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Journal of Experimental Botany, Vol. 52, Roots Special Issue, pp. 445±457, March 2001 Partitioning of nutrient transport processes in roots Mark Tester1 and Roger A. Leigh Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK Received 23 June 2000; Accepted 1 September 2000 Abstract Roots have a range of cell types that each contribute to the acquisition of nutrients and their subsequent transfer to the xylem. The activities of these cells must be coordinated to ensure that delivery of nutrients to the shoot occurs at a rate that matches the demands of growth. The partitioning of transport processes between different cell types is thus essential for roots to function effectively. This partitioning is considered at the level of proteins, organelles and cells in relation to the accepted concepts of how nutrients are taken up by roots and delivered to the xylem. Using K+ as an example, the evidence underpinning current concepts is examined, gaps in understanding identified and the contribution of some new approaches assessed. Key words: Root physiology, nutrient uptake, stress tolerance, cell-specific expression, transgenic plants. Introduction The ability of roots to obtain nutrients from the soil and to deliver these to the aerial tissues at a rate that matches the needs of growth is key to ensuring that the shoot has the resources it needs to function effectively. When roots fail to do this, plants show a range of visual symptoms indicative of nutrient de®ciencies (e.g. smaller stature, discolouration). However, even before such obvious symptoms appear, mismatches between the demands of the shoot and the supply from the roots can affect productivity. In agricultural systems this results in smaller yields, while in natural ecosystems it may affect the ability of plants to compete effectively. In general, it is understood how roots take up and transfer nutrients to the xylem for onward transport to 1 the shoot. They form highly branched structures that provide a large surface are for nutrient and water absorption and which ramify through the soil pro®le to tap new sources of nutrients. Further, this structure is dynamic; it can respond to local conditions, for example, becoming highly branched in regions of high nutrient availability (Drew and Saker, 1975), at least for some nutrients in some species (Robinson, 1994). The activity of roots also affects, in a number of ways, the availability of nutrients at the soil surface. Thus the rate at which nutrients and water are taken up is important in generating the driving forces for movement of nutrients through the soil either by mass ¯ow of soil solution or by diffusion (Tinker and Nye, 2000) while secreted chemicals can modify the root environment to increase nutrient solubility or promote the formation of bene®cial associations with micro-organisms such as rhizobia or mycorrhizal fungi. Once ions reach the root surface, they are transferred across the root to the xylem by apoplastic (extracellular) anduor symplastic (intracellular) pathways (Clarkson, 1993). The apoplastic route can be followed as far as the endodermis where the presence of a poorly ionpermeable secondary thickening in the cell wall (the Casparian Strip) restricts further movement. Thus any nutrient which reaches this point via the apoplastic pathway must, perforce, cross the plasma membrane of the endodermal cells to reach the stele. However, entry into the symplastic pathway is not restricted to the endodermis. It can also occur in root hairs, other epidermal cells, and in the cortex. Once in the stele, ions are secreted into the xylem for transfer to the shoot. Although this general picture is widely accepted, the molecular processes that mediate and control uptake and trans-root movement of nutrients are not well understood, nor are the activities of individual cells in these processes completely described. A key to understanding root function is to describe how processes that contribute To whom correspondence should be addressed. Fax: q44 1223 333953. E-mail: [email protected] ß Society for Experimental Biology 2001 446 Tester and Leigh to the uptake of nutrients and their transfer to the xylem are partitioned at different levels. This partitioning is considered in an hierarchical way here by examining it at the level of proteins within membranes, compartments within cells, and cell types within roots. For each level of organization present concepts and understanding are outlined, what evidence supports them is considered, what gaps in knowledge exist, and how new approaches could help to provide new insights and understanding. Particular emphasis has been placed on Kq, but the general concepts are applicable to other ions. Partitioning of transport between proteins within membranes: K+ uptake as a paradigm Roots are able to absorb nutrients over a wide range of external ion concentrations and, provided the rate of delivery to the root surface is high enough, plants can grow at maximal rates in the presence of very low external nutrient concentrations. This is exempli®ed by the experiments of Asher and Ozanne who grew a number of plants in rapidly ¯owing nutrient solutions (delivered at 1000 l pot 1 d 1) containing a range of Kq concentrations (Asher and Ozanne, 1967). Their results showed that individuals of all of the species they used could survive in 1 mM Kq, a few grew maximally in 8 mM Kq, while the majority of the remainder needed only 24 mM Kq for optimal growth. Further, very large changes in the external concentration were not matched by proportionately large changes in shoot or root Kq concentrations (Table 1). A 1000-fold increase in external Kq concentration (from 1 to 1000 mM) resulted in only a 6- to 14-fold change in the total Kq concentration in the roots and shoots. Additionally, the root and shoot Kq concentrations were comparable in all of the species, implying that Kq uptake and transfer to the shoot are regulated in a broadly similar way, at least in terms of the outcome (i.e. the ®nal root and shoot Kq concentrations) that is achieved. These observations indicate that Kq uptake by roots can operate at very low concentrations and that the rate of uptake is matched to growth of the shoot and the root to regulate tissue Kq concentrations. Net Kq uptake at the root plasma membrane is classically viewed as resulting from the operation of active and passive transporters, which have different af®nities for Kq and which make different contributions to uptake as the external concentration changes. Evidence for the operation of different Kq uptake systems came from the work of Epstein and co-workers (Epstein et al., 1963; Welch and Epstein, 1968) who suggested that at low external Kq concentrations (below approximately 1 mM), uptake of Kq is mediated by a high-af®nity saturable mechanism (so-called System 1) that is highly selective for Kq. At higher external Kq concentrations, a second, less selective system operates (System 2). De®ning the precise nature of the transport systems that are responsible for the observed patterns of uptake requires knowledge of the way electrochemical potential gradients for Kq respond to the changes in external Kq. Detailed studies in Arabidopsis roots (reviewed by Maathuis and Sanders, 1996) showed that growth and Kq uptake were possible at an external Kq concentration of 10 mM and that, at the prevailing membrane potential difference (PD) of 129 to 153 mV, and cytosolic Kq activity (80 mM), Kq uptake into root epidermal cells at this external Kq concentration has to be active (Maathuis and Sanders, 1993). Patch clamp studies showed that this uptake is probably mediated by a Kq: Hq symporter (Maathuis and Sanders, 1994). Uptake within the concentration range of System 2 is probably passive and mediated by inward Kq-selective channels. Patch clamp studies have indicated that such channels are present in epidermal and cortical cells from Table 1. Comparison of shoot and root Kq concentrations in plants growing in solutions containing 1 or 1000 mM Kq Values in brackets are the fold increase of Kq concentration in the shoots and roots of plants grown in 1000 mM Kq compared with those grown in 1 mM Kq. Data are from Asher and Ozanne (Asher and Ozanne, 1967), with shoot Kq concentration recalculated on the basis of tissue water from the results in their Tables 3 and 4. Species Trifolium subterraneum Trifolium hirtum Ornithopus sativus Vicia sativa Erodium botrys Lolium rigidum Vulpia myuros Hordeum vulgare Bromus rigidus Avena sativa Shoot Kq concentration (mM) Root Kq concentration (mM) wKxo 1 mM wKxo 1000 mM wKxo 1 mM wKxo 1000 mM 23 29 25 26 23 23 38 15 35 30 267 229 213 160 177 238 240 215 274 204 20 19 18 13 11 15 17 10 14 13 150 130 106 92 89 131 110 135 142 114 (11.6) (7.9) (8.5) (6.2) (7.7) (10.3) (6.3) (14.3) (7.8) (6.8) (7.5) (6.8) (5.9) (7.1) (8.1) (8.7) (6.5) (13.5) (10.1) (8.8) Partitioning of transport in roots a range of species (Schachtman et al., 1991; Maathuis and Sanders, 1995; Roberts and Tester, 1995; Gassmann and Schroeder, 1994). In Arabidopsis root cells there are at least two such channels with conductances of 5 pS and 20 pS, respectively, when measured with 10 mM Kq on each side of the membrane. The properties of the 5 pS channel are consistent with it being the dominant pathway for Kq uptake at mM external concentrations of Kq and its activity increases following Kq starvation (Maathuis and Sanders, 1995). Similar results have been observed in wheat roots where there was a 6- to 10-fold increase in a channel-mediated Kq transport following growth in Kq-free media (Buschmann et al., 2000), suggesting an important role for Kq channels in Kq nutrition. Other Kq-permeable channels have also been described in roots, for example, non-selective cation channels (Amtmann and Sanders, 1999; Davenport and Tester, 2000) and cyclic-nucleotide-gated channels (KoÈhler et al., 1999; Leng et al., 1999), but their signi®cance in overall Kq uptake into the root is unknown. The identi®cation of the proteins responsible for active and passive uptake of Kq has progressed rapidly in the last decade mainly through the use of heterologous expression systems both to clone genes and to characterize their products. A number of plant Kq transporters have been identi®ed by functional complementation of the Kq transport-de®cient trk1 trk2 mutant of yeast (Saccharomyces cerevisiae). This mutant is unable to grow at low external Kq concentrations and plant cDNAs encoding Kq transporters have been identi®ed by their ability to restore growth of the mutant on low Kq. Further characterization has been performed using a variety of heterologous expression systems such as yeast itself, Xenopus oocytes, and insect cells. Transporters that have been identi®ed include inwardly- and outwardlyrectifying Kq-selective channels, and high af®nity active transporters (Rodriguez-Navarro, 2000; Schachtman, 2000). Among those that have been shown to be expressed in roots are AKT1 (an inward rectifying channel; Sentenac et al., 1992; Lagarde et al., 1996), SKOR (an outward rectifying channel; Gaymard et al., 1998), HKT1 (initially identi®ed as a Hq-linked Kq symport, but now thought to function as a Naq: Kq or Naq: Naq symport; Schachtman and Schroeder, 1994; Rubio et al., 1995; Gassmann et al., 1996), and members of the KUPuHAK family (dual af®nity transporters with similarity to fungal Hq: Kq symporters; Kim et al., 1998; Fu and Luan, 1998; Santa-Maria et al., 1997; Rubio et al., 2000). AKT1 is a member of the Shaker superfamily of voltage-gated Kq-selective ion channels that in animal cells mediate outward Kq transport (Jan and Jan, 1997). AKT1 shares many of the structural features of the animal channels, including six membrane spanning 447 domains (S1±S6), a preponderance of positive charges in S4 that probably represents the voltage-sensing domain, and a hydrophilic domain (P-domain) between S4 and S5 that forms part of the ion-conducting pore and selectivity ®lter and contains a conserved GYGD motif (Sentenac et al., 1992). Despite this high level of structural similarity, electrophysiological studies of AKT1 expressed in insect cells indicate that it functions as an inward rather than an outward rectifying Kq channel (Gaymard et al., 1996). Localization using the GUS reporter gene indicate that AKT1 is expressed mainly in root epidermal and cortical cells and thus could catalyse passive Kq uptake (Lagarde et al., 1996). This was con®rmed using an Arabidopsis AKT1 knock-out mutant (akt1-1). Patch clamp analysis of root cells from this mutant showed that they lack inward Kq currents and are compromised in Kq uptake (Hirsch et al., 1998; Spalding et al., 1999). SKOR is structurally similar to AKT1, but has been shown to mediate outward rather than inward Kq transport and to be expressed speci®cally in stelar cells. It is thus thought to be involved in the passive transport of Kq out of the stelar cells into the xylem. Knockout mutants lacking SKOR have diminished Kq transport to the shoot and this defect can be complemented by reintroduction of the SKOR gene (Gaymard et al., 1998). HKT1 was cloned from wheat and, in roots, is preferentially expressed in cortical cells. Its gene product was initially characterized as a Hq : Kq symport that can mediate high af®nity active transport of Kq when expressed in Xenopus oocytes or yeast (Schachtman and Schroeder, 1994). However, subsequent work showed that it probably functions as a Naq: Kq symport at low external Naq concentrations and as a Naq: Naq symport at higher external Naq concentrations (Gassmann et al., 1996; Rubio et al., 1995). Thus its precise role in Kq transport remains somewhat equivocal. It has been shown that Naq-dependent Kq uptake does not occur in roots of terrestrial plants (Maathuis et al., 1996) and that an Arabidopsis HKT1 orthologue encodes a Naq transporter that is unaffected by Kq (Uozumi et al., 2000). Thus the prevailing evidence tends to support the view that HKT1 may be more important in Naq uptake. Nonetheless, its involvement in high af®nity Kq transport is supported by the observation that its expression is rapidly up-regulated in response to removal of external Kq and with a time-course that matches changes in Kq uptake (Wang et al., 1998). In addition, Spalding et al., using the Arabidopsis akt1-1 knockout mutant, found that high af®nity non-AKT1-dependent Kq uptake in the roots of these plants was sensitive to the external concentration of both Hq and Naq which might indicate a role for HKT1 in this process (Spalding et al., 1999). Sensitivity of HKT1 to NH4q is consistent with this suggestion, but it has not been de®nitely proven (Santa-Maria et al., 2000). 448 Tester and Leigh Potassium transporters of the KUPuHAK subtypes have been isolated from a variety of plants and their structural and sequence similarities indicate that they form a family of related transporters (Rubio et al., 2000). They have 12 membrane-spanning domains and a long cytosolic C-terminal domain (Kim et al., 1998; Fu and Luan, 1998; Santa-Maria et al., 1997; Rubio et al., 2000). Up to 13 related genes are present in Arabidopsis and members of the family have been detected in a number of other plants (Rubio et al., 2000). The proteins are predicted to be about 30% identical in amino acid sequence to both the KUP Kq transport system from E. coli and the HAK1 system from Schwanniomyces occidentalis. The fungal HAK1 gene products encode high af®nity Hq: Kq symporters (Haro et al., 1999) and the plant orthologues mediate high-af®nity Kq transport when expressed in yeast (Kim et al., 1998; Fu and Luan, 1998; Santa-Maria et al., 1997; Rubio et al., 2000). However, it is unclear whether the transport catalysed by the plant members of this family is active or passive as attempts to characterize the gene products by expression in Xenopus oocytes (which are more amenable to electrophysiology than yeast) have failed (Kim et al., 1998; Rubio et al., 2000). Evidence that this transport may be channel-like is suggested by the ability of KUP1 to catalyse high rates of Kq transport at mM concentrations, the sensitivity of the transport to tetraethylammonium (a blocker of Kq channels) and by the presence of an IYGD motif with homology to the GYGD that is characteristic of Kq channels (Fu and Luan, 1998). KUPuHAK transporters may also be involved in the NH4q-inhibited, high af®nity Kq transport because the activity of these transporters is sensitive to NH4q (SantaMaria et al., 1997, 2000), although they lack the Naq sensitivity that the NH4q-inhibited component was reported to have (Spalding et al., 1999). How Kq uptake is partitioned between different transporters remains unclear. The isolation of genes for both Kq channels and putative high af®nity active transporters appears to con®rm that the elements needed to explain Systems 1 and 2 are present. However, Sanders showed, using reaction kinetic modelling, that a single transporter can give rise to multiphasic uptake kinetics and so multiple transporters are not a prerequisite for such kinetics (Sanders, 1990). Similarly, energy-barrier models can also explain predict multiphasic kinetics (White and Ridout, 1995). A simple explanation that assigns transport by HKT1, HAK and KUP transporters to System 1 and that by AKT1 or other ion channels to System 2 is complicated by observations that suggest that all of these transporters may be capable of mediating transport at both mM and mM external Kq concentrations. For instance, using an AKT1 knockout mutant (akt1-1), evidence was found to suggest that this channel accounts for a signi®cant proportion of total Kq uptake by Arabidopsis roots at external Kq concentrations as low as 10 mM (Hirsch et al., 1998; Spalding et al., 1999). This need not indicate that AKT1 mediates an active transport process since passive in¯ux can occur at such low external concentrations if the PD is suf®ciently negative. With a cytosolic Kq activity of about 80 mM (Maathuis and Sanders, 1993; Walker et al., 1996), passive inward transport of Kq could be achieved from solutions containing 8 mM Kq if the PD was about 236 mV or more negative. Plant cells do not normally have such negative PDs (Maathuis and Sanders, 1993), but values have been reported of around 225 mV in Arabidopsis roots and so, under these conditions, AKT1 could mediate passive inward Kq transport from mM external Kq (Hirsch et al., 1998). Similarly, while transporters of the KUPuHAK family are capable of high af®nity Kq transport (Santa-Maria et al., 1997; Kim et al., 1998; Fu and Luan, 1998; Rubio et al., 2000), KUP1 has been reported to mediate high rates of Kq transport from solutions containing up to 150 mM Kq when expressed in yeast (Fu and Luan, 1998). However, the reliability of the yeast trk1 trk2 mutant for making Kq uptake measurements at high external Kq concentrations has been questioned because the mutant has a high intrinsic capacity for transport at these elevated concentrations (Rubio et al., 2000). These authors suggest that, under these conditions, assigning differences in Kq uptake to a heterologously expressed protein may be dif®cult. Therefore, the role of the HAKuKUP transporters in passive Kq uptake remains to be established. The importance of the PD in controlling directions of ion movement must also be emphasized. The activity of ion channels in catalysing inward and outward movements of Kq, and therefore the direction of rapid passive movements of Kq, are controlled by PD, a parameter which is, in turn, the output of a complex interaction of ®xed charges and membrane transport processes (Nobel, 1999). The role of PD in Kq transport has been examined for guard cells (Thiel et al., 1992), but there has been very little such work in roots (Wegner and de Boer, 1997). PD was measured across the plasma membrane of the cortical and stelar cells of maize roots and it was found that ABA caused the stelar cell PD to become more negative relative to the xylem apoplast (Roberts and Snowman, 2000). Thus, the driving force favouring movement of Kq out of the stelar cells is less with ABA (assuming there were no effects on the transmembrane Kq concentration gradient). These results are consistent with a role for PD in inhibiting the loading of solutes into the stelar apoplast, for subsequent transfer to the xylem and then shoot. This is in addition to the inhibition by ABA of the conductance of the plasma membrane to Kq (Roberts, 1998) and Cl (Gilliham and Tester, 2000). There are undoubtedly more Kq transporters to be discovered in plants and some of these will mediate Partitioning of transport in roots q K uptake processes in roots. The sequencing of the Arabidopsis genome raises the possibility of identifying all Kq transporters, at least where they share sequence homologies with those from other organisms. However, to characterize their physiological roles it will be necessary to functionally characterize each gene product in heterologous systems to gain knowledge of their properties, and then to con®rm their role in Kq acquisition by identifying which transporters are expressed in roots, which cells and membranes they are located in, what conditions they are expressed in, and what the consequences are of knocking out their expression using antisense, insertional mutagenesis, gene silencing or homologous recombination. 449 in the vacuole may re¯ect available supply rather than export across the tonoplast from a previously replete store. Therefore, experiments are needed to determine what happens to vacuolar Kq when replete cells are placed in de®cient conditions. For nitrate this does lead to a loss of nitrate from the vacuole (van der Leij et al., 1998), but this has not yet been tested for Kq. It is possible that limitations on Kq mobilization from the vacuole may be imposed by the availability of other cations to replace it because of the need to maintain vacuolar sap osmotic pressure (Leigh and Wyn Jones, 1984). If other cations are not available, Kq mobilization may be limited unless other solutes such as sugars can be transferred to the vacuole. However, when Kq is replaced by uncharged solutes, there must also be mobilization of Partitioning of nutrient ions between the cytosol and vacuole Compartmentation of ions in the vacuole has received much attention because of the importance of this organelle both as a permanent repository for toxic ions or as a temporary storage site for those nutrients taken up in excess of immediate needs (e.g. nitrate and phosphate). In relation to uptake and transfer of nutrients to the xylem, vacuoles are a side-branch off the main transport pathway. Nonetheless, the tonoplast has a potentially important role in regulating ion concentrations in the cytosol and hence in the symplastic pathway. Measurements with ion-selective microelectrodes indicate that the concentrations of both Kq (Walker et al., 1996) and NO3 (Miller and Smith, 1996) are regulated in the cytosol of root cells while concentrations in the vacuole vary with external supply, demonstrating the ¯exibility of the vacuole as a storage organelle (Walker et al., 1996; Zhen et al., 1991; van der Leij et al., 1998). In the case of Kq, vacuolar Kq activity (aK) in root cells is about 100 mM in Kq-replete cells, but falls to 10 mM or less in starved cells (Fig. 1; Walker et al., 1996). In contrast, the aK in the cytosol is relatively constant at about 80 mM over a wide range of Kq supplies, indicating the tight regulation that is exerted on this parameter (Leigh and Wyn Jones, 1984). However, the degree of control differs in different cell types so that at low tissue Kq concentrations, there is a larger decline in cytosolic aK in epidermal cells than in cortical cells (Fig. 1; Walker et al., 1996). How cytosolic aK is regulated remains unclear. It is uncertain whether it primarily involves plasma membrane or vacuolar transport systems or both. The maintenance of a relatively constant cytosolic aK while that in the vacuole declines might indicate that Kq is transferred out of the vacuole to maintain the level in the cytosol. However, most experiments are done by growing roots at different external Kq concentrations where the level Fig. 1. The effect of Kq starvation on Kq activity (aK) in the vacuole (open symbols) and cytosol (closed symbols) of (A) epidermal and (B) cortical cells of barley roots. Note that, in both cell types, aK in the vacuole decrease more than that in the cytosol, and that at low tissue Kq concentrations there is a larger decline in cytosolic aK in the epidermal cells than in the cortex. Measurements were made with Kq-selective microelectrodes (from Walker et al., 1996, where further details can be obtained). 450 Tester and Leigh a counteranion along with Kq to maintain electrical neutrality. One aspect of the accumulation of Kq in vacuoles that has received little attention is the observation that the maximum concentration to which it accumulates is different in roots and leaves. In many plants under K-replete conditions, the Kq concentration in the shoots is approximately twice that in the roots (data in Table 1 for plants grown in 1000 mM Kq). Measurements with Kq-selective microelectrodes indicate that cytosolic aK is similar in cells of roots (Walker et al., 1996) and leaves (T Cuin, SA Laurie, AJ Miller, RA Leigh, unpublished results) so the differences in the vacuole must result from differences in the regulation of Kq transport at the tonoplast. Unfortunately, investigations into the nature of this regulation must await identi®cation of the mechanisms responsible for Kq transport into the vacuole, which remain elusive (compare Davies et al., 1992; Ros et al., 1996). As the symplastic pathway of trans-root transport to the xylem involves the linked cytosols of adjacent cells, the concentrations of ions in this compartment presumably exert some in¯uence on the rate of transfer. However, although Kq and N are needed in the shoot in roughly equal proportions, the concentrations of nitrate and Kq in the cytosol are very different. Thus, whereas aK is about 80 mM (Fig. 1), nitrate activity is only about 4 mM (Zhen et al., 1991; Miller and Smith, 1996). Thus, assuming the two ions move at equal rates through the symplast (a process that will be primarily determined by cytoplasmic streaming and transpirationally-driven bulk ¯ow, not diffusional processes) and that the Kq ef¯ux mechanism controlling release to the xylem responds linearly to increasing Kq (which is possible, given the relatively low af®nity of some plant cation channels, e.g. White and Ridout, 1995), approximately 20-fold more Kq than nitrate would reach the xylem, at least in plants performing most of their nitrate reduction in the shoot. Clearly this level of imbalance does not occur so mechanisms must exist to modify the relative rates of loading of these ions into the xylem from the symplast. Partitioning between cell types within roots Root structure is complex, with a wide diversity of cell types apparent even with low resolution microscopy. This complexity is, of course, a necessary consequence of the growth and physiology of the root. Many cell types have particular roles in the uptake of ions and their transfer to the xylem and these will be discussed with particular reference to root hairs, and cells of the cortex and the stele. Trichoblasts, root epidermal cells with an elongated projection (`root hair') that extends into the soil, are the most obvious root cell type in which function is intuitively distinct. Root hairs appear to be specialized for the uptake of nutrients from the soil because they increase the surface area for absorption. The result of their activity is effectively to move the zone of depletion of nutrients further from the root surface (Tinker and Nye, 2000). This has the effect of increasing the volume of soil exploited for a given root length, whilst minimizing the input of resources needed to achieve this. However, the zones of depletion of individual root hairs overlap substantially indicating that more are produced than is theoretically needed to maximize nutrient availability. This may be because root hairs are short-lived and must maximize depletion before they die, but it could also indicate that they have other functions such as anchoring the root to enable the root tip to push forward through the soil without causing the backwards movement of mature root (Stolzy and Barley, 1968), maintaining physical contact between roots and soil (Russell, 1977), and in root exudation (Bhat et al., 1976). The relative importance of these functions has not been established. Now that mutants are available that are either compromised in root hair formation or that produce an overabundance of root hairs (Schiefelbein and Somerville, 1990; Grierson et al., 1997), it should be possible to take a more analytical approach to the role of root hairs and test theoretical predictions that their major role is in the uptake of poorly mobile nutrients. When there is a depletion zone around the root, the cortex is unlikely to play a signi®cant role in the initial uptake of nutrients, but when nutrients are at high concentrations (e.g. after fertilizer application, in a high nutrient patch, or with toxic concentrations of salts), solutes could also be taken up by cortical cells. However, the signi®cance of the exodermis in limiting apoplastic entry into the cortex is still uncertain, although it can clearly limit such ¯ow in some species (Peterson, 1987; Varney et al., 1993). Generalizing from such studies is, however, not possible, as there is clearly much variation in exodermal development (Canny and Huang, 1994). For example, salinity has been reported to induce the formation of an exodermis in cotton (Reinhardt and Rost, 1995). Although, in general, epidermal and cortical cells are treated as physiologically equal in many analyses of nutrient uptake, they may not be, and the epidermis in many situations will be quantitatively more important. It has been generally assumed that absorbed nutrients mostly move symplastically from epidermal cells through the cortex to the stele; the Casparian Strip of the endodermis providing a major barrier for the apoplastic movement of solutes and water into the stele. This barrier means that to enter the stele, any nutrient ions reaching the Casparian Strip must be taken up into endodermal cells. Thus all nutrients bound for the xylem, whichever route they follow to the endodermis, are in the symplast Partitioning of transport in roots at this point on the trans-root transfer pathway. To reach the xylem these ions must subsequently be secreted into the stelar apoplast. A corollary of this is that two protein-mediated steps are necessary for the delivery of solutes to the xylem one for uptake into the symplast (whether at the epidermis, cortex or endodermis) and one for release from stelar cells (Clarkson, 1993). Electrophysiological studies con®rm the presence of transport proteins in the plasma membrane of trichoblasts that could catalyse the uptake of some nutrients. The presence of inwardly rectifying Kq channels in wheat root hairs has been shown (Gassmann and Schroeder, 1994), while there has been strong evidence for the presence of Hq-coupled nitrate in¯ux (Meharg and Blatt, 1995). The higher density of plasmodesmata at the interface between the base of trichoblasts and the outer layer of cortical cells (Vakhmistrov and Kurkova, 1979; Vakhmistrov et al., 1981) is also consistent with a role for root hairs in the uptake, then subsequent symplastic transfer, of nutrients into the plant. Similarly, epidermal and cortical cells possess both low and high af®nity transport systems capable of catalysing uptake of a variety of different nutrients (Chrispeels et al., 1999; Maathuis and Sanders, 1999). The symplastic pathway of ion transfer from the soil to the stele requires transport of ions from cell to cell via plasmodesmata. Surprisingly, current knowledge of the pathway is remarkably sparse considering it underpins a widely-accepted hypothesis. Unlike leaves where maps of plasmodesmatal density (so-called `plasmodesmograms') have provided important indications of the role of symplastic and apoplastic pathways in phloem loading (van Bel, 1993), no detailed information is available on the extent of these connections in mature roots. This is urgently needed, if only to demonstrate that the pathway can potentially operate across the whole root. There is also a need to determine the extent to which disruption of the symplastic pathway affects uptake and transfer of ions to the xylem. With the likelihood that plasmodesmatal proteins will soon be identi®ed (Lee et al., 2000), it might be possible to down-regulate these proteins and so disrupt plasmodesmatal formation or structure. The consequences for trans-root movement of ions and water could then be investigated. The role of the Casparian Strip in limiting apoplastic movements of solutes has been mainly shown by incubating plants in membrane-impermeant compounds, and visualizing their build-up in the apoplast immediately adjacent to the endodermis (Nagahashi et al., 1974; Enstone and Peterson, 1992). Studies of the kinetics of appearance of labelled Ca2q and Mg2q in the cortex and stele of mycorrhizal spruce roots also supports the notion that a major barrier to apoplastic movement to the stele is located at the endodermis (Kuhn et al., 2000). However, there is evidence suggesting that the apoplastic 2q 451 in Arabidopsis roots is not limited movement of Ca by the Casparian Strip (White et al., 2000), signi®cantly at odds with dogma. Yeo et al. have also suggested that a major pathway for the entry of Naq into the xylem of rice roots is via an apoplastic pathway (Yeo et al., 1987). If these latter results prove to be more general (e.g. for different solutes, species and growing conditions; Schreiber et al., 1999), it will demand a signi®cant reconsideration of the view that membrane transport must occur for nutrients to enter the stele. Electrophysiological techniques have demonstrated the presence of ion channels that are likely to be responsible for the loading of solutes into the xylem. Patch clamping of protoplasts isolated from the xylem parenchyma of barley and the stele of Arabidopsis showed clearly the presence of both cation and anion channels with characteristics consistent with a role in the loading of solutes into the xylem (Wegner and Raschke, 1994; Wegner and de Boer, 1997; Maathuis et al., 1998; KoÈhler and Raschke, 2000). In a comparative study of Kq channels in the cortex and stele of maize roots, the existence of distinct channel types in the two cell types has been shown (Roberts and Tester, 1995). The plasma membrane of cortical cells was dominated by a Kq channel that would favour Kq in¯ux into the cells (and thus root), whereas the stelar cells were dominated by a Kq channel favouring Kq ef¯ux into the apoplast (and thus xylem: Fig. 2). Such clear-cut differences in channel activities were not seen in Arabidopsis, where similar activities of an outwardly-rectifying Kq channel was measured in protoplasts from both the cortex and stele, while inwardly-rectifying Kq channels were twice as frequent in stelar cells compared with cortical cells (Maathuis et al., 1998). However, given the clear effect of environment on these patterns of expression (see below), the signi®cance of such observations is uncertain. Underlining the likely physiological relevance of the different channel types observed in the cortex and stele of maize roots, Roberts found that addition of ABA to intact plants for 12 h inhibited the stelar Kq outwardly rectifying channel, but this treatment had no effect on the cortical inwardly rectifying channel (Fig. 3) (Roberts, 1998). This mirrors results in intact plants where the application of ABA rapidly inhibits the transfer of solutes to the shoot, whilst having little effect on their uptake into the root (Cram and Pitman, 1972). Furthermore, expression of SKOR, a gene encoding a stelar outwardlyrectifying Kq channel, has been shown to be rapidly inhibited by ABA (Fig. 4), whereas transcript from the gene encoding, AKT1, the channel responsible for at least some of the initial Kq uptake into the cortex (Hirsch et al., 1998) is unaffected (Gaymard et al., 1998). The results described above, at levels ranging from the gene to the whole plant, illustrate clearly the importance of cell-speci®c control of transport from the soil solution 452 Tester and Leigh Fig. 2. Demonstration of a Kq in¯ux channel in cortical cells and a Kq ef¯ux channel in stelar cells of maize roots. Patch clamping was used to measure Kq currents across the plasma membrane of isolated protoplasts (from Roberts and Tester, 1995, where further details can be obtained). to the xylem. This highlights the need for a more targeted approach for the genetic manipulation of nutrient uptake, whether done for further investigation of underlying processes or for plant improvement. Gene misexpression must be in speci®c cell types, rather than constitutively throughout the whole plant. Thus, either cell-speci®c promoters need to be catalogued and their wide availability ensured, or other systems need to be developed for manipulating gene expression in speci®c cells. One such system has been developed in Arabidopsis (see http:uuwww.plantsci.cam.ac.ukuHaseloffuHome.html). In this, GAL4, which is a strong transcriptional activator from yeast, has been modi®ed to provide high levels of activation of genes harbouring the upstream activation sequence (UAS) to which GAL4 binds (Brand and Perrimon, 1993). This has been used to create `enhancer trap' lines in Arabidopsis by random insertion into the plant genome of a construct that contains the GAL4 gene and a separate, UAS linked to a gene (mGFP5-ER) for ER-targeted green ¯uorescent protein (GFP) (Fig. 5A; J Haseloff, S Hodge, M Bauch, K Siemering, HM Goodman, unpublished results; Kiegle et al., 2000b). Insertion of this construct in fortuitous locations in the plant genome places the GAL4 expression under the control of endogenous plant promoters, resulting in its cell-speci®c expression which can be visualized by the presence of GFP resulting from the binding of GAL4 to the UAS linked to the mGFP5-ER gene. Lines with the cell-speci®c patterns of GFP expression can be selected from a library of such enhancer-trapped plants. Using this system, lines with cell-speci®c expression of GFP, and hence GAL4, in the epidermis, the elongation zone cortex, the endodermis, and the pericycle of the root have been obtained (Fig. 6). These lines not only give insights into the existence of interesting patterns of gene expression in roots, but the GFP is a useful cell marker that can be used to identify protoplasts derived from particular cell types (Kiegle et al., 2000a). However, the real power of the system is to use the presence of the exogenous transcription factor, GAL4, to activate transcription of any other genes transformed into the enhancer trap lines using a construct that contains the gene of interest downstream of a second GAL4 UAS (Fig. 5B). This leads to the activation of expression of the introduced gene in the GFP-marked cells in which the GAL4 gene product is also expressed. In work to test the utility of this system to drive targeted gene expression, transformation with a construct Partitioning of transport in roots 453 Fig. 4. Down-regulation by ABA of transcription of a gene encoding a stelar-speci®c outwardly rectifying Kq channel (SKOR), with no effects on an inwardly rectifying Kq channel gene (AKT1). Northern blot indicating transcript levels for the genes SKOR and AKT1 at various times after pre-treatment of Arabidopsis with 10 mM ABA (from Gaymard et al., 1998, where further details can be obtained). Fig. 3. A Kq ef¯ux channel in stelar cells is speci®cally inhibited by ABA. Mean current densities of whole cell inward and outward currents in protoplasts isolated from the cortex and stele of maize roots that had been well watered (WW), not watered for 60 h prior to protoplast isolation (WS), or watered with 20 mM ABA 12 h before protoplast isolation (ABA). Note the lack of inhibition by ABA of the inwardly rectifying Kq currents in cortical protoplasts, but the large inhibition of the outwardly rectifying currents in stelar protoplasts (from Roberts, 1998, where further details can be obtained). Fig. 5. Constructs used for GAL4-UAS transactivation of genes. (A) An enhancer trap construct for random insertion into the plant genome. Fortuitous expression of GAL4 driven by endogenous promoters at the point of insertion provides GAL4 protein which binds to the GAL4-UAS in the second part of the construct. This drives the production of GFP from mGFP5-ER, allowing visualization of cell-speci®c patterns of expression. (B) Construct for GAL4-dependent expression of a second gene (in this case a YFP-aequorin fusion). Introduction of this construct into the enhancer trap lines leads to the expression of the gene, driven by the GAL4 produced by the previously-introduced construct shown in part A (see Fig. 6 for examples, for details, see Kiegle et al., 2000b). 454 Tester and Leigh Fig. 6. Cell-speci®c expression of GAL4-mGFP5-ER and YFP-aequorin in Arabidopsis roots. (A) Cross-sectional diagram of an Arabidopsis root. (B±E) Laser scanning confocal microscopic images of aequorin in different Arabidopsis lines. Cell walls were stained with propidium iodide (red ¯uorescence). GFP and YFP are localised to (B) the epidermis, (C) the cortex of the elongation zone, (D) the endodermis, and (E) the pericycle. (F, G) High magni®cation localization of GFP and YFP in epidermal cells of line J0481. Scale bars for B±E 50 mM; F 25 mM (for details see Kiegle et al., 2000b). containing a yellow ¯uorescent protein (YFP)-aequorin fusion under the control of ®ve repeats of the GAL4 UAS (Fig. 5B) resulted in expression of YFP-aequorin in cells labelled with GFP (Fig. 6). These plants have been used to investigate the effects of stress on cytosolic Ca2q activities in various cell types (Kiegle et al., 2000b). Cell-speci®c expression of aequorin revealed cell-speci®c alterations in cytosolic Ca2q in response to NaCl or water stress, including clear oscillations that were not evident in plants expressing aequorin constitutively (Fig. 7). This system can be clearly used not only to monitor plant function, but also to manipulate it. Due to the Partitioning of transport in roots 455 when combined with the use of microarrays, will enable rapid identi®cation of cell-speci®c expression patterns. It is expected that, with these new technologies and perspectives, the coming years will bring many advances to the understanding of root function. Supplementary Material Supplementary material is available at JXB Online. References Fig. 7. Representative measurements of light emission from aequorin in roots of Arabidopsis plants. The aequorin was expressed (a, b) constitutively, (c, d) in the endodermis, or (e, f ) in the pericycle. Plants were subjected to (a, c, e) water stress by the addition of 440 mM mannitol or (b, d, e) salt stress by the addition of 220 mM NaCl (for details, see Kiegle et al., 2000b). importance of cell-speci®c processes in plant function, such manipulations will prove invaluable for future studies of the control of nutrient uptake. For example, the relative role of the cortex versus epidermis in nutrient uptake in a range of conditions can be tested directly by the manipulation of the expression of transporters in these different cell types. 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