Physiological and Molecular Plant Pathology (2000) 57, 101±110

doi:10.1006/pmpp.2000.0287, available online at http://www.idealibrary.com on

Phenolic-storing cells: keys to programmed cell death and periderm

formation in wilt disease resistance and in general defence responses
in plants?
CA R L H . B E C K M A N
Department of Plant Sciences, College of the Environment and Life Sciences, University of Rhode Island, Kingston, R. I.,
02881, U.S.A.

(Accepted for publication August 2000)

Specialized cells of plants synthesize phenolics and store them in their vacuoles during the normal
processes of di€erentiation. Such phenolic-storing cells are distributed within most tissues. In some tissues
they occur uniformly in all of the cells, whereas in other tissues they occur randomly scattered, and in still
others they appear to be strategically located at potential points of entry. Based on the evidence presented
herein, it is proposed that these cells can, ®rst, by decompartmentation, rapid oxidation of their phenolic
content, and the ensuing ligni®cation and suberization of cells, and cell death, seal o€ infections or injuries
at the immediate site of cellular penetration and, secondly, if this defence should fail and the stress persist,
these same processes promote the prolonged build-up of IAA and ethylene that cause a further metabolic
cascade in outlying cells that includes secondary metabolism and growth responses to produce a
peridermal defence in depth. A relationship between the strategic location of these phenolic-storing cells
within plant tissues and the defence sequence that ensues is proposed. *c 2000 Academic Press

Keywords: callose; ¯avanoids; hypersensitive reaction; ligni®cation; periderm; phenolic compounds;

plant defence; programmed cell death; suberization; tyloses; banana (Musa acuminata L.); castor bean
(Ricinus communis L.); cotton (Gossypium hirsutum L.); hops (Humulus lupulus L.); melon (Cucumis melo L.);
pea (Pisum sativum L.); potato (Solanum tuberosum L.); radish (Raphanus sativus L.); tomato (Lycopersicon
esculentum Mill.); Fusarium oxysporum Schlect. f. spp. Snyd. et Hans; Ralstonia solanacearum E. F. Smith;
Saccharomyces cerevisiae van der Walt; Verticillium albo-atrium Reine & Berth.

INTRODUCTION hydroxyl groups that may be variously elaborated with

Plant phenolics may be usefully thought of in two classes:
(1) those that are synthesized during the normal develop-
ment of plant tissues [30, 53] and (2) those that are
synthesized by plants in response to physical injury,
infection or other stress [50]. Other than their role in
inhibiting potential pathogens directly, we, as plant
pathologists, have not, intentionally studied the former
and their role in defence except in a few cases, e.g.
catechol and protocatechuic acid in onion [76]. It is these
innate phenolic substances, their occurrence in various
plant tissues, and their overall function in plant defence
that I am intrigued by and shall consider here.
Phenolics that occur innately in plants have long been
reported and have been intensively studied in taxonomic
and chemical terms [14]. They occur ubiquitously in
plants in a variety of tissues [30, 31, 68]. Indeed, more
than 50 000 secondary metabolites, including phenolics of
great diversity, have now been shown to be compartmen-
ted within plant cell vacuoles [53, 79]. All of the phenolics
have one or more benzene rings with one or more
0885-5765/00/090101+10 $35.00/00
methyl, methoxyl, amino or glycosyl groups. They are,
therefore, extremely diverse and di€er considerably from
species to species and from tissue to tissue within a species
[20, 56]. They are widely used to classify plants taxo-
nomically [14, 23] and some earlier researchers judged
that they are evolutionary remnants that have no current
function (Challice, pers. comm.). Others suggested that
they were metabolic end-products that are toxic to the
plant and are therefore stored away in vacuoles of
specialized cells (tannin cells), for safe keeping [53, 61].
Evidence that constitutive phenolics play a vital role in
plants is that many of them are recycled within the
metabolic stream of plants [2, 25, 70]. They have been
shown to serve as signalling molecules [79] and to
modulate the action of IAA [79]. It is more in keeping
with our evolutionary understanding that they provide
some clear bene®t to the plants that o€sets the cost of
maintaining the enzymatic pathways and expending the
resources and energy needed to produce and store them.
Yet, as stated in Salisbury and Ross [61], ``With
*
c 2000 Academic Press
102 Carl H. Beckman
important exceptions, the functions of most phenolics are
obscure. Many presently appear simply to be by-products
of metabolism, but this view likely re¯ects our still poor
knowledge of ecological biochemistry''.
Several plausible and clearly functional roles for some of
these compounds have been proposed and documented.
They include: (1) coloration of ¯owers that attracts
pollinating animals; (2) protection from injurious UV
radiation; (3) deterrence to grazing animals and feeding
insects because of their astringent, toxic nature; (4) pro-
viding a tanglefoot mechanism for trapping feeding
insects; (5) resistance to pathogens; and (6) volatiles that
a€ect the growth of other neighboring plants [28, 30, 79].
Thus the primary established roles are clearly ecological in
nature, some having dual or even multiple functions [77,
79]. Furthermore, in his insightful treatment of the role of
plant cell vacuoles in the storage of secondary metabolites
and xenobiotics, Wink [79] has pointed out that phenolics
are often stored at strategically important sites where they
play a signalling role, and often a direct role, in defence.
Herein I propose, ®rst, to document the occurrence of
phenolic-storing cells in plant tissues, then to ¯esh out
Wink's signalling hypothesis with respect to their role in
defence within xylem tissues, and, ®nally, to draw
together evidence regarding their role in triggering
defence responses in general, including programmed cell
death and periderm formation.
THE OCCURRENCE OF PHENOLIC-STORING
CELLS IN VARIOUS PLANT TISSUES
In 1963, Mace [38] examined the specialized ``tannin''
cells that were randomly distributed in the xylem
parenchyma tissues of freshly prepared roots of banana
(Musa acuminata L.). These cells did not contain poly-
merized or condensed ``tannins'' {see [67], pp. 169±197}
as earlier reported and generally assumed, but rather,
contained a free o-dihydroxyphenol (3-hydroxytyramine)
commonly known as dopamine. This was an important
®nding because phenolics that are in a free state are
normally oxidized and polymerize rapidly. Thus the
3-hydroxytyramine that Mace found in these specialized
phenolic-storing cells was apparently compartmented in
some way and maintained in a reduced state within the
vacuoles of the living cells that synthesized and stored
them. That mechanism is now clear. The major tonoplast
protein is an H ‡ -ATPase. The H ‡ concentration of the
vacuole is therefore ``orders of magnitude higher than that
in the cytoplasm'' [79] and serves to maintain the
hydroxyl groups of phenolics in a non-ionized, reduced
state within the vacuoles.
In closely-linked histochemical and biochemical studies
of phenolic-storing cells in roots of cotton (Gossypium
hirsutum L.), it was shown that two of the phenolics stored
are catechin and gallocatechin [39, 40]. These ¯avanoid
compounds were readily observed histochemically to be
uniformly present in all root cap and endodermal cells
and in cells around emerging secondary roots. They also
occur as randomly scattered cells, making up 5±10 % of
the cell population (unpublished work), within cortical,
ray, pith, and xylem parenchyma tissues, including the
paravascular contact cells that ensheath xylem vessels
[48]. After being cut o€ in the meristem of the root tip,
the endodermal cells in cotton di€erentiated into fully
developed phenolic-storing cells within 60±90 min [48].
COMPARTMENTATION OF ENZYMES
INVOLVED IN PHENOLIC SYNTHESIS
AND UTILIZATION
Mueller and Beckman [49] examined the localization of
the enzymes and structures by which these phenolics can
be synthesized and maintained in a reduced state, on the
one hand, and can be rapidly oxidized, on the other. In
young, healthy root and hypocotyl tissues of cotton, the
enzymes were located in di€erent cellular compartments.
Polyphenoloxidase (PPO) was con®ned to the thylakoids
of plastids, where the starch reserves are stored and the
phenolics are synthesized, a frequent observation [79].
Once synthesized, the phenolics are transferred to the cell
vacuoles where they are stored, also a common obser-
vation [79]. Catalase was con®ned to cytoplasmic
microbodies, whereas peroxidase was con®ned to the
cell walls of most epidermal, subepidermal, and endo-
dermal cells, as well as in randomly scattered cortical and
stelar parenchyma cells [49].
Thus, these phenolic-storing cells have a highly
specialized distribution within the plant tissues and an
elaborate organization that serves to synthesize the
phenolics and keep them compartmented and in a
reduced, poised state within their vacuoles, while at the
same time providing the means for their rapid oxidation
should decompartmentation occur. Such decompartmen-
tation can be triggered by a variety of environmental
stresses including wounding or infection by a variety of
organisms [5]. Even when freshly sectioned tissues are
exposed to strong substage lighting of a microscope, a
sudden release of phenolics can be seen (unpublished
personal observations).
It should be noted that many stored phenolics occur as
glycosides, in which form they are not toxic to plant cells.
Glycosidases, which would strip them of their glucose
moeity, are also numerous and highly speci®c with
respect to the aglycone involved [16, 20]. Because these
aglycones are highly reactive and toxic to the cell proto-
plasts, these processes must also be rigorously controlled
in normal plant functioning. This control is also achieved
by compartmentation. The storing site of the glycoside is
 Phenolic-storing cells
commonly in the cell vacuole, but the various glycosidase
enzymes are apparently located at or near the site of
ultimate utilization of the aglycone. Glycosidases associ-
ated with ligni®cation, for example, are con®ned to the
apoplast of cells, whereas another that is implicated in the
hydrolysis of phytohormone glycosides is con®ned to
plastids. Still others are con®ned to the exterior of the
tonoplast [20]. Although these glucoside/glucosidase
systems have been implicated in a variety of key
metabolic events and growth related responses, most of
the postulated functions have not as yet been rigorously
documented and elucidated [20].
PHENOLIC-STORING CELLS IN XYLEM
TISSUES AND THEIR ROLE IN DEFENCE
Xylem tissues a€ord a convenient and e€ective material
for study of host-parasite interactions because one can, by
severing roots under an aqueous medium and allowing
transpirational uptake through the vessels, quickly
introduce fungal spores, together with tracer particles,
at a speci®c time into a speci®c mass of tissue. These
tissues can then be easily excised for further study in a
time course fashion [4].
Xylem vessels, by their design and function, provide a
particularly dangerous avenue for extensive and rapid
distribution of parasites should a break in the system
occur. Perforation plates at the ends of vessels provide
check points that screen out fungal spores from the
transpiration stream, but organisms that can quickly
germinate, penetrate these end-walls and sporulate in
the next xylem element above present a serious threat to
the plant. Vascular pathogens do so. But it takes 2±3
days for a successful fungal vascular parasite to germi-
nate, grow through vessel end-walls and produce mature
spores that can abscise and be carried further upward
in the transpiration stream in the next vessel element
above. Thus the plant has a period of 2, and at most 3,
days in which to seal o€ the infection sites [8]. These
®ndings are presented in a model of the defence system in
xylem tissue (Fig. 1). It is the role of phenolic-storing cells
in programming several of these defence processes that I
shall present here.
ROLE OF STORED PHENOLICS IN
LIGNIFICATION
Histochemical, biochemical and ®ne-structural studies
have shown phenolic-storing cells to be present in the
xylem tissues of roots of several plant species, including
banana, cotton, potato (Solanum tuberosum L.) and tomato
(Lycopersicon esculentum Mill.) [38±40, 42, 47, 48, 75].
Following vascular infection of many species of higher
103
plants by any of a variety of organisms, including the
vascular pathogens Fusarium oxysporum Schlecht., f. spp.
Synd. and Hans, and Verticillium albo-atrum Reinke &
Berth, as well as non-pathogens including brewer's yeast
(Saccharomyces cerevisiae van der Walt) [5], these stored
phenolics have been shown to be released and to di€use
out of their vacuolar compartments into the cell at large.
Here they become oxidized and polymerize with each
other and with other host constituents, i.e. cellular
proteins and cell wall carbohydrates, to form ``ligni®ed''
structures. The capacity of a phenolic to infuse and
stabilize wall structures was veri®ed in vitro and in vivo [6].
Thus, it has been demonstrated that phenolics can help
to lock up the immediate sites of infection with
substances that kill the cells in question and, in the
process, create of the cells one large, durable, inert
macromolecule.
It is signi®cant to note that any organism that grew
within the vessels triggered a strong, rapid response,
except the host-speci®c pathogen which triggered a
slower, less pronounced, and less e€ective response [5].
ROLE OF STORED PHENOLICS IN
SIGNALLING FOR A LONGITUDINAL
VASCULAR DEFENCE IN DEPTH
Gordon and Paleg [24] also made the exciting discovery
that the oxidation of various phenolics having mono-
hydroxy, ortho di-hydroxy- and tri-hydroxy con®gur-
ations, mediated the conversion of typtophan to 3-indo-
leacetic acid (IAA). This ®nding was later con®rmed
using dopamine from banana by Mace and Solit [41] and
chlorogenic acid from tomato by Matta and Gentile [44].
Recent evidence has also demonstrated the release of IAA
from cellular conjugates by various hydrolytic enzymes
[1, 51]. It is clear then, as has long been observed, that
IAA can build up rapidly in plants when decompart-
mentation occurs.
Following decompartmentation, two other processes
could cause the level of IAA to rise sharply in the a€ected
xylem tissues. First, ¯avanols have been shown to inhibit
the ATPase pumps in the basal portion of vascular
parenchyma cells that provide for the downward polar
transport of IAA [37, 51]. The release of ¯avanols at the
site of infection would, therefore, cause an accumulation
of IAA above that point. Second, the oxidized phenolics
have been shown to inhibit the destructive oxidation of
IAA thus permitting a further, several-fold build-up of
IAA [63].
Such a build-up of IAA, of host origin, has been
demonstrated to occur in infected vascular tissues of
tomato infected with Fusarium and of tobacco infected
with Ralstonia (Pseudomonas) solanacearum, E. F. Smith
[43, 64] (Fig. 1). The action of IAA in a€ected tissues is
104 Carl H. Beckman
Model
1994
Host factors Pathogen factors
predominate predominate

Space - 1
(h) Tylose
growth
(36–96h)
occlusion-fusion
(optional)

Gel cleavage

2º spores
(48–72h)
Gel-Gum
(g)
(24<96h)
–IAA + ethylene

Hydrolytic
enzymes
Spore Endo-pl (24<72h)
trapping Endo-pg ?
(i)
(t0)
Phenolic
infusion
(9<72h)

(a) Recognition Diminished

Diminished
Cytoplasmic Diminished recognition
(b)

Space - 0
reorganization by
Callose Mimicry?
(c) deposition Inhibition?
Diminished
(4<48h)
(d) Wall coating
(6<42h) Diminished

2º metabolic Fungal invasion

(e) (24h)
synthesis
(18<48h)
Fungal inhibition Diminished
suberinization
Chitinase
(f) β-1,3 glucanase Diminished
(24<168h)

Spores
inoculum
tracer
particles

F I G . 1. A time±space model of longitudinal and lateral host±parasite interactions that occur within Space-0 (the initially
inoculated vessel element below a vessel ending, and the surrounding vascular parenchyma tissue) and in Space-1 (the next vessel
above and its surrounding parenchyma tissue). The left side of the model shows the various defence processes of the host (a)±(i) in
chronological order) and the times of their occurrence in Space-0 and Space-1 that, when they predominate, serve to inhibit a
vascular parasite and to localize the infection. The right side of the model shows the processes of the pathogen and the times of their
occurrence in Space-0 and Space-1 that, when they predominate, enable the parasite to escape Space-0 and traverse Space-1.
Inoculum is initially introduced through severed vessels (bottom of model) and drawn upward through Space-0 by transpirational
pull within 10 min (Time-0). Note that phenolic infusion from a phenolic-storing cell into the vessel is clearly visible by 9 h after
inoculation. The movement of IAA and ethylene upward from the point of phenolic release and oxidation (i) to initiate gel
extrusion and tylose growth is hypothetical, but many-fold increases in the concentrations of IAA and ethylene in infected vascular
tissues have been documented. Reproduced, with permission, from Beckman and Roberts, 1995 [8].
 Phenolic-storing cells
often indicated, in physiological terms, by an oxidative
burst [36, 54] and by the activation of H ‡ pumps and a
drop in apoplastic pH [15, 27]. Such a drop in pH ( from
the normal of 4.5 to 2.5±3.0) was found in the walls of
banana parenchyma cells in contact with Fusarium-
infected vessels [3]. Misaghi et al. [46] found no build-up
of cytokinin concentration in Verticillium-infected vascular
tissues of cotton, but an eventual decline occurred when
foliar symptoms appeared. These results indicate that
there is an early and dramatic shift in the IAA/cytokinin
balance in infected vascular tissues at the sites of
perturbation and immediately above them. Such a shift
in hormone balance has been shown to promote lateral
growth of cells in the a€ected region [21, 34] and
treatment of banana roots with IAA was shown to
promote such lateral growth in adjacent paravascular
parenchyma cells to form tyloses that, when enlarged,
wall o€ the lumina of vessels [41], and an oxidative burst
occurs during this period [4].
Such lateral growth of cells may not, however, be a
direct response to IAA [66]. McKeon et al. [45] have
demonstrated that IAA promoted ethylene production by
inducing the synthesis of ACC synthase, which can occur
in as little as 30 min [59]. Furthermore, treatment with
ethylene alone promotes the lateral growth of cells [55]. A
marked increase in ethylene levels has also been
demonstrated to occur in tomatoes following vascular
infections [18]. Thus the occurrence of lateral growth and
tylose formation likely involves both IAA and ethylene
(Fig. 1).
In the paravascular parenchyma cells that surround
xylem elements, such lateral growth of cells is expressed in
several ways, depending on the structure of the xylem
elements themselves. First, in the case of protoxylem
elements with annular or spiral thickenings, such lateral
growth serves to crush the xylem vessels, which have little
structural stability. In the case of metaxylem vessels in
which the elements have strong reticulate walls, growth
responses of adjacent parenchyma cells may occur in two
additional ways. Growth may occur by the invagination
of the pits and the formation of bladder-like outgrowths
(tyloses) that wall o€ the a€ected vessels above the site of
invasion. Talboys has demonstrated, however, that large,
vessel-occluding tyloses are formed in hops (Humulus
lupulus L.), only when pits of the vessel walls are large
enough to allow the nucleus of the growing parenchyma
cells to pass and enter the expanding portion of the cell in
the vessel in question [69]. He found that when pits were
too small to allow such movement, little cell enlargement
and vessel occlusion by tyloses occurred. Synthesis of wall
materials occurred, however, and they were extruded to
form gels in the vessel lumina. Beckman and Roberts [8]
reported that such invagination and tylose formation, as
well as occlusion by extruded gels, occurs in reticulate
vessels of banana, tomato and melon (Cucumis melo L.)
105
tissues in which pit apertures are large (40±50 mm2).
However, in vessels with simple or bordered pits having
apertures less than approx. 0.5 mm2, as in cotton, radish
(Raphanus sativus L.) and pea (Pisum sativum L.), gels but
no tyloses were formed. They too found that the
occluding gels were extruded from the paravascular
parenchyma cells through the pit regions and into the
vessel lumina. Vander Molen et al. [71] have demon-
strated, in addition, that such gels in banana consist of the
usual primary wall substances that are synthesized and
(except for cellulose ®bers) extruded into the vessel
lumina. Ethylene was found to induce the formation of
such gels in the vessels of castor bean (Ricinus communis L.)
[72]. Thus, in this third instance, where vessels are
occluded with gels alone, the synthetic processes of
adjacent parenchyma cells appear to continue, although
cell enlargement is prevented by physical constraints
(Fig. 1).
At the interface with the parasite (Fig. 1), all of these
structures (crushed elements, gels and tyloses) then
become ``ligni®ed'' by infusion with phenolics or ``sub-
erized'' by the additional infusion of lipids into the mole-
cular complex [9, 58, 75]. Thus, these tissue responses
share the anatomical features of wound periderm [12] as
well as heartwood formation [65]. They, as well as other
defence processes [11, 19], including de novo phytoalexin
synthesis, the hypersensitive reaction, programmed cell
death, senescence [52] and systemic resistance [74],
appear to be elicited in part, at least, by ethylene. All
apparently result from gene activation by ethylene [11,
52] which has been associated with DNA fragmentation
[59] and the accumulation of mRNAs [13].
There is substantial correlative evidence, then, that
these phenolic-storing cells, produced during normal
di€erentiation and strategically located and kinetically
poised in xylem parenchyma tissues, serve as a sensing
and defence-triggering system. In the event of injury or
infection, these cells ``explode'' with a chemical reaction
that serves to oxidize the phenolics, which then serve to
lignify and/or suberize the site of immediate disturbance
and, in the process, shift the IAA/ethylene/cytokinin
balance. These hormonal reactions serve to sound the
alarm and mobilize a periderm-like defense in depth,
including induced secondary metabolism, for several
centimeters beyond the point of immediate danger.
This sequence of responses, when successfully carried
out, serves to seal o€ the endangered vessels long-
itudinally for some distance. Above this zone of response,
the vessels again become functional by means of
anastomosis with una€ected vessels.
The role in defence of another phenolic, salicylic acid,
as reported by Ga€ney et al. [22] and Kauss and Jeblick
[35], among others, has not been explored in vascular
tissues.
106 Carl H. Beckman
THE LATERAL DEFENCE IN DEPTH IN
VASCULAR TISSUES
The above processes provide for a longitudinal defence in
depth in the xylem tissues. There is also a lateral defense
in depth within xylem parenchyma tissues in which cells
surrounding infected vessels react either by means of
hypersensitivity or the deposition of callose which then
becomes ligni®ed and/or suberized [10] (Fig. 2). Cells
responding with the HR were never found to be invaded.
The relative success or failure of the callose response to
limit tissue invasion varied with the genetic complement
of the host and parasite (Fig. 3). The longitudinal and
lateral components of defence and the approximate times
of their occurrence, together with the actions of a
potential pathogen are presented in the model (Fig. 1).
Although there is a considerable amount of evidence to
support the scenario presented here, the title of this
paper, with respect to the triggering action of phenolic-
storing cells, must end with a question mark. To resolve
that question, it must be shown that IAA and ethylene
(or ACC synthase) do indeed appear at relevant times
(minutes to hours after inoculation) at the speci®c sites of
interaction between parasites and the phenolic-storing
cells they encounter. With the immuno-histochemical
and cytological techniques now available such studies
appear to be feasible.
F I G . 2. Electron micrographs of longitudinal sections of tomato
hypocotyls showing vessels and adjacent xylem parenchyma
cells 3 days (a), (b) and 6 days (c) after inoculation with
A ROLE FOR PHENOLIC-STORING CELLS Fusarium oxysporum f. sp. Lycopersici, race 1 and tracer particles.
AND RESPONSES IN OTHER TISSUES? Red tracer particles were included with the inoculum to de®ne
the sites and time (510 min) of initial spore entrapment. (a) A
Volume 55 of PMPP included an editorial [33], a hypha (h) in a pit of a secondary xylem vessel with an adjacent
commentary [57], and a mini-review [26] on HR and parenchyma cell in which a callose apposition layer has been
programmed cell death (PCD), as well as a second deposited (arrow). The osmiophilic blackening suggests that the
commentary [29] exploring the processes by which callose has become ``ligni®ed'' by infusion with phenolics and
possibly ``suberized'' by the infusion of lipids. The protoplast of
induced resistance may be accomplished. Yet, a long
the parenchyma cell appears healthy, 35 000. (b) A hypha
standing enigma still remains, as stated by Heath [33], (h) in a primary xylem vessel with an apposition layer in an
``Why disease resistance in plants should be almost adjacent parenchyma cell that is particularly thick at the site of
ubiquitously associated with rapid, localized plant cell contact with the hypha (arrow), but extends along the entire
death has been a source of debate ever since the HR was wall of the large pit membrane, 35 000. (c) Four para-
clearly identi®ed by Stakman in 1915.'' It is this enigma vascular parenchyma cells adjacent to an infected secondary
that I wish to explore. xylem vessel. Note the fungal hyphae (h) and small tracer
particles ( p) in the vessel lumen and the occluded, darkened
Most epidermal and/or sub-epidermal cells of leaves
pits (arrows). Of the adjacent parenchyma cells, three show
and succulent stems contain anthocyanins or other ¯ava- layers of protective callose deposit (arrowheads) at the pits and
noids [62], as do the bulbous cells of hairs (trichomes) on along the cell walls adjacent to the infected vessel. The
the leaves and stems of many plants [7]. They are believed protoplasts of these cells are not invaded and appear healthy.
to protect the tissues from injurious UV radiation, but The fourth cell has responded with what appears to be a
they need not have a single function [79]. Dai et al. [17] hypersensitive reaction in which the entire protoplast has
have demonstrated that when cotton cotyledons were become disorganized and electron dense. It has not been
invaded. The tear in the protoplast of this cell, which is a
inoculated with Xanthomonas in an incompatible combi-
common result of sectioning phenolic-storing cells, presumably
nation, ¯avanoids became ®xed into the polysaccharides results from the infusion and incorporation of the phenolic into
of host cell walls, middle lamellae and callose-rich papillae the embedding polymer that changes its sectioning properties,
within 10 h after inoculation in an HR-type reaction. This (3500  ). (c) Reproduced, with permission, from Beckman,
early incorporation of ¯avanoids into cellular structures, Verdier and Mueller, 1989 [10].
 Phenolic-storing cells 107
as compared to de novo synthesis of terpenoids only after Those in hops have been shown to prevent the entry of
48 h, raises an interesting question: could it be that these Verticillium albo-atrum into the stelar tissues of roots [9].
¯avanoids or closely related precursers were already stored Pennell and Lamb [52] in discussing programmed cell
and poised for action in epidermal cells before inoculation death (PCD) in plants remind us that PCD is a process
took place? involved in the selective elimination of unwanted cells.
In higher plant roots, all of the endodermal cells have This process included senescence. By tying up the
been shown to store phenolics [73]. Those in cotton and contents of dying and dead cells, PCD may help prevent
tomato contained catechin and gallocatechin [40, 42]. the infection of a senescing organ or stressed tissue and
thus prevent spread of disease to living parts of the plant.
It is signi®cant to note that among those tissues that
100 are listed as sites in which PCD commonly occurs are
H.R 2/117 Not challenged (a)
Callosed Not root cap cells, trichomes and the bundle sheath cells
8/117 challenged surrounding veins in stems. The latter are more
90 Callosed
11/99 susceptible to death inducing signals than are mesophyll
H.R.
80 0/86 cells. Of these exact sites, the root cap cells [47, 48] and
Callosed the bulbous trichomes [7] have been consistently shown
7/86
70 to store phenolics, whereas the bundle sheath cells of the
stem are an extension of endodermis tissue of the roots
60 [73] which, as we have noted, also store phenolics. The
Cells affected (%)

bundle sheath cells do not store phenolics but do store

50 starch and may well be poised to synthesize phenolics,
(ii)

Infected Infected
107/117 82/99
which we have noted can occur within 1±2 h in
40 di€erentiating endodermal cells [48]. There is, then, a
Infected
60/86 strong correlation between cells that are noted for PCD
30 and those that store phenolics.
Furthermore, it is interesting to note that Wang et al.
20 [78], when examining cells of onion root tips have shown
that cleavage and fragmentation of DNA which precedes
10 PCD and HR [60] occurs only in cells near the surface of
the root caps, precisely the location of phenolic-storing
0 cells in cotton root caps. In inner tissues that are less
Contact 1º Adjacent 2º Adjacent exposed, phenolic-storing cells occur less frequently and
100 (b)
H.R
14/152 F I G . 3. The responses of xylem parenchyma cells of (a)
90 susceptible (ii gene) and (b) resistant (I-1 gene) tomato plants
to infection with the host-speci®c pathogen Fusarium oxysporum
f.sp. lycopersici, race 1. The cells designated ``HR'' responded in a
80
Not manner that resulted in highly osmiophilic and electron opaque
challenged cell walls as well as osmiophilic and opaque, but disorganized,
Callosed
70 59/152 host cell cytoplasm. These cells remained non-colonized by the
pathogen. The cells designated ``callosed'' had thick wall
Cells affected (%)

60 appositions adjacent to the inoculated vessels, beginning at

Not
challenged the challenged pit membranes. These appositions became
``marblized'' with osmiophilic material and were not colonized.
(II)

50
H.R. Cells in the ®rst and second adjacent layers that are labelled
40
21/136 ``not challenged'' were shielded from contact with the pathogen
by cells that had responsed with HR or callose-containing
depositions. The remaining cells became successively invaded
30 by hyphae of the pathogen, the paravascular cells most severely,
Infected Callosed
79/152 29/136
and adjacent cells less so. The fractions given represent the
20 number of cells found in each category (HR, callose, or
invaded) over the total number of cells observed within each
10
H.R. 4/121
layer of cells (contact, or ®rst or second adjacent cells). The
Infected Callosed di€erences in response between the susceptible interactions (a)
20/136 9/121
Infected 3/121
and the single-dominant-gene resistant interactions (b) were not
0
qualitative, but quantitative in nature. Reproduced, with
Contact 1º Adjacent 2º Adjacent permission, from Beckman, Verdier and Mueller, 1989 [10].
108 Carl H. Beckman
are randomly scattered throughout the tissues. These
include the mesophyll of leaves [62], cortical cells of stems
and roots [39], and pith cells ( personal observations).
There is, then, strong positional, biochemical and
physiological evidence to suggest that phenolic-storing
cells may be intimately involved in HR and PCD and the
cascade of processes that result in periderm formation,
that is, all of the defense strategies of plants that delimit
injuries and infections.
WHAT OF APPARENT ANOMOLIES?
Is it possible that the distribution and function of
phenolic-storing cells could account for some of the
di€erent responses shown by leaf epidermal and meso-
phyll cells to penetration by rust fungi as reported by
Heath [32]? In tissues such as the epidermis in which
penetration is direct, all cells encountered would,
presumably, contain phenolics (anthocyanins). Here
HR is the dominant response. On the other hand, when
entry of the parasite is through stomates, the ®rst cells
penetrated would be in mesophll tissues in which
phenolic-storing cells are relatively few and randomly
scattered [62]. Thus the number of phenolic-storing cells
encountered by the parasite in mesophyll tissues would be
relatively few and randomly scattered. The majority of
the cells in such a tissue would be non-phenolic-storing
and would respond, not with PCD or HR, but with a
callose response. If the callose encasement in xylem
tissues is a valid comparison, the success of this reaction
varies considerably depending upon the genetics of the
interactants and environmental circumstances (see
Fig. 3). The host responses that provide for resistance
here would of necessity occur at the tissue level, rather
than at the single cell level, and would result in ¯ecks,
spots or lesions of characteristic size and shape.
Thus, in response to the question raised by Nicholson
and Hammerschmidt [50] with respect to the role in
defence of constitutively produced phenolics, it appears
that phenolic-storing cells may well play key roles in the
defence strategies of plants and that their relative
distribution in various tissues can explain some of the
apparent variations that have been reported with respect
to these defence strategies.
This study constitutes Contribution No. 3685 of the
Rhode Island Agricultural Experiment Station.
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