Logistics of Water and Salt Transport Through the Plant: Structure and Functioning of the Xylem

Home , Symplast, Tracheid, Vessel element

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

1Vrije 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; PD, electric potential difference; SKOR, stelar potassium The xylem is a long-distance transport system that is unique outward rectifying channel; TRP, trans-root potential. to higher plants. It evolved into a very sophisticated plumbing system ensuring controlled loading/unloading of ions and water and their effective translocation to the required INTRODUCTION sinks. The focus of this overview will be the intrinsic inter- Like most multicellular organisms plants need structures relations between structural and functional features of the that enable the mass flow of solvents (mainly water) and xylem. Taken together the xylem is designed to prevent solutes such as ions, sugars, amino acids, hormones, etc. In cavitation (entry of air bubbles), induced by negative pres- analogy to the blood circulation system in animals, plants sures under transpiration and to repair the cavitated ves- use tube-like structures called the xylem and phloem. sels. Half-bordered pits between xylem parenchyma cells Whereas the study of the blood circulation system is tech- and xylem vessels are on the one hand the gates to the nically relatively easy, xylem and phloem transport path- vessels but on the other hand a serious ‘bottle-neck’ for ways are notably difficult to study due to their location deep transport. Hence it becomes evident that special transport inside the plant tissue, the presence of reinforced cell walls systems exist at the interface between the cells and vessels, and the large positive or negative pressures inside the tubes. which allow intensive fluxes of ions and water to and out of Despite, or maybe challenged by these hurdles, a large body the xylem. The molecular identification and biophysical/ of research has been devoted to unravelling the mecha- biochemical characterization of these transporters has just nisms that sustain the functioning of the plant long-distance started. Paradigms for the sophisticated mechanism of transport systems. For recent insights in phloem transport controlled xylem transport under changing environmental we refer to reviews by Patrick et al. (2001) and Oparka & +- conditions are SKOR, a Shaker-like channel involved in K Cruz (2000). loading and SOS1, a Na+/H+ antiporter with a proposed Here we try to link the structure–function relationship of dual function in Na+ transport. In view of the importance the dead xylem conduits and living adjacent parenchyma of plant water relations it is not surprising to find that water cells with a focus on the mechanisms that facilitate and channels dominate the gate of access to xylem. Future stud- control the flow of ions and water into (loading) and out of ies will focus on the mechanism(s) that regulate water chan- the xylem (unloading). Although it was hypothesized in the nels and ion transporters and on their physiological role in, early days of xylem research that, for example, xylem load- for example, the repair of embolism. Clearly, progress in ing in the root was a passive process due to the lack of this specific field of research will greatly benefit from an oxygen in the core of root tissue (Crafts & Broyer 1938), it integration of molecular and biophysical techniques aimed is clear now that we are dealing with sophisticated regula- to understand ‘whole-plant’ behaviour under the ever- tion mechanisms aimed to sustain growth and development changing environmental conditions in the daily life of all in highly dynamic environments. Since structure and func- plants. tion are intimately interwoven, the structure of the xylem transport system will be discussed first. Thereafter, current Key-words: cavitation repair; ion channels; ion transport; knowledge about a number of essential biophysical param- ion transporters; potassium; sodium; water transport; eters namely the electrical, pH and pressure gradients xylem. between the vessels and surrounding symplast/apoplast will be summarized. The molecular mechanisms of potassium, Correspondence: Dr A. H. de Boer. Fax: + 31 20 4447155; sodium, and water transport will be discussed in more detail e-mail: [email protected] at the end.

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 system 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 transpiration) the walls of the vessel or tracheary elements must be reinforced. On the one hand leakage of water and solutes out of the tubes must be minimized (so the walls must a be as leak-proof as possible), but on the other hand specialized structures in the walls must enable rapid exchange of water and solutes between the vessel interior and surrounding cells. Another feature that is vital for survival of the xylem is to prevent and repair the interruption of the continuous water columns by air bubbles (embolism).

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

Structural and functional features of xylem 89 concluded that ‘the phenomenon might be based upon lapsed xylem vessels were isolated as well (Turner & Som- swelling or shrinking of the vessel-to-vessel pit mem- erville 1997). In order to prevent ‘leakage’ of water and branes’. Van Ieperen et al. (2000) concluded that the pres- solutes from the vessels it seems logical that the lignified ence of small amounts of cations (even the effect of 10 µM secondary walls are relatively impermeable to these KCl was significant) and not the osmotic strength influ- compounds. enced the conductance. However, their conclusion that One of the few studies on xylem cell wall permeability is swelling and shrinking of vessel-to-vessel pit membranes the one by Wisniewski, Ashwort & Schaffer (1987) who played no role in this phenomenon was contradicted by studied deep supercooling in woody plants and used lan- Zwieniecki et al. (2001). The latter authors reached the thanum nitrate as an apoplastic tracer. Lanthanum pene- same conclusion as formulated by Zimmermann (1978) trates capillaries as small as 2 nm, so a cell wall without namely that the observed change in hydraulic resistance lanthanum staining is relatively impermeable. They found when changing the ionic composition of the xylem sap is that the primary cell wall of cortical tissues was very per- due to the pit membranes connecting one vessel to the meable to the lanthanum solution, but primary (except other. In these ‘membranes’ water flows through micro- parts associated with the pit membranes) and secondary channels of the modified primary cell wall, made up by walls of vessel elements were impermeable. Interestingly, cellulose microfibrils, hemicelluloses and pectins. It was the pit membranes composed of the middle lamella, pri- suggested that the size of the microchannels increases when mary walls of the adjoining cells and the protective layer the pectin matrix (D-galacturonic acid being a main com- (in xylem parenchyma cells and contact cells) were highly ponent) shrinks in response to the binding of ions to the permeable. These results are in line with the conclusion that negatively charged matrix. If charge compensation is the after occlusion of pit membranes, vessel elements can mechanism that determines the flow resistance, then it is assume a closed-cell structure and may have a permeability surprising that no significant effect of pH within the natural approaching zero (Siau 1984). pH range of xylem sap (5·8–8·0) was observed (Zwieniecki These permeability properties of the xylem walls are rel- et al. 2001), although pH affected swelling and shrinking of evant to the question whether water and solutes can directly isolated cell walls (Meychik & Yermakov 2001). Relevant enter the vessels from the stelar apoplast. If not, then they of course is the question whether these ion-mediated have to be taken up by stelar cells into the symplast and changes in hydraulic resistance do have a physiological role. loaded into the vessel lumen after passing the membrane The K+ concentrations having the strongest effect (0– of a xylem parenchyma cell underlying a pit membrane. For 20 mM; Zwieniecki et al. 2001), are within the range mea- example, the stelar potassium outward rectifying channel sured in transpiring intact plants (Herdel et al. 2001). How- (SKOR; activated at depolarizing voltages) is thought to ever, it remains to be seen whether these K+ variations in have a function in loading K+ into the xylem (Wegner & a constant background of, for example, divalent cations still Raschke 1994; Wegner & de Boer 1997a; Gaymard et al. have an effect on the hydraulic properties. 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 Reinforcement and waterproofing to withstand SKOR promoter region (Gaymard et al. 1998). Note also negative pressures and retain water that pericycle cells of barley roots stained strongly for the Tracheary elements not only have to withstand the com- plasma membrane ATPase (Samuels, Fernando & Glass pressive forces from surrounding cells, but especially the 1992). The pericycle cells do not directly border a xylem large negative pressures that develop in actively transpiring vessel. Therefore, the question is how K+ ions released from plants (see below). Therefore, secondary walls composed of the pericycle cells into the stelar apoplast do reach the cellulose and xylan are deposited on top of the primary cell xylem lumen if the xylem vessel walls are as impermeable wall (Fukuda 1996). Early-formed protoxylem elements of to ions and water as shown by Wisniewski et al. (1987). the primary xylem have ringlike (annular) or spiral (helical) thickenings that still allow a certain degree of cell elonga- Pit structures facilitate water-/solute (un)loading tion. In the late-formed metaxylem elements and elements and embolism repair of the secondary xylem the secondary cell walls cover the entire primary walls, except at pit membranes shared with Pits constitute the gates between vessels and from vessels adjoining tracheids/vessels or xylem parenchyma cells and to adjacent xylem parenchyma cells (Fig. 1). The total area contact cells. Subsequently, the secondary cell walls are of pit fields, their shape and pattern of lignification are of lignified: a network is formed that strengthens and water- large variation between species. For example, maize pit proofs the wall by the polymerization of aromatic com- fields constitute about 15% of the vessel area, with pit pounds called monolignols (McCann 1997). Not membranes having a diameter of about 2 µm with a hydrau- surprisingly, inhibitors of lignification result in a plant phe- lic conductivity about 10 times over that of the vessel wall notype with collapsed vessels (Smart & Amrhein 1985). (Peterson & Steudle 1993). Typically, between vessels bor- Primary cell wall polymers are also important for the sub- dered pits are found that are characterized by overarching sequent lignification and strengthening of a vessel, and walls that form a bowl-shaped chamber (Fig. 1b). It has mutants in cellulose deposition in the cell wall with col- 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 angiosperms and gymnosperms under certain conditions water columns) and subsequent embolism (filling of vessels (Tyree et al. 1986; Sperry et al. 1987; Tyree & Yang 1992; and tracheids with air) (Zimmermann 1983; Tyree & Sperry Cochard, Ewers & Tyree 1994). However, not all plants 1989). This is because the modified primary cell wall that seem to develop root pressure (20 out of 109 species studied constitutes the pit membrane is permeable to water, but had no root pressure, Fisher et al. 1997) and it must be kept does not allow the passage of even the tiniest air bubbles. in mind that root pressure is dissipated by gravity at a rate For this reason tracheids, wherein all elements are linked of 10 kPa per m height. Therefore, tall trees are unlikely to through pits are thought to be safer for plants than vessels develop positive pressures in the shoot as a consequence of although the latter elements are more efficient conductors pressure built-up in the root. So, how do plants dissolve of water. In some monocots, such as rye, in the root–shoot embolism without root pressure? This question is even junction tracheids separate the vessels in the root from more relevant in the light of recent studies reporting embo- those in the shoot to protect the vessels of the roots from lism repair under conditions when the threshold xylem − τ embolism originating in the shoot (Aloni & Griffith 1992). pressure (Px) (see Table 1) is much less than 2 /r (where However, pits are not only important structural elements τ is the surface tension of water in the vessel and r is the that prevent spreading of air bubbles through the water- radius of curvature of the air–water interface) (Salleo et al. conducting elements of plants. They are instrumental in 1996; Tyree et al. 1999). The only possible explanation for repairing the ‘broken water pipes’ as well (Tyree et al. 1999; this repair mechanism seems to be that locally (i.e. in the Holbrook & Zwieniecki 1999; Zwieniecki & Holbrook embolized vessel or tracheid element) the xylem pressure 2000). Repair means removal of the air bubble; how can is higher than −2τ/r. There are a number of intriguing prob- this be achieved? As we have seen above, an air bubble lems with this model: cannot be simply ‘flushed’ out of a conduit. The existing 1 The data suggest that this mechanism of embolism paradigm suggests that an air bubble is removed through removal is concurrent with transpiration (Salleo et al. gas dissolution in the surrounding water (see Table 1). An 1996; Canny 1997b; Tyree et al. 1999). If so, how can important structural demand is that in order to contain this xylem pressure increase in the embolized element while pressurization the perimeter of the embolized vessels must the adjacent vessels are still under tension? be sealed. As we have seen above, the secondary wall meets 2 What is the mechanism that generates locally a xylem this requirement since the thickness and lignification make pressure >−2τ/r? it waterproof. However, what about the pits in which the water permeability of the pit membrane is high (see The first problem was distinguished by Tyree et al. (1999) below)? Let us first consider the hypothesis that a plant has and Holbrook & Zwieniecki (1999) came up with a hypoth- to generate positive (above atmospheric) pressures in the esis as to why in two adjacent vessels (one embolized, the vessels. The best understood mechanism that generates pos- other filled and under tension) connected by pits, a pressure itive pressures is root pressure; namely pressure that is difference can be maintained until the embolized vessels generated in the root during the night or in the shoot in are water filled again (Holbrook & Zwieniecki 1999; Zwie- early spring when transpiration is low through osmotic niecki & Holbrook 2000). Without going into much detail, water flow into the vessels. There is good experimental the idea is that the structure of the bordered pits ensures evidence that this mechanism operates in a range of that the embolized element remains hydraulically isolated from the neighbouring vessels under tension until the moment that all air has been dissolved. Zwieniecki & Hol- Table 1. Threshold xylem pressure brook (2000) showed that the contact angle of water and the angle of the bordered pit chamber walls are such that 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 a convex gas–water interface forms in the pit chamber. The is proportional to the partial pressure of the gas species adjacent surface tension of this convex curvature opposes the hydro- to the water (Henry’s law). Water in plants is saturated with air at static pressure within the filling lumen as long as the pres- atmospheric pressure and therefore the pressure of the gas in the sure in the filling element does not exceed the force due to bubble must be above atmospheric for the gas to dissolve in the the surface tension. Clearly, this model deserves further surrounding water. experimental testing. What determines the pressure of the gas in the bubble (P )? g The second problem seems rather easy to solve. Namely, (1) The pressure inside the xylem element, Px; and (2) the pressure inside the gas bubble that develops due to the xylem conduits are generally in contact with numerous liv- surface tension at the air–water interface, 2τ/r, where r is the ing cells, the xylem parenchyma cells and contact cells radius of the curvature of the air–water interface and τ is the (Zimmermann 1983; Lachaud & Maurousset 1996). These surface tension of the water in the vessel. cells could be triggered by embolism to load osmotically τ Thus the pressure of the gas in the bubble equals: Pg = Px + 2 /r. active ions such as K+ and Cl– across the pit membrane into Therefore when the xylem pressure is greater than −2τ/r (the the vessel lumen. Such a mechanism was proposed for the threshold xylem pressure), the gas in an embolism will exceed first time by Grace (1993) who studied embolism repair in atmospheric pressure and start to dissolve. For a narrow vessel of tracheids from Pinus sylvestris. Water would then be driven 10 µm diameter this threshold pressure is around −30 kPa (−0·3 bar). See Tyree et al. (1999) for further details. by osmosis into the vessel lumen. This idea is consistent with the observation that convex drops protrude through

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 microanalysis gives a reliable estimate of the xylem sap ion concentration, then we need a new paradigm of how positive xylem pressures build-up in embolized vessels and how root pressure 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) spectrometry 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 measurement time drastically (Rokitta et al. 1999; Scheenen et al. 2000; Peuke et al. 2001). If the refilling of embolized vessels is not driven osmotically by salts loaded into the vessel lumen by the surround- Figure 2. Part of an embolized late metaxylem vessel in maize ing xylem parenchyma cells, then maybe reverse osmosis is root. Note the pits between the vessel and bordering xylem par- a feasible mechanism (McCully 1999; Tyree et al. 1999) as chyma cells. Arrows indicate droplets of liquid exuding through originally proposed by Canny (1997a, b). Reverse osmosis pits into the lumen (× 1580) [Reproduced from McCully et al. could occur if cells surrounding the xylem decrease their (1998), with permission of Blackwell Publishing, Oxford, UK]. 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- the pits into the lumen of embolized vessels (see Fig. 2) out ions) into the embolized vessel element. An auxin- from sunflower petioles (Canny 1997b), roots of maize induced loading of solutes in the phloem may be part of (McCully, Huang & Ling 1998) and Laurel nobilis branches this mechanism (Salleo et al. 1996; Tyree et al. 1999). How- (Tyree et al. 1999). However, thus far there is one major ever, Tyree et al. (1999) have pointed out the problems that problem: attempts to measure high concentrations of salts come with this mechanism. One important problem to in the water that enters an embolized vessel have failed solve is how to prevent water from flowing in all directions (Canny 1997b; McCully et al. 1998; McCully 1999; Tyree from the ‘squeezed’ parenchyma cells. In other words, how et al. 1999). All of the methods used to estimate the xylem to generate a direction in the flow of water (a problem sap ion concentration, rely on the technique of X-ray solved in the ‘osmosis’ hypothesis by specific loading of ions microanalysis except one (Tyree et al. 1999). In the latter into the embolized element across the pit membrane). One study ‘native’ xylem sap from well-watered and cavitated possibility is that water channels in the patch of membrane twigs was collected by perfusion. Treatments wherein cavi- underlying the pit membrane are activated, thus creating a tation recovery was high also showed a strong increase (up path of low resistance to waterflow into the vessel element to four-fold) in K+ concentration. This positive correlation (see section on water channels). does suggest that the increase in ion concentration has something to do with the refilling of embolized elements. If Vessel-associated cells are equipped for a task so, then the increase in concentration of solutes in the refill- in (un)loading ing vessels may be much higher since the sap recovered from the perfused stems will be diluted by water from the Living vessel-associated cells, the xylem parenchyma cells full vessels. So, the discussion on the mechanism of refilling 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 Xylem electrical potential and unloading of the xylem. In line with the energy demand of these (un)loading processes, structural features of cells The initial hypothesis postulated by Crafts & Broyer (1938) indicate high metabolic activities that could fuel massive that primary active transport (e.g. an ATP driven H+- transmembrane transport. These features are a dense cyto- ATPase) at the symplast/vessel boundary was precluded plasm, well-developed endoplasmic reticulum, numerous due to lack of oxygen, proved not to be correct (Okamoto, ribosomes, mitochondria and peroxisomes (Läuchli et al. Katou & Ichino 1979; De Boer et al. 1983; Clarkson, Will- 1974). The specialized transport function of these cells is iams & Hanson 1984). In roots of Plantago and hypocotyls evidenced by the development of cell wall invaginations in of Vigna unguiculata the electric potential difference (PD) the area underlying the pit membrane bordering the vessels between vessel and parenchyma cells contains an electro- (Kramer et al. 1977; Yeo et al. 1977; Kuo et al. 1980; Mueller genic component, which in both roots and hypocotyls can & Beckman 1984). This folding pattern increases the mem- amount to 70 mV. The primary H+-ATPase is thought to be brane surface area and is characteristic for transfer cells responsible for this electrogenic component because it dis- (Pate & Gunning 1972; Gunning 1977) and the develop- appears in the presence of inhibitors of the oxidative phos- ment is stimulated by salt stress (Kramer et al. 1977; Yeo phorylation (anoxia and azide) and increases with auxin et al. 1977). Relatively little is known thus far about the and fusicoccin, the activator of the plasma membrane H+- distribution of ion transporters in the membrane of vessel- ATPase (De Boer et al. 1985; De Boer 1997). It must be associated cells. Sauter, Iten & Zimmermann (1973) kept in mind that the electrical potential of the cortical and observed high ATP hydrolysing activity at the large pits of xylem (stelar) apoplast are not necessarily the same. An contact cells facing the vessels and suggested a role for this insulating layer formed by the ring of endodermis cells with enzymatic activity in sugar loading of the xylem. This activ- their Casparian strips, can maintain a PD between the two ity was only observed during starch mobilization in the rays apoplasts. Because the cortical, endodermal and stelar cells cells in spring or when starch mobilization was artifically are connected through plasmodesmata (forming the sym- induced by defoliation. Xylem parenchyma cells of barley plast) the apoplast PD is in fact the difference of two mem- and oat roots were strongly labelled by antibodies against brane potentials in series: (PDsymplast/cortical apoplast) the plasma membrane ATPase, but the resolution was minus (PDsymplast/xylem apoplast) (Okamoto et al. 1979; insufficient to discern differences in subcellular distribution De Boer et al. 1983; De Boer et al. 1985). This PD is also of the protein (i.e. patches on the vessel-facing side) called trans-root potential (TRP), but the same principle (Parets-Soler, Pardo & Serrano 1990; Samuels et al. 1992). holds for stems and other tissues. These results are in line with the localization of ATPase All measurements mentioned above were carried out on activity in barley xylem parenchyma cells as determined excised plants or perfused root and stem segments. This with a cytochemical lead precipitation method (Winter- approach can be criticized because in excised roots the Sluiter, Läuchli & Kramer 1977). turgor and intracellular osmotic pressure gradients collapse The external cytoplasmic layer of xylem parenchyma (Rygol et al. 1993). Therefore, measurements on intact cells has specific features at the point of contact pits (pits plants are necessary. Dunlop (1982) succeeded for the first between xylem parenchyma cells and xylem vessels). There, time to measure directly with a microelectrode in the ves- the network of actin filaments contains fewer cortical sels of intact Trifolium repens plants. The electrical poten- microtubules in comparison with other parts of the cyto- tial of the vessels was 90 mV negative with respect to the plasmic layer where an intensive cortical microtubule cortical apoplast. Because xylem parenchyma cells had a cytoskeleton is present (Chaffey, Barlow & Barnett 2000; membrane potential of −168 mV it can be calculated that Chaffey & Barlow 2001). The function of these structural the potential difference across the symplast/xylem bound- changes of the cortical cell layer may be two-fold: to pre- ary was around 80 mV (xylem positive). These values are vent the deposition of a thick secondary wall, a process close to the values measured in excised Plantago roots which depends on cortical microtubules, and the regulation where the electrogenic component of the symplast/xylem of ion and water channel activity. Actin involvement in ion PD was zero (De Boer et al. 1983). Recently, a new method channel regulation was shown for guard cells (Hwang et al. was introduced, using a modified pressure probe, enabling 1997). 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 THE PROTON MOTIVE FORCE AT THE XYLEM/ measurements was that the roots of intact transpiring plants SYMPLAST INTERFACE: DRIVING FORCE FOR have electrical properties similar to those of excised roots XYLEM (UN)LOADING (Wegner et al. 1999). These measurements also demon- The energy necessary to drive transport of ions, amino strated that the regulation of the trans-root electrical poten- acids, sugars, etc. across the membrane of vessel-associated tial is a dynamic process. Namely, a switch to a higher light cells into or out of the vessel lumen, is derived from the intensity induced strong oscillations in TRP that preceded electrical gradient across this membrane, the pH gradient oscillations in the negative xylem pressure. Interestingly, or both. Therefore, it is important to know the value of the depolarization of TRP correlated with the most nega- these two parameters and the dynamic regulation thereof. tive xylem pressure. Since the membrane potential of the

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101 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 detection (Gansert, Burgdorf & Lösch 2001).

Xylem sap pH The pH of the xylem sap is relevant for (un)loading processes 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 measurements 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- Figure 3. Effect of fusicoccin, activator of the plasma membrane how the xylem has a high capacity to maintain its pH to a H+-ATPase, on the ion concentration and ion fluxes into/out of the set-value (Clarkson et al. 1984; Senden et al. 1992). How- xylem as measured by means of a xylem perfusion technique. (a) ever, in response to changes in environmental conditions Perfusion of taproots from Plantago maritima and (b) of nodal the xylem sap pH does fluctuate. Under drought stress the roots from barley. In both roots, fusicoccin (10 µM) strongly acidi- xylem sap pH from Commelina communis increased from fies the xylem sap, reduces the flow of K+ into the xylem from the 6·1 to 6·7 (Wilkinson & Davies 1997) and iron stress condi- surrounding cells and stimulates the release of Na+ into the xylem tions reduced the pH from sugar beet xylem sap from 6·0 stream. [(a), De Boer unpubl. res.; (b), Volkov and De Boer unpubl. res.]. 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 ulating the closure of stomata (Wilkinson & Davies 1997). cation exchange capacity of the xylem cell walls due to the Moreover, K+ loading into the root xylem may be a pH- presence of negatively charged groups (Ferguson & Bollard sensitive process through modulation of the activity of 1976; Senden et al. 1992) and the presence of organic acids SKOR, the outward rectifying K+ channel involved in K+ such as succinate and malate (Schurr & Schulze 1995). But release into the xylem (Lacombe et al. 2000 and see below). 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 MOLECULAR MECHANISM OF SOLUTE AND example the H+-ATPase activator fusicoccin is perfused WATER (UN)LOADING through xylem vessels the pH of the emerging sap Potassium decreases within minutes after application, reaching values as low as 4·2 (Fig. 3). This effect is explained by the uptake Xylem loading of K+ is regulated separately from K+ uptake of fusicoccin by xylem parenchyma cells around the vessels. from the external solution (Cram & Pitman 1972; Engels & Fusicoccin then activates the plasma membrane H+- Marschner 1992). Studies at the cellular and molecular level ATPase (De Boer 1997), which results in a strong efflux of in recent years have shown two principal types of K+ chan- protons into the xylem sap. Further evidence for the nels in the root (Gaymard et al. 1998; Spalding et al. 1999): involvement of metabolically active cells is the observation AKT1 for K+ uptake from the medium and SKOR for K+ that the pH rapidly increases when roots are subjected to loading into the xylem. The in planta properties of SKOR anoxia (De Boer & Prins 1985; Lacan & Durand 1996). [formerly KORC in barley (Wegner & De Boer 1997a)] Several roles for changes in xylem sap pH have been pro- were first characterized through patch-clamp studies on posed. Thus, the increase in pH initiated by drought stress, xylem parenchyma protoplasts isolated from barley and is thought to increase the efficiency of abscisic acid in stim- maize roots (Wegner & Raschke 1994; Roberts & Tester

1995; Wegner & De Boer 1997a; Roberts 1998). These from K+ uptake from the external solution (Cram & Pitman outward rectifying channels activate in a time-dependent 1972; Pitman & Wellfare 1978; Engels & Marschner 1992) manner at membrane potentials that shift with and remain is underlined by the fact that the K+ channel (activity and + + slightly positive of the reversal potential of K (EK). They expression) responsible for K uptake into the root sym- are selective for K+ but do facilitate the passage of Ca++ as plast, AKT1 is not affected by ABA (Roberts 1998; Gay- well. They cannot be responsible for Na+ loading of the mard et al. 1998). xylem as they are virtually impermeable to Na+. Under Roberts (1998) also observed a short-term effect of ABA physiological conditions a depolarization of the membrane upon addition to the bath medium during a patch-clamp + potential to values positive of EK will result in a K efflux measurement: within 5 min after ABA addition the out- and an influx of Ca++. Therefore, a non-functional SKOR ward K+ current in maize stelar protoplasts declined by may result in a reduction of the amount of K+ and an about 50%. This effect might be mediated by changes in increase in the amount of Ca++ in the shoot (see below). cytosolic pH since a decrease in cytosolic pH from 7·4 to In addition to its sensitivity to membrane voltage and 7·2 induced a strong (80%) voltage-independent decrease apoplastic K+ concentration, the activity of SKOR is regu- of the macroscopic SKOR current in a heterologous expres- lated by cytosolic Ca++, the stress hormone abscisic acid sion system (oocytes) (Lacombe et al. 2000). However, both (ABA) and pH. A rise in the cytosolic Ca++ concentration in dark-grown coleoptiles and in barley aleurone proto- from 150 nM to 5 µM reduced the frequency at which SKOR plasts ABA induced an alkalinization of 0·1–0·2 units was recorded in barley xylem parenchyma cells (whole-cell (Gehring, Irving & Parish 1990; Van der Veen, Heimo- configuration) from 90 to 10% (Wegner & De Boer 1997a). varaa-Dijkstra & Wang 1992). If the same were true for As this effect was absent when inside-out patches were xylem parenchyma cells, then an ABA-induced alkaliniza- used, the involvement of a cytosolic intermediate was sug- tion of the cytoplasm would increase the outward K+ cur- gested. ABA has a dual effect on SKOR activity: a short- rent. It remains necessary to determine the pH sensitivity term effect measurable within minutes after addition of of SKOR in planta as well as the effect of ABA on the pH ABA and a long-term effect (Roberts 1998; Gaymard et al. of xylem parenchyma cells. 1998). The short-term effect was observed when ABA was The high level of expression of SKOR in the pericycle added to protoplasts during a patch-clamp experiment: cells raises some questions. As these cells are not in direct within minutes it reduced the outward current in 55% of contact with the xylem vessels, K+ ions released through the cells; the inward current was unaffected. Drought, or SKOR will end up in the stelar apoplast. If the ion perme- overnight pre-treatment of intact plants with ABA also ability of the secondary walls of the xylem vessels is low resulted in a strong reduction of the outward K+ current. In (see above) then loading of these ions into the xylem might line with earlier experiments (Cram & Pitman 1972; Pitman be preceded by uptake into the xylem parenchyma cells 1977) ABA pre-treatment had no effect on in- and out- followed by release into the vessels. This could explain the ward-currents in cortical root cells (Roberts 1998). It relatively large number of inward rectifying K+ channels remains to be shown if Ca++ plays a role as second messen- (i.e. channels that activate at hyperpolarizing voltages) in ger in the xylem parenchyma ABA signalling pathway, as barley xylem parenchyma cells, two of which are G-protein it does in guard cells (Allen et al. 2000). An indication regulated (Wegner & de Boer 1997b). The explanation for maybe that both Ca++ and ABA affect SKOR in a similar such a complex mechanism of loading is not immediately fashion. obvious, but in some plants sugar loading into the phloem The gene encoding the channel responsible for K+ load- also includes an apoplast unloading step followed by ing in the xylem, called SKOR, belongs to the Shaker super- uptake into the companion cells and symplast loading into family (Gaymard et al. 1998). SKOR expressed in Xenopus the sieve elements (Van Bel et al. 1992). These xylem paren- oocytes, exhibited most if not all of the characteristics of chyma K+ inward rectifying channels may also have a func- xylem parenchyma K+ channel characterized in barley and tion in the apoplast resorption of K+ ions released by the maize: sigmoidal activation, shift in activation potential phloem in the root stelar apoplast. Retranslocation of K+ ++ with EK, sensitivity to blockers, Ca permeability and from the shoot to the root in the phloem is a well-known increased magnitude of the outward current with increasing phenomenon and it has been speculated that the amount external K+ concentration. Northern blot analysis showed of K+ returning from the shoot acts as a signal for root K+ that SKOR is expressed exclusively in roots and there the uptake from the soil (White 1997; Jeschke & Hartung expression is limited to pericycle cells and cells surrounding 2000). the xylem vessels; hence the name stelar K+ outward recti- Xylem perfusion experiments, using tetraethylammo- fier. Treatment of intact plants with the stress hormone nium as blocker of SKOR, showed the in vivo function of abscisic acid resulted in a rapid decline in SKOR mRNA SKOR in loading the xylem with K+ (Wegner & De Boer abundance (Gaymard et al. 1998), thus shedding light on 1997a). The phenotype of the skor-1 knockout mutant ABA inhibition of xylem K+ loading in intact barley plants (Gaymard et al. 1998) indeed showed a reduced transport (Cram & Pitman 1972) and the activity of K+-outward rec- (50%) of K+ to the shoot of skor-1. Electrophysiological tifying channels in maize stelar protoplasts as measured characterization of the selectivity properties of SKOR in with the patch-clamp technique (Roberts 1998). The con- barley yielded a PCa++/PK+ ratio of 0·68 (Wegner & De Boer clusion that xylem loading of K+ is regulated separately 1997a). This outcome suggested a role for SKOR in Ca++

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101 Structural and functional features of xylem 95 unloading of the xylem in roots, which was conﬁrmed by (a) (b) the higher Ca++ content of the skor-1 leaves (Gaymard et al. root shoot 1998). 700 0 mM NaCl 1 mM NaCl + On the way up through the stem into the leaves K ions 25 mM NaCl 600 will be taken up into the symplast by cells in contact with ) the vessels and tracheids. The uptake of K+ in the elonga- 500 d.m. 1 – tion zone of the hypocotyl of Vigna unguiculata is stimu- 400 lated by the growth-promoting hormone auxin and fusicoccin (De Boer et al. 1985). The actual mechanism (ion 300 (me.kg

+ + + channel or H /K -symporter) of uptake is not known. Keu- 200 necke et al. (1997) used a patch-clamp approach to unravel Na the molecular mechanism of xylem unloading. In proto- 100 plasts isolated from xylem contact cells in leaves of Vicia 0 12 12 faba and maize two different types of K+ channels were P.media P.maritima P.media P.maritima identified, but a role in K+ unloading could not be assigned. The small veins of leaves are often surrounded by a layer Figure 5. Different strategies of Plantago media (glycophyte) + of compactly arranged cells, the bundle sheath. These cells and P. maritima (halophyte) towards the accumulation of Na ions isolate the veins from the intercellular spaces (the apoplast) in the root and shoot. The glycophyte has a reduced translocation of Na+ from the root to the shoot, whereas the halophyte prefer- of the leaf. Keunecke et al. (2001) showed that uptake of entially accumulates Na+ in the leaves. Plants were grown on sand K+ from the vessels of small veins in maize leaves occurs at supplied with nutrient solution with Na+ concentrations as indi- the xylem/bundle sheath cell interface. cated [from De Boer (1985), where further details can be An intriguing question is whether the flow of K+ (and obtained]. 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 transport step in nutrient distribution and the C/N economy example, rapidly growing buds/leaves that still have a rela- of the plant (Van Bel 1990; White 1997; Jeschke & Hartung tively low transpiration rate? Gunning, Pate & Green 2000). On the other hand, it seems contra-productive to (1970) showed that xylem transfer cells in stems of many transport large amounts of, for example, K+ to rapidly tran- plants were almost exclusively associated with departing spiring mature leaves which are not in need of K+. It will foliar traces (Fig. 4). They estimated that in these regions be interesting to study whether the flow of K+ into the the membrane surface area of cells surrounding the vessels petiole of these leaves is restricted by active resorption may be amplified by as much as 20-fold. This, suggests a from the transpiration stream entering that leaf. potential for very intensive lateral exchange of solutes. Fur- thermore, xylem–phloem exchange is a very important 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 halophytes the accumulation of Na+ is at the expense of K+ (Fig. 5b). Therefore it is to be expected that different trans- + XP port mechanisms for Na are present at the symplast/xylem boundary in the root where xylem loading takes place. Cer- V tain glycophytes (such as bean, maize and reed) have devel- V oped a xylem Na+ unloading mechanism in the basal part of the root and shoot where Na+ ions are taken up from the transpiration stream (Jacoby 1965; Johanson & Cheeseman XP 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. Figure 4. Conspicuous wall ingrowths of transfer cells lining the Sodium entry into the root symplast is probably medi- xylem of the cotelydonary traces of a seedling of Jacob’s ladder (Polemonium caeruleum) (× 600). V, xylem vessel; XP, xylem ated by weakly voltage-dependent non-selective cation parenchyma cell. [Reproduced from Gunning et al. (1970), with channels (Roberts & Tester 1995; Tyerman & Skerrett 1999; permission of Springer-Verlag Austria]. Davenport & Tester 2000; Demidchik, Davenport & Tester

2002; Demidchik & Tester 2002). The properties of these fungi (Shi et al. 2000; see also Hasegawa, Bressan & Pardo channels resemble those of cyclic nucleotide-gated chan- 2000). In the same article it was reported that SOS1 gene nels (CNGC) (Leng et al. 1999). Evidence for a role of expression is concentrated in the cells surrounding the CNGCs in Na+ uptake is two-fold: (i) the cngc1 mutant xylem, suggesting that SOS1 may function in loading Na+ shows enhanced Na+ tolerance when grown on moderate into the xylem for long-distance transport. Recently, the external Na+ (Talke et al. 2001); and (ii) membrane-perme- suggestion that SOS1 encodes a Na+/H+ antiporter was able cAMP/cGMP analogues reduced unidirectional Na+ experimentally substantiated using purified plasma mem- influx and overall Na+ accumulation in NaCl grown Ara- brane vesicles from salt-stressed Arabidopsis thaliana biodopsis (Maathuis & Sanders 2001). leaves (Qiu et al. 2001). Salt-treated sos1 mutant plants In the plasma membrane of barley xylem parenchyma showed a 60% lower Na+/H+ exchange activity in compari- cells a non-selective ion channel, called NORC, has been son with wild-type. SOS1 expression is up-regulated by characterized but in contrast to the CNGCs it is non-selec- NaCl stress and this up-regulation is abolished in plants with tive for both cations and anions and it is voltage dependent a mutation in the SOS3 gene encoding a phosphatase with (Wegner & Raschke 1994; Wegner & De Boer 1997a). significant sequence similarity with the calcineurin B sub- Although NORC is equally permeable to K+ and Na+, it unit from yeast (Liu & Zhu 1998). In the presence of Ca++ remains to be shown whether, under physiological condi- SOS3 interacts with SOS2 (a protein kinase) and activates tions, NORC has a function in loading of Na+ into the the kinase activity of SOS2 (Halfter, Ishitani & Zhu 2000). transpiration stream. NORC also passes glutamate (Weg- Apparently, SOS2 directly modulates SOS1 since in vitro ner & de Boer 1997a). In this respect it is noteworthy that addition of SOS2 to plasma membrane vesicles isolated a glutamate receptor gene, AtGluR2, is expressed in cells from NaCl stressed plants resulted in a two-fold increase in adjacent to the conducting vessels in Arabidopsis leaves the Na+/H+ exchange activity (Qiu et al. 2001). (Kim et al. 2001). AtGluR2 is homologous to mammalian During SOS1-facilitated loading of Na+ into the xylem ionotropic glutamate receptors that are non-selective cat- (Shi et al. 2002), one proton flowing from the xylem to the ion channels permeable to Ca++, K+ and Na+, but not Mg++ symplast (a ‘down-hill’ process energetically speaking) (Lam et al. 1998). The primary physiological function of drives the transport of one Na+ ion into the xylem vessels. these channels is thought to be Ca++ influx into cells (Den- Na+ loading into the xylem is unlike K+ loading probably nison & Spalding 2000). Over-expression of AtGluR2 not a channel-mediated process. Much remains to be resulted in Ca++-deficiency symptoms and according to Kim learned from a comparison of K+ and Na+ exchange across et al. (2001) this might be due to increased Ca++ unloading the symplast/xylem boundary in experiments in which the from the transpiration stream in the root and stem causing xylem of root or shoot segments is perfused with defined Ca++ deficiency in the young, growing parts of the plant. The solutions. As discussed above, the high K+ concentration in transgenic plants also exhibited hypersensitivity to Na+ and the xylem parenchyma cells (Drew, Webb & Saker 1990) K+ ionic stress, which might be due to reduced Ca++ uptake allows K+ loading down the electrochemical K+ gradient in the presence of excess Na+ or K+ in combination with facilitated by the K+-outward channel SKOR (Wegner & de increased Ca++ unloading on the way up to the top of the Boer 1997a; Gaymard et al. 1998). In line with the proper- plant. ties of SKOR (depolarization activated and inactivated by Na+ loading into the xylem of barley and maize roots is external acidification; Lacombe et al. 2000), xylem perfu- not mediated by the outward rectifying K+ channel of xylem sion of the H+-ATPase activator fusicoccin stops the K+ parenchyma cells since this channel is virtually imperme- loading process in roots of the halophyte Plantago maritima able to Na+ (Roberts & Tester 1995; Wegner & De Boer (Fig. 3a). Interestingly, the response of Na+ is very different 1997a). Heterologous expression of SKOR in Xenopus from that of K+: in the first 45 min after the start of fusic- oocytes corroborates this conclusion (Gaymard et al. 1998). occin perfusion the Na+ concentration in the xylem A prominent candidate gene encoding a Na+ transporter decreases (i.e. Na+ unloading of the xylem sap is stimu- involved in Na+ transport across the symplast/xylem bound- lated). This increase in uptake is unlikely to be caused by a ary is SOS1 (Ding & Zhu 1997; Shi et al. 2000, 2002). SOS1 pH change of the xylem sap (because this is still small), but was identified as a NaCl-hypersensitive Arabidopsis mutant rather due to the rapid hyperpolarization of the membrane and initially described as a mutant defective in high-affinity potential induced by fusicoccin (De Boer & Prins 1985; De potassium uptake (Wu, Ding & Zhu 1996). The 22Na-uptake Boer et al. 1985). This uptake may be a channel-mediated and efflux measurements indicated that the sos1 mutants process. However, when the xylem sap starts to acidify were impaired both in high-affinity K+-and low-affinity Na+- there is a turning point resulting in Na+ loading into the uptake (Ding & Zhu 1997). However, in these experiments xylem when fusicoccin has acidified the xylem sap pH with the whole seedling was exposed to the uptake/efflux solu- more than 1 unit (Fig. 3a). The large pH gradient between tion and this makes interpretation of the results in terms of xylem sap and the cytoplasm of adjacent parenchyma cells the SOS1 function in uptake and translocation in planta may now facilitate a Na+/H+ antiport system similar to hazardous. Subsequent sequencing of the locus showed that SOS1 (Shi et al. 2002). Similar fusicoccin-induced responses sos1 encodes a 127 kDa protein with 12 transmembrane of xylem sap pH and K+/Na+ (un)loading were observed in spanning domains and with significant sequence similarity xylem perfusion experiments using nodal roots of barley to plasma membrane Na+/H+ antiporters from bacteria and (Fig. 3b).

Shi et al. (2002) proposed a model where SOS1 plays a because water molecules have no charge, whereas ions do. dual role in Na+ loading and reabsorption from the xylem Water can move via the apoplast (within cell walls), sym- sap, depending on salinity and pH difference between symplast (moving via cells without leaving them) or via a cell- plast and xylem, in analogy to a model proposed by Lacan to-cell route (moving from one cell to another, but crossing & Durand (1996). The latter authors observed an increase the membranes, including the tonoplast, of the cells) (Steu- in Na+ unloading and an increase in K+ loading of the xylem dle 2000). Generations of physiologists before the 1980s sap when the initial pH of the perfusion solution was more had the firm opinion that water flow occurs mainly in the acidic. They conclude that Na+ is taken up from the xylem apoplast apart from the endodermis, where water has to through a Na+/H+ antiport mechanism, but now with H+ cross the endodermal barrier due to the suberized cell walls driven ‘uphill’ by a ‘down-hill’ Na+ gradient. However, a of endodermis, known as the Casparian strip (Schreiber mechanistic interpretation of their results is difficult since 1996). Modelling and hydration kinetic experiments, exper- it is not clear how the xylem pH changes in view of the iments with cell and root pressure probes and NMR studies strong pH-stat properties of the xylem (Clarkson et al. showed that water probably moves mainly via the transcel- 1984). Moreover, it is also not clear how the different initial lular pathway (= a combination of symplastic and cell-to- pH values of the perfusion solution affect the membrane cell pathways) and therefore via membranes. The argu- potential. The observed positive correlation between K+ ments in favour of a transcellular route were provided by loading and acidity of perfusion solution as reported by Newman (1976). In fact, the cross-section of apoplast is Lacan & Durand (1996) is not in line with a SKOR- small compared with that of the symplast, and water, having mediated loading mechanism since SKOR activity is very a fast equilibrium, would enter the cells even under rela- low at acidic external pH (Lacombe et al. 2000). It must be tively high flow rates in the apoplast. Much experimental kept in mind that perfusion of an acidic buffer will result in support for the transcellular pathway came from pressure an exchange of protons with other cations such as K+ at the probe measurements, in which the membrane resistance of negatively charged cell walls of the xylem and that this may one cell was compared with the resistance of the whole cause an apparent increase in K+ loading (Senden et al. root. The data demonstrated that the results for a root 1992). The reabsorption of Na+ from the upgoing transpira- agreed well with those, when modelling a root as a series tion stream in the basal parts of the root and shoot has been of membranes (Jones et al. 1988; Steudle & Peterson 1998). well documented both in intact plants and in perfused seg- The idea that water molecules not just diffuse through the ments (Jacoby 1965; Johanson & Cheeseman 1983; Lacan lipid bilayer of a membrane, but that proteins facilitate & Durand 1996; Ehwald 2001). However, the storage rapid exchange of water was corroborated 10 years ago by capacity of the stelar cells is limited and therefore a retrans- the cloning of genes encoding water channels or aquaporins location of Na+ by the phloem to the root and extrusion (for reviews see Maurel 1997; Kaldenhoff & Eckert 1999; into the medium has been suggested as a mechanism to Johansson et al. 2000; Maurel & Chrispeels 2001). Because increase the detoxification capacity. Although Van Bel et al. the water permeability of the lignified secondary walls of 1992) has already pointed to the importance of xylem– the xylem vessels is low, water very likely enters the vessels phloem exchange processes nothing is yet known about the across the pit membranes separating the xylem parenchyma mechanism of Na+ loading into the phloem and unloading cells and the vessels. At this point water has to pass the in the root. xylem parenchyma plasma membrane underlying the pit membranes. Rough calculations show that water flow across the xylem parenchyma/xylem interface increases Water one-hundred-fold compared with the flow into the epider- On its route to the root xylem, water flow is restricted by a mis cells (the vessel radius is about 50 times less than the number of morphological structures such as the epidermis, root radius, with pit fields occupying about 10–20% of the outer cortex layer (exodermis), several layers of cortical vessel’s surface). So, entry into the xylem is clearly a ‘bottle- cells, endodermis, pericycle, parenchyma cells of central neck’ in the plants plumbing system and it is to be expected cylinder, and cell walls of xylem vessels (Steudle & Peter- that the density of water channels in these membrane son 1998). Under conditions of high transpiration, water patches is high in order to create a pathway of low resis- flow is passively dragged across the roots at a high rate, tance to water flow. whereas under high humidity and absence of transpiration As early as 1991 it was demonstrated that a putative the ascending water flow to the shoot is generated by root plasma membrane aquaporin gene (TobRB27) is highly pressure with low flow rates. In the permanent fluctuating expressed in the central cylinder of roots (Yamamoto et al. river of water, the aim of a plant is to control ion fluxes to 1991). Since the cell-to-cell route of water transport the xylem and to keep most of them relatively independent includes passage through the vacuolar membranes (Steudle of water flow. In fact, transpiration may not be required for 2000) it is not surprising that the Arabidopis aquaporin γ- the supply of minerals to the shoot (Tanner & Beevers TIP was found to be highly expressed in the vascular bun- 2001). The latter authors provided evidence that xylem flow dles of roots and leaves (Ludevid et al. 1992). In roots of brought about by root pressure and Münch’s phloem coun- the common ice plant (Mesembryanthemum crystallinum) terflow, might be sufficient for long-distance mineral supply. the plasma membrane aquaporins MIP A and MIP B are The plant can discriminate well between water and ions expressed in the youngest portions of the xylem and in

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 87–101 98 A. H. de Boer & V. Volkov xylem parenchyma cells, respectively (Yamada et al. 1995, of Scholander’s assumption. American Journal of Botany 84, 1997). Kirch et al. (2000) corroborated by means of immu- 1217–1222. nolocalization that the MIP-B protein is present in xylem Canny M.J. (1997b) Vessel contents during transpiration- embo- lisms and refilling. American Journal of Botany 84, 1223–1230. parenchyma cells of Mesembryanthemum roots. An unex- Chaffey N.J. & Barlow P.W. (2001) The cytoskeleton facilitates a pected observation was the presence of MIP-B between three-dimensional symplasmic continuum in the long-lived ray xylem vessels, namely in places not expected to contain and axial parenchyma cells of angiosperm trees. Planta 213, 811– membranes. Moreover, in these areas and between vessels 823. and xylem parenchyma cells MIP-B was found in small Chaffey N.J., Barlow P.W. & Barnett J.R. (2000) A cytoskeletal areas in a patchy fashion. Recently, the tobacco plasma basis for wood formation in angiosperm trees: the involvement membrane aquaporin NtAQP1 was found to be expressed of microfilaments. Planta 210, 890–896. Clarkson D.T., Carvajal M., Henzler T., Waterhouse R.N., Smyth close to the xylem vessels in roots, stems and petioles (Otto A., Cooke D.T. & Steudle E. (2000) Root hydraulic conduc- & Kaldenhoff 2000). Electron microscopy analysis of a root tance: diurnal aquaporin expression and the effects of nutrient xylem cross-section showed an uneven distribution of the stress. Journal of Experimental Botany. 51, 61–70. NtAQP1 protein resembling pitch-like structures (Siefritz Clarkson D.T., Williams L. & Hanson J.B. (1984) Perfusion of et al. 2001). This could indicate that this aquaporin is con- onion root xylem vessels: a method and some evidence of concentrated at the contact pits between xylem parenchyma trol of the pH of the xylem sap. Planta 162, 361–369. Cochard H., Ewers F.W. & Tyree M.T. (1994) Water relations of cells and xylem vessels ensuring a polarity in the hydraulic a tropical vine-like bamboo (Rhipidocladum racemiflorum): resistance of cells adjacent to the vessels. root pressures, vulnerability to cavitation and seasonal changes Such a polar distribution of aquaporins could lend sup- in embolism. Journal of Experimental Botany 45, 1085–1089. port to the hypothesis put forward by Canny (1997a, b) Crafts A.S. & Broyer T.C. (1938) Migration of salts and water into describing the filling of embolized vessels by means of xylem of the roots of higher plants. American Journal of Botany reverse osmosis. Pressurization of cells bordering an embo- 25, 529–535. lized vessel could then direct the flow of water along the Cram W.J. & Pitman M.G. (1972) The action of abscisic acid on ion uptake and water flow in plant roots. Australian Journal of pathway of least resistance, namely across the pit mem- Biological Sciences 25, 1125–1132. brane into the vessel. Although there are still other prob- Davenport R.J. & Tester M. (2000) A weakly voltage-dependent, lems with this model, as indicated by Tyree et al. (1999), it non-selective cation channel mediates toxic sodium influx in seems worthwhile to study the behaviour of aquaporins in wheat. Plant Physiology 122, 823–834. cells bordering an embolized vessel. De Boer A.H. (1985) Xylem/symplast ion exchange: mechanism Fine-tuned control over the fluxes of water into, inside and function in salt-tolerance and growth. PhD Thesis, Univer- and out of the plant is vital for their struggle to survive in sity of Groningen, The Netherlands. De Boer A.H. (1997) Fusicoccin- a key to multiple 14–3−3 locks? ever-changing environmental conditions. Intense research Trends in Plant Science 2, 60–66. in the field of aquaporins during the last 10 years has shown De Boer A.H. & Prins H.B.A. (1984) Trans-root potential in roots that regulation of membrane water permeability by differ- of Plantago media L. as affected by hydrostatic pressure: the ential expression of a large number of aquaporin genes in induction of an O2 deficient root core. Plant and Cell Physiology combination with post-translational regulation are instru- 25, 643–655. mental for this fine-tuned control (Maurel 1997; Clarkson De Boer A.H. & Prins H.B.A. (1985) Xylem perfusion of tap root et al. 2000; Maurel & Chrispeels 2001). A very nice example segments of Plantago maritima: the physiological significance of electrogenic xylem pumps. Plant, Cell and Environment 8, 587– of such a dynamic regulation is the recently described 594. change in osmotic water permeability of leaf cells in De Boer A.H., Katou K., Mizuno A., Kojima H. & Okamoto H. response to the magnitude of the transpiration stream (1985) The role of electrogenic xylem pumps in K+ absorption (Morillon & Chrispeels 2001). from the xylem of Vigna unguiculata: the effects of auxin and fusicoccin. Plant, Cell and Environment 8, 579–586. De Boer A.H., Prins H.B.A. & Zanstra P.E. (1983) Biphasic com- ACKNOWLEDGMENTS position of trans-root potential of Plantago species: involvement of spatially separated electrogenic pumps. Planta 157, 259–266. We acknowledge support from the Commission of the Demidchik V. & Tester M. (2002) Sodium fluxes through nonse- European Union via contract FMRX-CT96-0007 (RYP- lective cation channels in the plasma membrane of protoplasts from Arabidopsis roots. Plant Physiology 128, 379–387. LOS) to A.H. de B. V.V acknowledges support by the Foun- Demidchik V., Davenport R.J. & Tester M. (2002) Nonselective dation for Single Cell Research and by an IAC fellowship. cation channels in plants. Annual Review of Plant Physiology and Plant Molecular Biology 53, 67–107. Dennison K.L. & Spalding E.P. (2000) Glutamate-gated calcium REFERENCES fluxes in Arabidopsis. Plant Physiology 124, 1511–1514. Ding L. & Zhu J.-K. (1997) Reduced Na+ uptake in the NaCl- Allen G.J., Chu S.P., Schumacher K., et al. (2000) Alteration of hypersensitive sos1 mutant of Arabidopsis thaliana. Plant Phys- stimulus-specific guard cell calcium oscillations and stomatal iology 113, 795–799. closing in Arabidopsis det3 mutant. Science 289, 2338–2342. Drew M.C., Webb J. & Saker L.R. (1990) Regulation of K+ uptake Aloni R. & Griffith M. (1992) Functional xylem anatomy in root- and transport to the xylem in barley roots: K+ distribution deter- shoot junctions of 6 cereal species. Planta 184, 123–129. mined by electron probe X-ray microanalysis of frozen-hydrated Canny M.J. (1997a) Vessel contents of leaves after excision – a test cells. Journal of Experimental Botany 41, 815–825.

Dunlop J. (1982) Membrane potentials in the xylem in roots of Jacoby B. (1965) Sodium retention in excised bean stems. Physio- intact plants. Journal of Experimental Botany 33, 910–918. logia Plantarum 18, 730–739. Ehwald R. (2001) Salt and water stress of common reed growing Jeschke W.D. & Hartung W. (2000) Root–shoot interactions in in wind-protected flat brackish locations. In International Sym- mineral nutrition. Plant and Soil 226, 57–69. posium ‘Plants Under Environmental Stress’, Moscow, October, Johanson J.G. & Cheeseman J.M. (1983) Uptake and distribution 23–28, pp. 66–67. Timiriazev Institute, Moscow, Russia. of sodium and potassium by corn seedlings. Role of the meso- Engels C. & Marschner H. (1992) Adaptation of potassium trans- cotyl in sodium exclusion. Plant Physiology 73, 153–158. location into the shoot of maize (Zea mays) to shoot demand: Johansson I., Karlsson M., Johanson U., Larsson C. & Kjellbom P. evidence for xylem loading as a regulating step. Physiologia (2000) The role of aquaporins in cellular and whole plant water Plantarum 86, 263–268. balance. Biochimica Biophysica Acta 1465, 324–342. Enns L.C., Canny M.J. & McCully M.E. (2000) An investigation Jones H., Leigh R.A., Wyn Jones R.G. & Tomos A.D. (1988) The of the role of solutes in the xylem sap and in the xylem paren- integration of whole-root and cellular hydraulic conductivities chyma as the source of root pressure. Protoplasma 211, 183–197. in cereal roots. Planta 174, 1–7. Ferguson I.B. & Bollard E.G. (1976) The movement of calcium in Kaldenhoff R. & Eckert M. (1999) Features and function of plant woody stems. Annals of Botany 40, 1057–1065. aquaporins. Journal of Photochemistry and Photobiology B 52, Fisher J.B., Angeles G., Ewers F.W. & Lopez-Portillo J. (1997) 1–6. Survey of root pressure in tropical vines and woody species. Keunecke M., Lindner B., Seydel U., Schulz A. & Hansen U.P. International Journal of Plant Science 158, 44–50. (2001) Bundle sheath cells of small veins in maize leaves are the Fukuda H. (1996) Xylogenesis: initiation, progression, and cell loction of uptake from the xylem. Journal of Experimental Bot- death. Annual Review of Plant Physiology and Plant Molecular any 52, 709–714. Biology 47, 299–325. Keunecke M., Sutter J.U., Sattelmacher B. & Hansen U.P. (1997) Gansert D., Burgdorf M. & Lösch R. (2001) A novel approach to Isolation and patch clamp measurements of xylem contact cells the in situ measurement of oxygen concentrations in the sap- for the study of their role in the exchange between apoplast and wood of woody plants. Plant, Cell and Environment 24, 1055– symplast of leaves. Plant and Soil 196, 239–244. 1064. Kim S.A., Kwak J.M., Jae S.-K., Wang M.-H. & Nam H.G. (2001) Gaymard F., Pilot G., Lacombe B., Bouchez D., Bruneau D., Overexpression of the AtGluR2 gene encoding an Arabidopsis Boucherez J., Michaux-Ferrière N., Thibaud J.-B. & Sentenac homolog of mammalian glutamate receptors impairs calcium H. (1998) Identification and disruption of a plant Shaker-like utilization and sensitivity to ionic stress in transgenic plants. outward channel involved in K+ release into the xylem sap. Cell Plant and Cell Physiology 42, 74–84. 94, 647–655. Kirch H.-H., Vera-Estrella R., Golldack D., Quigley F., Micha- Gehring C.A., Irving H.R. & Parish R.W. (1990) Effects of auxin lowski C.B., Barkla B.J. & Bohnert H.J. (2000) Expression of and abscisic acid on cytosolic calcium and pH in plant cells. water channel proteins in Mesembryanthemum crystallinum. Proceedings of the National Academy of Sciences USA 87, 9645– Plant Physiology 123, 111–124. 9649. Kramer D., Läuchli A., Yeo A.R. & Gullasch J. (1977) Transfer Grace J. (1993) Refilling of embolized xylem. In Water Transport cells in roots of Phaseolus coccineus: ultrastructure and possible in Plants Under Climatic Stress (eds M. Borghetti, J. Grace, & function in exclusion of sodium from the shoot. Annals of Bot- A. Raschi), pp. 52–62. Cambridge University Press, Cambridge, any 41, 1031–1040. UK. Kuo J., Pate J.S., Rainbird R.M. & Atkins C.A. (1980) Internodes Gunning B.E.S. (1977) Transfer cells and their roles in transport of grain legumes – new location for xylem parenchyma transfer of solutes in plants. Scientific Progress, Oxford 64, 539–568. cells. Protoplasma 104, 181–185. Gunning B.E.S., Pate J.S. & Green L.W. (1970) Transfer cells in Lacan D. & Durand M. (1996) Na+-K+ exchange at the xylem/ the vascular system of stems: taxonomy, association with nodes, symplast boundary. Plant Physiology 110, 705–711. and structure. Protoplasma 71, 147–171. Lachaud S. & Maurousset L. (1996) Occurrence of plasmodesmata Halfter U., Ishitani M. & Zhu J.-K. (2000) The Arabidopsis SOS2 between differentiating vessels and other cells in Sorbus tormi- protein kinase physically interacts with and is activated by the nalis L. Crantz and their fate during xylem maturation. Proto- calcium-binding protein SOS3. Proceedings of the National plasma 191, 220–226. Academy of Sciences USA 97, 3735–3740. Lacombe B., Pilot G., Gaymard F., Sentenac H. & Thibaud J.-B. Hasegawa M., Bressan R. & Pardo J.M. (2000) The dawn of plant (2000) pH control of the plant outward-rectifying potassium salt tolerance genetics. Trends in Plant Science 8, 317–319. channel SKOR. FEBS Letters 466, 351–354. Herdel K., Schmidt P., Feil R., Mohr A. & Schurr U. (2001) Lam H.-M., Chiu J., Hieh M.-H., Meisel L., Oliveira I.C., Shin M. Dynamic of concentrations and nutrient fluxes in the xylem of & Coruzzi G. (1998) Glutamate-receptor genes in plants. Nature Ricinus communis – diurnal course, impact of nutrient availabil- 396, 125–126. ity and nutrient uptake. Plant, Cell and Environment 24, 41–52. Läuchli A., Kramer D., Pitman M.G. & Lüttge U. (1974) Ultra- Holbrook N.M. & Zwieniecki M.A. (1999) Embolism repair and structure of xylem parenchyma cells of barley roots in relation xylem tension: do we need a miracle? Plant Physiology 120, 7– to ion transport to the xylem. Planta 119, 85–99. 10. Leng Q., Mercier R.W., Yao W. & Berkowitz G.A. (1999) Cloning Holbrook N.M., Ahrens E.T., Burns M.J. & Zwieniecki M.A. and functional characterization of a plant nucleotide-gated cat- (2001) In vivo observation of cavitation and embolism repair ion channel. Plant Physiology 121, 753–761. using magnetic resonance imaging. Plant Physiology 126, 27–31. Liu J. & Zhu J.-K. (1998) A calcium sensor homolog required for Huber B. & Merz W. (1958) Über die Bedeutung des Hoftüpfelv- plant salt tolerance. Science 280, 1943–1945. erschlusses für die axiale Wasserleitfähigkeit von Nadelhölzern. López-Millán A.F., Morales F., Abadía A. & Abadía J. (2000) Planta 51, 645–672. Effects of iron deficiency on the composition of the leaf apoplas- Hwang J.-U., Suh S., Kim J. & Lee Y. (1997) Actin filaments tic fluid and xylem sap in sugar beet. Implications for iron and modulate both stomatal opening and inward K+-channel carbon transport. Plant Physiology 124, 873–884. activities in guard cells of Vicia faba L. Plant Physiology 115, Ludevid D., Höfte H., Himmelblau E. & Chrispeels M.J. (1992) 335–342. The expression pattern of the tonoplast intrinsic γ-TIP in Ara-

bidopsis thaliana is correlated with cell enlargement. Plant Phys- Qiu Q.-S., Guo Y., Dietrich M.A., Schumaker K.S. & Zhu J.-K. iology 100, 1633–1639. (2001) Characterization of the plasma membrane Na+/H+ Maathuis F.J.M. & Sanders D. (2001) Sodium uptake in Arabidop- exchanger in Arabidopsis thaliana. In The 12th International sis roots is regulated by cyclic nucleotides. Plant Physiology 127, Workshop Plant Membrane Biology, Madison, p. 235. University 1617–1625. of Wisconsin, Madison, WI, USA. Maurel C. (1997) Aquaporins and water permeability of plant Roberts S.K. (1998) Regulation of K+ channels in maize roots by membranes. Annual Review of Plant Physiology and Plant water stress and abscisic acid. Plant Physiology 116, 145–153. Molecular Biology 48, 399–429. Roberts K. & McCann M.C. (2000) Xylogenesis: the birth of a Maurel C. & Chrispeels M.J. (2001) Aquaporins. A molecular corpse. Current Opinion in Plant Biology 3, 517–522. entry into plant water relations. Plant Physiology 125, 135– Roberts S.K. & Tester M. (1995) Inward and outward K+-selective 138. currents in the plasma membrane of protoplasts from maize root McCann M.C. (1997) Tracheary element formation: building up to cortex and stele. Plant Journal 8, 811–825. a dead end. Trends in Plant Science 2, 333–338. Rokitta M., Peuke A.D., Zimmermann U. & Haase A. (1999) McCully M.E. (1999) Root xylem embolisms and refilling. Rela- Dynamic studies of phloem and xylem flow in fully differenti- tion to water potentials of soil, roots, and leaves, and osmotic ated plants by fast nuclear-magnetic-resonance microimaging. potentials of root xylem sap. Plant Physiology 119, 1001–1008. Protoplasma 209, 126–131. McCully M.E., Huang C.X. & Ling L.E.C. (1998) Daily embolism Rygol J., Pritchard J., Zhu J.J. & Zimmermann U. (1993) Transpi- and refilling of xylem vessels in the roots of field-grown maize. ration induces radial turgor pressure gradients in wheat and New Phytologists 138, 327–342. maize roots. Plant Physiology 103, 493–500. Meychik N.R. & Yermakov I.P. (2001) Swelling of root cell walls Salleo S., Lo Gullo M.A., De Paoli D. & Zippo M. (1996) Xylem as an indicator of their functional state. Biochemistry (Moscow) recovery from cavitation-induced embolism in young plants of 66, 178–187. Laurus nobilis: a possible mechanism. New Phytologist 132, 47– Morillon R. & Chrispeels M.J. (2001) The role of ABA and the 56. transpiration stream in the regulation of the osmotic water per- Samuels A.L., Fernando M. & Glass A.D.M. (1992) Immunofluo- meability of leaf cells. Proc. Natl. Acad. Sci. USA 98, 14138– rescent localization of plasma membrane H+-ATPase in barley 14143. roots and effects of K nutrition. Plant Physiology 99, 1509– Mueller W.C. & Beckman C.H. (1984) Ultrastructure of the cell 1514. wall of vessel contact cells in the xylem of tomato stems. Annals Sauter J.J., Iten W. & Zimmermann M.H. (1973) Studies on the of Botany 53, 107–114. release of sugar into the vessels of sugar maple (Acer saccha- Newman E.I. (1976) Water movement through root systems. rum). Canadian Journal of Botany 51, 1–8. Philosophical Transactions of Royal Society London. Series B. Scheenen T.W.J., Van Dusschoten D., De Jager P.A. & Van As H. 273, 463–478. (2000) Quantification of water transport in plants with NMR Okamoto H., Katou K. & Ichino K. (1979) Distribution of electri- imaging. Journal of Experimental Botany 51, 1751–1759. cal potential and ion transport in the hypocotyl of Vigna ses- Schreiber L. (1996) Chemical composition of Casparian strips iso- quipedalis. VI. The dual structure of radial electrogenic activity. lated from Clivia miniata Reg. roots: evidence for lignin. Planta Plant and Cell Physiology 20, 103–114. 199, 596–601. Oparka K.J. & Cruz S.S. (2000) The great escape: phloem trans- Schurr U. (1998) Xylem sap sampling – new approaches to an old port and unloading of macromolecules. Annual Review of Plant topic. Trends in Plant Science 3, 293–298. Physiology and Plant Molecular Biology 51, 323–347. Schurr U. & Schulze E.-D. (1995) The concentration of xylem sap Otto B. & Kaldenhoff R. (2000) Cell-specific expression of the constituents in root exudate, and in sap from intact, transpiring mercury-insensitive plasma-membrane aquaporin NtAQP1 castor bean plants (Ricinus communis L.). Plant, Cell and Envi- from Nicotiana tabacum. Planta 211, 167–172. ronment 18, 409–420. Parets-Soler A., Pardo J.M. & Serrano R. (1990) Immunolocaliza- Senden M.H.M.N., Van Paassen F.J.M., van der Meer A.J.G.M. & tion of plasma membrane H+-ATPase. Plant Physiology 93, Wolterbeek H.Th. (1992) Cadmium – citric acids – xylem cell 1654–1658. wall interactions in tomato plants. Plant, Cell and Environment Pate J.S. & Gunning B.E.S. (1972) Transfer cells. Annual Review 15, 71–79. of Plant Physiology 23, 173–196. Shi H., Ishitani M., Kim C. & Zhu J.-K. (2000) The Arabidopsis Patrick J.W., Zhang W.H., Tyerman S.D., Offler C.E. & Walker thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ N.A. (2001) Role of membrane transport in phloem transloca- antiporter. Proceedings of the National Academy of Sciences tion of assimilates and water. Australian Journal of Plant Phys- USA 97, 6896–6901. iology 13, 695–707. Shi H., Quintero F.J., Pardo J.M. & Zhu J.-K. (2002) The putative Peterson C.A. & Steudle E. (1993) Lateral hydraulic conductivity plasma membrane Na+/H+ antiporter SOS1 controls long-dis- of early metaxylem vessels in Zea mays L. roots. Planta 189, 288– tance Na+ transport in plants. Plant Cell. 14, 465–477. 297. Siau J.F. (1984) Transport Processes in Wood. Springer, Berlin. Peuke A.D., Rokitta M., Zimmermann U., Schreiber L. & Haase Siefritz F., Biela A., Eckert M., Otto B., Uhlein N. & Kaldenhoff A. (2001) Simultaneous measurement of water flow velocity R. (2001) The tobacco plasma membrane aquaporin NtAQP1. and solute transport in xylem and phloem of adult plants of Journal of Experimental Botany 52, 1953–1957. Ricinus communis over a daily time course by nuclear magnetic Smart C.C. & Amrhein N. (1985) The influence of lignification on resonance spectrometry. Plant, Cell and Evironment 24, 491– the development of vascular tissue in Vigna radiata L. Proto- 503. plasma 124, 87–95. Pitman M.G. (1977) Ion transport into the xylem. Annual Review Spalding E.P., Hirsch R.E., Lewis D.R., Qi Z., Sussman M.R. & of Plant Physiology 28, 71–88. Lewis B.D. (1999) Potassium uptake supporting plant growth in Pitman M.G. & Wellfare D. (1978) Inhibition of ion transport in the absence of AKT1 channel activity. Inhibition by ammonium excised barley roots by abscisic acid: relation to water perme- and stimulation by sodium. Journal of General Physiology 113, ability of the roots. Journal of Experimental Botany 29, 1125– 909–918. 1138. Sperry J.S., Holbrook N.M., Zimmermann M.H. & Tyree M.T.

(1987) Spring filling of xylem vessels in wild grapevine. Plant parenchyma of barley roots. Plant Physiology 105, 799– Physiology 83, 414–417. 813. St. Aubin G., Canny M.J. & McCully M.E. (1986) Living vessel Wegner L.H. & Zimmermann U. (1998) Simultaneous recording elements in the late metaxylem of sheathed maize roots. Annals of xylem pressure and trans-root potential in roots of intact of Botany 58, 577–588. glycophytes using a novel xylem pressure probe technique. Steudle E. (2000) Water uptake by plant roots: an integration of Plant, Cell and Environment 2, 849–865. views. Plant and Soil 226, 45–56. Wegner L.H., Sattelmacher B., Läuchli A. & Zimmermann U. Steudle E. & Peterson C.A. (1998) How does water get through (1999) Trans-root potential, xylem pressure, and root cortical roots? Journal of Experimental Botany 49, 775–788. membrane potential of ‘low-salt’ maize plants as influenced by Talke I.N., Bouché N., Bouchez D., Fromm H., Sanders D. & nitrate and ammonium. Plant, Cell and Environment 22, 1549– Maathuis F.J.M. (2001) Characterization of CNGC1, a putative 1558. cyclic nucleotide-gated channel from Arabidopsis thaliana. In White P.J. (1997) The regulation of K+ influx into roots of rye The 12th International Workshop Plant Membrane Biology, (Secale cereale L.) seedlings by negative feedback via the K+ flux Madison, p. 69. University of Wisconsin, Madison, WI, USA. from the shoot to root in the phloem. Journal of Experimental Tanner W. & Beevers H. (2001) Transpiration, a prerequisite for Botany 48, 2063–2073. long-distance transport of minerals in plants? Proceedings of the Wilkinson S. & Davies W.J. (1997) Xylem sap pH increase: a National Academy of Sciences USA 98, 9443–9447. drought signal received at the apoplastic face of the guard cell Turner S.R. & Somerville C.R. (1997) Collapsed xylem phenotype that involves the suppression of saturable abscisic acid uptake of Arabidopsis identifies mutants deficient in cellulose deposi- by the epidermal symplast. Plant Physiology 113, 559–573. tion in the secondary cell wall. Plant Cell 9, 689–701. Winter-Sluiter E., Läuchli A. & Kramer D. (1977) Cytochemical Tyerman S.D. & Skerrett I.M. (1999) Root ion channels and salin- localization of K+-stimulated adenosine-triphosphate activity in ity. Scientia Horticulturae 78, 175–235. xylem parenchyma of barley roots. Plant Physiology 60, 923– Tyree M.T. & Sperry J.S. (1989) Vulnerability of xylem to cavita- 927. tion and embolism. Annual Review of Plant Physiology and Wisniewski M., Ashworth E. & Schaffer K. (1987) The use of Plant Molecular Biology 40, 19–38. lanthanum to characterize cell wall permeability in relation to Tyree M.T. & Yang S. (1992) Hydraulic conductivity recovery depp supercooling and extracellular freezing in woody plants. I. versus water pressure in xylem of Acer saccharum. Plant Phys- Intergeneric comparisons between Prunus, Cornus and Salix. iology 100, 669–676. Protoplasma 139, 105–116. Tyree M.T., Fiscus S.D., Wullschleger M.A. & Dixon M.A. (1986) Wu S.-J., Ding L. & Zhu J.-K. (1996) SOS1, a genetic locus essen- Detection of xylem cavitation in corn under field conditions. tial for salt tolerance and potassium acquisition. Plant Cell 8, Plant Physiology 82, 597–599. 617–627. Tyree M.T., Salleo S., Nardini A., Lo Gullo M.A. & Mosca R. Yamada S., Katsuhara M., Kelly W., Michalowski C. & Bohnert (1999) Refilling of embolized vessels in young stems of Laurel. H. (1995) A family of transcripts encoding the water channel Do we need a new paradigm? Plant Physiology 120, 11–21. proteins: tissue specific expression in the common ice plant. Van Bel A.J.E. (1990) Xylem-phloem exchange via the rays: the Plant Cell 7, 1129–1142. undervalued route of transport. Journal of Experimental Botany Yamada S., Nelson D.E., Ley E., Marquez S. & Bohnert H.J. 41, 631–644. (1997) The expression of an aquaporin promoter from Mesem- Van Bel A.J.E., Gamalei Y.V., Ammerlaan A. & Bik L.P.M. bryanthemum crystallinum in tobacco. Plant and Cell Physiology (1992) Dissimilar phloem loading in leaves with symplastic or 38, 1326–1332. apoplastic minor-vein configurations. Planta 186, 518–525. Yamamoto Y.T., Taylor C.G., Acedo G.N., Cheng C.L. & Con- Van der Veen R., Heimovaraa-Dijkstra S. & Wang M. (1992) Cyto- kling M.A. (1991) Characterization of cis-acting sequences reg- solic alkalinization mediated by abscisic acid is necessary, but ulating root-specific gene expression in tobacco. Plant Cell 3, not sufficient, for abscisic acid-induced gene expression in barley 371–382. aleurone protoplasts. Plant Physiology 100, 699–705. Yeo A.R., Kramer D., Läuchli. A. & Gullasch J. (1977) Ion distri- Van Ieperen W., Van Meeteren U. & van Gelder H. (2000) Fluid bution in salt-stressed mature Zea mays roots in relation to composition influences hydraulic conductance of xylem con- ultrastructure and retention of sodium. Journal of Experimental duits. Journal of Experimental Botany 51, 769–776. Botany 28, 17–29. Watson R., Pritchard J. & Malone M. (2001) Direct measurement Zimmermann M.H. (1978) Hydraulic architecture of some diffuse- of sodium and potassium in the transpiration stream of salt- porous trees. Canadian Journal of Botany 56, 2286–2295. excluding and non-excluding varieties of wheat. Journal of Zimmermann M.H. (1983) Xylem Structure and the Ascent of Sap. Experimental Botany 52, 1873–1881. Berlin: Springer-Verlag. Wegner L.H. & De Boer A.H. (1997a) Properties of two outward- Zwieniecki M.A. & Holbrook N.M. (2000) Bordered pit structure rectifying channels in root xylem parenchyma cells suggest a role and vessel wall surface properties. Implications for embolism in K+ homeostasis and long-distance signaling. Plant Physiology repair. Plant Physiology 123, 1015–1020. 115, 1707–1719. Zwieniecki M.A., Melcher P.J. & Holbrook N.M. (2001) Hydrogel Wegner L.H. & De Boer A.H. (1997b) Two inward K+ channels in control of xylem hydraulic resistance in plants. Science 291, the xylem parenchyma cells of barley roots are regulated by G- 1059–1062. protein modulators through a membrane-delimited pathway. Planta 203, 506–516. Received 11 December 2001; received in revised form 15 May 2002; Wegner L.H. & Raschke K. (1994) Ion channels in the xylem accepted for publication 20 May 2002