Radial Transport of Nutrients: The Plant Root as a Polarized Epithelium

Different routes underlie radial transport of nutrients in plant roots and are influenced by the endodermis permeability and the polarity of transporters.

Abstract

In higher plants, roots acquire water and soil nutrients and transport them upward to their aerial parts. These functions are closely related to their anatomical structure; water and nutrients entering the root first move radially through several concentric layers of the epidermis, cortex, and endodermis before entering the central cylinder. The endodermis is the innermost cortical cell layer that features rings of hydrophobic cell wall material called the Casparian strips, which functionally resemble tight junctions in animal epithelia. Nutrient uptake from the soil can occur through three different routes that can be interconnected in various ways: the apoplastic route (through the cell wall), the symplastic route (through cellular connections), and a coupled trans-cellular route (involving polarized influx and efflux carriers). This Update presents recent advances in the radial transport of nutrients highlighting the coupled trans-cellular pathway and the roles played by the endodermis as a barrier.

In terms of function, the root can be seen as an inverted gut with the two epithelial functions (selective uptake and diffusion barrier) split between the epidermis and endodermis (Fig. 1). The plant epidermis, cortex, and endodermis are polarized cells, with an outer (peripheral) and inner (central) plasma membrane (PM) domain similar to the apical-basolateral polarity in animals (Fig. 1). The outermost epidermis and its root hairs are in direct contact with the soil and appear to mediate most of the selective uptake and functionally resemble the brush border in epithelia. The endodermis is the innermost cortical cell layer that surrounds the central vasculature and forms a barrier for the free diffusion of solutes from the soil to the stele. This parallel between the plant endodermis and animal epithelia has already been mentioned in several reviews (Clarkson, 1993; Alassimone et al., 2012; Geldner, 2013). In many plants, the exodermis, a cell layer below the epidermis, forms a second barrier for the transport of solutes in roots (Hose et al., 2001). This cell layer shares many features with the endodermis; however, it will not be discussed in this Update.

Figure 1.

The plant root as a polarized epithelium. A schematic comparison between animal intestinal epithelium (left) and plant roots (right) is shown. In terms of function, the plant root can be seen as an inverted gut with the two epithelial functions, selective uptake and barrier for diffusion, split between the epidermis, cortex, and endodermis. Functional and structural similarities between animal epithelium and plant root are represented with the same color code (red-like color indicates acquisition from outside, and green-like color indicates transport inside the corresponding vascular systems). Note that animal epithelium displays an apical-basolateral polarity, while plant root presents an outer-inner polarity.

What current textbooks tell us regarding the radial transport of nutrients in plant roots is that there are two different routes from the soil to the stele: one passive (the apoplastic pathway) and one active (the symplastic pathway; Marschner, 1995). However most of our knowledge regarding nutrients on their way from the soil to the stele are concerned with their selective uptake into the cell. Recent decades have seen the identification and characterization of the major transporters and channels involved in the uptake of nutrients from the soil. In Arabidopsis (Arabidopsis thaliana), the NRT1.1, NRT1.2, and NRT2.1 transporters for nitrate (Tsay et al., 1993; Huang et al., 1999; Filleur et al., 2001), the SULTR1;2 transporter for sulfate (Shibagaki et al., 2002; El Kassis et al., 2007), the Arabidopsis K⁺ Transporter1 (AKT1; Hirsch et al., 1998), the IRON-REGULATED TRANSPORTER1 (IRT1) for metals (Henriques et al., 2002; Varotto et al., 2002; Vert et al., 2002), and the boric acid influx channel NOD26-like intrinsic protein5 (NIP5;1; Takano et al., 2006) have been particularly well characterized. This is mainly due to the fact that single loss-of-function mutants for the corresponding genes display strong phenotypes that are often hiding the effect of mutations of downstream effectors. A good illustration is sulfate, where different laboratories performed a genetic screen for selenate-resistant mutants (toxic analog to sulfate) that all lead to the identification of several alleles of the same SULTR1;2 gene encoding the main sulfate transporter (Rose, 1997; Shibagaki et al., 2002; El Kassis et al., 2007). What happens to nutrients after uptake into root cells? Several channels and transporters have been reported to play important roles in xylem loading, such as Stelar K⁺ Outward Rectifier for potassium (Gaymard et al., 1998), PHO1 for phosphate (Hamburger et al., 2002), or BOR1 for boric acid/borate (Takano et al., 2002). However, the processes involved between nutrient uptake from the soil and loading to the xylem are generally poorly understood. In particular, the importance played by the endodermis as a major diffusion barrier is not yet clear, although it is discussed in every model of plant nutrient uptake (Geldner, 2013). This is mainly due to the fact that, until recently, the molecular actors controlling endodermis differentiation were not identified. Moreover, recent advances in the cellular imaging of protein localization and trafficking indicate that several transporters are polarized in root cell layers. These discoveries indicate that current textbook descriptions of the radial transport of nutrients, with their simplistic subdivisions into an apoplastic and a symplastic pathway, are insufficient and that a third route can be conceived: a coupled trans-cellular pathway. Moreover, those three different pathways can be combined along the root cell layers, making the model of radial transport more complex. In this context, this Update presents new advances regarding the radial transport of nutrients in root cell layers, with a special focus on the roles played by the endodermis as a barrier and the lateral polarity of transporters.

THREE ROUTES FOR NUTRIENTS

The Apoplastic Pathway

The apoplastic pathway provides a route for mass flow and the diffusion of water and nutrients toward the stele through free spaces and cell walls of the epidermis and cortex (Fig. 2A). The rate of diffusion depends upon the ionic gradient between the external solution and the apoplastic free space. Primary cell walls consist of a network of cellulose, hemicellulose, and glycoproteins. In this network, a variable proportion of pectins act as cation exchangers. This is due to the negatively charged nature of the carboxylic groups (R.COO⁻) in the galacturonic acid monomers of pectin. Therefore, cations from the external solution can accumulate through a nonmetabolic process in the root apoplast, whereas anions are repelled. The cation-binding capacity of the apoplast can contribute significantly to the total cation content in roots, as shown by studies of the uptake of cations such as aluminum, silicon, and iron (Lobreaux et al., 1992; Wang et al., 2004).

Figure 2.

Transport of nutrients in roots: three different pathways. A, Schematic view of the three different pathways involved in the transport of nutrients from the soil to the endodermis. The symplastic pathway (in gray) requires at first a selective uptake into a cell and then transport from one cell to the other through plasmodesmata. The coupled trans-cellular pathway (in red) involves influx (in yellow) and efflux (in purple) carriers to transport nutrients from one cell to the other. The apoplastic pathway (in blue) corresponds to a passive transport in the extracellular space and is blocked by the CS at the level of the endodermis. B, Magnification of the circled area in A, focusing on the transport of nutrients from the apoplast to the endodermis. Transport routes through the endodermis involve a short symplastic pathway (in black) and a single trans-cellular pathway (in pink), restricted at the level of the endodermis. Co, Cortex; En, endodermis; Ep, epidermis; Pe, pericycle.

The Casparian strips (CS) are lignin-like structures deposited as a ring in the transverse section of endodermal cells and block the passive flow of water and solutes in the apoplast (see below). This property to interrupt the cell wall continuity was already investigated in 1910, with a study of the penetration of some salts into the root that provided evidence that several salts needed to pass through the endodermis symplast in order to reach the stele (de Rufz de Lavison, 1910). Later, many studies reported that the diffusion of fluorescent dyes or ions is blocked at the level of the endodermis and that this coincides with the presence of the CS (Clarkson and Sanderson, 1969; Robards and Robb, 1974; Singh and Jacobson, 1977; Peterson, 1987). Recently, studies in Arabidopsis employed propidium iodide (PI) as a tracer for the apoplastic route (Alassimone et al., 2010). PI allows the whole-mount staining of living roots and has been shown to be a powerful tool for the characterization of CS functionality in vivo (Alassimone et al., 2010; Naseer et al., 2012; Hosmani et al., 2013; Lee et al., 2013).

In consequence, a purely apoplastic pathway from the soil to the stele can occur only before CS differentiation (i.e. in the young parts of the roots) or at sites where the CS barrier is disrupted, for example at sites of lateral root emergence. Once CS are differentiated, nutrients in the apoplast can reach the stele only by being transported into the endodermal symplast through influx carriers present at the endodermal PM (Fig. 2B). This transport through the endodermis would then correspond to a short symplastic transport (Fig. 2B), which can be prolonged through the pericycle, as has been suggested for calcium uptake (Ferguson and Clarkson, 1976). Alternatively, it could constitute a single trans-cellular transport, if both influx and efflux occur at the endodermis (Fig. 2B).

The Symplastic Pathway

The symplastic route to the vasculature involves cell-to-cell transport via plasmodesmata (Fig. 2A). Plant cells are surrounded by a cell wall, creating a challenge for individual cells to directly communicate and exchange material. This is overcome by plasmodesmata, channels providing cytoplasmic continuity between each cell and its immediate neighbor (Burch-Smith and Zambryski, 2012). The rate of symplastic transport, therefore, should depend upon the size exclusion limit and the frequency of plasmodesmata between the cells, features that can be modulated by certain stress conditions. This has been particularly well described in pea (Pisum sativum) roots, where increased symplastic transport during osmotic stress correlates with changes in plasmodesmata dimensions (Schulz, 1994, 1995). One of the factors controlling plasmodesmata permeability is callose, a β-1,3-linked homopolymer of Glc, whose accumulation restricts the channel aperture to inhibit the cell-to-cell transport of macromolecules. In contrast, down-regulation of its accumulation allows for more macromolecular trafficking (Zavaliev et al., 2011). Callose deposition at plasmodesmata in roots is induced by various abiotic stresses, including silicon, cadmium, and aluminum application, suggesting that this corresponds to a mechanism to modulate the transport of nutrients (Sivaguru et al., 2000; Ueki and Citovsky, 2005; Narro and Arcos, 2010; Shetty et al., 2012).

In the symplastic pathway, the PM acts as a selective surface at the soil-plant interface controlling the uptake of nutrients (Marschner, 1995). Once entered in a cell, their mobility can be modulated by chelation with macromolecules or subcompartmentalization. Zinc is a good illustration of the complexity of the symplastic pathway (Olsen and Palmgren, 2014). IRT1 and IRT2 are the main transporters mediating the uptake of zinc to the symplast of epidermis and cortex (Vert et al., 2001, 2002). Once in the symplast, zinc is prone to bind a multitude of organic molecules, which severely restrict its mobility. Therefore, zinc traffics from one cell to its immediate neighbor when bound to a symplastic metal chelator such as nicotianamine (Deinlein et al., 2012). The zinc-nicotianamine complex diffusion in the root symplast is restricted by sequestration for storage into the vacuole. This import requires active transporters such as the two metal tolerance proteins MTP1 and MTP3 and the Heavy Metal-Transporting P-type ATPase3 (HMA3; Desbrosses-Fonrouge et al., 2005; Arrivault et al., 2006; Kawachi et al., 2009; Morel et al., 2009).

However, while the role of plasmodesmata for the transport of large molecules such as RNA, proteins, and viral RNA-protein complexes is increasingly understood (Burch-Smith and Zambryski, 2012), not much is known regarding their interaction with metal-chelate complexes. Considering that the transit through plasmodesmata is dependent upon regulated size exclusion limits, the transport of metal-chelate complexes between cells could be a fine-tuned and regulated process. Hence, plasmodesmata could modulate metal-chelate transport either by blocking them or facilitating their transport in a selective way.

The Coupled Trans-Cellular Pathway

The coupled trans-cellular pathway corresponds to a trans-epithelial transport (paracellular) with influx and efflux transporters, transporting nutrients from one cell to another in a vectorial fashion (Fig. 2). In this scenario, transporters are polarly distributed, not only in the endodermis but also in cortical and epidermal cells (Figs. 1 and 2). Nutrients would then be transported from one cell to the other, passing repeatedly from symplast to apoplast, in a mechanism similar to auxin polar transport (Löfke et al., 2013). Such a mechanism would have the advantage of providing directional long-distance transport toward the stele over multiple cell layers, regardless of the mass flow of nutrients. Curiously, the possibility of a coupled trans-cellular pathway is often disregarded in current models of root radial transport. This is mainly due to the fact than an essential feature for achieving this transport, a polar localization of influx and efflux carriers along the different root cell layers, is difficult to visualize and was discovered only recently in plants (see below). Another strong support for a coupled trans-cellular pathway in roots comes from the characterization of the boric acid efflux transporter BOR1, which localizes to the inner domain not only in endodermis and stele but also in outer cell layers, a feature not required for a symplastic or apoplastic pathway (Takano et al., 2010).

THE ENDODERMIS AS A BARRIER

One Cell Layer, Two Barriers

Endodermal specification is controlled by the interaction of two transcriptional factors, SHORTROOT (SHR) and SCARECROW (SCR). SHR and SCR interaction requires the movement of SHR from the stele to the cortical endodermal initials, where together they trigger the periclinal division of the cortical endodermal initial cells, leading to the formation of two cell layers, the cortex and the endodermis (Helariutta et al., 2000; Nakajima et al., 2001; Gallagher et al., 2004). While endodermal specification in roots is a well-described mechanism, its differentiation started to be investigated only recently (Alassimone et al., 2010; Roppolo et al., 2011; Naseer et al., 2012; Hosmani et al., 2013; Lee et al., 2013). In Arabidopsis, endodermal differentiation is characterized by two developmental stages (Fig. 3): the establishment of the CS characterizes the primary differentiation stage of an endodermal cell. This is later followed by a deposition of suberin lamellae that eventually coat the entire endodermal cell. Some endodermal cells remain at the primary stage even late in root development. These cells are called endodermal passage cells (Alassimone et al., 2010; Naseer et al., 2012; Geldner, 2013; Robbins et al., 2014).

Figure 3.

Endodermis differentiation. The schematic representation shows the succession of events during endodermal differentiation (not to scale). The different stages are represented in a longitudinal view and in three-dimensional perspective to illustrate specific developmental hallmarks. Endodermal differentiation starts with the deposition of CS islands (in green) in the anticlinal walls of endodermal cells (early differentiation), which fuse into a CS ring (differentiation state I). Later, suberin lamellae (in yellow) cover the entire endodermal cells except in the passage cells (differentiation state II).

CS

The CS are localized impregnations of the primary cell wall that surrounds the endodermal cells, forming a ring, functionally equivalent to the tight junctions in animal epithelium (Fig. 1). This ring is established in a periclinal fashion along the endodermal cell’s meridian (Fig. 3). Electron micrographs show that the CS occupies the entire space between two adjacent endodermal cells (Bonnett, 1968; Roppolo et al., 2011; Hosmani et al., 2013). The CS are established close to the root meristem, forming a discontinuous ring of CS islands at first (string of pearls stage) that eventually fuse to form a continuous ring (Roppolo et al., 2011). In the time frame of endodermal differentiation, the apoplastic barrier (scored as a block in PI penetration) establishment correlates very well with CS formation (Alassimone et al., 2010). Experimental manipulation of lignin and suberin production in Arabidopsis combined with histological, pharmacological, and chemical analysis demonstrated that CS have a lignin-like nature (Naseer et al., 2012). Contrary to tight junctions in animal epithelia, the establishment of CS cannot be achieved by protein-mediated cell-cell interaction but requires the localized impregnation of the cell wall, guided by protein platforms at the Casparian strip domain (CSD). The CS membrane domain proteins (CASP1–CASP5) localize to and define this membrane domain. They form a platform that is thought to control the localized recruitment of proteins involved in CS deposition and fusion into a ring (Roppolo et al., 2011). Corroborating this model, recent works indicate that the localized lignin polymerization in CS is achieved by the combination of a localized production of reactive oxygen species and localized peroxidase activity brought together by the scaffolding activity of the CASPs (Lee et al., 2013). Loss of the NADPH oxidase Respiratory Burst Oxidase Homolog F results in a strong delay in CS establishment, while the peroxidase PER64 was shown to strictly colocalize with CASP1 and to depend upon CASPs (Lee et al., 2013). The dirigent domain-containing protein ENHANCER OF SUBERIN1 (ESB1) is another player in the machinery localizing CS lignin deposition (Hosmani et al., 2013). ESB1 localizes to the CS, and its mutation leads to an ectopic suberization in roots (the mutant esb1 was named after this phenotype) and a delay in CS establishment. Once the CS is formed in esb1-1, they are broader, apparently due to ectopic lignin disposition, comparable to the phenotype of the casp1-casp3 mutant (Baxter et al., 2009; Hosmani et al., 2013). Recently, a genetic screen for mutants with an impaired apoplastic barrier (J. Alassimone and N. Geldner, unpublished data) led to the identification of SCHENGEN3 (SGN3), a Leu-rich repeat receptor kinase that is necessary for the fusion of CS islands into a continuous ring (Pfister et al., 2014).

While their role as a physical apoplastic barrier is doubtless, the actual requirement of the CS as a barrier during radial nutrient transport is poorly supported by evidence and is mainly based on correlations. This is mainly due to the fact that, until recently, there were no mutants that specifically affected CS establishment that would have allowed us to study the consequences of a lack of CS for root function. The mutants esb1 and casp1-casp3 potentially provide a first starting point for studying the importance of the CS for plant nutrition (Baxter et al., 2009; Roppolo et al., 2011; Hosmani et al., 2013). However, as these mutants only delay CS formation and present at the same time an ectopic suberization, the nutritional phenotypes observed are difficult to interpret (Baxter et al., 2009; Hosmani et al., 2013). Therefore, the identification of sgn3, specifically affected for CS establishment and not affected in suberization, resulting in a nonfunctional apoplastic barrier in the entire root, represents a promising new tool for studying CS function (Pfister et al., 2014). Analysis of this mutant revealed a weaker than expected effect of impaired CS on plant nutrition, the main consequences being an increased water transport, sensitivity to temperature, and a potassium deficiency (Pfister et al., 2014). This first characterization of the sgn3 mutant challenges our current views regarding CS function and highlights the need for further physiological characterization of CS mutants.

Suberin Lamellae

Suberin is a hydrophobic polymer consisting of aliphatic polyesters with minor amounts of hydroxycinnamic acids (mainly ferulic acid) and glycerol. The enzymes involved in suberin biosynthesis are well characterized (Beisson et al., 2012). In Arabidopsis, the ω-hydroxylases HORST and RALPH belonging to the CYP86 subfamily of cytochrome P450 monooxygenases and the glycerol-3-phosphate acyltransferase GPAT5 have been particularly characterized (Beisson et al., 2007; Li et al., 2007; Höfer et al., 2008; Molina et al., 2009; Yang et al., 2010). The suberin lamellae correspond to a secondary cell wall deposition that forms in the inner surface of primary cell walls and eventually covers the entire endodermal cell. In contrast to CS, suberin lamellae do not affect the apoplastic transport, as mutants with ectopic suberization or lines with no suberin lamella demonstrated no effect on PI blocking by the endodermis (Naseer et al., 2012; Hosmani et al., 2013). The presence of suberin lamellae could modulate the direct uptake of nutrients from the apoplast into endodermal cells (Geldner, 2013; Robbins et al., 2014), which in turn would impact the coupled trans-cellular pathway as well as the single trans-cellular pathway at the level of the endodermis (Fig. 2).

Contrary to the CS, suberin’s role in plant nutrition is better understood in particular regarding water and salt stress. Analysis of several rice (Oryza sativa) cultivars demonstrated a negative correlation between suberin accumulation in roots and sodium uptake in shoot and plant survival of salt stress (Krishnamurthy et al., 2009, 2011). In Arabidopsis, the horst mutant, with lower suberin content and delayed suberin lamellae formation, displays a higher hydraulic conductivity (Höfer et al., 2008). On the other hand, the esb1 mutant, with ectopic suberization, displays increased water use efficiency (Baxter et al., 2009; Hosmani et al., 2013). Hence, it appears that suberin forms a bidirectional barrier to prevent the entry of sodium from the soil and to retain water in the root. Regarding other minerals, ionomic analysis of the esb1 mutant reveals that ectopic suberization is associated with a decrease in the accumulation of calcium, manganese, and zinc and an increase in the accumulation of sodium, sulfur, potassium, arsenic, molybdenum, and selenium (Baxter et al., 2009; Hosmani et al., 2013). These results demonstrate that suberin plays different roles depending on the substrate. One explanation could be that suberin plays a role as a barrier to the entry of some elements (such as calcium and manganese) and might rather act to retain others within the stele (such as sulfur and potassium). Future work concerning uptake and export activity measurements combined with imaging of nutrients in suberized versus nonsuberized roots will help us to understand the mechanisms underlying this differential role of suberin.

OUTER AND INNER POLARITY OF TRANSPORTERS

Lateral Polarity of Transporters

An important characteristic of animal epithelial cells is that they exhibit an apical-basolateral polarity marked by distinct membrane domains: an apical domain in contact with the external environment or body cavities and a basolateral domain adjoined to the underlying tissues (Fig. 1). By separating the external from the internal environment, epithelial cells perform specialized functions such as trans-cellular water, ion, and peptide transport. This is achieved by a battery of influx and efflux carriers that are specifically localized to the apical or basolateral PM domain of epithelial cells. Mechanisms for epithelial polarity establishment and maintenance have been extensively studied in animals (Mellman and Nelson, 2008; Apodaca and Gallo, 2013). Epithelial polarity requires the coordinated interaction of machineries modifying protein trafficking and distribution (Mellman and Nelson, 2008; Apodaca and Gallo, 2013). Intrinsic sorting signals such as Tyr-based motifs or di-Leu signals are well described to promote the directional targeting of proteins to the PM. Moreover, protein complexes associated differentially to PM domains reinforce membrane asymmetry, and cell-cell and cell-substrate adhesion orient the direction of membrane traffic (such as the tight junction providing a diffusion barrier to the apical and basolateral PM domains). Illustrating its essential role, defects in polarity establishment and maintenance are associated with various metabolic diseases (Mellman and Nelson, 2008; Apodaca and Gallo, 2013).

In plants, recent advances have led to the identification of an increasing number of influx and efflux transporters for nutrients that are localized in a polar fashion in root cells, whether to the outer PM domain (also called the peripheral or distal domain) or to the inner PM domain (also called the central or proximal domain; Fig. 1; Table I). This lateral polarity was first described in potato (Solanum tuberosum) roots, with the phosphate transporter StPT2 localizing to the outer PM domain of epidermal cells, consistent with its function in phosphate uptake (Gordon-Weeks et al., 2003). However, lateral polarity became recognized as a general feature in root cells only after the characterization of the rice silicic acid uptake channel Low-Silicon1 (Lsi1) and the silicic acid exporter Lsi2, reciprocally localizing to the outer and inner PM domains of both exodermis and endodermis in roots (Ma et al., 2006, 2007). This suggested for the first time a mechanism of nutrient transport with an influx transporter on one side and an efflux transporter on the other side of a cell to permit an effective, directional trans-cellular transport of nutrients in plants. In Arabidopsis, the same opposite polarization was described for the boric acid influx channel NIP5;1 and the boric acid/borate efflux transporter BOR1, localizing to the outer and inner PM domains of endodermal cells, respectively (Alassimone et al., 2010; Takano et al., 2010). Recently, the rice manganese transporter NRAMP5 (for natural resistance-associated macrophage protein) was also reported to localize to the outer PM domain in both exodermis and endodermis, but in this case no corresponding efflux transporter localized to the inner PM domain has been identified so far (Sasaki et al., 2012). Interestingly, lateral polarity of transporters is not specific to the endodermis, supporting the existence of coupled trans-cellular pathways for nutrients within the root (Fig. 2; Table I). Hence, when expressed under the control of their native promoters, NIP5;1 localized to the outer domain of elongated epidermal cells while BOR1 localized to the inner domain of elongated epidermal and endodermal cells (Takano et al., 2010). Most transporters displaying a lateral polarity localize to the outer PM of root epidermal cells, such as the nitrate high-affinity transporter NRT2;4 and the metal transporter IRT1 in Arabidopsis, consistent with their physiological roles in the uptake of nutrient from the soil (Kiba et al., 2012; Barberon et al., 2014). Moreover, lateral polarity appears to be important not only for the uptake of ions but also for their export. The boron exporter BOR4, for example, localizes to the outer PM domain of epidermal cells, mediating a directional export of boron from the roots to the soil under high boron concentrations (Miwa et al., 2007).

Table I.

Nutrient transporters displaying lateral polarity in roots

Protein	Function	PM Domain	Tissues/Cells	Species	References
StPT2	Phosphate uptake	Outer	Phosphate-deficient root elongating epidermis	Potato	Gordon-Weeks et al. (2003)
Lsi1	Silicon uptake	Outer	Root exodermis and endodermis	Rice	Ma et al. (2006)
Lsi2	Arsenic/silicon efflux in xylem	Inner	Root exodermis and endodermis	Rice	Ma et al. (2007)
BOR4	Boron efflux to the soil	Outer	Root epidermis	Arabidopsis	Miwa et al. (2007)
Zm Yellow-Stripe1	Ferric-phytosiderophore uptake	Outer	Epidermis of iron-deficient crown and lateral roots	Maize (Zea mays)	Ueno et al. (2009)
HvLsi1	Silicon uptake	Outer	Seminal root epidermis and cortex; lateral root hypodermis	Barley (Hordeum vulgare)	Chiba et al. (2009)
ZmLsi1	Silicon uptake	Outer	Seminal and crown root epidermis and hypodermis; lateral root cortex	Maize	Mitani et al. (2009)
BOR1	Boron efflux in xylem	Inner	Boron-deficient root epidermis and endodermis	Arabidopsis	Alassimone et al. (2010); Takano et al. (2010)
NIP5;1	Boron uptake	Outer	Boron-deficient root epidermis and endodermis	Arabidopsis	Alassimone et al. (2010); Takano et al. (2010)
NRAMP5	Manganese/cadmium uptake	Outer	Root exodermis and endodermis	Rice	Sasaki et al. (2012)
NRT2.4	Nitrate uptake	Outer	Nitrogen-starved root epidermis	Arabidopsis	Kiba et al. (2012)
IRT1	Metala uptake	Outer	Metal-deficient root epidermis	Arabidopsis	Barberon et al. (2014)

^aIRT1 metal substrates refer to iron, zinc, manganese, cobalt, cadmium, and nickel (Eide et al., 1996; Korshunova et al., 1999; Rogers et al., 2000; Vert et al., 2002; Nishida et al., 2011).

Cellular Mechanisms of Lateral Polarity in Root Cells

While apical-basal polarity has been extensively studied for the last 15 years in plant roots, in particular regarding its role in polar auxin transport (Löfke et al., 2013), the mechanisms involved in lateral polarity establishment and maintenance are poorly understood. This is mainly due to the relatively recent discovery of lateral polarization in root cells and the low number of proteins known to display such a localization in Arabidopsis (Table I).

Apical and basal polar domains in root cells are characterized by the localization of several polar cargos, such as the PIN-FORMED (PIN) efflux carrier for the plant hormone auxin, and is achieved by nonpolar secretion, subsequent internalization, and polarized recycling (Löfke et al., 2013). Interestingly, lateral polarization of proteins is achieved by different mechanisms and does not require known molecular components of apical or basal targeting, such as the ADP-Ribosylation Factor-Guanine Nucleotide Exchange Factor GNOM, the endoplasmic reticulum-localized Auxin-Resistant4 protein, or the protein kinase PINOID (Langowski et al., 2010). Fluorescence recovery after photobleaching experiments indicate that, unlike the known apical and basal cargos, the polar localization of outer lateral cargos is already established during the polar secretion of newly synthesized proteins (Langowski et al., 2010).

As the CS together with the CSD constitute a barrier for lateral diffusion of the PM domain, they could play a pivotal role in lateral polarity establishment and maintenance. Hence, in differentiated endodermal cells, BOR1 and NIP5;1 are localized to the inner and outer PM domains, respectively, and are strictly excluded from the CSD (Alassimone et al., 2010). Arguments against this hypothesis come from the observation that BOR1 and NIP5.1 lateral polarity is established early during embryogenesis, prior to CSD differentiation, and in nonendodermal cell layers (Alassimone et al., 2010; Takano et al., 2010). Moreover, an interrupted CSD does not interfere with the localization of a well-established outer polar marker (Pfister et al., 2014). Still, an important feature is that in the absence of CSD (such as in epidermal cells or undifferentiated endodermis), the BOR1 and NIP5.1 inner and outer markers localized to their respective polar domains, but their localizations are less restricted and overlap in apical-basal domains (Alassimone et al., 2010). This suggests that the CSD is not needed for the proper establishment or maintenance of polarity but rather acts to refine and separate partially overlapping polar domains.

As mentioned above, intrinsic sorting signals in protein sequences are an important component in PM protein delivery and maintenance to PM domains. Tyr-based motifs like YxxΦ (where Y is Tyr, x is any amino acid, and Φ is any bulky hydrophobic residue) are well established as signals for endocytosis, sorting to lysosomes, and also polar PM sorting in mammals (Bonifacino and Traub, 2003; Mellman and Nelson, 2008). The Tyr-based motifs are recognized by a medium (µ) subunit of adaptor protein complexes for sorting into clathrin-coated vesicles. Three Tyr residues (Tyr-373, Tyr-398, and Tyr-405) are important for BOR1 polar localization, and their mutation to Ala results in a nonpolar localization at the PM (Takano et al., 2010). However, clathrin-mediated endocytosis does not appear to be a general mechanism controlling lateral polarity of the PM, as its interference stabilizes IRT1 at the PM but does not affect its localization to the outer polar domain (Barberon et al., 2014).

Function of Lateral Polarity

The increasing number of transporters found to localize to the outer and inner PM domains in plant roots suggests a general mechanism of a coupled trans-cellular pathway for the radial transport of nutrients from the soil to the xylem. However, this is mainly based on the match between transporter localization and their respective functions in terms of transport. Until recently, there was no functional link between lateral polarity of a transporter and the consequence for the transport of its substrate in root cell layers. The characterization of the metal transporter IRT1, which localizes to endocytic compartments but stabilizes to the outer PM domain of the root epidermis under low-metal conditions, opens new perspectives (Barberon et al., 2014). Plants overexpressing FYVE1, a FYVE domain protein recruited to early and late endosomes, display an apolar IRT1 localization to the epidermal PM, associated with iron deficiency symptoms and low metal accumulation, suggesting that IRT1 polarity is a component for its function (Barberon et al., 2014; Zelazny and Vert, 2014).

PERSPECTIVES

The last decades represented a great advance in the understanding of root function in the transport of nutrients, with the identification of the main actors involved in uptake from the soil and loading to the xylem. Although they were described 150 years ago, the CS functions as a selective barrier are poorly understood (Caspary, 1865). This was mainly due to the lack of mutants affected in CS establishment and function. With the discovery of the first CS-associated proteins (CASP1–CASP5) and the identification of mutants with interrupted CS, we are now entering a new era (Roppolo et al., 2011; Hosmani et al., 2013; Lee et al., 2013). However, establishing the roles of CS in plant nutrition appears to be more challenging than expected. First, most mutants affected in CS formation are also affected in suberin lamellae deposition, putting the specificity of the observed phenotypes in question. Second, a mutant specifically affected for CS formation displays rather selective nutritional disorders that are difficult to rationalize (Pfister et al., 2014). The combination of CS mutants with mutants affected in nutrient uptake and distribution in roots will be instrumental to obtain clear genetic evidence of the roles played by CS as a barrier for nutrients.

On the other hand, the discovery of lateral polarization of transporters in endodermis but also in other root cell layers constitutes a great advance. While surprising at first, this feature appears to be conserved in different plant species and to concern different families of transporters, indicating a general mechanism of coupled trans-cellular transport for nutrients (Table I). In this context, the boric acid influx channel NIP5.1 and the boric acid/borate efflux transporter BOR1, localizing reciprocally to the outer and inner PM domains, are a model of choice (Alassimone et al., 2010; Takano et al., 2010). However, the underlying mechanisms of this polarity are still poorly understood. Further identification of mutations affecting lateral polarity of transporters, combined with high-resolution imaging of nutrients in root cell layers, should pave the way to a better understanding of the function of lateral polarity in plant nutrition in the coming years.

Glossary

PM

plasma membrane

CS

Casparian strips

PI

propidium iodide

CSD

Casparian strip domain