Journal of Experimental Botany, Vol. 52, No. 90001, pp. 445-457,
March 2001
© 2001 Oxford University Press
Partitioning of nutrient transport processes in roots
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Received 23 June 2000; Accepted 1 September 2000
 |
Abstract |
|---|
 Top Abstract IntroductionPartitioning of transport...Partitioning of nutrient ions...Partitioning between cell types...ConclusionsReferences |
|---|
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 co-ordinated 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 |
|---|
|
TopAbstractIntroductionPartitioning of transport...Partitioning of nutrient ions...Partitioning between cell types...ConclusionsReferences |
|---|
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 deficiencies (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 the shoot. They form highly branched structures that provide a large surface are for nutrient and water absorption and which ramify through the soil profile 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 flow 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 beneficial 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) and/or symplastic (intracellular) pathways (Clarkson, 1993). The apoplastic route can be followed as far as the endodermis where the presence of a poorly ion-permeable 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 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 K+, but the general concepts are applicable to other ions.
|
Partitioning of transport between proteins within membranes: K+ uptake as a paradigm |
|---|
|
TopAbstractIntroductionPartitioning of transport...Partitioning of nutrient ions...Partitioning between cell types...ConclusionsReferences |
|---|
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 exemplified by the experiments of Asher and Ozanne who grew a number of plants in rapidly flowing nutrient solutions (delivered at 1000 l pot-1 d-1) containing a range of K+ concentrations (Asher and Ozanne, 1967
). Their results showed that individuals of all of the species they used could survive in 1 µM K+, a few grew maximally in 8 µM K+, while the majority of the remainder needed only 24 µM K+ for optimal growth. Further, very large changes in the external concentration were not matched by proportionately large changes in shoot or root K+ concentrations (Table 1). A 1000-fold increase in external K+ concentration (from 1 to 1000 µM) resulted in only a 6- to 14-fold change in the total K+ concentration in the roots and shoots. Additionally, the root and shoot K+ concentrations were comparable in all of the species, implying that K+ uptake and transfer to the shoot are regulated in a broadly similar way, at least in terms of the outcome (i.e. the final root and shoot K+ concentrations) that is achieved. These observations indicate that K+ 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 K+ concentrations.
|
Net K+ uptake at the root plasma membrane is classically viewed as resulting from the operation of active and passive transporters, which have different affinities for K+ and which make different contributions to uptake as the external concentration changes. Evidence for the operation of different K+ 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 K+ concentrations (below approximately 1 mM), uptake of K+ is mediated by a high-affinity saturable mechanism (so-called System 1) that is highly selective for K+. At higher external K+ concentrations, a second, less selective system operates (System 2).
Defining 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 K+ respond to the changes in external K+. Detailed studies in Arabidopsis roots (reviewed by Maathuis and Sanders, 1996) showed that growth and K+ uptake were possible at an external K+ concentration of 10 µM and that, at the prevailing membrane potential difference (PD) of -129 to -153 mV, and cytosolic K+ activity (80 mM), K+ uptake into root epidermal cells at this external K+ concentration has to be active (Maathuis and Sanders, 1993). Patch clamp studies showed that this uptake is probably mediated by a K+:H+ symporter (Maathuis and Sanders, 1994). Uptake within the concentration range of System 2 is probably passive and mediated by inward K+-selective channels. Patch clamp studies have indicated that such channels are present in epidermal and cortical cells from 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 K+ on each side of the membrane. The properties of the 5 pS channel are consistent with it being the dominant pathway for K+ uptake at mM external concentrations of K+ and its activity increases following K+ 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 K+ transport following growth in K+-free media (Buschmann et al., 2000), suggesting an important role for K+ channels in K+ nutrition. Other K+-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 (Köhler et al., 1999; Leng et al., 1999), but their significance in overall K+ uptake into the root is unknown.
The identification of the proteins responsible for active and passive uptake of K+ 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 K+ transporters have been identified by functional complementation of the K+ transport-deficient trk1 trk2 mutant of yeast (Saccharomyces cerevisiae). This mutant is unable to grow at low external K+ concentrations and plant cDNAs encoding K+ transporters have been identified by their ability to restore growth of the mutant on low K+. 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 identified include inwardly- and outwardly-rectifying K+-selective channels, and high affinity 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 identified as a H+-linked K+ symport, but now thought to function as a Na+:K+ or Na+:Na+ symport; Schachtman and Schroeder, 1994; Rubio et al., 1995; Gassmann et al., 1996), and members of the KUP/HAK family (dual affinity transporters with similarity to fungal H+:K+ 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 K+-selective ion channels that in animal cells mediate outward K+ transport (Jan and Jan, 1997). AKT1 shares many of the structural features of the animal channels, including six membrane spanning 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 filter 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 K+ 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 K+ uptake (Lagarde et al., 1996). This was confirmed using an Arabidopsis AKT1 knock-out mutant (akt1-1). Patch clamp analysis of root cells from this mutant showed that they lack inward K+ currents and are compromised in K+ 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 K+ transport and to be expressed specifically in stelar cells. It is thus thought to be involved in the passive transport of K+ out of the stelar cells into the xylem. Knockout mutants lacking SKOR have diminished K+ 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 H+:K+ symport that can mediate high affinity active transport of K+ when expressed in Xenopus oocytes or yeast (Schachtman and Schroeder, 1994). However, subsequent work showed that it probably functions as a Na+:K+ symport at low external Na+ concentrations and as a Na+:Na+ symport at higher external Na+ concentrations (Gassmann et al., 1996; Rubio et al., 1995). Thus its precise role in K+ transport remains somewhat equivocal. It has been shown that Na+-dependent K+ uptake does not occur in roots of terrestrial plants (Maathuis et al., 1996) and that an Arabidopsis HKT1 orthologue encodes a Na+ transporter that is unaffected by K+ (Uozumi et al., 2000). Thus the prevailing evidence tends to support the view that HKT1 may be more important in Na+ uptake. Nonetheless, its involvement in high affinity K+ transport is supported by the observation that its expression is rapidly up-regulated in response to removal of external K+ and with a time-course that matches changes in K+ uptake (Wang et al., 1998). In addition, Spalding et al., using the Arabidopsis akt1-1 knockout mutant, found that high affinity non-AKT1-dependent K+ uptake in the roots of these plants was sensitive to the external concentration of both H+ and Na+ which might indicate a role for HKT1 in this process (Spalding et al., 1999). Sensitivity of HKT1 to NH4+ is consistent with this suggestion, but it has not been definitely proven (Santa-Maria et al., 2000).
Potassium transporters of the KUP/HAK 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 K+ transport system from E. coli and the HAK1 system from Schwanniomyces occidentalis. The fungal HAK1 gene products encode high affinity H+:K+ symporters (Haro et al., 1999) and the plant orthologues mediate high-affinity K+ 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 K+ transport at mM concentrations, the sensitivity of the transport to tetraethylammonium (a blocker of K+ channels) and by the presence of an IYGD motif with homology to the GYGD that is characteristic of K+ channels (Fu and Luan, 1998). KUP/HAK transporters may also be involved in the NH4+-inhibited, high affinity K+ transport because the activity of these transporters is sensitive to NH4+ (Santa-Maria et al., 1997, 2000), although they lack the Na+ sensitivity that the NH4+-inhibited component was reported to have (Spalding et al., 1999).
How K+ uptake is partitioned between different transporters remains unclear. The isolation of genes for both K+ channels and putative high affinity active transporters appears to confirm 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 µM and mM external K+ concentrations. For instance, using an AKT1 knockout mutant (akt1-1), evidence was found to suggest that this channel accounts for a significant proportion of total K+ uptake by Arabidopsis roots at external K+ concentrations as low as 10 µM (Hirsch et al., 1998; Spalding et al., 1999). This need not indicate that AKT1 mediates an active transport process since passive influx can occur at such low external concentrations if the PD is sufficiently negative. With a cytosolic K+ activity of about 80 mM (Maathuis and Sanders, 1993; Walker et al., 1996), passive inward transport of K+ could be achieved from solutions containing 8 µM K+ 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 K+ transport from µM external K+ (Hirsch et al., 1998). Similarly, while transporters of the KUP/HAK family are capable of high affinity K+ 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 K+ transport from solutions containing up to 150 mM K+ when expressed in yeast (Fu and Luan, 1998). However, the reliability of the yeast trk1 trk2 mutant for making K+ uptake measurements at high external K+ 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 K+ uptake to a heterologously expressed protein may be difficult. Therefore, the role of the HAK/KUP transporters in passive K+ 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 K+, and therefore the direction of rapid passive movements of K+, are controlled by PD, a parameter which is, in turn, the output of a complex interaction of fixed charges and membrane transport processes (Nobel, 1999). The role of PD in K+ 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 K+ out of the stelar cells is less with ABA (assuming there were no effects on the transmembrane K+ 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 K+ (Roberts, 1998) and Cl- (Gilliham and Tester, 2000).
There are undoubtedly more K+ transporters to be discovered in plants and some of these will mediate K+ uptake processes in roots. The sequencing of the Arabidopsis genome raises the possibility of identifying all K+ 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 confirm their role in K+ 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.
|
Partitioning of nutrient ions between the cytosol and vacuole |
|---|
|
TopAbstractIntroductionPartitioning of transport...Partitioning of nutrient ions...Partitioning between cell types...ConclusionsReferences |
|---|
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 K+ (Walker et al., 1996) and 
(Miller and Smith, 1996) are regulated in the cytosol of root cells while concentrations in the vacuole vary with external supply, demonstrating the flexibility 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 K+, vacuolar K+ activity (aK) in root cells is about 100 mM in K+-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 K+ 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 K+ 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 K+ is transferred out of the vacuole to maintain the level in the cytosol. However, most experiments are done by growing roots at different external K+ concentrations where the level in the vacuole may reflect available supply rather than export across the tonoplast from a previously replete store. Therefore, experiments are needed to determine what happens to vacuolar K+ when replete cells are placed in deficient 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 K+. It is possible that limitations on K+ 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, K+ mobilization may be limited unless other solutes such as sugars can be transferred to the vacuole. However, when K+ is replaced by uncharged solutes, there must also be mobilization of a counteranion along with K+ to maintain electrical neutrality.
One aspect of the accumulation of K+ 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 K+ concentration in the shoots is approximately twice that in the roots (data in Table 1
for plants grown in 1000 µM K+). Measurements with K+-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 K+ transport at the tonoplast. Unfortunately, investigations into the nature of this regulation must await identification of the mechanisms responsible for K+ 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 influence on the rate of transfer. However, although K+ and N are needed in the shoot in roughly equal proportions, the concentrations of nitrate and K+ 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 flow, not diffusional processes) and that the K+ efflux mechanism controlling release to the xylem responds linearly to increasing K+ (which is possible, given the relatively low affinity of some plant cation channels, e.g. White and Ridout, 1995), approximately 20-fold more K+ 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 |
|---|
|
TopAbstractIntroductionPartitioning of transport...Partitioning of nutrient ions...Partitioning between cell types...ConclusionsReferences |
|---|
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 over-abundance 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 significant 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 significance of the exodermis in limiting apoplastic entry into the cortex is still uncertain, although it can clearly limit such flow 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 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 confirm the presence of transport proteins in the plasma membrane of trichoblasts that could catalyse the uptake of some nutrients. The presence of inwardly rectifying K+ channels in wheat root hairs has been shown (Gassmann and Schroeder, 1994), while there has been strong evidence for the presence of H+-coupled nitrate influx (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 affinity 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 identified (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 Ca2+ and Mg2+ 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 movement of Ca2+ in Arabidopsis roots is not limited by the Casparian Strip (White et al., 2000), significantly at odds with dogma. Yeo et al. have also suggested that a major pathway for the entry of Na+ 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 significant 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; Köhler and Raschke, 2000). In a comparative study of K+ 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 K+ channel that would favour K+ influx into the cells (and thus root), whereas the stelar cells were dominated by a K+ channel favouring K+ efflux 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 K+ channel was measured in protoplasts from both the cortex and stele, while inwardly-rectifying K+ 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 significance 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 K+ 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 outwardly-rectifying K+ 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 K+ 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-specific control of transport from the soil solution 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 specific cell types, rather than constitutively throughout the whole plant. Thus, either cell-specific promoters need to be catalogued and their wide availability ensured, or other systems need to be developed for manipulating gene expression in specific cells.
One such system has been developed in Arabidopsis (see http://www.plantsci.cam.ac.uk/Haseloff/Home.html). In this, GAL4, which is a strong transcriptional activator from yeast, has been modified 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 fluorescent 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-specific 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-specific patterns of GFP expression can be selected from a library of such enhancer-trapped plants. Using this system, lines with cell-specific 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 containing a yellow fluorescent protein (YFP)-aequorin fusion under the control of five 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 Ca2+ activities in various cell types (Kiegle et al., 2000b). Cell-specific expression of aequorin revealed cell-specific alterations in cytosolic Ca2+ 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 importance of cell-specific 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.
|
Conclusions |
|---|
|
TopAbstractIntroductionPartitioning of transport...Partitioning of nutrient ions...Partitioning between cell types...ConclusionsReferences |
|---|
For appropriate root function, it is clear that there must be the controlled and appropriate partitioning of processes at a range of levels. The tendency in the past has been to treat roots as uniform organs, but the key challenge now is to try to explain root function in terms of the activities of individual cells. This will be facilitated by new tools to access cells, such as single cell sampling, in situ hybridization, in situ PCR, and cell-specific misexpression. The construction of expression libraries from single cell types is also now possible (Galbraith, 2000
), which, when combined with the use of microarrays, will enable rapid identification of cell-specific 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.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +44 1223 333953. E-mail: Mat10{at}cus.cam.ac.uk
|
References |
|---|
|
TopAbstractIntroductionPartitioning of transport...Partitioning of nutrient ions...Partitioning between cell types...ConclusionsReferences |
|---|
Amtmann A, Sanders D. 1999. Mechanisms of Na+ uptake by plant cells. Advances in Botanical Research 29, 75–112.
Asher CJ, Ozanne PG. 1967. Growth and potassium content of plants in solution cultures maintained at constant potassium concentrations. Soil Science 103, 155–161.
Bhat KKS, Nye PH, Baldwin JP. 1976. Diffusion of phosphate to plant roots in soil. IV. The concentration distance profile in the rhizosphere of roots with root hairs in a low-P soil. Plant and Soil 44, 63–72.
Brand AH, Perrimon N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.[Abstract]
Buschmann PH, Vaidyanathan R, Gassmann W, Schroeder JI. 2000. Enhancement of Na+ uptake currents, time-dependent inward-rectifying K+ channel currents, and K+ transcripts by K+ starvation in wheat root cells. Plant Physiology 122, 1387–1397.
Canny MJ, Huang CX. 1994. Rates of diffusion into roots of maize. New Phytologist 126, 11–19.[ISI]
Clarkson DT. 1993. Roots and the delivery of solutes to the xylem. Philosophical Transactions of the Royal Society, London, Series B 341, 5–17.
Chrispeels MJ, Crawford NM, Schroeder JI. 1999. Proteins for transport of water and minerals across membranes of plant cells. The Plant Cell 11, 661–675.
Cram WJ, Pitman MG. 1972. The action of abscisic acid on ion uptake and water flow in plant roots. Australian Journal of Biological Sciences 25, 1125–1132.
Davenport RJ, Tester M. 2000. A weakly voltage-dependent, non-selective cation channel mediates toxic sodium influx in wheat. Plant Physiology 122, 823–834.
Davies JM, Poole RJ, Rea PA, Sanders D. 1992. Potassium transport into plant vacuoles energized directly by a proton-pumping inorganic pyrophosphatase. Proceedings of the National Academy of Sciences, USA 89, 11701–11705.
Drew MC, Saker LR. 1975. Nutrient supply and the growth of the seminal root systems of barley. II. Localized compensatory increases in lateral growth and rates of nitrate uptake. Journal of Experimental Botany 26, 79–90.
Enstone DE, Peterson CA. 1992. A rapid fluorescence technique to probe the permeability of the root apoplast. Canadian Journal of Botany 70, 1493–1501.
Epstein E, Rains DW, Elzam OE. 1963. Resolution of dual mechanisms of potassium absorption by barley roots. Proceedings of the National Academy of Sciences, USA 49, 684–692.
Fu HH, Luan, S. 1998. AtKup1: a dual-affinity K+ transporter from Arabidopsis. The Plant Cell 10, 63–73.
Galbraith DW. 2000. Applications of DNA microarrays in the analysis of the regulation of gene expression in higher plants. Journal of Experimental Botany 51, Supplement, 21.
Gassmann W, Rubio F, Schroeder JI. 1996. Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1. The Plant Journal 10, 869–882.[ISI][Medline]
Gassmann W, Schroeder JI. 1994. Inward-rectifying K+ channels in root hairs of wheat. A mechanism for aluminum-sensitive low-affinity K+ uptake and membrane potential control. Plant Physiology 105, 1399–1408.[Abstract]
Gaymard F, Cerutti M, Horeau C, Lemaillet G, Urbach S, Ravallec M, Devauchelle G, Sentenac H, Thibaud JB. 1996. The baculovirus/insect cell system as an alternative to Xenopus oocytes. First characterization of the AKT1 K+ channel from Arabidopsis thaliana. Journal of Biological Chemistry 271, 22863–22870.
Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J, Michaux-Ferrière N, Thibaud J-B, Sentenac H. 1998. Identification and disruption of a plant Shaker-like outward channel involved in K+ release into the xylem sap. Cell 94, 647–655.[ISI][Medline]
Gilliham M, Tester M. 2000. Anion channels in maize root stelar cells and their regulation by ABA. Journal of Experimental Botany 51, Supplement, 45.
Grierson CS, Roberts K, Feldmann KA, Dolan L. 1997. The COW1 locus of Arabidopsis acts after RHD2 and in parallel with RHD3 and TIP1, to determine the shape, rate of elongation, and number of root hairs produced from each site of hair formation. Plant Physiology 115, 981–990.[Abstract]
Haro R, Sainz L, Rubio F, Rodriguez-Navarro A. 1999. Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae. Molecular Microbiology 31, 511–520.[ISI][Medline]
Hirsch RE, Lewis BD, Spalding EP, Sussman MR. 1998. A role for the AKT1 potassium channel in plant nutrition. Science 280, 918–921.
Jan LY, Jan YN. 1997. Cloned potassium channels from eukaryotes and prokaryotes. Annual Review of Neuroscience 20, 91–123.[ISI][Medline]
Kiegle E, Gilliham M, Haseloff J, Tester M. 2000a. Hyperpolarization-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots. The Plant Journal 21, 225–229.[ISI][Medline]
Kiegle E, Moore C, Haseloff J, Tester M, Knight M. 2000b. Cell-type specific calcium responses to drought, NaCl and cold in Arabidopsis root: a role for endodermis and pericycle in stress signal transduction. The Plant Journal (in press).
Kim EJ, Kwak JM, Uozumi N, Schroeder JI. 1998. AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity. The Plant Cell 10, 51–62.
Köhler B, Raschke K. 2000. The delivery of salts to the xylem. Three types of anion conductance in the plasmalemma of the xylem parenchyma of roots of barley. Plant Physiology 122, 243–254.
Köhler C, Merkle T, Neuhaus G. 1999. Characterization of a novel gene family of putative cyclic nucleotide- and calmodulin-regulated ion channels in Arabidopsis thaliana. The Plant Journal 18, 97–104.[ISI][Medline]
Kuhn AJ, Scroder WH, Bauch J. 2000. The kinetics of calcium and magnesium entry into mycorrhizal spruce roots. Planta 210, 488–496.[ISI][Medline]
Lagarde D, Basset M, Lepetit M, Gaymard F, Astruc S, Grignon C. 1996. Tissue-specific expression of Arabidopsis AKT1 gene is consistent with a role in K+ nutrition. The Plant Journal 9, 195–203.[ISI][Medline]
Lee J-Y, Yoo B-C, Lucas WJ. 2000. Parallels between nuclear-pore and plasmodesmal trafficking of information molecules. Planta 210, 177–187.[ISI][Medline]
Leigh RA, Wyn Jones RG. 1984. A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant cell. New Phytologist 97, 1–13.
Leng Q, Mercier RW, Yao WZ, Berkowitz GA. 1999. Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel. Plant Physiology 121, 753–761.
Maathuis FJM, Sanders D. 1993. Energization of potassium uptake in Arabidopsis thaliana. Planta 191, 302–307.[ISI]
Maathuis FJM, Sanders D. 1994. Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 91, 9272–9276.
Maathuis FJM, Sanders D. 1995. Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana. Planta 197, 456–464.[ISI][Medline]
Maathuis FJM, Sanders D. 1996. Mechanisms of potassium absorption by higher plant roots. Physiologia Plantarum 96, 158–168.
Maathuis FJM, Sanders D. 1999. Plasma membrane transport in context—making sense out of complexity. Current Opinion in Plant Biology 2, 236–243.[ISI][Medline]
Maathuis FJM, Verlin D, Smith FA, Sanders D, Fernandez JA, Walker NA. 1996. The physiological relevance of Na+-coupled K+-transport. Plant Physiology 112, 1609–1616.[Abstract]
Maathuis FJM, May ST, Graham NS, Bowen HC, Jelitto TC, Trimmer P, Bennett MJ, Sanders D, White PJ. 1998. Cell marking in Arabidopsis thaliana and its application to patch-clamp studies. The Plant Journal 15, 843–851.[ISI][Medline]
Meharg AA, Blatt MR. 1995. NO3- transport across the plasma membrane of Arabidopsis thaliana root hairs: kinetic control by pH and membrane voltage. Journal of Membrane Biology 145, 49–66.[ISI][Medline]
Miller AJ, Smith SJ. 1996. Nitrate transport and compartmentation in cereal root cells. Journal of Experimental Botany 47, 843–854.
Nagahashi G, Thomson WW, Leonard RT. 1974. The Casparian strip as a barrier to the movement of lanthanum in corn roots. Science 183, 670–671.
Nobel PS. 1999. Physicochemical and environmental plant physiology, 2nd edn. San Diego: Academic Press.
Peterson CA. 1987. The exodermal Casparian band of onion roots blocks the apoplastic movement of sulphate ions. Journal of Experimental Botany 38, 2068–2081.
Reinhardt DH, Rost TL. 1995. Salinity accelerates endodermal development and induces an exodermis in cotton seedling roots. Environmental and Experimental Botany 35, 563–574.
Roberts SK. 1998. Regulation of K+ channels in maize roots by water stress and abscisic acid. Plant Physiology 116, 145–153.
Roberts SK, Snowman BN. 2000. The effects of ABA on channel-mediated K+ transport across higher plant roots. Journal of Experimental Botany 51, 1585–1594.
Roberts SK, Tester M. 1995. Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele. The Plant Journal 8, 811–825.[ISI]
Robinson D. 1994. The responses of plants to non-uniform supplies of nutrients. New Phytologist 127, 635–74.