671
13
Hawes MC, Brigham LA (1992) Impact of root border cells on
microbial populations in the rhizosphere. Adv Plant Pathol
8:119–148
Hawes MC, Smith LY, Howarth AJ (1988) Agrobacterium
tumefaciens mutants deficient in chemotaxis to root exudates. Mol Plant Microbe Int 1:182–186
Hawes MC, Brigham LA, Wen F, Woo HH, Zhu Y (1998)
Function of root border cells in plant health: pioneers in
the rhizosphere. Ann Rev Phytopathol 36:311–327
Hawes MC, Gunawardena U, Miyasaka S, Zhao X (2000) The
role of root border cells in plant defense. Trends Plant Sci
5:128–133
Hawes MC, Bengough G, Cassab G, Ponce G (2003) Root caps
and rhizosphere. J Plant Growth Regul 21:352–367
Hawes MC, Curlango-Rivera G, Wen F et al (2011) Extracellular DNA: the tip of root defenses. Plant Sci 180:741–745
Hinsinger P (2001) Bioavailability of soil inorganic P in the
rhizosphere as affected by root-induced chemical changes:
a review. Plant Soil 237:173–195
Hinsinger P, Brauman A, Devau N et al (2011) Acquisition of
phosphorus and other poorly mobile nutrients by roots.
Where do plant nutrition models fail? Plant Soil 348:29–61
Hirsch AM (2004) Plant-microbe symbioses: a continuum from
commensalism to parasitism. Symbiosis 37:345–363
Humphris SN, Bengough AG, Griffiths BS et al (2005) Root
cap influences root colonisation by Pseudomonas fluorescens SBW25 on maize. FEMS Microbiol Ecol 54:123–130
Iijima M, Griffiths B, Bengough AG (2000) Sloughing of cap
cells and carbon exudation from maize seedling roots in
compacted sand. New Phytol 145:477–482
Iijima M, Sako Y, Rao TP (2003) A new approach for the
quantification of root cap mucilage exudation in the soil.
Plant Soil 255:399–407
Izano EA, Amarante MA, Kher WB, Kaplan JB (2008) Differential roles of poly-N-acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus aureus and S.
epidermidis biofilms. Appl Environ Microbiol 74:470–476
Jaroszuk-Scisel J, Kurek E, Rodzik B, Winiarczyk K (2009)
Interactions between rye (Secale cereale) root border cells
(RBCs) and pathogenic and nonpathogenic rhizosphere
strains of Fusarium culmorum. Mycol Res 113:1053–1061
Jones DD, Morre DJ (1973) Golgi apparatus mediated polysaccharide secretion by outer root cap cells of Zea mays. III.
Control by exogenous sugars. Physiol Plant 29:68–75
Jones DL, Nguyen C, Finlay RD (2009) Carbon flow in the
rhizosphere: carbon trading at the soil-root interface. Plant
Soil 321:5–33
Kawasaki H, Iwamuro S (2008) Potential roles of histones in
host defense as antimicrobial agents. Infect Disord Drug
Targets 8:195–205
Knee EM, Gong FC, Gao MS et al (2001) Root mucilage from
pea and its utilization by rhizosphere bacteria as a sole
carbon source. Mol Plant Microbe Int 14:775–784
Knox OGG, Vadakattu GVSR (2005) Evaluation of border cell
number and cry protein expression from root tips of Gossypium hirsutum. In: Cote JC, Otvos IS, Schwartz JL (eds)
Pacific rim conference on the biotechnology of Bacillus
thuringiensis and its environmental impact
Knox OGG, Gupta VVSR, Nehl DB, Stiller WN (2007) Constitutive expression of cry proteins in roots and border cells
of transgenic cotton. Euphytica 154:83–90
14
Knox OGG, Gupta VVSR, Lardner R (2009) Cotton cultivar
selection impacts on microbial diversity and function.
Aspects Appl Biol 98:1–8
Knudson L (1917) The toxicity of galactose and mannose for
green plants and the antagonistic action of other sugars
toward these. Am J Bot 4:430–437
Knudson L (1919) Viability of detached root cap cells. Am J Bot
6:309–310
Kraszewska EK, Bjerknes CA, Lamm SS, Van’t Hopf J (1985)
Extrachromosomal DNA of pea-root (Pisum sativum) has
repeated sequences and ribosomal genes. Plant Mol Biol
5:353–361
Kubista M, Akerman B, Norden B (1987) Characterization of
interaction between DNA and 4,6-diamidino- 2- phenylindole by optical spectroscopy. Biochemistry 26:4545–4553
Kuzyakov YV (2001) Tracer studies of carbon translocation by
plants from the atmosphere into the soil. Eurasian Soil Sci
34:28–42
Kwon C, Bednarek P, Schulze-Lefert P (2008) Secretory pathways
in plant immune responses. Plant Physiol 147:1575–1583
Lagopodi AL, Ram AFJ, Lamers GEM et al (2002) Novel
aspects of tomato root colonization and infection by Fusarium oxysporum f. sp radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green
fluorescent protein as a marker. Mol Plant Microbe Int
15:172–179
Lee A, Hirsch AM (2006) Signals and responses: choreographing the complex interaction between legumes and alphaand beta-rhizobia. Plant Signal Behavior 1:161–168
Levy-Booth DJ, Campbell RG, Gulden RH et al (2007) Cycling
of extracellular DNA in the soil environment. Soil Biol
Biochem 39:2977–2991
Liu B, Zeng Q, Yan F et al (2005) Effects of transgenic plants on
soil microorganisms. Plant Soil 271:1–13
Liu J, Yu M, Wang W, Feng Y (2007) Influence of boron and
aluminum on production and viability of root border cells
of pea. Adv Plant Animal Boron Nutrition, pp 67–74
Loh J, Pierson EA, Pierson LS III, Stacy G, Chatterjee A (2002)
Quorum sensing in plant-associated bacteria. Curr Opin
Plant Biol 5:1–5
Lundegarth H, Stenlid G (1944) On the exudation of nucleotides
and flavonone from living roots. Arkiv f Bot 31A:10
Luster J, Gottlein A, Nowack B, Sarret G (2009) Sampling,
defining, characterizing and modeling the rhizosphere—
the soil science tool box. Plant Soil 321:457–482
Lynch JM, Whipps JM (1990) Substrate flow in the rhizosphere.
Plant Soil 129:1–10
Lynch MA, Staehelin LA (1995) Immunocytochemical localization of cell wall polysaccharides in the root tip of Avena
sativa. Protoplasma 188:115–127
Marschner P, Crowley D, Rengel Z (2011) Rhizosphere interactions between microorganisms and plants govern iron
phosphorus acquisition along the root axis—model and
research methods. Soil Biol Biochem 43:883–894
Matsuyama T, Satoh H, Yamada Y, Hashimoto T (1999) A
maize glycine-rich protein is synthesized in the lateral root
cap and accumulates in the mucilage. Plant Physiol
120:665–674
Maxwell CA, Phillips DA (1990) Concurrent synthesis and
release of nod gene inducing flavonoids from alfalfa roots.
Plant Physiol 93:1552–1558
Plant Soil (2012) 355:1–16
McDougall BM, Rovira AD (1970) Sites of exudation of C-14labelled compounds from wheat roots. New Phytol 69:999
Medina E (2009) Neutrophil extracellular traps: a strategic tactic
to defeat pathogens with potential consequenes for the
host. J Innate Immun 1:176–179
Miki NK, Clarke KJ, McCully ME (1980) A histological and
histochemical comparison of the mucilages on the root tips
of several grasses. Can J Bot 58:2581–2593
Mitroulis I, Kambas K, Chrysanthopoulou A et al (2011) Neutrophil extracellular trap formation is associated with IL1beta and autophagy-related signaling in gout. PLoS One
6:e29318
Miyasaka S, Hawes MC (2001) Possible role of root border cells
in detection and avoidance of aluminum toxicity. Plant
Physiol 125:1978–1987
Moody SF, Clarke AE, Bacic A (1988) Structural analysis of
secreted slime from wheat and cowpea roots. Phytochemical 27:2857–2861
Moore R, Fondren WM (1986) The possible involvement of
root-cap mucilage in gravitropism and calcium movement
across root tips of Allium cepa L. Ann Bot 58:381–387
Morris CE, Monier J-M (2003) The ecological significance of
biofilm formation by plant-associated bacteria. Ann Rev
Phytopathol 41:429–453
Newcomb EH (1967) Fine structure of protein-storing plastids
in bean root tips. J Cell Biol 33:143–163
Oades JM (1978) Mucilage at the root surface. J Soil Sci 29:1–
16
Odell RE, Dumlao MR, Samar D, Silk WK (2008) Stagedependent border cell and carbon flow from roots to rhizosphere. Am J Bot 95:441–446
Pan J, Ye D, Wang L et al (2004) Root border cell development
is a temperature-insensitive and Al-sensitive process in
barley. Plant Cell Physiol 45:751–760
Park SJ, Pai KS, Kim JH, Shin JI (2012) ANCA-associated glomerulonephritis in a patient with infections endocarditis: the
role of neutrophil extracellular traps? Revue Med Int 33:57
Patat SA, Carnegie RB, Kingsbury C et al (2004) Antimicrobial
activity of histones from hemocytes of the pacific white
shrimp. Eur J Biochem 271:4825–4833
Patel S, Kumar S, Jyoti A et al (2010) Nitric oxide donors
release extracellular traps from human neutrophils by augmenting free radical generation. Nitric Oxide 22:226–234
Peters NK, Long SR (1988) Alfalfa root exudates and compounds which promote or inhibit induction of Rhizobium
meliloti nodulation genes. Plant Physiol 88:396–400
Phillips HL, Torrey JG (1971) Deoxyribonucleic acid synthesis
in root cap cells of cultured roots of Convolvulus. Plant
Physiol 48:213–218
Pierson LS III, Pierson EA (2007) Roles of diffusible signals in
communication among plant-associated bacteria. Phytopathology 97:227–232
Pietramellara G, Ascher J, Borgogni F et al (2009) Extracellular
DNA in soil and sediment: fate and ecological relevance.
Biol Fertil Soils 45:219–235
Pilszik FH, Salina D, Poon KK et al (2010) A novel mechanism of
rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185:7413–7425
Pinton R, Varanini Z, Nanipieri P, eds (2007) The rhizosphere:
biochemistry and organic substances at the soil-plant interface. Marcel Dekker, Inc. New York, Basel
Plant Soil (2012) 355:1–16
Ponce G, Barlow PW, Feldman LJ, Cassab GI (2005) Auxin and
ethylene interactions control mitotic activity of the quiescent centre, root cap size, and pattern of cap cell differentiation in maize. Plant Cell Environ 28:719–732
Read DB, Gregory PJ, Bell AE (1999) Physiological properties
of axenic maize root mucilage. Plant Soil 211:87091
Rogers HT, Pearson RW, Pierre WH (1942) The source and
phosphatase activity of exoenzyme systems of corn and
tomato roots. Soil Sci 54:353–365
Rovira AD (1969) Plant root exudates. Bot Rev 35:35–57
Rovira AD (1991) Rhizosphere research: 85 years of progress
and frustration. In: Keister DL, Cregan PB (eds) The rhizosphere and plant growth. Kluwer, Dordrecht, pp 3–13
Saxena D, Stotzky G (2001) Bacillus thuringiensis (Bt) toxin
released from root exudates and biomass of Bt corn has no
apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol Biochem 33:1225–1230
Schroth MN, Snyder WC (1961) Efect of host exudates on
chlamydospore germination of the bean root rot fungus
Fusarium solani f sp phaseoli in soil. Phytopathology
52:279–285
Sealey LJ, McCully ME, Canny MJ (1995) The expansion of
maize root cap mucilage during hydration. I. Kinetics.
Physiol Plant 93:38–46
Sherwood RT (1987) Papilla formation in corn root cap cells
and leaves inoculated with Colletotrichum graminicola.
Phytopathology 77:930–934
Smucker AJM, Erickson AE (1987) Anaerobic stimulation of
root exudates and disease of peas. Plant Soil 99:423–433
Somasundaram S, Fukuzono S, Iijima M (2008) Dynamics of root
border cells in rhizosphere soil of Zea mays L.: crushed cells
during root penetration, survival in soil, and long term soil
compaction effect. Plant Prod Sci 11:440–446
Stenlid G (1944) Physicochemical properties of the surface of
growing plant cells. Nature 153:618–619
Stubbs VEC, Standing D, Knox OGG et al (2004) Root border
cells take up and release glucose-C. Ann Bot 93:221–224
Sylvia DM, Fuhrmann JJ, Hartel PG, Zuberer DA (1998) Principles
and applications of soil microbiology. Prentice Hall, USA
Tamas L, Budikova S, Huttova J et al (2005) Aluminuminduced cell death of barley root border cells is correlated
with peroxidase and oxalate oxidase-mediated hydrogen
peroxide production. Plant Cell Rep 24:189–194
Tapp H, Stotzky G (1997) Monitoring the fate of insecticidal
toxins from Bacillus thuringiensis in soil with flow cytometry. Can J Microbiol 43:1074–1078
Urban CF, Lourido S, Zychlinsky A (2006) How do microbes
evade neutrophil killing? Cell Microbiol 8:1687–1696
Uren NC (2001) Types, amounts, and possible functions of
compounds released into the rhizosphere by soil-grown
plants. In: Pinton R, Varanini P, Nannipieri P (eds) The
rhizosphere: biochemistry and organic substances at the
soil-plant interface. Marcel Dekker, Inc., New York, Basel.
pp 19–40
VanEgeraat AWSM (1975) Exudation of ninhydrin-positive
compounds by pea seedling roots: a study of the sites of
exudation and of the composition of the exudate. Plant Soil
42:37–47
Van’t Hopf J, Bjerknes CA (1982) Cells of pea (Pisum sativum)
that differentiate from G2 phase have extrachromosomal
DNA. Mol Cell Biol 2:339–345
15
Vermeer J, McCully ME (1982) The rhizosphere in Zea: new
insight into its structure and development. Planta 156:45–
61
Vicre M, Santaella C, Blanchet S, Gateau A, Driouich A (2005)
Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with rhizobacteria.
Plant Physiol 138:998–1008
Vlassov VV, Laktionov PP, Rykova EY (2007) Extracellular
nucleic acids. Bioessays 29:654–667
Voeller BR, Ledbetter MC, Porter KR (1964) The plant cell:
aspects of its form and function. In: Bracket J, Minsky EE
(eds) The cell, vol 6. Academic, London, pp 245–312
Wang Y, Li M, Stadler S, Correll S et al (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184:205–
213
Wardini AB, Guimaraes-Costa AB, Nascimento MT et al (2010)
Characterization of neutrophil extracellular traps in cats
naturally infected with feline leukemia virus. J Gen Virol
91:259–264
Watt M, McCully ME, Jeffree CE (1993) Plant and bacterial
mucilages of the maize rhizosphere: comparison of their
soil binding properties and histochemistry in a model system. Plant Soil 151:151–165
Watt M, Silk WK, Passioura J (2006) Rates of root and organism growth, soil conditions and temporal and spatial development of the rhizosphere. Ann Bot 97:839–855
Weller DM (1988) Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu Rev Phytopathol 26:379–407
Wen F, Zhu Y, Brigham LA, Hawes MC (1999) Expression of an
inducible pectinmethylesterase gene is required for border
cell separation from roots of pea. Plant Cell 11:1129–1140
Wen F, Curlango-Rivera G, Hawes MC (2007a) Proteins among
the polysaccharides: a new perspective on root cap slime.
Plant Signal Behavior 2:1–3
Wen F, VanEtten HD, Tsaprailis G, Hawes MC (2007b) Extracellular proteins in pea root tip and border cell exudates.
Plant Physiol 143:773–783
Wen F, Woo HH, Pierson EA et al (2008) Synchronous elicitation of development in root caps induces transient gene
expression changes common to legume and gymnosperm
species. Plant Mol Biol Rep 27:58–68
Wen F, White GJ, VanEtten HD et al (2009) Extracellular DNA
is required for root tip resistance to fungal infection. Plant
Physiol 151:820–829
Wen F, Shen A, Choi A, Shi J (2012) A xenograft pancreatic
cancer mouse model to study the function of extracellular
DNA in metastasis. Proceedings, American Association of
Cancer Research
Whipps J, Lynch JM (1983) Substrate flow and utilization in the
rhizosphere of cereals. New Phytol 95:605–623
Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS (2002)
Extracellular DNA required for bacterial biofilm formation.
Science 295:1487
Woo HH, Hirsch AM, Hawes MC (2004) Altered susceptibility
to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle. Plant Cell
Rep 12:967–973
Wood RKS (1967) Physiological plant pathology. Blackwell,
Oxford
16
Wuyts N, Maung ZTZ, Swennen R, De Waele D (2006) Banana
rhizodeposition: characterization of root border cell production and effects on chemotaxis and motility of the parasitic
nematode Radopholus similis. Plant Soil 283:217–228
Xu J, Zhang X, Pelayo R, Monestier M et al (2009) Extracellular histones are major mediators of death in sepsis. Nat
Med 15:1318–1321
Yost CC, Cody MJ, Harris ES et al (2009) Impaired NET
formation: a novel innate immune deficiency of human
neonates. Blood 113:6419–6427
Young RL, Malcolm KC, Kret JE, Caceres SM et al (2011)
Neutrophil extracellular trap (NET)-mediated killing of
Pseudomonas aeruginosa: evidence of acquired resistance
within the cystic fibrosis airway, independent of CFTR.
PLoS One 6:e23637
Plant Soil (2012) 355:1–16
Zentmyer G (1963) Biological control of Phytophthora root rot
of avocado with alfalfa meal. Phytopathology 53:1383
Zhao X, Misaghi IJ, Hawes MC (2000) Stimulation of border
cell production in response to increased carbon dioxide
levels. Plant Physiol 122:181–188
Zhu M, Ahn S, Matsumoto H (2003) Inhibition of growth and
development of root border cells in wheat by Al. Physiol
Plant 117:359–367
Zhu Y, Pierson LS, Hawes MC (1997) Induction of microbial
genes for pathogenesis and symbiosis by chemicals from
root border cells. Plant Physiol 115:1691–1698
Zobel RW, Wright SF (2005) Roots and soil management:
interactions between roots and the soil. American Society
of Agronomy, Inc., Crop Science Society of America, Inc.,
Soil Science Society of America, Inc. Madison WI, USA