3.6.4  Barriers to apoplasmic flow

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Figure 3.23 (a) Scanning electron micrograph of an isolated endodermis (large cells) and pericycle (small cells) prepared from a barley root. Radial walls of the endodermal cells are tightly appressed because of the apoplasmic discontinuity created by suberisation of these walls. Scale bar = 40 µm. (b) Transverse sections of endodermal cells of barley roots taken 4 cm from the root apex. Roots were plasmolysed to reveal the attachnxent of plasma membrane to Casparian bands (CB) which is typical of State I endodermis. Apoplasmic discontinuity is achieved by the hydrophobic barrier Casparian strips impose in radial walls. Roots were fixed in glutaraldehyde then osrnium tetroxide, dehydrated and embedded in epoxy resin. Scale bar = 0.5 µm (Both figures courtesy A.W.R. Robards; reproduced with permission from Academic Press)

Ionic composition of soil solution is not strongly modified while passing through cell walls. Weak charge fields around wall polymers adsorb some divalent cations but monovalent cations and anions pass through largely free of interactions. However, hydrophobic layers in specialised cell walls force ions to cross membranes and provide important sites for selectivity. Ions either follow electrochemical gradients into cells via channels in membranes or are pumped via energy-dependent carriers located in membranes (Section 4.2). Rapid ion uptake is possible through channels when electrochemical gradients strongly favour influx (e.g. calcium) whereas influx of cations such as potassium (at low concentrations) and anions such as nitrate and orthophosphate is achieved by energy-dependent transporters (Chapter 4).

(a)  Endodermis

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Figure 3.24 Electron micrograph of a transverse section pf Puccinellia peionis root showing a mature endodermal cell (TE) and adjacent passage cell (PC). Chloride ions supplied to roots while they were intact were subsequently precipitated using silver ions (Ag+), leaving electron-dense AgCl at the barrier imposed by suberin (S). Chloride ions arriving from the inner cortex (IC) must travel either symplasmically to the stele or cross through a relatively scarce passage cell (PC). This root was grown in a non—aerated solution containing 200 mM NaCl then sectioned 1 cm from the apex. The root was fixed in buffer containing osmium tetroxide and embedded in uranyl acetate. Scale bar = 1 µm (Photograph courtesy R. Stelzer)

An endodermal cell layer constitutes the prime barrier to apoplasmic flow in roots (Figure 3.23a). State I endodermis forms within a few millimetres of the root apex when a single layer of cells around the stele lay down hydrophobic polymers of suberin and lignin in transverse and radial walls, leaving longitudinal walls unchanged (Figure 3.23b). Phenolics in these deposits can be stained to reveal the Casparian bands. Even in this early phase, Casparian strips begin to lower the permeability of cell walls to water and solutes, especially helped by tight adherence of cytoplasm to the suberised walls. Further differentiation of Casparian strips occurs within centimetres from the apex, where suberin lamellae tighten the apoplasmic seal (State II endodermis). Ultimately State III endodermis forms by deposition of cellulose around endodermal cells. In grasses, outer tangential walls have less cellulose than other walls but often all endodermal walls are thickened leaving only pits to preserve plasmodesmal continuity between cortex and stele. The effectiveness of this seal is shown when Ag+ is used to precipitate Cl ions supplied to roots of Puccinellia peisonis (Figure 3.24). Suberised regions of the endodermal cell wall prevent further progress of Cl towards the stele. The endodermis minimises both passive leakage of ions out of the stele (when concentrations exceed those in the cortex) and unrestricted apoplasmic flow of ions into the stele.

The importance of root structure and the endodermis, in particular, for ion uptake was recognised early this century and led Crafts and Broyer to propose in 1938 that ion transport entailed a passive leakage of ions into xylem vessels after an initial concentration step in the symplasm. Ion leakage was attributed to O2 deficiency in the stele. This model recognised structural features of roots and specifically coupled the endodermis as a barrier to ion movement with its capacity to impede O2 supply to respiring stelar cells. This mechanism has never been verified experimentally but it has attractive features such as an ‘anaerobic core’ which has since been identified in roots growing at diminished O2 levels (Section 18.2). The concept of an anaerobic core has been exploited to show that Cl influx into roots exposed to hypoxia was suppressed much less than Cl transport to xylem vessels, providing good evidence that an energy-dependent step was involved in Cl transport across the stele to xylem vessels (Table 3.3). This does not support passive leakage and is potent evidence for energy-dependent unloading of ions into xylem.

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Carrier molecules and channels are central to ion transport and help explain ion release into xylem vessels described by Pitman (1972). Much early experimental evidence for energy dependence of ion transport came from the use of respiratory inhibitors. Inhibitors, however, reveal nothing about sites of active ion uptake because they perfuse the whole root and alter metabolism generally by ATP deprivation. Membrane damage and associated ion leakage from cells are a common outcome of inhibitor treatment. Other ways to study the net influx of ions into roots have been through radiotracers such as 32P, 36Cl and 86Rb (a potassium analogue) but these experiments have limitations in that penetration of the isotopes deep into root tissues is too slow to allow firm conclusions on sites or rates of ion uptake in steady-state conditions. Discriminating short-term influx from longer-term net flux (influx minus efflux) is often fraught because optimal labelling times are tissue specific and therefore highly empirical. Approaches in which carrier and channel proteins are immunolocalised might reveal sites of uptake but tell little about ion fluxes at those sites in intact roots. In view of the specialised role of stelar cells in ion efflux, it might be possible to immunolocalise proteins involved in ion unloading separately from those which load ions into the cortex. A similar approach has led to immunolocalisation of ATPases involved specifically in phloem unloading in bean seed coats.

(b)  Exodermis

The exodermis is a second layer of root cells which imposes a barrier to radial transport processes in most species studied (Perumalla and Peterson 1985). As in an endodermis, Casparian strips restrict radial apoplasmic movement of ions but the exodermis forms in a layer of cortical cells beneath the epidermis (Figure 3.22). Exodermal layers become functionally mature 20–120 mm from the apex, where lateral roots are initiated, and therefore only constitute a barrier to apoplasmic ion flow in root zones where an endodermis is already present. In a similar way to the endodermis, maturation of an exodermis involves further deposition of cellulosic wall material, further impeding flow of solution through walls.

Individual passage cells (Figure 3.24) in both endodermal and exodermal layers allow apoplasmic passage of ions and therefore provide points of low radial resistance (Clarkson 1996). How and why passage cells form is unknown. Some families (e.g. irises) have large numbers of passage cells while others have very few.

Studies cited here describe roots with only primary tissues. Secondary thickening dramatically alters rates of water and nutrient influx because endodermal and cortical tissues of dicotyledonous plants are replaced by secondary phloem and a cork-like layer covered with bark. Permeability changes are discussed in Section 3.6.6. Woody roots might not even take up water or nutrients, or may only do so when supply to younger roots is severely limited or gradients into the root are very steep. Monocotyledonous roots have no secondary thickening but none the less form a thorough seal from soil by maturation of an endodermis and degradation of cortical cells.

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