Xylem cell death: Emerging understanding of regulation and function

Abstract

Evolutionary, as well as genetic, evidence suggests that vascular development evolved originally as a cell death programme that allowed enhanced movement of water in the extinct protracheophytes, and that secondary wall formation in the water-conducting cells evolved afterwards, providing mechanical support for effective long-distance transport of water. The extant vascular plants possess a common regulatory network to coordinate the different phases of xylem maturation, including secondary wall formation, cell death, and finally autolysis of the cell contents, by the action of recently identified NAC domain transcription factors. Consequently, xylem cell death is an inseparable part of the xylem maturation programme, making it difficult to uncouple cell death mechanistically from secondary wall formation, and thus identify the key factors specifically involved in regulation of cell death. Current knowledge suggests that the necessary components for xylem cell death are produced early during xylem differentiation, and cell death is prevented through the action of inhibitors and storage of hydrolytic enzymes in inactive forms in compartments such as the vacuole. Bursting of the central vacuole triggers autolytic hydrolysis of the cell contents, which ultimately leads to cell death. This cascade of events varies between the different xylem cell types. The water-transporting tracheary elements rely on a rapid cell death programme, with hydrolysis of cell contents taking place for the most part, if not entirely, after vacuolar bursting, while the xylem fibres disintegrate cellular contents at a slower pace, well before cell death. This review includes a detailed description of cell morphology, function of plant growth regulators, such as ethylene and thermospermine, and the action of hydrolytic nucleases and proteases during cell death of the different xylem cell types.

Full text

Journal of Experimental Botany, Vol. 63, No. 3, pp. 1081–1094, 2012
doi:10.1093/jxb/err438 Advance Access publication 2 January, 2012
DARWIN REVIEW
Xylem cell death: emerging understanding of regulation and
function
Benjamin Bollho
¨ner, Jakob Prestele and Hannele Tuominen*
Umea
˚Plant Science Centre, Department of Plant Physiology, Umea
˚University, SE-90187 Umea
˚, Sweden
* To whom correspondence should be addressed. E-mail: hannele.tuominen@plantphys.umu.se
Received 5 October 2011; Revised 6 December 2011; Accepted 9 December 2011
Abstract
Evolutionary, as well as genetic, evidence suggests that vascular development evolved originally as a cell death
programme that allowed enhanced movement of water in the extinct protracheophytes, and that secondary wall
formation in the water-conducting cells evolved afterwards, providing mechanical support for effective long-
distance transport of water. The extant vascular plants possess a common regulatory network to coordinate the
different phases of xylem maturation, including secondary wall formation, cell death, and finally autolysis of the cell
contents, by the action of recently identified NAC domain transcription factors. Consequently, xylem cell death is an
inseparable part of the xylem maturation programme, making it difficult to uncouple cell death mechanistically from
secondary wall formation, and thus identify the key factors specifically involved in regulation of cell death. Current
knowledge suggests that the necessary components for xylem cell death are produced early during xylem
differentiation, and cell death is prevented through the action of inhibitors and storage of hydrolytic enzymes in
inactive forms in compartments such as the vacuole. Bursting of the central vacuole triggers autolytic hydrolysis of
the cell contents, which ultimately leads to cell death. This cascade of events varies between the different xylem cell
types. The water-transporting tracheary elements rely on a rapid cell death programme, with hydrolysis of cell
contents taking place for the most part, if not entirely, after vacuolar bursting, while the xylem fibres disintegrate
cellular contents at a slower pace, well before cell death. This review includes a detailed description of cell
morphology, function of plant growth regulators, such as ethylene and thermospermine, and the action of hydrolytic
nucleases and proteases during cell death of the different xylem cell types.
Key words: Autolysis, ethylene, fibre, metacaspase, protease, thermospermine, tracheary element, VND.
Introduction
A large proportion of the biomass on earth consists of dead
but nevertheless functioning cells, the xylem elements. The
death and complete clearing of xylem vessel elements and
tracheids, commonly known as tracheary elements (TEs), is
a prerequisite for the transport of water. Hollow conduits,
supported by secondary wall thickenings, have been detected
in fossils from the Mid-Silurian period, ;430 million years
ago, and the water transport in these is believed to be one of
the most important factors for the evolutionary success of
vascular plants (Raven, 1993). A further improvement in
water transport capacity occurred when the vascular cam-
bium emerged, which allowed extensive lateral growth, in
the form of the secondary xylem, in woody plant species. The
gymnosperm secondary xylem is composed of tracheids,
which contribute to both physical support and water trans-
port. In angiosperms, functional diversification has occurred
between different cell types of the secondary xylem. Water
transport takes place in the vessel elements, whereas mechan-
ical support is mainly provided by the predominant cell type
of the xylem, the libriform or sclereid fibres (Esau, 1965).
Although fibres do not transport water, they undergo cell
death and continue to fulfil their structural purpose decades,
or even centuries, after their cellular death.
The main pattern of cellular differentiation is identical
between the different xylem cell types and involves initiation
in the vascular cambium, rapid cell expansion, deposition of
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the secondary cell wall, and finally cell death (Fukuda,
1996;De
´jardin et al., 2010). However, there are significant
differences in, for instance, cell morphology and the timing
of these processes between the different xylem cell types
(Fig. 1). Whereas vessel elements differentiate rapidly and
die within a couple of days after their specification in the
vascular cambium, fibres and tracheids stay alive substan-
tially longer. The lifetime of both libriform fibres and
tracheids was estimated to be ;1 month in trembling aspen
(Populus tremula) and Norway spruce (Picea abies), re-
spectively (JP, BB, and HT, unpublished). Further, whereas
the final autolysis of the cell contents is rapid in the xylem
vessel elements, it is slow in both the conifer tracheids and
angiosperm fibres (Wodzicki, 1971;Skene, 1972;Courtois-
Moreau et al., 2009). It therefore seems that conifer
tracheids and angiosperm fibres share at least some parts of
the cell death programme. In contrast, the vessel elements,
despite being classed as TEs, seem to have evolved a distinct
programme for cell death that is quite different from the
ancient programme present in tracheids. Lastly, ray paren-
chyma cells contribute only marginally to the total number
of xylem cells, but they are often the only living cells in the
fully mature secondary xylem and can, depending on
species, stay alive for decades before undergoing cell death
(Nakaba et al.,2006,2011). This review focuses on cell
death of the water-transporting TEs and xylary fibres.
Fig. 1. Cell morphology in different stages of tracheary element (TE) and fibre (F) differentiation. TE differentiation includes: early
differentiation in the cambial zone (TE1), cell expansion (TE2), secondary wall formation (TE3), changes in tonoplast permeability and
vacuolar rupture (TE4), DNA degradation (TE5), final autolysis (TE6), and partial hydrolysis of the non-lignified primary cell walls (TE7).
Fibre differentiation includes: early differentiation in the cambium (F1), cell expansion (F2), secondary wall formation (F3), loss of turgor
(F4), disappearance of the organelles, starting autolysis and DNA degradation (F5), swelling of the remaining organelles and continued
autolysis (F6), autolysis after vacuolar rupture (F7), and final clearing of the cell (F8). (n) nucleus, (v) vacuole, (o) organelles, (w) cell wall.
Vacuolar disintegration seems to play a less important role in the death of fibres than in that of TEs because most of the cellular
hydrolysis in xylem fibres occurs before the tonoplast breaks.
1082 |Bollho
¨ner et al.

Morphological changes during xylem cell
death
Tracheary elements
Early light microscopy analyses revealed that vessel ele-
ments or ‘wood pores’ were dead cells, but it was only with
the invention of electron microscopy that detailed analyses
of xylem differentiation and maturation became possible.
Esau et al. (1963) were the first to describe the degradation
of organelles and the protoplast in Cucurbita vessel
elements. In maturing pine xylem, Wodzicki and Brown
(1973) observed a gradual uptake of cell components into
the vacuole, which they defined as autolysis that finally
leads to breakdown of the vacuole. Autolysis of maturing
vessels has since been documented in several different
species (Srivastava and Singh, 1972;Esau and Charvat,
1978;Burgess and Linstead, 1984a). A detailed understand-
ing of the autolytic processes of TEs has been provided
by studies of cultures of Zinnia (Zinnia elegans) mesophyll
cells, which can be induced to transdifferentiate into
TEs in vitro in a semi-synchronized manner (Fukuda and
Komamine, 1980). In this system, auxin and cytokinin are
added to isolated mesophyll cells that initially dedifferenti-
ate during the so-called stage I into procambial-like cells,
followed by differentiation into TE precursor cells during
stage II, and finally differentiation into TEs, including
secondary wall formation and cell death, during stage III
(Fukuda, 1996).
Morphologically, the first obvious indication of incipient
cell death in Zinnia TEs is swelling of the vacuole, followed
by changes in tonoplast permeability (Kuriyama, 1999)and
rapid collapse of the vacuole (Groover et al., 1997).
Although the cytoplasm has been reported to become less
dense already during secondary wall biosynthesis (Groover
et al., 1997), autolytic events are not believed to occur to
any significant degree before vacuolar collapse. Cytoplasmic
streaming ceases when the vacuole collapses, which is
therefore considered as being the moment of cell death.
Release of hydrolytic enzymes from the vacuole and
activation of cytoplasmic enzymes by acidification of the
cytoplasm is believed to induce swelling of organelles such
as the endoplasmic reticulum (ER) and Golgi, and finally
degradation of cellular contents (Fukuda, 1997).
The pattern of DNA degradation is used for animal
systems as well as plants as a basis to classify different types
of cell death. The genetically programmed type of cell
death, especially animal apoptotic cell death and plant
vacuolar or autolytic cell death (van Doorn, 2011;van
Doorn et al., 2011), often involves nuclear fragmentation,
DNA degradation into a multitude of 180 bp fragments
that generates the so-called ‘DNA ladder’, as well as
positive staining with assays such as the TdT-mediated
dUTP nick end labelling (TUNEL) assay. In the Zinnia TE
system, nuclear DNA fragmentation has been visualized by
TUNEL staining (Mittler and Lam, 1995;Groover et al.,
1997;Twumasi et al., 2010). However, it has been demon-
strated in Zinnia TEs that nuclear DNA is degraded very
rapidly within 10–20 min after the rupture of the vacuole
(Obara et al., 2001), suggesting that positive TUNEL
staining of TEs might be related to post-mortem DNA
degradation rather than any controlled degradation of
DNA prior to cell death. It was also shown that the nucleus
maintained a spherical shape while DNA degradation
occurred, suggesting that no nuclear fragmentation occurs
in Zinnia TEs in vitro (Obara et al.,2001). DNA laddering
does not seem to occur either (Fukuda, 2000). In planta,
electron microscopic analyses have revealed lobing of the
nuclei in differentiating TEs (Esau et al., 1963;Lai and
Srivastava, 1976;Burgess and Linstead, 1984b)(Fig. 2A).
Fragmentation of the nucleus has also been observed prior
to final autolysis in protoxylem elements of Arabidopsis
roots expressing a nuclear-localized proAtMC9::GFP
reporter construct (Fig. 2B). A positive TUNEL signal has
been observed in the xylem vessel elements of pea roots
(Mittler and Lam, 1995) and tomato leaves (Wang et al.,
1996), but not in the secondary xylem of Populus stems
(Courtois-Moreau et al., 2009). Based on the results of the
Zinnia in vitro experiments and the in planta evidence, it
appears that even though nuclear changes can be readily
detected, DNA degradation is not causally related to TE
cell death but occurs post-mortem during final autolysis of
the cell contents.
After cellular hydrolysis, cell walls of TEs are modified by
enzymes that are resistant to the highly lytic environment of
dying TEs. Lignin deposition, that has been initiated prior
to cell death, continues after TE cell death (Stewart, 1966,
and references therein; Hergert, 1977;Burgess and Linstead,
1984b;Pesquet et al., 2010). In addition, primary walls are
at least partially hydrolysed after loss of the plasma
membrane in locations that are not protected by lignified
secondary walls (O’Brien and Thimann, 1967;O’Brien,
1970;Burgess and Linstead, 1984a,b). This is also reflected
as increased activity of cell wall hydrolytic enzymes of
maturing Zinnia TEs in vitro (Ohdaira et al., 2002). Primary
wall hydrolysis of lateral cell walls is believed to allow
passive stretching of protoxylem vessel elements (O’Brien,
1970), while it is required in the end walls of all types of
vessel elements for formation of the perforation plates.
Fibres
Xylary fibres die in a coordinated fashion, which suggests
an underlying genetic programme (Courtois-Moreau et al.,
2009). Prior to cell death, the fibres deposit extensive
secondary cell walls and, similar to TEs in vitro, the
majority of lignification seems to occur after cell death
(Fig. 2C–F). The main difference seems to be the rate of
differentiation. An analysis of cellular ultrastructure, as well
as nuclear integrity, has revealed that xylary fibres exhibit
DNA breaks, most probably due to degradative processes
in the nucleus, long before the cells die (Courtois-Moreau
et al., 2009). This is in contrast to TEs, where DNA
degradation is initiated only after vacuolar collapse (Obara
et al., 2001). Moreover, the cytoplasmic contents of the
fibres start to be hydrolysed gradually well ahead of
Xylem cell death |1083

vacuolar disintegration, as opposed to the rapid degrada-
tion observed in xylem vessels after bursting of the vacuole
(Courtois-Moreau et al., 2009). The slow degradation
pattern of the cytoplasmic contents is suggestive of autoph-
agy, which has been supported by studies showing a high
level of expression of genes homologous to yeast autophagy
genes (Courtois-Moreau et al., 2009). Autophagy is a cellu-
lar degradation process which involves formation of auto-
phagic bodies, called autophagosomes, that enclose
cytoplasmic contents and transport them to the vacuole for
degradation. In yeast, this was first demonstrated as
a response to starvation. The function of autophagy in
plants is not quite clear (Love et al., 2008,2009;Cacas and
Diamond, 2009), but it seems that it has a dual role in
protecting cells against harmful compounds and proteins,
and in promoting cell death after the cells are committed to
death (Hofius et al., 2011). In fibres, it is plausible that
autophagy allows efficient remobilization of nutrients from
the cytoplasm, as has been observed in senescing leaves
(Doelling et al., 2002;Hanaoka et al., 2002;Yoshimoto
et al., 2004). Autophagy has recently been implicated in TE
differentiation as well (Kwon et al., 2010). One of the
signals for induction of autophagy could be a dramatic
reduction in sucrose concentration during maturation
of xylem elements, which has been observed in a high-
resolution analysis of carbohydrate metabolism (Uggla
et al., 2001), although other hormonal signals, such as
indole-3-acetic acid and ethylene, which also show drastic
changes during maturation, cannot be excluded (Tuominen
et al., 1997;Pesquet and Tuominen, 2011).
The final stages of fibre cell death are accompanied by
changes in turgor, as reflected by relaxation of the condensed
nuclei, swelling of the remaining organelles, including a highly
dilated ER, bursting of the vacuole, and mega-autolysis of
the remaining cell contents (Fig. 2G;Courtois-Moreau et al.,
2009). Cellular debris is often retained for a long time in the
cell lumen, but ultimately the fibres are cleared completely
(Courtois-Moreau et al.,2009).
Xylem cell death-related signalling
The signals related to initiation and execution of xylem cell
death are poorly understood. This is partly due to difficulties
in identifying signalling that is specifically related to cell death
and not secondary cell wall formation. Most pharmacological
agents that block xylem cell death also block secondary cell
wall formation (Yamamoto et al.,1997;Groover and Jones,
1999;Yu et al.,2002;Twumasi et al.,2010), suggesting that
the two processes are co-regulated. Co-regulation of xylem
maturation has indeed recently been demonstrated to occur
Fig. 2. Specific characteristics of xylem cell death. (A) Electron
transmission micrograph of an Arabidopsis vessel element showing
lobing of the nucleus. (B) Nuclear fragmentation in Arabidopsis root
protoxylem cell, visualized by confocal microscopy analysis of
a nuclear-localized green fluorescent protein (GFP) under transcrip-
tional control of the AtMC9 promoter. Arrows indicate fragments of
the nucleus in a late maturing protoxylem cell. The arrowhead
indicates an intact nucleus in the neighbouring protoxylem cell.
Roots were counterstained with propidium iodide that reveals all cell
walls as well as the spiral/annular secondary wall thickenings of
protoxylem elements. (C–F) Bulk lignification and cell death in
Populus fibres. Microscopy images of cross-sections of the stem
show living cells of the stem staining blue after a viability staining (C)
(Courtois-Moreau et al., 2009), lignin autofluorescence (D), lignin
deposition by phloroglucinol staining (E), and a bright field image (F).
Death of the fibres, indicated by an arrow in C, coincides with
a massive increase in lignin autofluorescence (D), and phlorogluci-
nol-detectable lignin accumulation (E) as well as occurrence of air in
the dead fibres (F). (G) Appearance of nuclei in a radial section of
Populus xylem stained with 4’,6-diamidino-2-phenylindole (DAPI)
and visualized by epifluorescence microscopy. Progressing fibre
maturation (from the left to the right) leads to longitudinally elongated
nuclei that show relaxation, indicated by the asterisk, before they
disappear. Ray cell nuclei are relaxed and elongated in the radial
direction. (H) Absence of cell death in xylem fibres of Arabidopsis
hypocotyls. An epifluorescence micrograph shows a DAPI-stained
cross-section of a 2-month-old Arabidopsis Ler hypocotyl, where
fibres develop thick secondary walls but retain their nuclei. Bars
indicate 2 lm (A), 50 lm (B, G, H), or 200 lm (C–F).
1084 |Bollho
¨ner et al.

via the action of NAC transcription factors, as described later
in this review. However, it is clear that even though the
different phases of xylem maturation are jointly regulated by
a few master switches, it is likely that the individual processes
have separate controls as well. For instance, bursting of the
vacuole must involve unique regulatory aspects to allow the
correct timing of cellular autolysis in response to endogenous
and exogenous stimuli.
Plant growth regulators
Auxins and cytokinins are prerequisites for TE differentia-
tion in vitro (Fukuda and Komamine 1980), but it seems
that their only function is the early reprogramming of
mesophyll cells into the TE differentiation programme
(Milioni et al., 2001). Brassinosteroids, on the other hand,
are believed to play a role during late xylem maturation
based on experiments with Zinnia TEs in vitro. Brassinoste-
roid precursors have been shown to accumulate during TE
differentiation, whereas inhibition of brassinosteroid syn-
thesis in TE cultures undergoing differentiation prevented
cells from maturing and undergoing cell death (Yamamoto
et al., 1997). Ethylene is another hormone that deserves
special attention based on its crucial function in other cell
death processes (He et al., 1996;Tuominen et al.,2004). It
has been shown that maturing Zinnia TEs accumulate
ethylene (Pesquet and Tuominen, 2011), whereas blocking
ethylene signalling using silver thiosulphate (STS) appears
to block TE maturation (E. Pesquet and H. Tuominen,
unpublished results). STS-induced changes in TE matura-
tion are unique in the sense that TEs develop cellulosic
secondary walls but do not lignify or die. It has been shown
recently that cell death precedes bulk lignification in TEs in
vitro (Pesquet et al., 2010), which means that the STS-
mediated arrest of TE maturation is most probably due to
blocking of cell death, which in turn blocks lignification.
Therefore, it can be concluded on the basis of these
experiments in the Zinnia in vitro system that ethylene
seems to interfere with the cell death programme also in
TEs. This conclusion is, however, not supported by
Arabidopsis mutant analyses because no developmental
defects have been reported for any of the dominant ethylene
receptor or downstream signalling mutants, even though
complete removal of ethylene biosynthesis is reportedly
lethal (Tsuchisaka et al., 2009). It has to be emphasized that
inhibitors like STS are never specific to one pathway
(Strader et al., 2009). Nevertheless, it is possible that the in
vitro system actually reveals some basic regulatory aspects
of xylem differentiation that are masked or compensated for
by the cellular context in intact vascular tissues.
Polyamines are implicated in several different processes
during xylem differentiation, including cell wall formation,
lignin biosynthesis, and auxin–cytokinin signalling (Ge et al.,
2006;Cui et al.,2010;Vera-Sirera et al.,2010). Interestingly,
ACAULIS5 (ACL5), which encodes the biosynthetic enzyme
for the synthesis of a recently identified tetra-amine, thermo-
spermine, is specifically expressed in Arabidopsis vessel
elements prior to secondary wall deposition (Mun˜iz et al.,
2008). acl5 loss-of-function mutants exhibit incorrect or
incomplete secondary cell wall formation as well as early
expression of xylem cell death markers, and consequently
early vessel cell death compared with the wild type, suggest-
ing that thermospermine has a protective role against
premature xylem maturation and cell death (Mun˜iz et al.,
2008). Exogenous thermospermine has been shown to inhibit
Zinnia TE differentiation almost completely (Kakehi et al.,
2010), which could be due to accentuated protection against
premature TE maturation, resulting in complete arrest of TE
differentiation. Genetic analyses have further indicated
a basic helix–loop–helix (bHLH) transcription factor SUP-
PRESSOR OF ACAULIS51 (SAC51) as a target of ACL5
function. ACL5 or thermospermine is believed to activate
translation of SAC51 by inhibiting one of the negatively
acting upstream open reading frames of SAC51 (Imai et al.,
2006,2008). SAC51 has been shown to be a direct target of
one of the NAC transcription factors (VND7) (Zhong et al.,
2010b), and it is possible that SAC51 coordinates signals
coming from the NAC transcription factors and ACL5 to
control the rate of differentiation specifically in differentiat-
ing TEs (Fig. 3).
Other signalling components
It has been suggested that calcium ions regulate vacuolar
integrity during TE maturation. An increase in Ca
2+
influx
appears to accompany cell death of differentiating TEs.
Both chelation of extracellular Ca
2+
and blocking of Ca
2+
influx channels have been shown to suppress vacuolar
Fig. 3. Transcriptional regulation of tracheary element cell death.
Cell death is regulated as an integral part of the xylem maturation
programme by the activity of the NAC transcription factors VND6
and VND7 that induce expression of both cell death- and
secondary wall-related genes. Thermospermine synthase ACL5 is
proposed to impede the rate of xylem maturation by activating
translation of SAC51 (Vera-Sirera et al., 2010) even though it is not
clear how SAC51 mediates inhibition of xylem maturation.
Expression of SAC51 as well as XND1, that is another rate-
inhibitory factor, is induced by VND7 (Zhong et al., 2010b).
Lignification requires activity of second level transcription factors
(Zhong et al., 2010a) that are activated by the NAC master
switches. TE cell death is required for bulk lignification (Pesquet
et al., 2010).
Xylem cell death |1085

rupture and DNA degradation in differentiating Zinnia TEs
(Groover and Jones, 1999). It has also been proposed that
Ca
2+
influx is controlled by the activity of a secreted 40 kDa
serine protease, which continuously accumulates in the
extracellular space, inducing a massive Ca
2+
influx and TE
cell death above a certain critical level of the protease
(Groover and Jones, 1999). Reactive oxygen species (ROS)
are important signalling compounds in various cell death
processes both in animals and in plants. In Zinnia in vitro
cultures, it has been found that differentiating TEs are
constantly exposed to a highly oxidative environment, but
no bursts of rapidly increasing ROS levels seem to occur
(Groover et al., 1997;Go
´mez Ros et al., 2006). Inhibition of
ROS production by diphenyleneiodonium does not in-
fluence TE cell death either (Groover et al., 1997). However,
ROS levels have been correlated with the extent of xylem
lignification in planta (Srivastava et al., 2007), and ROS
production has been shown to be required for lignification
in Zinnia TEs (Karlsson et al., 2005). It seems, therefore,
that ROS are involved in regulation of TE lignification but
not cell death.
During apoptotic cell death in animals, changes in Ca
2+
,
pH, and ROS production can trigger formation of the
mitochondrial permeability transition pore (PTP), leading
to release of proteins, such as cytochrome c, from the
intermembrane space (Danial and Korsmeyer, 2004). Mito-
chondrial depolarization and morphological changes have
also been observed as fast responses to various cell death-
inducing conditions in plants (Logan, 2008). Also in
differentiating Zinnia TEs, it has been reported that
mitochondrial membranes are depolarized and cytochrome
cis released into the cytosol prior to vacuolar rupture (Yu
et al.,2002). However, cyclosporin A, which blocks
apoptosis in animals probably by disrupting the PTP, has
been shown to inhibit Zinnia TE formation and to block
DNA degradation and cell death induced by the anticancer
drug BetA without blocking cytochrome crelease (Yu et al.,
2002). It can therefore be concluded that cytochrome
calone is not sufficient to induce DNA degradation and
that the mitochondrial changes, observed prior to vacuolar
bursting, appear to be a side effect rather than an
evolutionarily conserved trigger of TE cell death.
Autolytic processes during xylem cell death
During differentiation of xylem elements, a large array of
proteases, lipases, and nucleases are believed to be produced
and stored in various compartments, such as the vacuole,
until they are released, activated, and mixed with the
cytoplasmic contents, resulting in hydrolysis of cellular
contents. Experimental evidence for this cascade of events
is rather scarce, but it has been shown in Zinnia cell cultures
that proteinase activity increases during TE differentiation
(Beers and Freeman, 1997), and several serine and cysteine
proteases with TE-specific expression patterns have also
been identified (Minami and Fukuda, 1995;Ye and Varner,
1996;Beers and Freeman, 1997;Yamamoto et al., 1997;
Groover and Jones, 1999). Furthermore, pharmacological
inhibition of cysteine protease activity in Zinnia cell cultures
reportedly blocks both secondary wall formation and cell
death if added at the start of the culture (Fukuda, 2000), or
delays degradation of the cell contents if added after
commitment to TE differentiation (Woffenden et al., 1998).
On the basis of these results, and others described in more
detail below, it seems that the final execution of xylem cell
death is similar to apoptotic cell death in the sense that
degradative enzymes act downstream of a signalling cas-
cade. An interesting aspect in plants is the participation of
several different organelles in the pre-death storage of the
degradative enzymes. Whereas the vacuole is believed to be
the most important storage organelle (see, for instance,
Funk et al., 2002), hydrolytic enzymes have also been
shown to be localized in the ER in xylem elements and
senescing cells (Farage-Barhom et al., 2011) as well as in
dying endosperm cells (Schmid et al., 1999).
Cysteine proteases XCP1, XCP2, and VPE
XYLEM CYSTEINE PEPTIDASE1 (XCP1) and XCP2
were originally identified in an Arabidopsis xylem cDNA
library (Zhao et al., 2000), and later shown to be expressed
specifically in xylem vessel elements (Funk et al., 2002).
XCP1 and XCP2 are papain-like cysteine proteases, which
have 71% similarity to each other at the nucleotide level and
60–65% similarity to other members of the papain-like
cysteine protease subfamily C1A. It has been shown that
XCP1 is synthesized with a pro-domain that is autocatalyt-
ically cleaved at an optimal pH of 5.5 to gain full enzymatic
activity (Zhao et al., 2000). Both XCP1 and XCP2 are
located in the vacuole where they function redundantly to
control micro-autolysis in the intact vacuole and mega-
autolysis of cellular contents after rupture of the tonoplast
(Avci et al., 2008).
Vacuolar-processing enzymes (VPEs) are cysteine proteases
that are responsible for maturation of vacuolar proteins
(Hara-Nishimura et al.,1991). VPEs have been shown to
contribute to resistance against several different types of
pathogens in both tobacco and Arabidopsis (Hatsugai et al.,
2004;Rojo et al.,2004;van Baarlen et al.,2007), and dVPE
has been implicated in a developmental cell death pro-
gramme during seed coat formation in Arabidopsis (Nakaune
et al.,2005). One of the four Arabidopsis VPE genes, aVPE,
was shown to be expressed specifically in developing xylem
elements of the root (Kinoshita et al.,1999). In addition, it
has been observed that several different VPE genes are
expressed in late-maturing xylem fibres of Populus stem
(Moreau et al.,2005;Courtois-Moreau et al.,2009), support-
ing the idea that VPEs are also involved in xylem cell death.
Strikingly, both the Arabidopsis cVPE and tobacco VPEs
were shown to have caspase-1-like activity and to be
inhibited by caspase-1 inhibitors (Hatsugai et al.,2004;Rojo
et al.,2004). Therefore, it is believed that a large proportion
of, if not all, caspase-1-like activities in plants can be
attributed to the activity of VPEs. Furthermore, Hatsugai
et al. (2004) demonstrated that full VPE activity is required
1086 |Bollho
¨ner et al.

for tonoplast rupture during tobacco mosaic virus induced
cell death, suggesting that VPEs control tonoplast integrity
during vacuolar cell death. This is particularly interesting
with regard to TE cell death, where tonoplast rupture is
clearly critical for the activities of many hydrolytic enzymes.
Identification of VPE targets is needed to shed further light
on the signalling pathways involved in the control of
tonoplast integrity.
Metacaspases and caspase-like activities
The cysteine protease family of caspases are central
constituents of an apoptotic cell death pathway that is
conserved in mammals, flies, and nematodes. Caspases are
required either for the cell death process itself (Ellis and
Horvitz, 1986) or for triggering the final degradative
processes (Riedl and Shi, 2004). For a long time, caspase
homologues were assumed to exist in plants as well, based
on the ability of some plant extracts to cleave synthetic
caspase substrates (for a summary, see Bonneau et al., 2008)
and the effectiveness of various apoptotic effectors in
modulating plant cell death processes (del Pozo and Lam,
2003;Danon et al., 2004;Rotari et al., 2005). However,
sequencing of plant genomes has revealed that plants do not
have homologues of caspases, and, more recently, several
plant enzymes have been identified as being responsible for
the caspase-like activities observed in plants. These include
the cysteine proteases VPE (Hatsugai et al., 2004;Rojo
et al., 2004) and a proteasome subunit (Hatsugai et al.,
2009), as well as the subtilisin-like serine proteases saspase
(Coffeen and Wolpert, 2004) and phytaspase (Chichkova
et al., 2010). There is, however, no evidence of caspase-like
activities during xylem cell death, but synthetic caspase
inhibitors as well as proteasome inhibitors have been shown
to reduce Zinnia TE differentiation (Woffenden et al., 1998;
Twumasi et al., 2010).
The search for plant caspase genes resulted in the
discovery of structurally related proteins, called metacas-
pases (Uren et al., 2000). Metacaspases have a cleavage site
specificity towards arginine or lysine in the P1 position, in
contrast to caspases, which have a strict requirement for an
aspartate residue in the cleavage site (Vercammen et al.,
2004;Bozhkov et al.,2005;Watanabe and Lam, 2005).
However, similar to caspases, metacaspases have a conserved
cysteine–histidine dyad. They are also separated into two
groups depending on the presence (type I metacaspases) or
absence of a pro-domain (type II metacaspases). Further-
more, the function of metacaspases appears to be analogous
to those of caspases. A spruce metacaspase (mcIIPa)has
been shown to be required for differentiation and cell death
in the embryo suspensor (Suarez et al.,2004). In Arabidopsis,
several different type II metacaspases have been implicated in
the control of various cell death processes, such as disease
resistance towards a fungal pathogen (van Baarlen et al.,
2007), UV radiation- and hydrogen peroxide-induced cell
death (He et al.,2008), and biotic and abiotic stress
(Watanabe and Lam, 2011). The type I Arabidopsis meta-
caspases AtMC1 and AtMC2 have been identified as positive
and negative regulators, respectively, of the hypersensitive
cell death response (Coll et al.,2010). The Arabidopsis
AtMC9 is specifically expressed in differentiating xylem
elements (Turner et al.,2007;Ohashi-Ito et al.,2010), and
ahomologueofAtMC9 was specifically up-regulated during
xylem maturation in Populus stem (Courtois-Moreau et al.,
2009). Populus has two homologues of AtMC9, which are
both expressed in the xylem. One of them is specifically
expressed in late maturing vessel elements, whereas the other
is expressed in both vessel elements and maturing fibres prior
to cell death (Fig. 4D, E). The function of AtMC9 or its
homologues has not yet been demonstrated, but the expres-
sion at the very last stages of xylem maturation strongly
suggests an involvement in xylem cell death.
Nucleases
In metazoa, apoptotic cell death involves various
nucleases, including Ca
2+
/Mg
2+
-, Mg
2+
-dependent, and
cation-independent forms that are responsible for a gradual
degradation of genomic DNA. After initial cleavage into
high molecular weight fragments, DNA is cut internucleo-
somally into fragments with multiples of 180 bp, causing
characteristic DNA laddering, before it is finally degraded
(Samejima and Earnshaw, 2005). There is no evidence for
this kind of DNA degradation pattern during xylem cell
death (Fukuda, 2000), even though it seems to occur
during some forms of plant cell death (Reape and
McCabe, 2008). In Zinnia cell cultures, three main
nucleases—a 24 kDa Ca
2+
/Mg
2+
-dependent nuclease and
two Zn
2+
-dependent nucleases with approximate sizes of
40 kDa and 43 kDa—appear specifically in differentiating
TEs (Ito and Fukuda, 2002). The 43 kDa Zn
2+
-dependent
nuclease has been identified as the S1-type nuclease
ZINNIA ENDONUCLEASE1 (ZEN1), which is most
probably localized to the vacuole (Thelen and Northcote,
1989;Aoyagi et al., 1998). Interestingly, a knock-down of
ZEN1 expression reduced, or at least delayed, degradation
of the nucleus in differentiating TEs in vitro, without
affecting the moment of cell death (Ito and Fukuda, 2002),
demonstrating the importance of ZEN1 in post-mortem
DNA degradation. ZEN1 is most similar to the Arabidop-
sis BIFUNCTIONAL NUCLEASE1 (BFN1), which
belongs to a small gene family composed of five members
(Ito and Fukuda, 2002). BFN1 is expressed in tissues
undergoing senescence and developmental cell death
(Pe
´rez-Amador et al.,2000;Farage-Barhom et al., 2008).
A recent study has shown that BFN1 is localized to special
ER compartments and moves towards the nucleus in
senescing leaf mesophyll cells, finally co-localizing with
fragmented nuclei of dead cells (Farage-Barhom et al.,
2011). Similarly, an ER-localized tomato LX ribonuclease
is expressed in senescing cells and differentiating xylem
elements (Lehmann et al., 2001). These results suggest that
degradation of nuclear DNA and RNA takes place in TEs
only after cell death, which is supported by morphological
analyses of TE nuclei. The situation is somewhat different
in the xylem fibres where at least partial DNA degradation
Xylem cell death |1087

occurs well before cell death, and therefore other nucleases
are expected to be involved, such as those proposed
recently (Courtois-Moreau et al., 2009).
What are the hydrolytic enzymes needed for in TEs?
Mutations in the genes encoding hydrolytic enzymes in
xylem TEs were expected to have serious effects on the
normal growth pattern of the plants because complete and
rapid autolysis of TEs is believed to be required for normal
water transport (Groover and Jones, 1999). However,
several studies have indicated this is not the case. A knock-
down of the tomato LX ribonuclease was found to have no
effect under optimal growth conditions, but merely delayed
senescence (Lers et al., 2006). Similarly, it has been shown
that loss of function of the Arabidopsis S1-nuclease BFN1
does not affect the overall pattern of plant growth (BB and
HT, unpublished results). The xcp1 xcp2 double mutant
reportedly grows completely normally (Avci et al., 2008),
and even complete abolishment of the whole VPE gene
family in Arabidopsis appears to cause no obvious alter-
ations in the vegetative growth pattern of the plants (Gruis
et al., 2004). Except for the xcp1 xcp2 double mutant, none
of these mutants has been characterized in detail with
regard to xylem death, and it is possible that minor changes
in xylem cell death occur in these mutants without affecting
the water transport capacity or overall growth pattern.
However, there are other possible reasons for the lack of
phenotypic alterations in these mutants. It is quite likely
that functional redundancy occurs, not just within each
gene family but also more widely within the large
protease families. Also, phenotypic changes may only be
apparent when the plants are exposed to competition for
nutrients and to biotic or abiotic stresses under natural
conditions.
It cannot be excluded that the hydrolytic enzymes are
present in the TEs for purposes other than or additional to
the autolysis of cellular contents. This is supported by
a recent finding on the inhibitory effect of the Cladosporium
fulvum virulence protein Avr2 on the activities of XCP1 and
XCP2 (van Esse et al., 2008). Cladosporium fulvum is
a biotrophic pathogen that spreads in the apoplast of
leaves. Its virulence factors interact with plant proteins,
such as proteases, to prevent the defence responses of the
plant. The interaction of Avr2 with XCP1 and XCP2
suggests that they have a role in resistance against xylem
resident pathogens. It is possible that XCP1 and XCP2
remain in the xylem sap even after the death of the xylem
vessel elements as a safety mechanism against eventual
pathogen invasion. Proteomics studies have identified
XCP1/XCP2 in apoplastic preparations (Boudart et al.,
2005) and in the xylem sap of Brassica napus (Kehr et al.,
2005). To elucidate the function of the hydrolytic enzymes
further, it is important to create mutants where whole
protease gene families are knocked out. An alternative
approach is to study xylem maturation in response to
xylem-specific expression of protease inhibitors, such as the
Avr2 virulence protein, which are expected to suppress the
activities of several different cysteine proteases (Rooney
et al., 2005;van Esse et al., 2008).
Fig. 4. Expression patterns of cell death-related genes during xylem maturation. (A–C) Histochemical GUS (b-glucuronidase) staining of
Arabidopsis (Col-0) hypocotyls undergoing extensive secondary growth and fibre formation. GUS activity is detected in developing
vessels of proBFN1::GUS (A), proAtMC9::GUS (B), and proXCP2::GUS (C), but not in fibres of any of the lines. (D and E) Histochemical
GUS staining of transgenic Populus stems, carrying pro::GUS fusions of the two Populus AtMC9 homologues. One homologue
(POPTR_0016s02510) is expressed specifically in vessel elements undergoing cell death (D), while the other (POPTR_0006s02730) is
expressed in both vessel elements and fibres (indicated with arrowheads) undergoing cell death (E), suggesting functional diversification
of the two AtMC9 homologues in Populus. The vertical bars indicate 100 lm.
1088 |Bollho
¨ner et al.

Transcriptional regulation
Positive regulators
TE cell death appears to be co-regulated with secondary
wall formation based on a large number of genetic and
pharmacological experiments where it has been impossible
to separate these two processes. However, occurrence of TE
cell death without any secondary wall formation has been
reported in at least two Arabidopsis mutants. Dead cells, or
rather empty holes, have occasionally been observed in
locations where TEs are supposed to be formed in a gapped
xylem (gpx) mutant (Turner and Hall, 2000). Premature
death of provascular root cells which do not form
secondary cell walls has been observed in the wee1 mutant
during replication stress (Cools et al., 2011). The opposite,
secondary wall formation that is not accompanied by cell
death, has been observed in TEs in response to STS
treatment, as described above, and in ectopic TEs of
Arabidopsis plants overexpressing the NAC transcription
factor genes NST1,NST2,andNST3/SND1 (Mitsuda et al.,
2005;Zhong et al.,2006). Therefore, it seems probable that
secondary wall formation and cell death are induced through
a common signalling cascade, diverting at some point to
control the two processes separately. Further evidence for
this kind of cascade has come from the analyses of the
NAM, ATAF1/2, CUC2 (NAC) domain transcription fac-
tors VASCULAR-RELATED NAC-DOMAIN6 (VND6),
VND7, and the NAC SECONDARY WALL THICKEN-
ING PROMOTING FACTOR1 (NST1), NST2, and NST3/
SECONDARY WALL-ASSOCIATED NAC DOMAIN
PROTEIN1 (SND1). VND6,VND7,andSND1 are all
expressed in TEs, and overexpression of each of them
induces ectopic secondary wall formation in a striated
pattern that is reminiscent of TEs (Kubo et al.,2005;
Mitsuda et al.,2005,2007;Zhong et al.,2006;Yamaguchi
et al.,2008). Vice versa, dominant repression of each of these
factors leads to inhibition of secondary wall formation in
specific cell types (Kubo et al.,2005;Zhong et al.,2006,
2007;Mitsuda et al.,2007). These data, together with the
ability of these factors to induce expression of secondary
wall-related genes, have clearly established these transcrip-
tion factors as master switches of secondary wall formation.
Surprisingly, post-translational activation of both VND6 and
VND7 resulted in transcriptional activation of genes in-
volved in not only secondary wall formation but also cell
death, such as XCP1,XCP2,andAtMC9 (Ohashi-Ito et al.,
2010;Yamaguchi et al.,2010,2011;Zhong et al.,2010b).
The activation was mediated by the binding of VND6 and
VND7 to promoter regions carrying the tracheary-element-
regulating cis-element (TERE) (Ohashi-Ito et al.,2010;
Yamaguchi et al.,2011). These results suggest that, firstly, in
addition to secondary wall formation, the NAC master
switches VND6 and VND7 also regulate TE cell death and,
secondly, that cell death is an integral part of the TE
maturation programme. This programme seems to be highly
conserved between different tracheophytes (Zhong et al.,
2010a,c;Ohtani et al.,2011).
The xylem fibres of Arabidopsis normally do not die (Fig.
2H), and genes encoding cell death-related hydrolases are
not expressed in these cell types (Fig. 4A–C). Hence, it is
not clear whether Arabidopsis possesses a transcriptional
programme for cell death in xylary fibres. Nevertheless, it
seems clear that the secondary wall formation of xylary
fibres is controlled by SND1. SND1 is highly expressed in
xylary fibres and both a dominant repression of SND1
function and a simultaneous knock-out in SND1 and NST1
inhibit secondary wall formation specifically in xylary fibres
(Zhong et al., 2006;Mitsuda et al., 2007;Zhong et al.,
2007). As expected, based on its function in non-dying cells,
post-translational activation of SND1 does not induce
expression of cell death-related hydrolytic enzymes in
Arabidopsis xylogenic cultures (Ohashi-Ito et al., 2010).
Zhong et al. (2010b), however, have demonstrated that
SND1 induces expression of some hydrolytic enzymes,
albeit to a much lower level than VND7, and mapped the
secondary wall NAC-binding element (SNBE) as the
consensus sequence necessary for SND1 binding in Arabi-
dopsis protoplasts. Hence, it is possible that xylary fibres in
Arabidopsis have the capacity to express genes required for
cell death by the activity of SND1 but that their expression
and therefore cell death is normally inhibited in planta by
still unknown factors. Identification of Arabidopsis mutants
or ecotypes with pronounced fibre cell death would help to
clarify this issue.
Negative regulators
VND-INTERACTING2 (VNI2) is a NAC transcription
factor that seems to inhibit TE cell death on the basis of its
inhibitory effect on the expression of XCP1 (Yamaguchi
et al., 2010). An inhibitory role in TE cell death has also been
assigned to yet another NAC transcription factor, XYLEM
NAC DOMAIN1 (XND1). Overexpression of XND1 has
been shown to suppress secondary wall formation and cell
death of vessel elements, suggesting a role for XND1 in
negative regulation of terminal TE differentiation (Zhao
et al., 2008). A loss-of-function mutation in XND1 results in
somewhat shorter vessel length, supposedly due to an
enhanced rate of differentiation. Similar changes in cell size
and rate of differentiation have been observed in the acl5
mutant (Mun˜iz et al., 2008), suggesting that XND1 and
ACL5 function in the same pathway. XND1 has been
demonstrated to be a direct target of SND1 and VND7 in
Arabidopsis protoplasts (Zhong et al., 2010b). It contains
a predicted bHLH consensus sequence in its promoter (http://
www.Arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl), and
it is therefore possible that ACL5 and its putative target
SAC51, a bHLH transcription factor, act upstream of XND1
to control the rate of TE differentiation.
Is it a programme for secondary wall formation or cell
death?
The coupling of secondary wall formation and cell death
makes sense, as neither a thin-walled dead cell nor a thick-
Xylem cell death |1089

walled living cell would be able to serve in long-distance
water transport. However, whereas living cells are com-
pletely inefficient with regard to water transport, dead cells
with thin walls are able to function as conduits in short-
distance water transport where only a small negative
pressure is generated, as for example in hydroids of mosses
and thin-walled conduits of extinct protracheophytes
(Sperry, 2003). Although phylogenetic relationships be-
tween mosses, protracheophytes, and extant vascular plants
are somewhat unclear (Kenrick, 2000), it is tempting to
speculate that cell death may have preceded secondary wall
formation during the evolution of vascular plants, leading
to a scenario where the regulation of secondary wall
formation was adopted into an existing cell death pro-
gramme. One could imagine that when secondary wall
formation evolved, the cell death process had to adapt and
slow down to enable appropriate thickening of the cell wall,
a function that has been suggested for thermospermine
synthase ACL5 (Mun˜iz et al., 2008). Finally, what is
observed after completion of the secondary wall could just
be organized disposal of the cell contents, completing a cell
death programme that had been switched on far earlier.
Concluding remarks
Regarding cell morphology, xylem vessel cell death in
angiosperms can clearly be defined as a vacuolar (van
Doorn et al., 2011) or autolytic type of cell death (van
Doorn, 2011). Vessel elements typically contain large
central vacuoles, which are involved in both storage of pro-
death factors and the actual killing of the cell after rupture
of the vacuolar membrane. The central vacuole is also
important for angiosperm xylem fibres and the ancient type
of TEs, the gymnosperm tracheids, even though loss of its
integrity is not causally related to cell death in these
particular cell types since substantial amounts of degrada-
tion and/or remobilization of the cell contents occurs prior
to the vacuolar bursting. Concerning cell death morpho-
types, vessel elements seem to have adopted a cell death
programme, which is probably not the most energetically
efficient way of remobilizing cellular contents but ensures
rapid cell death. In contrast, fibres seem to utilize autoph-
agy to remobilize the cellular contents perhaps in a more
controlled fashion.
Recent data on transcriptional regulation of xylem
differentiation have indicated that cell death is transcrip-
tionally regulated as a part of an overall xylem maturation
programme, which includes secondary cell wall formation.
This emphasizes the importance of cell death inhibition
during xylem maturation. If the components required for
cell death are synthesized simultaneously with the enzymes
responsible for secondary cell wall synthesis, it is crucial for
the cell to prevent premature activity of these components
efficiently. Some of the mechanisms for this, such as
inhibition of the hydrolytic enzymes by specific inhibitors
and storage in inactive forms in safe compartments, are
known. However, there must be additional mechanisms at
play, for example to keep the vacuole intact until the
secondary walls are correctly assembled in vessel elements
and until the cell contents are appropriately remobilized in
xylem fibres. Currently, little is known about the factors
controlling timing of the vacuolar rupture. In xylem fibres,
the time of vacuolar rupture varies significantly depending
on the activity of the cambium and several external factors,
and the lifetime of fibres is dramatically increased, for
instance, during tension wood formation. In some species,
xylem fibres retain their protoplast indefinitely (Fahn and
Leshem, 1963). Identification of factors that allow such
dramatic increases in the longevity of the xylem fibres is
likely to reveal completely new factors with the potential of
inhibiting cell death even after completion of secondary
walls.
Acknowledgements
The authors thank The Swedish Research Council VR and
The Swedish Governmental Agency for Innovation Systems
Vinnova (support of UPSC Berzelii Centre for Forest
Biotechnology), The Swedish Research Council Formas
(support of FuncFiber and BioImprove programs), and
Umea
˚University for financial support to HT. Eric Beers is
thanked for the gift of the Arabidopsis proXCP2::GUS
seeds.
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