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33 Ohkawa, H. et al. (1998) Molecular mechanisms of herbicide resistance. Rev.
Toxicol. 2, 245–252
34 Inui, H. et al. (1999) Herbicide metabolism and cross-tolerance in transgenic
potato plants expressing human CYP1A1. Pestic. Biochem. Physiol. 64, 33–46
35 Lacour, T. and Ohkawa, H. (1999) Engineering and biochemical
characterization of the rat microsomal cytochrome P450 to ferredoxinNADP(1) reductase from plant chloroplasts. Biochim. Biophys. Acta 1433,
87–102
36 Pierrel, M.A. et al. (1994) Catalytic properties of the plant cytochrome P450
CYP73 expressed in yeast. Substrate specificity of a cinnamate hydroxylase.
Eur. J. Biochem. 224, 835–844
37 Cabello-Hurtado, F. et al. (1998) Cloning, expression in yeast and functional
characterization of CYP81B1, a plant P450 which catalyses in-chain
hydroxylation of fatty acids. J. Biol. Chem. 273, 7260–7267

38 Robineau, T. et al. (1998) The chemically inducible plant cytochrome P450
CYP76B1 actively metabolizes phenylurea and other xenobiotics. Plant
Physiol. 118, 1049–1056
39 Siminsky, B. et al. (1999) Expression of a soybean cytochrome P450
monooxygenase cDNA in yeast and tobacco enhances the metabolism of

phenylurea herbicides. Proc. Natl. Acad. Sci. U. S. A. 96, 1750–1755

Danièle Werck-Reichhart*, Alain Hehn and Luc Didierjean are
at the Dept of Cellular and Molecular Enzymology, Institute of Plant
Molecular Biology, Centre National de la Recherche Scientifique
UPR 406, 28 rue Goethe, 67083 Strasbourg Cedex, France.
*Author for correspondence (tel 133 3 88 35 83 32;
fax 133 3 88 35 84 84; e-mail daniele.werck@ibmp-ulp.u-strasbg.fr).

Programmed cell death and
aerenchyma formation in roots
Malcolm C. Drew, Chuan-Jiu He and Page W. Morgan
Lysigenous aerenchyma contributes to the ability of plants to tolerate low-oxygen soil
environments, by providing an internal aeration system for the transfer of oxygen from the
shoot. However, aerenchyma formation requires the death of cells in the root cortex. In maize,
hypoxia stimulates ethylene production, which in turn activates a signal transduction
pathway involving phosphoinositides and Ca21. Death occurs in a predictable pattern, is
regulated by a hormone (ethylene) and provides an example of programmed cell death.

R

oots, rhizomes and other plant organs in the soil usually
obtain O2 for respiration directly from their immediate
environment – the soil gaseous atmosphere. But when the
soil becomes excessively wet, transfer of O2 from the air into the
soil is effectively blocked because the larger soil pores, which are
usually air-filled, are water-filled instead. Without replenishment
from the air, any dissolved O2 remaining in the soil is quickly consumed by microorganisms and plants, and the soil is then no
longer able to supply O2. Aerenchyma – tissue comprising a high
proportion of gas-filled spaces or lacunae – provides the plant
with an alternative strategy for obtaining O2. The interconnected
lacunae, extending from below the ground up into the stems and
leaves, make up an internal aeration system, enabling parts of
the plant to survive or grow for a time in environments that are
O2-deficient or even completely devoid of O2.
Spaces form within aerenchymatous organs, either by cell
separation at the middle lamella during development (shizogeny),
or by cell death and dissolution (lysigeny). Sometimes, as in
Sagittaria lancifolia, both processes occur, with lysigenous
aerenchyma in the roots and shizogenous aerenchyma in the

leaf petiole1. In lysigenous aerenchyma formation in the root
cortex of maize, rice and Sagittaria, cell death is first detected
at a distance of a cm or less from the root apical meristem, in the
zone where cell elongation has just been completed2–4. The
space created by the death of cells becomes increasingly prominent in older zones behind the tip. The system of gas-filled
lacunae therefore develops acropetally, extending into the root
towards the tip, simultaneous with the root’s extension into
the soil.

Aerenchyma function

Lysigenous aerenchyma provides not only an internal pathway for
O2 transfer, but also simultaneously reduces the number of O2consuming cells, a feature that might assist in low O2 environments. It has been suggested that aerenchymatous organs are often
water- or fluid-filled5, a feature that would prevent them from
functioning in O2 transfer. However, evidence from the measurement of gas-filled porosity, microscopic observation (Fig. 1) and
O2 transport6 is overwhelming – the lacunae are indeed usually
gas-filled and therefore are able to transfer gases by convection
and diffusion.
Aerenchyma formation is inducible by flooding in maize7,8, in
the coastal grass Spartina patens9 and in many other native

species, both monocot and dicot, that occupy wetland habitats10.
By contrast, in the maize relatives Tripsacum dactyloides (eastern
gamagrass) and Zea luxurians (teosinte)11, as well as in wetland
species, such as rice3,12 and S. lancifolia1,4, aerenchyma forms constitutively, apparently without any requirement for an external
stimulus. Eastern gamagrass possesses notable tolerance to
drought and flooding, which is thought to be because of its deep
rooting pattern and constitutive aerenchyma13. The roots have
been found to exceed 1.8 m in depth, penetrating a resistant clay
pan at or below 0.90 m, apparently when the soil was wet and
mechanically least resistant. Therefore aerenchyma was likely to
have assisted oxygenation of the root at that time. In general, soil
compaction can lower the oxygen concentration of the soil (fewer
large, gas-filled pores for gas exchange with the atmosphere).
Therefore, aerenchyma could be potentially beneficial for growth or
function of roots in a densely packed soil layer. These considerations

1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01570-3

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Fig. 1. Scanning electron micrograph of an aerenchymatous nodal
root of maize. The root was transversely fractured at liquid nitrogen
temperature, and the image of the frozen-hydrated tissue was collected under vacuum without any sublimation of ice. The micrograph shows the hydrated (frozen) cells, and prominent, sap-filled
metaxylem, with lacunae in the cortex that are free of ice. Scale
bar 5 0.1 mm. Micrograph: Malcolm Drew and John Sargent.

at warm temperatures, can outpace the rate of supply of O2 to the
respiring cells. Microsensors for O2 have shown steep concentration gradients across roots, from 20.6% O2 (the concentration
in air) at the epidermal surface of a maize root at 298C, to less
than half that in the center of the stele near the root apex18. Nonaerenchymatous maize roots in a low-O2 environment and receiving O2 principally from the shoot via intercellular spaces, had
steep longitudinal as well as radial gradients, with only 2.5% O2 in
the cortex and 1.0% in the stele at 70 mm below the stem base19.
Thus, conditions conducive to aerenchyma formation might be
common.
The involvement of ethylene in aerenchyma formation is well

documented for maize6; under well-oxygenated (normoxic) conditions, low concentrations of ethylene induce cell death precisely
among those cells that would succumb to hypoxia. Inhibitors of
ethylene action or ethylene biosynthesis block aerenchyma formation in hypoxic roots, whereas in ethylene-treated roots only
inhibitors of ethylene action are effective. The effect of hypoxia is
to stimulate ethylene biosynthesis, and the activities of 1aminocyclopropane-1-carboxylic acid (ACC) synthase and of
ACC oxidase increase in extracts from hypoxic roots following a
lag of ~3 h (Ref. 14). Mechanical impedance also increases the

might explain the functional significance
of the induction of root aerenchyma by
mechanical impedance in maize14. In addition, they suggest that constitutive aerenchyma development, in contrast with
induction after stress, might be an advantage in some environments. Constitutive
aerenchyma formation has been transferred
successfully from eastern gamagrass and
teosinte to maize by hybridization followed
by backcrossing11. This study found a major
gene for aerenchyma formation on a specific chromosome arm in eastern gamagrass. It will be interesting to see whether
improved resistance to flooding is associated with the constitutive formation of
aerenchyma in these lines. In crops such as
soybean15, and in Ranunculus species growing in river floodplains16, aerenchyma

formation is associated with flooding tolerance. However, formation of a prominent
aerenchyma by shizogeny or lysigeny is
not an invariable requirement for the adequate internal transfer of O2. In Carex
pseudocyperus17, the cortical cells remain
intact, and fine intercellular spaces extend
throughout the root and into the apical
zone, the average porosity of 20% providing sufficient O2 for the species to tolerate
anaerobic environments.
Induction of lysigenous aerenchyma

Hypoxia under laboratory conditions readily
induces aerenchyma formation in the roots
of maize (Fig. 2), and hypoxia probably
accounts for the induction of aerenchyma
in roots in flooded soils. Hypoxia might be a
much more common occurrence within roots
than has been supposed. Even in welloxygenated surroundings, the rate of O2
consumption during respiration, especially
124

March 2000, Vol. 5, No. 3

Fig. 2. Transverse section of maize nodal roots in a four-day-old zone. (a) Well-oxygenated (normoxic) control root, lacking aerenchyma. Note the radial packing of cells in the inner cortex and
hexagonal packing in the outer cortex. (b) Hypoxic root, with lysigenous aerenchyma in the mid
cortex. (c) Well-oxygenated root treated for four days with okadaic acid, an inhibitor of protein
phosphatases. Note that aerenchyma is beginning to form. (d) Hypoxic root treated for four days
with EGTA [ethylene glycol-bis (b-aminoethyl ether) N,N,N9,N9-tetraacetic acid] to complex
Ca21, thereby eliminating aerenchyma formation. (e) Hypoxic root stained with neutral red with
color showing pH ,7.0. Cell acidification can be seen in the mid cortex, which later dies and
degrades. (f) Hypoxic root showing uptake of Evans blue by cells in the mid cortex, indicating a
loss of ability to exclude this molecule at an early stage in cell degeneration. Scale bars = 0.25 mm.

trends in plant science
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Trends in Plant Science

activities of these enzymes, and hypoxia,
together with mechanical impedance, has
a synergistic effect that is unexplained.

Hypoxia
However, the burst of extra ethylene with
hypoxia or with mechanical impedance
probably accounts for aerenchyma forACC synthase
ACC oxidase
mation. In maize roots, it is not known
whether the relatively rapid increase in the
Ethylene
activity of enzymes involved in ethylene
Mechanical
biosynthesis is as a result of the activation of
impedance
pre-existing enzymes, or if there are changes
in the transcript level of the corresponding
?
Deficiency
genes. In roots of tomato20,21 and rice22, speReceptor
N
or
P

cific genes in the ACC synthase gene family
?
are induced by O2 deficiency.
Aerenchyma
In maize, aerenchyma formation can be
formation
Signaling cascade
induced under well-oxygenated conditions
in nutrient solution by the transient shortage
of either a N or a P source (Fig. 3). This is
ethylene-dependent, and does not require the
Cell death
augmentation of ethylene production, but
rather an increase in sensitivity to ethylene23.
Fig. 3. Model of induction of programmed cell death (PCD) in cortical cells of maize roots
There is uncertainty whether induction of
by environmental factors. PCD is viewed here as part of a feedback loop to acclimate roots
cell death in aerenchyma formation is related
to sub-optimal soil conditions. For hypoxia and mechanical impedance, the supply of oxyto root cortical death (RCD), a form of nongen allowed by aerenchyma helps sustain aerobic metabolism and growth. For nutrient depathogenic death that begins in the cortex
ficiency, cell death and lysis might provide essential inorganic nutrients and metabolites

24
and spreads towards the endodermis . RCD
to maintain meristematic cells.
has been detected in roots of many species in
the grass family, including wheat, barley and
maize. It is characterized by a loss of nuclear
staining with acridine orange, a symptom that coincides with the loss been confirmed by exposing roots to K252a, an inhibitor of protein
of both membrane permeability and esterase activity, with fluores- kinases (especially of protein kinase C), that blocks cell death in
cein diacetate as a substrate. However, RCD has not been examined hypoxic roots. Neomycin is also effective in preventing death durin relation to its inducibility by ethylene.
ing root growth under hypoxia by interfering in phosphoinositide
Although aerenchyma formation in rice is thought to be consti- metabolism and both EGTA [ethylene glycol-bis (b-aminoethyl
tutive12, in some varieties additional cell death occurs in response ether) N,N,N9,N9-tetraacetic acid] and ruthenium red prevent death
to ethylene or hypoxia3,25. Rice and other wetland species have by lowering the Ca21 concentration in the cytosol (Fig. 2). By conseveral anatomical adaptations that help minimize loss of O2 to the trast, reagents that tend to raise cytosolic Ca21, such as thapsigarsurrounding soil, thereby conserving more for respiration by the gin or caffeine, promoted death in well-oxygenated roots. Based on
apical meristem. These structures include a suberized hypoder- these observations, it is possible to tentatively construct a signal
mis, and a layer of lignified cells immediately interior to the hypo- transduction pathway for ethylene signaling in cortical cell death
dermis, both of which are only slightly gas
permeable. Apart from restricting the loss
of O2, these cell layers are likely to hold
Ethylene
in ethylene as well, even if it is slowly
synthesized. The question remains whether
the constitutive formation of aerenchyma
G protein
Phosphatidyl inositol
in wetland species is really a response to
4,5 bisphosphate
entrapped ethylene.
Phospholipase C

Induction of cell death in the root cortex of
maize has been examined in relation to the
possible pathway downstream of ethylene26.
A variety of agonists and antagonists of particular steps in signal transduction have been
used in this pharmacological approach. In
well-oxygenated roots, cell death can be initiated by GTP-g-S, which activates heterotrimeric G proteins. Death was also induced
by okadaic acid (Fig. 2), which inhibits protein phosphatases, thereby tending to maintain proteins in a phosphorylated state. The
importance of protein phosphorylation has

Diacylglycerol

Inositol 1,4,5 trisphosphate

Protein kinase C

Cytosolic Ca2+

Protein phosphorylation

Protein kinase
(Ca2+- or Ca-CaM-dependent)

Trends in Plant Science

Ethylene signal transduction pathway

Fig. 4. Proposed ethylene signal transduction pathway initiating cell death in cortical cells in
maize roots.

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(Fig. 4). As evidence that the reagents interfered with the ethylene
signal transduction pathway, rather than with a hypoxia pathway
per se, it was checked that the treatments that blocked cell death in
hypoxic roots were also effective in well-oxygenated roots exposed
to ethylene (C-J. He and M.C. Drew, unpublished). None of the
reagents inhibited ethylene production in hypoxic roots26.
The possibility that Ca21 might be involved in cell signaling
under low O2 conditions was established in cell cultures of maize27.
Anoxia quickly raised cytosolic Ca21 and induced alcohol dehydrogenase (ADH) activity. These responses could be simulated in
well-oxygenated cells by manipulating cytosolic Ca21 using ruthenium red or caffeine. However, the relationship between anoxia and
Ca21 is probably independent of ethylene because ethylene synthesis is blocked in the absence of O2 (anoxia) and, in addition, anoxic
cells fail to respond to ethylene6. It is not known whether ethylene
and anoxia share part of the same signal transduction pathway,
other than at the level of Ca21. Equally uncertain is the extent to
which ethylene signal transduction in different species follows the
same pathway. In Arabidopsis, molecular genetic studies have provided a tentative sequence in which putative ethylene-receptors,
and downstream, several classes of protein kinases, have a defined
role28,29 – comparable information is not available for other species.
Part of the proposed scheme (Fig. 4) for the initiation of cell
death in the ethylene signal transduction pathway of maize roots
has been tested. Changes in phopholipase activity and in inositol
1,4,5 trisphosphate (IP3) in maize roots following either the imposition of hypoxia or of exogenous ethylene have been examined
(C-J. He, P.W. Morgan and M.C. Drew, unpublished). Phospholipase C uses plasma membrane-bound phosphatidyl inositol bisphosphate as a substrate, releasing IP3 to the cytosol, where it
gates Ca21 channels at the endoplasmic reticulum or tonoplast,
thereby raising cytosolic Ca21. Measurements were made of the
apical 5 mm zone, which was well ahead of the first signs of cell
death or lysis detected under the light microscope at .10 mm
from the apical tip. Phospholipase C activity in the plasma membrane fraction rose sharply with hypoxia or ethylene, with an
approximate doubling of activity in 4 h (C-J. He, P.W. Morgan
and M.C. Drew, unpublished). IP3 also showed a corresponding
rise, which is consistent with the higher level of enzyme activity
and with the proposed rise in cytosolic Ca21 in response to IP3.
These results are compatible with a role for a phosphoinositide
signal transduction pathway in cell death in maize roots (Fig. 4).
Programmed cell death

Programmed cell death (PCD) can be defined as death resulting
from the activation of a specific biochemical pathway that is
under the genetic control of the cell, and therefore it amounts to
cellular suicide30,31. Apoptosis is a particular form of PCD, which
is defined in animal cells by a combination of changes. These
include:
• Nuclear condensation.
• Activation of Ca21-dependent endonucleases with DNA fragmentation into characteristic, internucleosomal lengths of
180–200 bp or multiples thereof.
• Nuclear and plasma membrane blebbing with expulsion of
apoptotic bodies.
By contrast, necrosis refers to ‘accidental’ cell death in response
to an injurious environmental factor, such as heat stress or a toxin;
it is not normally associated with development, and it does not
require the activation of signal transduction pathways. Necrosis
(also known as oncosis) is characterized typically by a loss of
plasma membrane integrity, sometimes with cellular swelling,
followed by organelle degeneration, with DNA degradation taking place relatively late in the process.
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March 2000, Vol. 5, No. 3

Cell death during lysigenous aerenchyma formation occurs in a
precise, predictable pattern during differentiation, and in some
species is induced by ethylene, supporting the PCD idea, but
information on nDNA degradation in this context has not been
published. Because cell death is not synchronous in the root cortex and occurs among a population of normal cells, DNA laddering might not give unambiguous answers. An alternative approach
would be to detect DNA fragmentation in individual cells in situ,
using the TUNEL reaction to label the exposed 39 ends of the
DNA, a method that has already been used with roots to demonstrate PCD in cap cells32. In S. lancifolia, electron microscopy
reveals nuclear condensation and nuclear blebbing in lysigenous
aerenchyma suggesting that PCD is involved4. In rice and maize3,
early signs of cell degeneration are staining with neutral red and
permeation of Evans blue – a dye that is normally excluded by the
plasma membrane of healthy cells (Fig. 2).
It has been suggested that cell death in rice roots, which always
begins among cells in the mid-cortex (as it does in maize) takes
place by PCD, and that the subsequent death of cells, which
spreads out to surrounding cortical cells, is by necrosis3. It is
thought that a factor diffuses symplastically from the initiating
cells to the remainder of the cortex in a radial direction, which is
the direction that most plasmodesmatal connections occur in roots,
but what that factor might be is obscure. The pattern of cell death
and lysis in maize aerenchyma formation begins in the mid-cortex
but unlike rice, spreads tangentially as well as radially. In Carex
species, as well as in Eriophorum angustifolium and Lysimachia
nummularia, cell death spreads only in a tangential direction10. Cell
packing also has a marked influence on whether shizogenous or
lysigenous aerenchyma forms or not. Aerenchyma develops preferentially where the pre-aerenchyma packing of cells in the cortex
is radial in transverse section, which is the pattern found in many
wetland species10. By contrast, aerenchyma rarely occurs where
there is non-radial, hexagonal packing, which is chiefly found in
non-wetland species, or in the outer cortical layers that do not participate in aerenchyma formation in typical wetland species. These
differences in the pattern of aerenchyma formation are difficult to
explain in terms of diffusion of a necrotic factor.
One of the earliest signs of cell death in maize roots is the rupture
of the tonoplast2. The release of hydrolytic enzymes sequestered in
the vacuole and the sudden cytoplasmic acidosis caused by the liberation of the acidic vacuolar sap might be sufficient to kill the ethylene-responsive cell. In rice roots also, cells in the mid-cortex that are
the first to die, are also the first to show signs of acidosis as detected
by neutral-red staining3. There is an obvious similarity here with the
death of xylem cells (tracheary elements, TE), which die at the completion of their differentiation. In Zinnia, in cell culture, induction of
cell death during TE formation is associated with a serine protease
that appears to activate Ca21 influx into the cytosol, the Ca21 then
triggering nDNA fragmentation as part of the PCD process33. Rupture of the tonoplast is also an early event in TE death, therefore the
parallel to cell death in the root cortex is striking.
Death of root cortical cells is followed by degradation and total
lysis of the cytoplasm, with only a few remnants of the cell wall
remaining2. This has to be accomplished by a whole suite of
enzymes, but this aspect has received little attention. One enzyme
that is closely involved is cellulase (b1,4-endoglucanase), which
is always tightly coupled to cell death; promotors of cell death
(ethylene, Ca21, okadaic acid) increase cellulase activity, whereas
inhibitors of cell death block any rise in its activity26. It appears as if
death cannot take place without subsequent degradation. Another
potentially degradative enzyme associated with aerenchyma
formation is xyloglucan endo-transglycosylase, which might be
involved in wall loosening34. Proteases, lipases, as well as DNAases

trends in plant science
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(and indeed many other enzymes) must also be involved in the
degradation process, and it is important to distinguish those that are
involved in degradation from those that are involved in the signaling
of cell death.
An intriguing question concerns the highly localized pattern of
cell death in the root cortex. In maize, the epidermis, hypodermis,
endodermis and stele are unaffected. Even in the cortex, some cells
remain intact and healthy, forming radial bridges immediately
adjacent to lysing cells. If lytic enzymes are freely released from
dying cells, what limits their spread? The same question arises in
the highly localized lysis of the primary cell wall and cytoplasmic
contents of TEs in the vicinity of healthy parenchymatous cells33.
Conclusions

Aerenchyma formation by cell death and lysigeny provides an
ideal model system for examining the initiation of PCD by environmental factors or by the phytohormone, ethylene. Information
is accumulating on the signal transduction pathway, only parts of
which may have features in common with that in Arabidopsis. A
rise in cytosolic Ca21 and rupture of the tonoplast are early events
common to death of TEs and root cortical cells, and it will be
interesting to explore the similarities between these two systems
further. If it is shown conclusively that PCD is the mechanism
involved in lysigenous aerenchyma formation, it will be of interest to determine the extent to which the process resembles that of
apoptosis in animal cells.
Acknowledgements

Research on aerenchyma formation in M.C.D.’s and P.M.W.’s
laboratories is supported by a competitive grant from the US Dept
of Agriculture, National Research Initiative program.
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Malcolm C. Drew* and Chuan-Jiu He are in the Dept of
Horticultural Sciences, Texas A&M University, College Station, TX
77843, USA; Page W. Morgan is in the Dept of Soil and Crop
Sciences, Texas A&M University, College Station, TX 77843, USA.
*Author for correspondence (tel 11 409 845 4462;
e-mail mcdrew@tamu.edu).

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