Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002

26
Original Article

A. H. de Boer & V. VolkovStructural and functional features of xylem

Plant, Cell and Environment (2003) 26, 87–101

Logistics of water and salt transport through the plant:

structure and functioning of the xylem
A. H. DE BOER1 & V. VOLKOV2,3

1
Vrije Universiteit, Faculty of Earth and Life Sciences, Department of Developmental Genetics, Section Mol. Plant Physiol. &
Biophysics, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands, 2K.A. Timiriazev Institute of Plant Physiology, Root
Physiology Laboratory, Botanischeskaya 35, 127276 Moscow, Russia and 3Horticultural Production Chains Group,
Wageningen University, Marijkeweg 22, 6709 PG Wageningen, The Netherlands

ABSTRACT Abbreviations: CNGC, cyclic nucleotide gated channels;

The xylem is a long-distance transport system that is unique
to higher plants. It evolved into a very sophisticated plumb-
ing system ensuring controlled loading/unloading of ions
and water and their effective translocation to the required
sinks. The focus of this overview will be the intrinsic inter-
relations between structural and functional features of the
xylem. Taken together the xylem is designed to prevent
cavitation (entry of air bubbles), induced by negative pres-
sures under transpiration and to repair the cavitated ves-
sels. Half-bordered pits between xylem parenchyma cells
and xylem vessels are on the one hand the gates to the
vessels but on the other hand a serious ‘bottle-neck’ for
transport. Hence it becomes evident that special transport
systems exist at the interface between the cells and vessels,
which allow intensive fluxes of ions and water to and out of
the xylem. The molecular identification and biophysical/
biochemical characterization of these transporters has just
started. Paradigms for the sophisticated mechanism of
controlled xylem transport under changing environmental
conditions are SKOR, a Shaker-like channel involved in K+-
loading and SOS1, a Na+/H+ antiporter with a proposed
dual function in Na+ transport. In view of the importance
of plant water relations it is not surprising to find that water
channels dominate the gate of access to xylem. Future stud-
ies will focus on the mechanism(s) that regulate water chan-
nels and ion transporters and on their physiological role in,
for example, the repair of embolism. Clearly, progress in
this specific field of research will greatly benefit from an
integration of molecular and biophysical techniques aimed
to understand ‘whole-plant’ behaviour under the ever-
changing environmental conditions in the daily life of all
plants.
Key-words: cavitation repair; ion channels; ion transport;
ion transporters; potassium; sodium; water transport;
xylem.
Correspondence: Dr A. H. de Boer. Fax: + 31 20 4447155;
e-mail: ahdeboer@bio.vu.nl
PD, electric potential difference; SKOR, stelar potassium
outward rectifying channel; TRP, trans-root potential.
INTRODUCTION
Like most multicellular organisms plants need structures
that enable the mass flow of solvents (mainly water) and
solutes such as ions, sugars, amino acids, hormones, etc. In
analogy to the blood circulation system in animals, plants
use tube-like structures called the xylem and phloem.
Whereas the study of the blood circulation system is tech-
nically relatively easy, xylem and phloem transport path-
ways are notably difficult to study due to their location deep
inside the plant tissue, the presence of reinforced cell walls
and the large positive or negative pressures inside the tubes.
Despite, or maybe challenged by these hurdles, a large body
of research has been devoted to unravelling the mecha-
nisms that sustain the functioning of the plant long-distance
transport systems. For recent insights in phloem transport
we refer to reviews by Patrick et al. (2001) and Oparka &
Cruz (2000).
Here we try to link the structure–function relationship of
the dead xylem conduits and living adjacent parenchyma
cells with a focus on the mechanisms that facilitate and
control the flow of ions and water into (loading) and out of
the xylem (unloading). Although it was hypothesized in the
early days of xylem research that, for example, xylem load-
ing in the root was a passive process due to the lack of
oxygen in the core of root tissue (Crafts & Broyer 1938), it
is clear now that we are dealing with sophisticated regula-
tion mechanisms aimed to sustain growth and development
in highly dynamic environments. Since structure and func-
tion are intimately interwoven, the structure of the xylem
transport system will be discussed first. Thereafter, current
knowledge about a number of essential biophysical param-
eters namely the electrical, pH and pressure gradients
between the vessels and surrounding symplast/apoplast will
be summarized. The molecular mechanisms of potassium,
sodium, and water transport will be discussed in more detail
at the end.

© 2003 Blackwell Publishing Ltd 87

88 A. H. de Boer & V. Volkov

HOW STRUCTURAL FEATURES OF THE XYLEM

MEET THE DEMANDS OF SHORT- AND LONG-
DISTANCE TRANSPORT
The main function of the xylem is to transport large quan-
tities of water and solutes. This requires a continuous sys-
tem of interconnected tubes with a relatively low resistance
to the flow of water. As the driving force for mass flow is a
pressure gradient (positive with respect to the atmospheric
pressure as during root pressure, or negative during tran-
spiration) the walls of the vessel or tracheary elements must
be reinforced. On the one hand leakage of water and sol-
utes out of the tubes must be minimized (so the walls must a
be as leak-proof as possible), but on the other hand special-
ized structures in the walls must enable rapid exchange of
water and solutes between the vessel interior and surround-
ing cells. Another feature that is vital for survival of the
xylem is to prevent and repair the interruption of the con-
tinuous water columns by air bubbles (embolism).

Low resistance allows high water flows

The principal conducting cells of the xylem are the trache-
ary elements of which there are two types: the tracheids
and the vessel elements. Both tracheary elements develop
normally from proliferating cells from the procambium and
vascular cambium. When fully differentiated the end walls
of the vessel elements are perforated or removed (what is
left is called the perforation plate) and the cells undergo
programmed cell death (Roberts & McCann 2000). Thus,
files of open interconnected cells are formed. Tracheids also
lack protoplasts at maturity, but in contrast to vessels the
end walls are not open but contain pits, which are areas
lacking the secondary wall. The so-called pit membrane, a
modified thin primary cell wall consisting of a dense net-
work of hydrophilic cellulose polymers, is water permeable
(Holbrook & Zwieniecki 1999). Water flowing through ves-
sels sometimes has to pass pit membranes as well, as vessels
are not continuous over the entire length of the plant and
are interconnected through pits or perforation plates
(Fig. 1). The principal water-conducting cells in
angiosperms are tracheids, whereas vessels are found both
in angiosperms and gymnosperms.
Important determinants of the hydraulic resistance are
the diameter of the vessels and the pit membranes or per-
foration plates. In xylem vessels, water is assumed to flow
according to Poiseuille’s law with a hydraulic conductivity
proportional to the fourth power of vessel’s radius. Thus, a
small change in the radius will give a large change in resis-
tance. The radius of a vessel may also be related to a func-
tion in solute loading. For example, in the first 10–20 cm of
roots of monocots only vessels having a small diameter are
functional in transport. The reason may be that narrow
vessels with a high surface-to-volume ratio are more suit-
able for controlling the composition of the xylem sap than
wide vessels. The large late metaxylem vessels in the apical
10–20 cm of a maize root do not conduct water (St. Aubin,
Canny & McCully 1986), because they mature just in the
pm
b
Figure 1. Bordered pit pairs interconnect xylem vessels and half-
bordered pits connect the vessels to xylem parenchyma cells. (a)
Cryo-planed transverse section through the secondary xylem of a
chrysanthemum stem. (b) Bordered pit pair in high magnification
(length of pit membrane is 3·7 µm). V, vessel; bp, bordered pit; hp,
half-bordered pit; pc, parenchyma cell; lf, libriform fibre; sp, simple
pit; pm, pit membrane (Micrograph kindly provided by Dr J.
Nijsse, Wageningen University).
upper zones of the root. In these upper zones, the wide
vessels collect the sap and function as simple conductive
elements, whereas narrow xylem vessels function not only
as conductive elements but also as xylem sap-controlling
elements.
The prevailing view that tracheary elements have either
a constant or a zero (after embolism) hydraulic conduc-
tance has recently been challenged (Van Ieperen, Van Mee-
teren & van Gelder 2000; Zwieniecki et al. 2001) although
the observation was already made by Zimmermann (1978)
in a nice example of serendipity. Observing a decreasing
flow rate of distilled water through a piece of stem (a prob-
lem already described by Huber & Merz 1958), Zimmer-
man hypothesized that tiny gas bubbles obstructed pit
pores. Therefore he replaced the distilled water with tap
water (assuming that tap water contained more bubbles
than distilled water), but contrary to expectation the flow
rate instantaneously increased. He discovered that this
effect was due to the presence of ions in the tap water and

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101

concluded that ‘the phenomenon might be based upon
swelling or shrinking of the vessel-to-vessel pit mem-
branes’. Van Ieperen et al. (2000) concluded that the pres-
ence of small amounts of cations (even the effect of 10 µM
KCl was significant) and not the osmotic strength influ-
enced the conductance. However, their conclusion that
swelling and shrinking of vessel-to-vessel pit membranes
played no role in this phenomenon was contradicted by
Zwieniecki et al. (2001). The latter authors reached the
same conclusion as formulated by Zimmermann (1978)
namely that the observed change in hydraulic resistance
when changing the ionic composition of the xylem sap is
due to the pit membranes connecting one vessel to the
other. In these ‘membranes’ water flows through micro-
channels of the modified primary cell wall, made up by
cellulose microfibrils, hemicelluloses and pectins. It was
suggested that the size of the microchannels increases when
the pectin matrix (D-galacturonic acid being a main com-
ponent) shrinks in response to the binding of ions to the
negatively charged matrix. If charge compensation is the
mechanism that determines the flow resistance, then it is
surprising that no significant effect of pH within the natural
pH range of xylem sap (5·8–8·0) was observed (Zwieniecki
et al. 2001), although pH affected swelling and shrinking of
isolated cell walls (Meychik & Yermakov 2001). Relevant
of course is the question whether these ion-mediated
changes in hydraulic resistance do have a physiological role.
The K+ concentrations having the strongest effect (0–
20 mM; Zwieniecki et al. 2001), are within the range mea-
sured in transpiring intact plants (Herdel et al. 2001). How-
ever, it remains to be seen whether these K+ variations in
a constant background of, for example, divalent cations still
have an effect on the hydraulic properties.
Reinforcement and waterproofing to withstand
negative pressures and retain water
Tracheary elements not only have to withstand the com-
pressive forces from surrounding cells, but especially the
large negative pressures that develop in actively transpiring
plants (see below). Therefore, secondary walls composed of
cellulose and xylan are deposited on top of the primary cell
wall (Fukuda 1996). Early-formed protoxylem elements of
the primary xylem have ringlike (annular) or spiral (helical)
thickenings that still allow a certain degree of cell elonga-
tion. In the late-formed metaxylem elements and elements
of the secondary xylem the secondary cell walls cover the
entire primary walls, except at pit membranes shared with
adjoining tracheids/vessels or xylem parenchyma cells and
contact cells. Subsequently, the secondary cell walls are
lignified: a network is formed that strengthens and water-
proofs the wall by the polymerization of aromatic com-
pounds called monolignols (McCann 1997). Not
surprisingly, inhibitors of lignification result in a plant phe-
notype with collapsed vessels (Smart & Amrhein 1985).
Primary cell wall polymers are also important for the sub-
sequent lignification and strengthening of a vessel, and
mutants in cellulose deposition in the cell wall with col-
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
Structural and functional features of xylem 89
lapsed xylem vessels were isolated as well (Turner & Som-
erville 1997). In order to prevent ‘leakage’ of water and
solutes from the vessels it seems logical that the lignified
secondary walls are relatively impermeable to these
compounds.
One of the few studies on xylem cell wall permeability is
the one by Wisniewski, Ashwort & Schaffer (1987) who
studied deep supercooling in woody plants and used lan-
thanum nitrate as an apoplastic tracer. Lanthanum pene-
trates capillaries as small as 2 nm, so a cell wall without
lanthanum staining is relatively impermeable. They found
that the primary cell wall of cortical tissues was very per-
meable to the lanthanum solution, but primary (except
parts associated with the pit membranes) and secondary
walls of vessel elements were impermeable. Interestingly,
the pit membranes composed of the middle lamella, pri-
mary walls of the adjoining cells and the protective layer
(in xylem parenchyma cells and contact cells) were highly
permeable. These results are in line with the conclusion that
after occlusion of pit membranes, vessel elements can
assume a closed-cell structure and may have a permeability
approaching zero (Siau 1984).
These permeability properties of the xylem walls are rel-
evant to the question whether water and solutes can directly
enter the vessels from the stelar apoplast. If not, then they
have to be taken up by stelar cells into the symplast and
loaded into the vessel lumen after passing the membrane
of a xylem parenchyma cell underlying a pit membrane. For
example, the stelar potassium outward rectifying channel
(SKOR; activated at depolarizing voltages) is thought to
have a function in loading K+ into the xylem (Wegner &
Raschke 1994; Wegner & de Boer 1997a; Gaymard et al.
1998). Besides expression in xylem parenchyma cells, clear
GUS-staining of root pericycle cells was observed in plants
expressing the GUS reporter gene under the control of the
SKOR promoter region (Gaymard et al. 1998). Note also
that pericycle cells of barley roots stained strongly for the
plasma membrane ATPase (Samuels, Fernando & Glass
1992). The pericycle cells do not directly border a xylem
vessel. Therefore, the question is how K+ ions released from
the pericycle cells into the stelar apoplast do reach the
xylem lumen if the xylem vessel walls are as impermeable
to ions and water as shown by Wisniewski et al. (1987).
Pit structures facilitate water-/solute (un)loading
and embolism repair
Pits constitute the gates between vessels and from vessels
to adjacent xylem parenchyma cells (Fig. 1). The total area
of pit fields, their shape and pattern of lignification are of
large variation between species. For example, maize pit
fields constitute about 15% of the vessel area, with pit
membranes having a diameter of about 2 µm with a hydrau-
lic conductivity about 10 times over that of the vessel wall
(Peterson & Steudle 1993). Typically, between vessels bor-
dered pits are found that are characterized by overarching
walls that form a bowl-shaped chamber (Fig. 1b). It has
long been known that these pits are vital structural ele-
90 A. H. de Boer & V. Volkov
ments in the plant’s defence against cavitation (rupture of
water columns) and subsequent embolism (filling of vessels
and tracheids with air) (Zimmermann 1983; Tyree & Sperry
1989). This is because the modified primary cell wall that
constitutes the pit membrane is permeable to water, but
does not allow the passage of even the tiniest air bubbles.
For this reason tracheids, wherein all elements are linked
through pits are thought to be safer for plants than vessels
although the latter elements are more efficient conductors
of water. In some monocots, such as rye, in the root–shoot
junction tracheids separate the vessels in the root from
those in the shoot to protect the vessels of the roots from
embolism originating in the shoot (Aloni & Griffith 1992).
However, pits are not only important structural elements
that prevent spreading of air bubbles through the water-
conducting elements of plants. They are instrumental in
repairing the ‘broken water pipes’ as well (Tyree et al. 1999;
Holbrook & Zwieniecki 1999; Zwieniecki & Holbrook
2000). Repair means removal of the air bubble; how can
this be achieved? As we have seen above, an air bubble
cannot be simply ‘flushed’ out of a conduit. The existing
paradigm suggests that an air bubble is removed through
gas dissolution in the surrounding water (see Table 1). An
important structural demand is that in order to contain this
pressurization the perimeter of the embolized vessels must
be sealed. As we have seen above, the secondary wall meets
this requirement since the thickness and lignification make
it waterproof. However, what about the pits in which the
water permeability of the pit membrane is high (see
below)? Let us first consider the hypothesis that a plant has
to generate positive (above atmospheric) pressures in the
vessels. The best understood mechanism that generates pos-
itive pressures is root pressure; namely pressure that is
generated in the root during the night or in the shoot in
early spring when transpiration is low through osmotic
water flow into the vessels. There is good experimental
evidence that this mechanism operates in a range of
Table 1. Threshold xylem pressure
Removal of air from an embolized vessel must occur by dissolution
of the gas in the surrounding water. The solubility of a gas in water
is proportional to the partial pressure of the gas species adjacent
to the water (Henry’s law). Water in plants is saturated with air at
atmospheric pressure and therefore the pressure of the gas in the
bubble must be above atmospheric for the gas to dissolve in the
surrounding water.
What determines the pressure of the gas in the bubble (Pg)?
(1) The pressure inside the xylem element, Px; and
(2) the pressure inside the gas bubble that develops due to the
surface tension at the air–water interface, 2τ/r, where r is the
radius of the curvature of the air–water interface and τ is the
surface tension of the water in the vessel.
Thus the pressure of the gas in the bubble equals: Pg = Px + 2τ/r.
Therefore when the xylem pressure is greater than −2τ/r (the
threshold xylem pressure), the gas in an embolism will exceed
atmospheric pressure and start to dissolve. For a narrow vessel of
10 µm diameter this threshold pressure is around −30 kPa
(−0·3 bar). See Tyree et al. (1999) for further details.
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
angiosperms and gymnosperms under certain conditions
(Tyree et al. 1986; Sperry et al. 1987; Tyree & Yang 1992;
Cochard, Ewers & Tyree 1994). However, not all plants
seem to develop root pressure (20 out of 109 species studied
had no root pressure, Fisher et al. 1997) and it must be kept
in mind that root pressure is dissipated by gravity at a rate
of 10 kPa per m height. Therefore, tall trees are unlikely to
develop positive pressures in the shoot as a consequence of
pressure built-up in the root. So, how do plants dissolve
embolism without root pressure? This question is even
more relevant in the light of recent studies reporting embo-
lism repair under conditions when the threshold xylem
pressure (Px) (see Table 1) is much less than −2τ/r (where
τ is the surface tension of water in the vessel and r is the
radius of curvature of the air–water interface) (Salleo et al.
1996; Tyree et al. 1999). The only possible explanation for
this repair mechanism seems to be that locally (i.e. in the
embolized vessel or tracheid element) the xylem pressure
is higher than −2τ/r. There are a number of intriguing prob-
lems with this model:
1 The data suggest that this mechanism of embolism
removal is concurrent with transpiration (Salleo et al.
1996; Canny 1997b; Tyree et al. 1999). If so, how can
xylem pressure increase in the embolized element while
the adjacent vessels are still under tension?
2 What is the mechanism that generates locally a xylem
pressure > −2τ/r?
The first problem was distinguished by Tyree et al. (1999)
and Holbrook & Zwieniecki (1999) came up with a hypoth-
esis as to why in two adjacent vessels (one embolized, the
other filled and under tension) connected by pits, a pressure
difference can be maintained until the embolized vessels
are water filled again (Holbrook & Zwieniecki 1999; Zwie-
niecki & Holbrook 2000). Without going into much detail,
the idea is that the structure of the bordered pits ensures
that the embolized element remains hydraulically isolated
from the neighbouring vessels under tension until the
moment that all air has been dissolved. Zwieniecki & Hol-
brook (2000) showed that the contact angle of water and
the angle of the bordered pit chamber walls are such that
a convex gas–water interface forms in the pit chamber. The
surface tension of this convex curvature opposes the hydro-
static pressure within the filling lumen as long as the pres-
sure in the filling element does not exceed the force due to
the surface tension. Clearly, this model deserves further
experimental testing.
The second problem seems rather easy to solve. Namely,
xylem conduits are generally in contact with numerous liv-
ing cells, the xylem parenchyma cells and contact cells
(Zimmermann 1983; Lachaud & Maurousset 1996). These
cells could be triggered by embolism to load osmotically
active ions such as K+ and Cl– across the pit membrane into
the vessel lumen. Such a mechanism was proposed for the
first time by Grace (1993) who studied embolism repair in
tracheids from Pinus sylvestris. Water would then be driven
by osmosis into the vessel lumen. This idea is consistent
with the observation that convex drops protrude through

Figure 2. Part of an embolized late metaxylem vessel in maize
root. Note the pits between the vessel and bordering xylem par-
chyma cells. Arrows indicate droplets of liquid exuding through
pits into the lumen (× 1580) [Reproduced from McCully et al.
(1998), with permission of Blackwell Publishing, Oxford, UK].
the pits into the lumen of embolized vessels (see Fig. 2)
from sunflower petioles (Canny 1997b), roots of maize
(McCully, Huang & Ling 1998) and Laurel nobilis branches
(Tyree et al. 1999). However, thus far there is one major
problem: attempts to measure high concentrations of salts
in the water that enters an embolized vessel have failed
(Canny 1997b; McCully et al. 1998; McCully 1999; Tyree
et al. 1999). All of the methods used to estimate the xylem
sap ion concentration, rely on the technique of X-ray
microanalysis except one (Tyree et al. 1999). In the latter
study ‘native’ xylem sap from well-watered and cavitated
twigs was collected by perfusion. Treatments wherein cavi-
tation recovery was high also showed a strong increase (up
to four-fold) in K+ concentration. This positive correlation
does suggest that the increase in ion concentration has
something to do with the refilling of embolized elements. If
so, then the increase in concentration of solutes in the refill-
ing vessels may be much higher since the sap recovered
from the perfused stems will be diluted by water from the
full vessels. So, the discussion on the mechanism of refilling
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
Structural and functional features of xylem 91
embolized vessels seems to hinge on the question whether
droplets entering an embolized element through the pits do
contain high enough levels of solutes. If X-ray microanaly-
sis gives a reliable estimate of the xylem sap ion concentra-
tion, then we need a new paradigm of how positive xylem
pressures build-up in embolized vessels and how root pres-
sure in general is generated (Enns, Canny & McCully
2000).
Clearly, there is a need for reliable methods for sampling
and analysis of ‘native’ xylem sap. In the literature many
methods have been described, as summarized by Schurr
(1998), none of which is without its own specific problems.
The methods range from the collection of xylem sap under
root pressure to such an unusual method as using xylem
feeding Philaenus spumarius (spittlebugs) to get the sap
sample (Watson, Pritchard & Malone 2001). As a general
rule, it is important to define the question to be addressed
and then define the requirements a sampling method must
meet. Ideally, the method of analysis does not disturb the
plant in general and the conditions of transpiration in par-
ticular. Non-invasive techniques such as nuclear magnetic
imaging (NMRi), nuclear magnetic resonance (NMR) spec-
trometry and magnetic resonance imaging (MRI) do in
principle meet these requirements (Scheenen et al. 2000;
Peuke et al. 2001; Holbrook et al. 2001). Spatial and tempo-
ral resolution was initially an important disadvantage of
these methods, but recent methods have decreased mea-
surement time drastically (Rokitta et al. 1999; Scheenen
et al. 2000; Peuke et al. 2001).
If the refilling of embolized vessels is not driven osmoti-
cally by salts loaded into the vessel lumen by the surround-
ing xylem parenchyma cells, then maybe reverse osmosis is
a feasible mechanism (McCully 1999; Tyree et al. 1999) as
originally proposed by Canny (1997a, b). Reverse osmosis
could occur if cells surrounding the xylem decrease their
osmotic potential (through ion uptake or conversion of
starch to sugars), built up a higher turgor pressure, pressur-
ize xylem parenchyma cells that then squeeze water (with-
out ions) into the embolized vessel element. An auxin-
induced loading of solutes in the phloem may be part of
this mechanism (Salleo et al. 1996; Tyree et al. 1999). How-
ever, Tyree et al. (1999) have pointed out the problems that
come with this mechanism. One important problem to
solve is how to prevent water from flowing in all directions
from the ‘squeezed’ parenchyma cells. In other words, how
to generate a direction in the flow of water (a problem
solved in the ‘osmosis’ hypothesis by specific loading of ions
into the embolized element across the pit membrane). One
possibility is that water channels in the patch of membrane
underlying the pit membrane are activated, thus creating a
path of low resistance to waterflow into the vessel element
(see section on water channels).
Vessel-associated cells are equipped for a task
in (un)loading
Living vessel-associated cells, the xylem parenchyma cells
and contact cells, are the cells that have direct access to the
92 A. H. de Boer & V. Volkov
vessel lumen. Therefore, they play a key role in the loading
and unloading of the xylem. In line with the energy demand
of these (un)loading processes, structural features of cells
indicate high metabolic activities that could fuel massive
transmembrane transport. These features are a dense cyto-
plasm, well-developed endoplasmic reticulum, numerous
ribosomes, mitochondria and peroxisomes (Läuchli et al.
1974). The specialized transport function of these cells is
evidenced by the development of cell wall invaginations in
the area underlying the pit membrane bordering the vessels
(Kramer et al. 1977; Yeo et al. 1977; Kuo et al. 1980; Mueller
& Beckman 1984). This folding pattern increases the mem-
brane surface area and is characteristic for transfer cells
(Pate & Gunning 1972; Gunning 1977) and the develop-
ment is stimulated by salt stress (Kramer et al. 1977; Yeo
et al. 1977). Relatively little is known thus far about the
distribution of ion transporters in the membrane of vessel-
associated cells. Sauter, Iten & Zimmermann (1973)
observed high ATP hydrolysing activity at the large pits of
contact cells facing the vessels and suggested a role for this
enzymatic activity in sugar loading of the xylem. This activ-
ity was only observed during starch mobilization in the rays
cells in spring or when starch mobilization was artifically
induced by defoliation. Xylem parenchyma cells of barley
and oat roots were strongly labelled by antibodies against
the plasma membrane ATPase, but the resolution was
insufficient to discern differences in subcellular distribution
of the protein (i.e. patches on the vessel-facing side)
(Parets-Soler, Pardo & Serrano 1990; Samuels et al. 1992).
These results are in line with the localization of ATPase
activity in barley xylem parenchyma cells as determined
with a cytochemical lead precipitation method (Winter-
Sluiter, Läuchli & Kramer 1977).
The external cytoplasmic layer of xylem parenchyma
cells has specific features at the point of contact pits (pits
between xylem parenchyma cells and xylem vessels). There,
the network of actin filaments contains fewer cortical
microtubules in comparison with other parts of the cyto-
plasmic layer where an intensive cortical microtubule
cytoskeleton is present (Chaffey, Barlow & Barnett 2000;
Chaffey & Barlow 2001). The function of these structural
changes of the cortical cell layer may be two-fold: to pre-
vent the deposition of a thick secondary wall, a process
which depends on cortical microtubules, and the regulation
of ion and water channel activity. Actin involvement in ion
channel regulation was shown for guard cells (Hwang et al.
1997).
THE PROTON MOTIVE FORCE AT THE XYLEM/
SYMPLAST INTERFACE: DRIVING FORCE FOR
XYLEM (UN)LOADING
The energy necessary to drive transport of ions, amino
acids, sugars, etc. across the membrane of vessel-associated
cells into or out of the vessel lumen, is derived from the
electrical gradient across this membrane, the pH gradient
or both. Therefore, it is important to know the value of
these two parameters and the dynamic regulation thereof.
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
Xylem electrical potential
The initial hypothesis postulated by Crafts & Broyer (1938)
that primary active transport (e.g. an ATP driven H+-
ATPase) at the symplast/vessel boundary was precluded
due to lack of oxygen, proved not to be correct (Okamoto,
Katou & Ichino 1979; De Boer et al. 1983; Clarkson, Will-
iams & Hanson 1984). In roots of Plantago and hypocotyls
of Vigna unguiculata the electric potential difference (PD)
between vessel and parenchyma cells contains an electro-
genic component, which in both roots and hypocotyls can
amount to 70 mV. The primary H+-ATPase is thought to be
responsible for this electrogenic component because it dis-
appears in the presence of inhibitors of the oxidative phos-
phorylation (anoxia and azide) and increases with auxin
and fusicoccin, the activator of the plasma membrane H+-
ATPase (De Boer et al. 1985; De Boer 1997). It must be
kept in mind that the electrical potential of the cortical and
xylem (stelar) apoplast are not necessarily the same. An
insulating layer formed by the ring of endodermis cells with
their Casparian strips, can maintain a PD between the two
apoplasts. Because the cortical, endodermal and stelar cells
are connected through plasmodesmata (forming the sym-
plast) the apoplast PD is in fact the difference of two mem-
brane potentials in series: (PDsymplast/cortical apoplast)
minus (PDsymplast/xylem apoplast) (Okamoto et al. 1979;
De Boer et al. 1983; De Boer et al. 1985). This PD is also
called trans-root potential (TRP), but the same principle
holds for stems and other tissues.
All measurements mentioned above were carried out on
excised plants or perfused root and stem segments. This
approach can be criticized because in excised roots the
turgor and intracellular osmotic pressure gradients collapse
(Rygol et al. 1993). Therefore, measurements on intact
plants are necessary. Dunlop (1982) succeeded for the first
time to measure directly with a microelectrode in the ves-
sels of intact Trifolium repens plants. The electrical poten-
tial of the vessels was 90 mV negative with respect to the
cortical apoplast. Because xylem parenchyma cells had a
membrane potential of −168 mV it can be calculated that
the potential difference across the symplast/xylem bound-
ary was around 80 mV (xylem positive). These values are
close to the values measured in excised Plantago roots
where the electrogenic component of the symplast/xylem
PD was zero (De Boer et al. 1983). Recently, a new method
was introduced, using a modified pressure probe, enabling
the simultaneous measurement of the trans-root potential
and the xylem pressure in intact transpiring plants (Wegner
& Zimmermann 1998). One of the conclusions of these
measurements was that the roots of intact transpiring plants
have electrical properties similar to those of excised roots
(Wegner et al. 1999). These measurements also demon-
strated that the regulation of the trans-root electrical poten-
tial is a dynamic process. Namely, a switch to a higher light
intensity induced strong oscillations in TRP that preceded
oscillations in the negative xylem pressure. Interestingly,
the depolarization of TRP correlated with the most nega-
tive xylem pressure. Since the membrane potential of the
 Structural and functional features of xylem 93

cortical cells showed no oscillations, the oscillations in TRP

must be the result of a change in ion transport activity
across the symplast/xylem boundary or as suggested by
Wegner et al. (1999) by a change in streaming potential due
to variations in the transpiration flow. However, thus far it
is not clear whether streaming potentials contribute signif-
icantly to the potential differences within a xylem vessel or
between the xylem and cortical apoplast.
Although we now know that xylem transport processes
are not simply passive, the early suggestion that the supply
of oxygen to the xylem might be a limiting factor (Crafts &
Broyer 1938; De Boer & Prins 1984) deserves renewed
attention in the light of recent analysis of the oxygen supply
to sapwood using a novel optical method for oxygen detec-
tion (Gansert, Burgdorf & Lösch 2001).

Xylem sap pH
The pH of the xylem sap is relevant for (un)loading pro-
cesses for two reasons: (i) as driving force for antiport and
symport processes (see below) and (ii) as regulator of ion
transporters or signalling molecules. There are no measure-
ments of the xylem sap pH in intact plants as far as we are
aware. Measurements on xylem sap collected from root
exudates or by means of centrifugation agree on a slightly
acidic pH in the range 5·5–6·5 (Schurr & Schulze 1995;
Wilkinson & Davies 1997; López-Millán et al. 2000). Some-
how the xylem has a high capacity to maintain its pH to a
set-value (Clarkson et al. 1984; Senden et al. 1992). How-
ever, in response to changes in environmental conditions
the xylem sap pH does fluctuate. Under drought stress the
xylem sap pH from Commelina communis increased from
6·1 to 6·7 (Wilkinson & Davies 1997) and iron stress condi-
tions reduced the pH from sugar beet xylem sap from 6·0
to 5·7 (López-Millán et al. 2000). What is the mechanism
that on the one hand buffers and on the other hand changes
the xylem pH? Buffering is certainly a consequence of the
cation exchange capacity of the xylem cell walls due to the
presence of negatively charged groups (Ferguson & Bollard
1976; Senden et al. 1992) and the presence of organic acids
such as succinate and malate (Schurr & Schulze 1995). But
the physiological changes may be due to regulation of the
activity of the H+-ATPase and/or H+-exchange systems in
the plasma membrane of vessel associated cells. If for
example the H+-ATPase activator fusicoccin is perfused
through xylem vessels the pH of the emerging sap
decreases within minutes after application, reaching values
as low as 4·2 (Fig. 3). This effect is explained by the uptake
of fusicoccin by xylem parenchyma cells around the vessels.
Fusicoccin then activates the plasma membrane H+-
ATPase (De Boer 1997), which results in a strong efflux of
protons into the xylem sap. Further evidence for the
involvement of metabolically active cells is the observation
that the pH rapidly increases when roots are subjected to
anoxia (De Boer & Prins 1985; Lacan & Durand 1996).
Several roles for changes in xylem sap pH have been pro-
posed. Thus, the increase in pH initiated by drought stress,
is thought to increase the efficiency of abscisic acid in stim-
Figure 3. Effect of fusicoccin, activator of the plasma membrane
H+-ATPase, on the ion concentration and ion fluxes into/out of the
xylem as measured by means of a xylem perfusion technique. (a)
Perfusion of taproots from Plantago maritima and (b) of nodal
roots from barley. In both roots, fusicoccin (10 µM) strongly acidi-
fies the xylem sap, reduces the flow of K+ into the xylem from the
surrounding cells and stimulates the release of Na+ into the xylem
stream. [(a), De Boer unpubl. res.; (b), Volkov and De Boer
unpubl. res.].
ulating the closure of stomata (Wilkinson & Davies 1997).
Moreover, K+ loading into the root xylem may be a pH-
sensitive process through modulation of the activity of
SKOR, the outward rectifying K+ channel involved in K+
release into the xylem (Lacombe et al. 2000 and see below).
MOLECULAR MECHANISM OF SOLUTE AND
WATER (UN)LOADING
Potassium
Xylem loading of K+ is regulated separately from K+ uptake
from the external solution (Cram & Pitman 1972; Engels &
Marschner 1992). Studies at the cellular and molecular level
in recent years have shown two principal types of K+ chan-
nels in the root (Gaymard et al. 1998; Spalding et al. 1999):
AKT1 for K+ uptake from the medium and SKOR for K+
loading into the xylem. The in planta properties of SKOR
[formerly KORC in barley (Wegner & De Boer 1997a)]
were first characterized through patch-clamp studies on
xylem parenchyma protoplasts isolated from barley and
maize roots (Wegner & Raschke 1994; Roberts & Tester

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
94 A. H. de Boer & V. Volkov
1995; Wegner & De Boer 1997a; Roberts 1998). These
outward rectifying channels activate in a time-dependent
manner at membrane potentials that shift with and remain
slightly positive of the reversal potential of K+ (EK). They
are selective for K+ but do facilitate the passage of Ca++ as
well. They cannot be responsible for Na+ loading of the
xylem as they are virtually impermeable to Na+. Under
physiological conditions a depolarization of the membrane
potential to values positive of EK will result in a K+ efflux
and an influx of Ca++. Therefore, a non-functional SKOR
may result in a reduction of the amount of K+ and an
increase in the amount of Ca++ in the shoot (see below).
In addition to its sensitivity to membrane voltage and
apoplastic K+ concentration, the activity of SKOR is regu-
lated by cytosolic Ca++, the stress hormone abscisic acid
(ABA) and pH. A rise in the cytosolic Ca++ concentration
from 150 nM to 5 µM reduced the frequency at which SKOR
was recorded in barley xylem parenchyma cells (whole-cell
configuration) from 90 to 10% (Wegner & De Boer 1997a).
As this effect was absent when inside-out patches were
used, the involvement of a cytosolic intermediate was sug-
gested. ABA has a dual effect on SKOR activity: a short-
term effect measurable within minutes after addition of
ABA and a long-term effect (Roberts 1998; Gaymard et al.
1998). The short-term effect was observed when ABA was
added to protoplasts during a patch-clamp experiment:
within minutes it reduced the outward current in 55% of
the cells; the inward current was unaffected. Drought, or
overnight pre-treatment of intact plants with ABA also
resulted in a strong reduction of the outward K+ current. In
line with earlier experiments (Cram & Pitman 1972; Pitman
1977) ABA pre-treatment had no effect on in- and out-
ward-currents in cortical root cells (Roberts 1998). It
remains to be shown if Ca++ plays a role as second messen-
ger in the xylem parenchyma ABA signalling pathway, as
it does in guard cells (Allen et al. 2000). An indication
maybe that both Ca++ and ABA affect SKOR in a similar
fashion.
The gene encoding the channel responsible for K+ load-
ing in the xylem, called SKOR, belongs to the Shaker super-
family (Gaymard et al. 1998). SKOR expressed in Xenopus
oocytes, exhibited most if not all of the characteristics of
xylem parenchyma K+ channel characterized in barley and
maize: sigmoidal activation, shift in activation potential
with EK, sensitivity to blockers, Ca++ permeability and
increased magnitude of the outward current with increasing
external K+ concentration. Northern blot analysis showed
that SKOR is expressed exclusively in roots and there the
expression is limited to pericycle cells and cells surrounding
the xylem vessels; hence the name stelar K+ outward recti-
fier. Treatment of intact plants with the stress hormone
abscisic acid resulted in a rapid decline in SKOR mRNA
abundance (Gaymard et al. 1998), thus shedding light on
ABA inhibition of xylem K+ loading in intact barley plants
(Cram & Pitman 1972) and the activity of K+-outward rec-
tifying channels in maize stelar protoplasts as measured
with the patch-clamp technique (Roberts 1998). The con-
clusion that xylem loading of K+ is regulated separately
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
from K+ uptake from the external solution (Cram & Pitman
1972; Pitman & Wellfare 1978; Engels & Marschner 1992)
is underlined by the fact that the K+ channel (activity and
expression) responsible for K+ uptake into the root sym-
plast, AKT1 is not affected by ABA (Roberts 1998; Gay-
mard et al. 1998).
Roberts (1998) also observed a short-term effect of ABA
upon addition to the bath medium during a patch-clamp
measurement: within 5 min after ABA addition the out-
ward K+ current in maize stelar protoplasts declined by
about 50%. This effect might be mediated by changes in
cytosolic pH since a decrease in cytosolic pH from 7·4 to
7·2 induced a strong (80%) voltage-independent decrease
of the macroscopic SKOR current in a heterologous expres-
sion system (oocytes) (Lacombe et al. 2000). However, both
in dark-grown coleoptiles and in barley aleurone proto-
plasts ABA induced an alkalinization of 0·1–0·2 units
(Gehring, Irving & Parish 1990; Van der Veen, Heimo-
varaa-Dijkstra & Wang 1992). If the same were true for
xylem parenchyma cells, then an ABA-induced alkaliniza-
tion of the cytoplasm would increase the outward K+ cur-
rent. It remains necessary to determine the pH sensitivity
of SKOR in planta as well as the effect of ABA on the pH
of xylem parenchyma cells.
The high level of expression of SKOR in the pericycle
cells raises some questions. As these cells are not in direct
contact with the xylem vessels, K+ ions released through
SKOR will end up in the stelar apoplast. If the ion perme-
ability of the secondary walls of the xylem vessels is low
(see above) then loading of these ions into the xylem might
be preceded by uptake into the xylem parenchyma cells
followed by release into the vessels. This could explain the
relatively large number of inward rectifying K+ channels
(i.e. channels that activate at hyperpolarizing voltages) in
barley xylem parenchyma cells, two of which are G-protein
regulated (Wegner & de Boer 1997b). The explanation for
such a complex mechanism of loading is not immediately
obvious, but in some plants sugar loading into the phloem
also includes an apoplast unloading step followed by
uptake into the companion cells and symplast loading into
the sieve elements (Van Bel et al. 1992). These xylem paren-
chyma K+ inward rectifying channels may also have a func-
tion in the apoplast resorption of K+ ions released by the
phloem in the root stelar apoplast. Retranslocation of K+
from the shoot to the root in the phloem is a well-known
phenomenon and it has been speculated that the amount
of K+ returning from the shoot acts as a signal for root K+
uptake from the soil (White 1997; Jeschke & Hartung
2000).
Xylem perfusion experiments, using tetraethylammo-
nium as blocker of SKOR, showed the in vivo function of
SKOR in loading the xylem with K+ (Wegner & De Boer
1997a). The phenotype of the skor-1 knockout mutant
(Gaymard et al. 1998) indeed showed a reduced transport
(50%) of K+ to the shoot of skor-1. Electrophysiological
characterization of the selectivity properties of SKOR in
barley yielded a PCa++/PK+ ratio of 0·68 (Wegner & De Boer
1997a). This outcome suggested a role for SKOR in Ca++

unloading of the xylem in roots, which was confirmed by
the higher Ca++ content of the skor-1 leaves (Gaymard et al.
1998).
On the way up through the stem into the leaves K+ ions
will be taken up into the symplast by cells in contact with
the vessels and tracheids. The uptake of K+ in the elonga-
tion zone of the hypocotyl of Vigna unguiculata is stimu-
lated by the growth-promoting hormone auxin and
fusicoccin (De Boer et al. 1985). The actual mechanism (ion
channel or H+/K+-symporter) of uptake is not known. Keu-
necke et al. (1997) used a patch-clamp approach to unravel
the molecular mechanism of xylem unloading. In proto-
plasts isolated from xylem contact cells in leaves of Vicia
faba and maize two different types of K+ channels were
identified, but a role in K+ unloading could not be assigned.
The small veins of leaves are often surrounded by a layer
of compactly arranged cells, the bundle sheath. These cells
isolate the veins from the intercellular spaces (the apoplast)
of the leaf. Keunecke et al. (2001) showed that uptake of
K+ from the vessels of small veins in maize leaves occurs at
the xylem/bundle sheath cell interface.
An intriguing question is whether the flow of K+ (and
other solutes) to the different parts of the shoot is simply
determined by the transpiration stream or whether there is
at certain points a ‘special filter’ diverting more K+ to, for
example, rapidly growing buds/leaves that still have a rela-
tively low transpiration rate? Gunning, Pate & Green
(1970) showed that xylem transfer cells in stems of many
plants were almost exclusively associated with departing
foliar traces (Fig. 4). They estimated that in these regions
the membrane surface area of cells surrounding the vessels
may be amplified by as much as 20-fold. This, suggests a
potential for very intensive lateral exchange of solutes. Fur-
thermore, xylem–phloem exchange is a very important
XP
V tain glycophytes (such as bean, maize and reed) have devel-
V oped a xylem Na+ unloading mechanism in the basal part
XP
Figure 4. Conspicuous wall ingrowths of transfer cells lining the
xylem of the cotelydonary traces of a seedling of Jacob’s ladder
(Polemonium caeruleum) (× 600). V, xylem vessel; XP, xylem
parenchyma cell. [Reproduced from Gunning et al. (1970), with
permission of Springer-Verlag Austria].
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
Structural and functional features of xylem 95
(a) root (b) shoot
0 mM NaCl
700
1 mM NaCl
600
25 mM NaCl
Na (me.kg d.m.)
500
–1
400
300
+
200
100
0 1 2 1 2
P.media P.maritima P.media P.maritima
Figure 5. Different strategies of Plantago media (glycophyte)
and P. maritima (halophyte) towards the accumulation of Na+ ions
in the root and shoot. The glycophyte has a reduced translocation
of Na+ from the root to the shoot, whereas the halophyte prefer-
entially accumulates Na+ in the leaves. Plants were grown on sand
supplied with nutrient solution with Na+ concentrations as indi-
cated [from De Boer (1985), where further details can be
obtained].
transport step in nutrient distribution and the C/N economy
of the plant (Van Bel 1990; White 1997; Jeschke & Hartung
2000). On the other hand, it seems contra-productive to
transport large amounts of, for example, K+ to rapidly tran-
spiring mature leaves which are not in need of K+. It will
be interesting to study whether the flow of K+ into the
petiole of these leaves is restricted by active resorption
from the transpiration stream entering that leaf.
Sodium
Salt-sensitive (glycophytes) and salt-tolerant (halophytes)
plants use different strategies when exposed to high Na+
levels in their root medium. In general, glycophytes try to
reduce net Na+ uptake from the soil and translocation of
Na+ to the shoot, whereas halophytes rapidly absorb and
translocate Na+ and sequester it in the vacuole where it is
used as ‘cheap’ osmoticum (see Fig. 5a). Especially in halo-
phytes the accumulation of Na+ is at the expense of K+
(Fig. 5b). Therefore it is to be expected that different trans-
port mechanisms for Na+ are present at the symplast/xylem
boundary in the root where xylem loading takes place. Cer-
of the root and shoot where Na+ ions are taken up from the
transpiration stream (Jacoby 1965; Johanson & Cheeseman
1983; Lacan & Durand 1996; Ehwald 2001). Although the
capacity of this system is limited, it may play a role in
avoiding salt damage in the young growing parts of the
shoot at moderate salt stress.
Sodium entry into the root symplast is probably medi-
ated by weakly voltage-dependent non-selective cation
channels (Roberts & Tester 1995; Tyerman & Skerrett 1999;
Davenport & Tester 2000; Demidchik, Davenport & Tester
96 A. H. de Boer & V. Volkov
2002; Demidchik & Tester 2002). The properties of these
channels resemble those of cyclic nucleotide-gated chan-
nels (CNGC) (Leng et al. 1999). Evidence for a role of
CNGCs in Na+ uptake is two-fold: (i) the cngc1 mutant
shows enhanced Na+ tolerance when grown on moderate
external Na+ (Talke et al. 2001); and (ii) membrane-perme-
able cAMP/cGMP analogues reduced unidirectional Na+
influx and overall Na+ accumulation in NaCl grown Ara-
biodopsis (Maathuis & Sanders 2001).
In the plasma membrane of barley xylem parenchyma
cells a non-selective ion channel, called NORC, has been
characterized but in contrast to the CNGCs it is non-selec-
tive for both cations and anions and it is voltage dependent
(Wegner & Raschke 1994; Wegner & De Boer 1997a).
Although NORC is equally permeable to K+ and Na+, it
remains to be shown whether, under physiological condi-
tions, NORC has a function in loading of Na+ into the
transpiration stream. NORC also passes glutamate (Weg-
ner & de Boer 1997a). In this respect it is noteworthy that
a glutamate receptor gene, AtGluR2, is expressed in cells
adjacent to the conducting vessels in Arabidopsis leaves
(Kim et al. 2001). AtGluR2 is homologous to mammalian
ionotropic glutamate receptors that are non-selective cat-
ion channels permeable to Ca++, K+ and Na+, but not Mg++
(Lam et al. 1998). The primary physiological function of
these channels is thought to be Ca++ influx into cells (Den-
nison & Spalding 2000). Over-expression of AtGluR2
resulted in Ca++-deficiency symptoms and according to Kim
et al. (2001) this might be due to increased Ca++ unloading
from the transpiration stream in the root and stem causing
Ca++ deficiency in the young, growing parts of the plant. The
transgenic plants also exhibited hypersensitivity to Na+ and
K+ ionic stress, which might be due to reduced Ca++ uptake
in the presence of excess Na+ or K+ in combination with
increased Ca++ unloading on the way up to the top of the
plant.
Na+ loading into the xylem of barley and maize roots is
not mediated by the outward rectifying K+ channel of xylem
parenchyma cells since this channel is virtually imperme-
able to Na+ (Roberts & Tester 1995; Wegner & De Boer
1997a). Heterologous expression of SKOR in Xenopus
oocytes corroborates this conclusion (Gaymard et al. 1998).
A prominent candidate gene encoding a Na+ transporter
involved in Na+ transport across the symplast/xylem bound-
ary is SOS1 (Ding & Zhu 1997; Shi et al. 2000, 2002). SOS1
was identified as a NaCl-hypersensitive Arabidopsis mutant
and initially described as a mutant defective in high-affinity
potassium uptake (Wu, Ding & Zhu 1996). The 22Na-uptake
and efflux measurements indicated that the sos1 mutants
were impaired both in high-affinity K+-and low-affinity Na+-
uptake (Ding & Zhu 1997). However, in these experiments
the whole seedling was exposed to the uptake/efflux solu-
tion and this makes interpretation of the results in terms of
the SOS1 function in uptake and translocation in planta
hazardous. Subsequent sequencing of the locus showed that
sos1 encodes a 127 kDa protein with 12 transmembrane
spanning domains and with significant sequence similarity
to plasma membrane Na+/H+ antiporters from bacteria and
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
fungi (Shi et al. 2000; see also Hasegawa, Bressan & Pardo
2000). In the same article it was reported that SOS1 gene
expression is concentrated in the cells surrounding the
xylem, suggesting that SOS1 may function in loading Na+
into the xylem for long-distance transport. Recently, the
suggestion that SOS1 encodes a Na+/H+ antiporter was
experimentally substantiated using purified plasma mem-
brane vesicles from salt-stressed Arabidopsis thaliana
leaves (Qiu et al. 2001). Salt-treated sos1 mutant plants
showed a 60% lower Na+/H+ exchange activity in compari-
son with wild-type. SOS1 expression is up-regulated by
NaCl stress and this up-regulation is abolished in plants with
a mutation in the SOS3 gene encoding a phosphatase with
significant sequence similarity with the calcineurin B sub-
unit from yeast (Liu & Zhu 1998). In the presence of Ca++
SOS3 interacts with SOS2 (a protein kinase) and activates
the kinase activity of SOS2 (Halfter, Ishitani & Zhu 2000).
Apparently, SOS2 directly modulates SOS1 since in vitro
addition of SOS2 to plasma membrane vesicles isolated
from NaCl stressed plants resulted in a two-fold increase in
the Na+/H+ exchange activity (Qiu et al. 2001).
During SOS1-facilitated loading of Na+ into the xylem
(Shi et al. 2002), one proton flowing from the xylem to the
symplast (a ‘down-hill’ process energetically speaking)
drives the transport of one Na+ ion into the xylem vessels.
Na+ loading into the xylem is unlike K+ loading probably
not a channel-mediated process. Much remains to be
learned from a comparison of K+ and Na+ exchange across
the symplast/xylem boundary in experiments in which the
xylem of root or shoot segments is perfused with defined
solutions. As discussed above, the high K+ concentration in
the xylem parenchyma cells (Drew, Webb & Saker 1990)
allows K+ loading down the electrochemical K+ gradient
facilitated by the K+-outward channel SKOR (Wegner & de
Boer 1997a; Gaymard et al. 1998). In line with the proper-
ties of SKOR (depolarization activated and inactivated by
external acidification; Lacombe et al. 2000), xylem perfu-
sion of the H+-ATPase activator fusicoccin stops the K+
loading process in roots of the halophyte Plantago maritima
(Fig. 3a). Interestingly, the response of Na+ is very different
from that of K+: in the first 45 min after the start of fusic-
occin perfusion the Na+ concentration in the xylem
decreases (i.e. Na+ unloading of the xylem sap is stimu-
lated). This increase in uptake is unlikely to be caused by a
pH change of the xylem sap (because this is still small), but
rather due to the rapid hyperpolarization of the membrane
potential induced by fusicoccin (De Boer & Prins 1985; De
Boer et al. 1985). This uptake may be a channel-mediated
process. However, when the xylem sap starts to acidify
there is a turning point resulting in Na+ loading into the
xylem when fusicoccin has acidified the xylem sap pH with
more than 1 unit (Fig. 3a). The large pH gradient between
xylem sap and the cytoplasm of adjacent parenchyma cells
may now facilitate a Na+/H+ antiport system similar to
SOS1 (Shi et al. 2002). Similar fusicoccin-induced responses
of xylem sap pH and K+/Na+ (un)loading were observed in
xylem perfusion experiments using nodal roots of barley
(Fig. 3b).

Shi et al. (2002) proposed a model where SOS1 plays a
dual role in Na+ loading and reabsorption from the xylem
sap, depending on salinity and pH difference between sym-
plast and xylem, in analogy to a model proposed by Lacan
& Durand (1996). The latter authors observed an increase
in Na+ unloading and an increase in K+ loading of the xylem
sap when the initial pH of the perfusion solution was more
acidic. They conclude that Na+ is taken up from the xylem
through a Na+/H+ antiport mechanism, but now with H+
driven ‘uphill’ by a ‘down-hill’ Na+ gradient. However, a
mechanistic interpretation of their results is difficult since
it is not clear how the xylem pH changes in view of the
strong pH-stat properties of the xylem (Clarkson et al.
1984). Moreover, it is also not clear how the different initial
pH values of the perfusion solution affect the membrane
potential. The observed positive correlation between K+
loading and acidity of perfusion solution as reported by
Lacan & Durand (1996) is not in line with a SKOR-
mediated loading mechanism since SKOR activity is very
low at acidic external pH (Lacombe et al. 2000). It must be
kept in mind that perfusion of an acidic buffer will result in
an exchange of protons with other cations such as K+ at the
negatively charged cell walls of the xylem and that this may
cause an apparent increase in K+ loading (Senden et al.
1992). The reabsorption of Na+ from the upgoing transpira-
tion stream in the basal parts of the root and shoot has been
well documented both in intact plants and in perfused seg-
ments (Jacoby 1965; Johanson & Cheeseman 1983; Lacan
& Durand 1996; Ehwald 2001). However, the storage
capacity of the stelar cells is limited and therefore a retrans-
location of Na+ by the phloem to the root and extrusion
into the medium has been suggested as a mechanism to
increase the detoxification capacity. Although Van Bel et al.
1992) has already pointed to the importance of xylem–
phloem exchange processes nothing is yet known about the
mechanism of Na+ loading into the phloem and unloading
in the root.
Water
On its route to the root xylem, water flow is restricted by a
number of morphological structures such as the epidermis,
outer cortex layer (exodermis), several layers of cortical
cells, endodermis, pericycle, parenchyma cells of central
cylinder, and cell walls of xylem vessels (Steudle & Peter-
son 1998). Under conditions of high transpiration, water
flow is passively dragged across the roots at a high rate,
whereas under high humidity and absence of transpiration
the ascending water flow to the shoot is generated by root
pressure with low flow rates. In the permanent fluctuating
river of water, the aim of a plant is to control ion fluxes to
the xylem and to keep most of them relatively independent
of water flow. In fact, transpiration may not be required for
the supply of minerals to the shoot (Tanner & Beevers
2001). The latter authors provided evidence that xylem flow
brought about by root pressure and Münch’s phloem coun-
terflow, might be sufficient for long-distance mineral supply.
The plant can discriminate well between water and ions
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101
Structural and functional features of xylem 97
because water molecules have no charge, whereas ions do.
Water can move via the apoplast (within cell walls), sym-
plast (moving via cells without leaving them) or via a cell-
to-cell route (moving from one cell to another, but crossing
the membranes, including the tonoplast, of the cells) (Steu-
dle 2000). Generations of physiologists before the 1980s
had the firm opinion that water flow occurs mainly in the
apoplast apart from the endodermis, where water has to
cross the endodermal barrier due to the suberized cell walls
of endodermis, known as the Casparian strip (Schreiber
1996). Modelling and hydration kinetic experiments, exper-
iments with cell and root pressure probes and NMR studies
showed that water probably moves mainly via the transcel-
lular pathway (= a combination of symplastic and cell-to-
cell pathways) and therefore via membranes. The argu-
ments in favour of a transcellular route were provided by
Newman (1976). In fact, the cross-section of apoplast is
small compared with that of the symplast, and water, having
a fast equilibrium, would enter the cells even under rela-
tively high flow rates in the apoplast. Much experimental
support for the transcellular pathway came from pressure
probe measurements, in which the membrane resistance of
one cell was compared with the resistance of the whole
root. The data demonstrated that the results for a root
agreed well with those, when modelling a root as a series
of membranes (Jones et al. 1988; Steudle & Peterson 1998).
The idea that water molecules not just diffuse through the
lipid bilayer of a membrane, but that proteins facilitate
rapid exchange of water was corroborated 10 years ago by
the cloning of genes encoding water channels or aquaporins
(for reviews see Maurel 1997; Kaldenhoff & Eckert 1999;
Johansson et al. 2000; Maurel & Chrispeels 2001). Because
the water permeability of the lignified secondary walls of
the xylem vessels is low, water very likely enters the vessels
across the pit membranes separating the xylem parenchyma
cells and the vessels. At this point water has to pass the
xylem parenchyma plasma membrane underlying the pit
membranes. Rough calculations show that water flow
across the xylem parenchyma/xylem interface increases
one-hundred-fold compared with the flow into the epider-
mis cells (the vessel radius is about 50 times less than the
root radius, with pit fields occupying about 10–20% of the
vessel’s surface). So, entry into the xylem is clearly a ‘bottle-
neck’ in the plants plumbing system and it is to be expected
that the density of water channels in these membrane
patches is high in order to create a pathway of low resis-
tance to water flow.
As early as 1991 it was demonstrated that a putative
plasma membrane aquaporin gene (TobRB27) is highly
expressed in the central cylinder of roots (Yamamoto et al.
1991). Since the cell-to-cell route of water transport
includes passage through the vacuolar membranes (Steudle
2000) it is not surprising that the Arabidopis aquaporin γ-
TIP was found to be highly expressed in the vascular bun-
dles of roots and leaves (Ludevid et al. 1992). In roots of
the common ice plant (Mesembryanthemum crystallinum)
the plasma membrane aquaporins MIP A and MIP B are
expressed in the youngest portions of the xylem and in
98 A. H. de Boer & V. Volkov
xylem parenchyma cells, respectively (Yamada et al. 1995,
1997). Kirch et al. (2000) corroborated by means of immu-
nolocalization that the MIP-B protein is present in xylem
parenchyma cells of Mesembryanthemum roots. An unex-
pected observation was the presence of MIP-B between
xylem vessels, namely in places not expected to contain
membranes. Moreover, in these areas and between vessels
and xylem parenchyma cells MIP-B was found in small
areas in a patchy fashion. Recently, the tobacco plasma
membrane aquaporin NtAQP1 was found to be expressed
close to the xylem vessels in roots, stems and petioles (Otto
& Kaldenhoff 2000). Electron microscopy analysis of a root
xylem cross-section showed an uneven distribution of the
NtAQP1 protein resembling pitch-like structures (Siefritz
et al. 2001). This could indicate that this aquaporin is con-
centrated at the contact pits between xylem parenchyma
cells and xylem vessels ensuring a polarity in the hydraulic
resistance of cells adjacent to the vessels.
Such a polar distribution of aquaporins could lend sup-
port to the hypothesis put forward by Canny (1997a, b)
describing the filling of embolized vessels by means of
reverse osmosis. Pressurization of cells bordering an embo-
lized vessel could then direct the flow of water along the
pathway of least resistance, namely across the pit mem-
brane into the vessel. Although there are still other prob-
lems with this model, as indicated by Tyree et al. (1999), it
seems worthwhile to study the behaviour of aquaporins in
cells bordering an embolized vessel.
Fine-tuned control over the fluxes of water into, inside
and out of the plant is vital for their struggle to survive in
ever-changing environmental conditions. Intense research
in the field of aquaporins during the last 10 years has shown
that regulation of membrane water permeability by differ-
ential expression of a large number of aquaporin genes in
combination with post-translational regulation are instru-
mental for this fine-tuned control (Maurel 1997; Clarkson
et al. 2000; Maurel & Chrispeels 2001). A very nice example
of such a dynamic regulation is the recently described
change in osmotic water permeability of leaf cells in
response to the magnitude of the transpiration stream
(Morillon & Chrispeels 2001).
ACKNOWLEDGMENTS
We acknowledge support from the Commission of the
European Union via contract FMRX-CT96-0007 (RYP-
LOS) to A.H. de B. V.V acknowledges support by the Foun-
dation for Single Cell Research and by an IAC fellowship.
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Received 11 December 2001; received in revised form 15 May 2002;
accepted for publication 20 May 2002