An Analytical Microscopical Study on the Role of the Exodermis in Apoplastic Rb+(K+) Transport in Barley Roots

An analytical microscopical study on the role of the exodermis in apoplastic Rb+(K+) transport in barley roots

M. Gierth∗, R. Stelzer and H. Lehmann Institut für Tierökologie und Zellbiologie, Tierärztliche Hochschule Hannover, Bünteweg 17d, D-30559 Hannover, Germany

Received: 29 June 1998. Accepted in revised form: 7 December 1998

Key words: Cryosectioning, endodermis, ion localisation, ion transport, rhizodermis, X-ray microanalysis

Abstract The paper investigates how the apoplastic route of ion transfer is affected by the outermost cortex cell layers of a primary root. Staining of hand-made cross sections with aniline blue in combination with berberine sulfate demonstrated the presence of casparian bands in the endo- and exodermis, potentially being responsible for hindering apoplastic ion movement. The use of the apoplastic dye Evan’s Blue allowed viewing under a light microscope of potential sites of uncontrolled solute entry into the apoplast of the root cortex which mainly consisted of injured rhizodermis and/or exodermis cells. The distribution of the dye after staining was highly comparable to EDX analyses on freeze-dried cryosectioned roots. Here, we used Rb+ as a tracer for K+ in a short-time application on selected regions of intact roots from intact plants. After subsequent quench-freezing with liquid propane the distribution of K+ and Rb+ in cell walls was detected on freeze-dried cryosections by their specific X-rays resulting from the incident electrons in a SEM. All such attempts led to a single conclusion, namely, that the walls of the two outermost living cell sheaths of the cortex largely restrict passive solute movements into the apoplast. The ring of turgescent living rhizodermis cells in the root tip region forms the first barrier. With increasing distance to the root tip, in the course of their maturation resp. degradation, this particular function of the rhizodermis cells is replaced by the hypodermis resp. exodermis. Furthermore, the restriction of apoplastic ion flow by the outermost cortex cell layers is rather effective but not complete. Thus, the solute transfer into the stele is mainly restricted by the casparian bands of the endodermis. The overall conclusion is that the resistances of the rhizodermis and exodermis are additive to the endodermis in their role of regulating the apoplastic solute movement across roots.

Abbreviations: EDXA – Energy Dispersive X-ray Analysis; EM – Electron Microscopy; LMX – Late Metaxylem; SEM – Scanning Electron Microscopy; STEM – Scanning Transmission Electron Microscopy

Introduction dermis, are very difficult. The root pressure probe provides a tool for measuring the total flow across Efficient barriers against apoplastic solute flows ori- the root. But neither between the three components of ginate in the deposition of hydrophobic substances, water flow described in the “composite flow model” e.g. suberin, in hypodermal and endodermal cell walls (Steudle, 1989) nor between different resistances e.g. of roots. The presence of casparian band like struc- of the exo- and endodermis can be properly distin- tures in the anticlinal walls of hypodermal root cells, guished. Most of the studies on that subject were done reflects the similar functions of both, the hypodermis with invasive techniques e.g. isolated hypodermis and the endodermis (Esau, 1969 and literature cited sleeves (Robards et al., 1979; Kochian and Lucas, therein). Studies on the permeability of the root cortex 1983; Shone and Clarkson, 1988) and sequential punc- cell walls, in particular of the exodermis and endo- turing of tissues from excised roots combined with the root pressure probe (Steudle et al., 1993; Peterson et ∗ E-mail: [email protected] al., 1993). In parallel, the casparian bands in the ra- 210 dial walls of these physiological sheaths were nicely Materials and methods demonstrated by their characteristic fluorescence visible under UV excitation after staining root cross sec- Plant growth tions with berberine sulfate/aniline blue (Brundrett et al., 1988). This technique has been successfully used Barley seeds (Hordeum vulgare L. cv. Alexis) were · −3 to screen for more plant species with an exodermis germinated for 7 days on aerated 0.2 mol m CaSO4 in their roots (Damus et al., 1997, Peterson, 1989, solution. After that, seedlings were transferred as Peterson and Perumalla, 1990) but also for testing single plants on aerated nutrient solutions (5 plants hydraulic conductivities (Cruz et al., 1992, Peterson per 3L solution and pot) of the subsequent compos- · −3 et al., 1993). Barley roots have also been investig- ition (data given in mol m ): 0.5 NH4NO3,0.7 ated for this anatomical feature, but were judged to K2SO4, 0.1 KCl, 2.0 Ca(NO3)2,0.5MgSO4,0.1 · −3 · −4 lack an exodermis (Perumalla et al., 1990). However, KH2PO4,0.02Fe-EDTA,1 10 H3BO4,5 10 · −4 · −4 · −5 · in the present study, barley roots were successfully MnSO4,2 10 CuSO4,1 10 ZnSO4,1 10 tested for an exodermis. In matured roots with an (NH4)6Mo7O24. exodermis, the diffusion of membrane-impermeable The solutions were renewed weekly. The condi- tracers used as markers for the apoplastic route of tions in a temperature and light controlled growth solute flows, generally ends at the “casparian bands” cabinet were 16 h fluorescent light (approx. 10,000 of the hypodermal cell layer (Peterson, 1987; Peterson Lux) and 8 h dark period. The plants had developed and Emanuel 1983; Peterson et al. 1978; Moon et al, 3–5 nodal roots up to 15 cm length after 21 days of 1984). This blockage of diffusion of large organic mo- cultivation (inclusive 7 d on calcium solution). lecules by hypodermal suberin incrustations appears to be representative for the smaller hydrated inorganic Light microscopy cations, such as potassium (Marschner, 1995; Clark- The water soluble stain Evan’s Blue (SIGMA) was son, 1996). Recent investigations on the properties of used as a marker for the apoplastic pathway of ra- radial apoplastic water transport in corn roots combin- dial solute transport across the root (Taylor and West, ing the application of the apoplastic dye PTS1 and the 1980). The root systems of intact plants were incub- root pressure probe showed that water transport was ated for 2–4 h in a nutrient solution containing 0,5% affected by the maturation of an exodermis (Zimmer- (w/v) Evan’s Blue. Afterwards the roots were briefly mann and Steudle, 1998). The permeation of the dye, rinsed and hand made cross sections, taken from the however, was uninfluenced by an exodermis leading tip regions (2–3mm) and the basis (8–10cm) were to the conclusion that PTS was not suitable for tracing viewed with a light microscope (ZEISS, AXIOLAB) water movement across roots. and photographed on an EKTACHROME 64T colour Until now, there are no direct measurements con- reversal film (KODAK). cerning the exodermis’ influence on the access of In combination with the sample preparation for inorganic ions to the root apoplast. Cryo-fixation of cryosectioning, root segments taken from above the roots in combination with EDX-analyses provide a excision zone were used for testing hypodermal fluor- tool allowing “direct measurements” in situ (Echlin, escence (Brundrett et al., 1988). Hand made cross sec- 1992; Newbury et al., 1986; Zierold, 1988). Using tions were kept for 60 min. in 0.1 % (w/v) berberine- these techniques, we analysed whether the exodermis sulfate solution, transferred to 0.5 % (w/v) aniline blue actually retains diffusive inorganic ions, especially solution for 30 min. and embedded in glycerine (50 % K+, from their passage into the apoplast of the root w/v) containing 0.1 % (w/v) FeCl . The sections were cortex of barley. To avoid difficulties in distinguishing 3 viewed and photographed under 450-490 nm UV-light. among cellular K+ and K+ being externally applied + Rb was used as a tracer. The validity of this method Cryo-preparation has already been confirmed by Kochian and Lucas + (1983) for investigating the main sites of K uptake One nodal root without laterals was pierced through in corn roots. the central hole of an Al-cryo-microtome holder. The holder was carefully moved towards the root basis (> 10 cm behind the tip) or to the root apex respectively (1–1.5cm behind the tip). Approximately 10 µl − 1 trisodium 3-hydroxy-5,8-10-pyrenetrisulfonate of a 60 mol · m 3 RbCl solution were supplied for 211

120 s to the root close to the surface of the holder. ical segments of roots treated with RbCl). Of every The last few seconds were used to remove the surface root different sections were analysed until at least 5 adhering solution with a filter paper, to cut the root measurements existed for each particular cell wall loc- approximately 1.5–2 mm above the holder surface, ation. The means of p/b ratios at each position were and finally to submerge the assembly into liquid pro- pooled and standard deviations of the means were cal- pane. In addition to every set of 5 replications, control culated between different roots of every experimental roots without RbCl were frozen and analysed in the set. One given p/b-ratio includes the mean of 15–25 same way. For cryo-sectioning the sample holder with single measurements for a particular cell wall location. the frozen root segment was locked in the precooled microtome holder (ULTRACUT FC 4 E, REICHERT, Preparation of the EDX-standards Austria). The temperatures for knife and sample were The procedure aimed at obtaining standards with sizes kept at 228 K and for the chamber at 163 K. First of formed ice crystals being of similar size as those in of all, approximately 250µm of the root stub was the analysed tissues. To limit ice crystal growth during removed stepwise with a glass knife. After this, the freezing, barley root cell walls were used as a frame- sample was trimmed with a metal knife so that the flat ◦ work. First the roots were killed by freezing in liquid tip of the “pyramid” showed only a 90 sector of a nitrogen and afterwards thawed in distilled water at transversely sectioned root. A diamond knife was used 293K. Prepared root ghosts were cut into segments, for making 300 nm sections, to be used for analytical washed several times in distilled water and vacuum EM purposes. A number of 10–15 frozen-hydrated infiltrated with solutions containing 20% (w/v) Dex- sections were picked up with an eye-lash and trans- tran 500 (SERVA, Heidelberg) plus 0, 25, 50, 100 ferred to the polished surface of a precooled Cu-stub. mol · m−3 RbCl and KNO . Dextran was added to A deeply cooled 150 mesh Cu-grid was mounted onto 3 give an organic matrix comparable to that of the cell the sections and with the polished surface of a cooled walls (Frey et al., 1997). The infiltration of the root metal rod this assembly was pressed onto the surface ghosts with the solutions was performed in an exsic- of the solid Cu-stub. After removing the Cu-grid the cator by 5–10 periodic evacuating/aerating cycles. The Cu-stub with the adhering cryo-sections was stored root segments remained in the infiltration solutions in liquid nitrogen until examination. The transfer of over night and were frozen in liquid propane the next the cryo-sections to the precooled cryo-stage (77 K) morning. Sectioning, transfer, and analyses were done of the SEM (ETEC-AUTOSCAN) occurred under li- in the same way as described for the samples. quid nitrogen by means of self made equipment which largely excluded contact with atmospheric water va- pour to prevent ice crystal contamination. The sample Results was freeze-dried under electron optical control within the column of the SEM. The X-ray spectra were taken Under UV light the basal segments of nodal roots, for 45 s (dead time corrected) from cell walls and from taken from above the nutrient solution level, showed standards respectively by an X-ray detector (KEVEX) fluorescent radial walls in the exodermis (Figure 1). and evaluated by a multichannel analyser (NORAN, Apart from this, the radial and inner tangential walls TN 5402). For Acquiring EDX spectra from cell walls of the endodermis, the cell walls in the stele, except the electron beam was focussed on an area not larger those of phloem and pericycle, did also show fluores- than 0.3–0.4 µm2 at 15 000 × magnification in the re- cence. In contrast, there was no fluorescent exodermis duced area mode (TV-scan). The accelerating voltage in younger root portions close to the root tip and in was 20 kV with a penetration depth of the electron basal root portions, which were permanently covered beam of ≥1000 nm for organic material (Newbury with nutrient solution (Figure 2). Fluorescent stelar et al., 1986). The quantitative evaluations of X-ray tissues were the same as in the basal root portions. spectra were based on elemental peak/background ra- The number and locations of late metaxylem vessels tios from samples in comparison to those of especially (LMX) were used to distinguish between cross sec- prepared standards. tions from different roots with a light microscope. Seventeen roots of different plants (one root per Matured nodal roots comprised several LMX vessels plant) were included into the study (5 basal segments circularly allocated in the stele (Figures 1–3) whilst of control roots, 5 basal segments of roots treated seminal roots generally exhibited a solitary LMX ves- with RbCl, 4 apical segments of control roots, 3 ap- sel within the centre. Figure 3 shows an intermediate 212

Figure 1–5. The fluorescence micrographs (1–3) show hand made cross sections of nodal barley roots stained with berberine sulfate/aniline blue to demonstrate the presence of casparian bands in the exodermis. Figure 1. Approx. 100 mm behind the root apex. The radial walls of the exodermis and endodermis show bright fluorescence indicating the presence of casparian bands. Figure 2. Approx. 10 mm behind the root apex. Note that there is no fluorescence, particularly not in the radial cell walls of the hypodermis. Figure 3. Micrograph showing an intermediate state of exodermis maturation. Only part of the radial hypodermal cell walls show fluorescence i.e. casparian band formation. The bright-field micrographs (4–5) show hand made cross sections of nodal barley roots stained for 2 h. with the apoplastic dye Evan’s Blue. RHD: rhizodermis EX: exodermis (hypodermis). Figure 4. Section from approx. 1 mm behind the root apex shows the dye preferentially localised within the mucigel layer on the root surface. On places where particular cells are ruptured (arrow) the dye invaded the outer tangential walls of the hypodermis (EX). Figure 5. Section taken from approx. 100 mm behind the apex shows that the dye generally has moved towards the radial walls of the exodermis (arrow). Note the gaps among the rhizodermis cells (RHD). 213

Figure 6. Locations on tissue sections for taking X-ray measurements. (a) Secondary electron micrograph showing the surface of a hydrated 300 nm cryosection across a barley root taken from approx. 100 mm behind the tip. Some intercellular spaces, cell wall thickenings and especially cell walls of the rhizodermis (RHD), the exodermis (EX) and parenchymatous cortex cells are visible. The vacuoles contain large ice crystals. Bar = 20µm; (b) The same section as in a) after partial freeze-drying in the vacuum of the SEM. Bar = 20µm. (c) Schematic representation of a root cross section showing the positions of the electron beam during EDX-analyses. 1: rhizodermis; outer tangential wall 2: rhizodermis; inner tangential wall 3: hypodermis; inner tangential wall 4: wall of the ﬁrst cortex cell 5: wall of the second cortex cell 6: endodermis; inner tangential wall 7: wall of the metaxylem vessel state of exodermis maturation where not every radial of the rhizodermal radial walls were clearly marked. exodermis wall shows ﬂuorescence. Occasionally, in the proximity of an injured cell the The results from Evan’s Blue staining for mark- outer tangential walls of the hypodermis and the in- ing potentially available apoplastic pathways of solute tercellular spaces were excessively stained by the dye movement are shown in Figures 4 and 5. The root tip (arrow in Figure 4). In basal root segments possessing region, close behind the meristematic zone, is shown an exodermis the walls of the rhizodermis cells and the in Figure 4. The mucous rhizosphere, the outer tan- outer tangential and portions of the radial walls of the gential cell walls, and the spaces towards the middle exodermis were marked by the dye (Figure 5). 214

Surveys of cryosections as they were used for EDX-analytical purposes are shown in Figure 6. The fully hydrated cryosection in Figure 6a exhibits the walls of the rhizodermis, hypodermis and some root cortex cells. The interior of the cells is filled with more or less regularly shaped ice crystals and makes an orientation rather difficult. After freeze drying (Fig- ure 6b) it is much easier to recognize all the sites in the cells and tissues of interest e.g. for positioning the electron beam for sampling the element specific X-rays. The summary of all electron beam positions for taking X-ray spectra is shown in Figure 6c. The encircled numbers correspond to those given on the abscissas in Figure 7 and 8. Highest p/b ratios for Rb+, representing clearly detectable Rb+ concentrations (30–35 mol · m−3,Table 1), were found in the outer tangential walls of the rhizodermis from both the basal (Figure 7a) and the apical root regions (Figure 7b). The p/b ratios for Rb+ decline sharply between the rhizodermis and hypodermis in the basal region (Figure 7a) and in the apical region almost between the outer and inner tangential wall of the same cell layer, namely of the rhizodermis Figure 7. p/b ratios of Rb in different cell walls of freeze-dried root cross sections. a: basal root segments; b: apical root segments. The (Figure 7b). Other p/b ratios given in Figure 7 were outermost cell wall is the outer tangential wall of the rhizodermis + not significantly different from the Rb blank (0,025, (distance to the root surface = 0). The horizontal line marks the p/b − horizontal line in Figure 7) including the walls of basal ratio of standards containing 0 mol · m 3 Rb (blank). The numbers and apical segments from control roots. The p/b ra- on the abscissa refer to the positions as shown in Figure 6c. Mean + values and standard deviations (vertical bars). n = 3–5 (see materials tios for K were always above the blank (0.015 in and methods). Figure 8). In the basal root zones they showed transient increases culminating at the locations 4 for the controls or 5 for the RbCl treated varieties respect- Discussion ively (Figure 8a). In the apical root segments the RbCl treated variety showed a sharp transient increase cul- The application of large organic molecules as e.g. minating at the inner tangential wall of the rhizodermis Evan’s Blue to delineate the apoplastic pathway is a i.e. location 2 in Figure 8b. In basal segments of the rapid method which provides illustrative results. Un- + control roots, however, the p/b ratios for K decreased fortunately, this technique has also some drawbacks steadily from location 1 down to number 7. so the results are not as specific as it would be desired + + The Rb and K concentrations, estimated by (Zimmermann and Steudle, 1998). One technical comparing p/b ratios of the samples with those of problem is the damage of cells during cutting. The the dextran standards, are shown in Table 1. In the other one arises from the fact that the diffusivity of outermost cell walls of both the basal and apical large uncharged or negatively charged dyes might be + root segments the Rb concentrations ranged between quite different from that of hydrated inorganic ions. − + 31–36 mol · m 3 (Table 1). The calculated K con- (Bayliss et al., 1996). For these reasons the micro- centrations, however, varied in a wide range showing graphs of dye distribution can merely be used to minimum values in the inner tangential walls of the illustrate that during an exposure time of 2–4 h the − endodermis (∼20 mol · m 3, Table 1) and maximum diffusion of large organic molecules is restricted by values in the cell walls of the inner cortex (50–100 mol the radial cell walls of intact i.e. living and turgescent − · m 3,Table1). rhizodermis cells (Figure 4). In the case that rhizodermis cells were ruptured by cutting or had already reached the end of their natural life cycle, the dye was excessively retained by the radial walls of the 215

Table 1. Rb and K concentrations in different cell walls of freeze-dried barley root cross sections as estimated by comparison with dextran standards. Numbers refer to the positions of measurements explained in Figure 6c. Identical letters mark signiﬁcantly different concentrations (α=0.05). Statistical analyses were performed using the SAS program package.

Rubidium Controls RbCl incubated − [mol ·m 3] basal apical basal apical rhd otw 1 – – 36 31 hd otw 2 – – 21 – hd itw 3 – – – – cc 1 4 – – – – cc 2 5 – – – – ted itw 6 – – – – mx/lmx 7 – – – –

Potassium Controls RbCl incubated − [mol ·m 3] basal apical basal apical rhd otw 1 29a 70 28f 22 hd otw 2 37bm 59 54gil 94lm hd itw 3 40c 65 67j 57 Figure 8. p/b ratios of K in different cell walls of freeze-dried root cc 1 4 78abde 68 84fk 69 cross sections. (a) basal root segments; (b) apical root segments. c ghn n Outermost cell wall is the rhizodermal outer tangential wall (dis- cc 2 5 69 –9738 d hij tance to root surface = 0). The horizontal line marks the p/b ratio ted itw 6 23 –19– − of standards containing 0 mol · m 3 K (blank). Numbers on the mx/lmx 7 29e 43 35k 36 abscissa refer to the positions of measurements shown in Figure 6c. Mean values and standard deviations (vertical bars). n = 3–5 (see materials and methods). hypodermis (=exodermis) (Figure 5). For the access stage (93 K) of the SEM. Furthermore, cell structures of inorganic ions like K+ or Rb+ to the root apoplast as walls, vacuolar contents and cytoplasmic structures these results should not be overestimated. To obtain sustained the excessive electron bombardments, un- more reliable results a direct and non-invasive tech- avoidably occurring during EDX-analyses, better on nique, the electron probe microanalysis in a SEM was a solid metal stub than on a grid. A sufficient lateral employed. The application of cryotechniques avoids resolution for EDX-analyses obtainable with cryosec- the dislocation of soluble ions and it allows the use tions has its price in the decrease of the signal to noise of intact plants and defined time intervals between ratios (p/b) for the elements caused by the decrease solute application and fixation (freezing). The use of in mass thickness. According to our present experi- cryosections instead of bulk-frozen material gives a ence, the shown p/b ratios from 300 nm cryosections better lateral resolution for analysing structures smal- in average were 8–10 times lower than in bulk frozen ler than 1 µm in diameter, e.g. root cortex cell walls, material. by EDX (Zierold, 1986; Frey et al., 1997). In this All detected K+ concentrations vary considerably. study cryosections of plant tissues were fixed on the The averaged K+concentrations in the cell walls of the polished surface of a deeply cooled metal stub and controls range between 20 and 80 mol · m−3 (Table analysed in a SEM. One advantage in comparison with 1). In the apical root segments the variations among the use of a metal grid is the better temperature ex- the various sites of measuring were not significantly change between the cryosections and the solid metal different (Table 1). In the basal root portions, however, block. Thus, we could investigate the cryosections in the K+ concentrations increased significantly between a fully frozen-hydrated state for more than one hour outer cell walls (sites 1 and 2) and the inner cor- under the vacuum conditions (1.30 mPa) on the cryo- tex cell walls (sites 4 and 5) with absolutely lowest 216

K+ concentrations in the outer tangential walls of the ates that the apoplastic Rb+ trespass in young roots rhizodermis (Table 1). was prevented in the radial walls of the rhizodermis It is possible that the observed K+ concentrations or in the radial walls of the exodermis with preceding in the cell walls may be influenced by elastic scattered differentiation respectively. Additionally it can be ex- electrons, e.g. deriving from the holder surface be- cluded from this result that substantial symplastic ion low the section which cause emission of K-specific transport occurred under the experimental conditions. X-rays from adjacent cytoplasm. This is supported by Our direct measurements on freeze-dried cryosections the commonly accepted opinion that high K+ con- largely support recent studies on corn, onion and centrations are mainly confined to intracellular com- sorghum using diffusive fluorescent dyes to mark the partments and indicate the presence of intact living apoplastic route for radial ion movement (Peterson, cells (Leigh and Wyn Jones, 1984). In this respect the 1988; Enstone and Peterson, 1992; Cruz et al., 1992). measured K+ concentrations might rather reflect the Kochian and Lucas (1983) concluded from their protoplast vitality than the factual K+ concentration results with 2 cm corn root segments that have been in the cell wall. So, in the case that apoplastic con- exposed to toxic [203Hg]PCMBS1 and NEM2 for up centrations are focussed at, the cryosections should be to 20 minutes that the exodermis is no barrier to mounted on a Cu-grid. The price for a better lateral solute movement because in root cross sections of resolution, however, is a decrease of its persistency freeze-substituted roots they found the complete root during X-ray measurements. labelled with 203Hg. Nevertheless they concluded that In this study, the most reliable information were K+ influx into the root symplast is limited to the root obtained from the Rb measurements. To detect periphery at least at low K+ concentrations. Rb+ specific X-rays exclusively from the cell walls, It was surprising to see that in apical root segments a steep Rb+ concentration gradient (0 : 60 mol · m−3) the entry of Rb+ into the apoplast was already restric- and a short time of application (120 s) were chosen. ted on its way from the outer to the inner tangential The distance Rb+ should have moved by diffusion wall of the rhizodermis (Figure 7b). It appears very during that time can be estimated from calculations likely that in apical regions of the root, where the exo- using Fick’s second law [s = (t · D)1/2, where s is the dermis is not yet matured, the restriction of free apo- distance, t is the time of application and D is the diffu- plastic ion movement is achieved by the living cells of sion coefficient]. The diffusion coefficients for Rb+ in the rhizodermis. The mechanism of this ion blockage, water (Deq) and in plant tissue (Dtiss) were taken from however, is unknown. Presumably mucilages adher- −9 2 Aikman et al. (1980) (Daq =2.1· 10 m /s; Dtiss = ing to the surface e.g. mucopolysaccharides and pectic 0.19 · 10−9 m2/s). If the movement of Rb+ within the substances could be involved in that (Marschner, 1995; root apoplast was due to free diffusion than it should McCully and Boyer, 1997). have penetrated approximately 500 µm into the tissue. We conclude from the comparisons of the two Even if a ten times lower coefficient is assumed, as it developmental stages that during maturation the exo- was found for the tissue of sugar beet root by Aikman dermis increasingly supports, completes and replaces et al. (1980), Rb+ penetration should have progressed some particular ionselective functions of the rhizo- up to a depth of 160 µm, i.e. approximately between dermis. In the apical part of the root the uncontrolled the the fourth and fifth cell row beneath the root sur- en try of Rb+ as well as the entry of Evan’s Blue into face. It was also assumed that the Rb+ accumulation in the apoplast of the cortex are mainly restricted by the the protoplast did not exceed detectable concentrations living cells of the rhizodermis. The nature resp. the within that short time. substances hindering ion diffusion into the cortex cell Both, the p/b ratios of the blanks consisting of wall system, however, are not known. This type of cryosectioned dextran-solutions without Rb+ and of an ion blockage probably is necessarily not very ef- standards containing 20 mol · m−3 Rb+ vary in the fective because the early rhizodermis is a temporary range of 0.025. Most likely, the data below the ho- structure. In the older, more basal localised root seg- rizontal line in Figure 7a are below the detection ments the degradation of rhizodermis cells proceeds threshold of our assay. Rubidium was exceptionally and consequently the ion blockage by its living cells present in the outer tangential walls of the rhizo- becomes more and more precarious. This goes in hand dermis from young roots and up to the outer tangential walls of the exodermis from old roots. The absence 1 + p-chloromercuribenzene sulfonic acid of Rb from the more inner cortex cell walls indic- 1 N-ethyl maleimide 217 with the exodermal maturation in particular by differ- Kochian L V and Lucas W J 1983 Potassium transport in corn roots. entiation of casparian bands and encrusting their cell II. The significance of the root periphery. Plant Physiol. 73, 208– walls with suberin. Along this line of arguments the 215. + Leigh R A and Wyn Jones R G 1984 A hypothesis relating crit- externally applied Rb ions became detectable in the ical potassium concentrations for growth to the distribution and outer and inner tangential cell walls of the rhizodermis functions of this ion in the plant cell. New Phytol. 97, 1–13. but not in the inner tangential wall of the exodermis Marschner H 1995 Mineral nutrition of higher plants. Academic Press, London, San Diego, New York, Boston, Sydney, Toronto. (Figure 7a). For the whole plant the resistance of the pp. 537–595. rhizodermis and exodermis to solute movement across McCully M E and Boyer J S 1997 The expansion of maize root- the root apoplast are considered to be additive. They cap mucilage during hydration. 3. Changes in water potential and are supported by and/or support the endodermis in its water content. Physiol. Plant. 99, 169–177. Moon G J, Peterson C A and Peterson R L 1984 Structural, chemical particular function on the radial ion movement. None and permeability changes following wounding in onion roots. of these facilities alone might provide a perfect barrier Can. J. Bot. 62, 2253–2259. to uncontrolled ion entry into the stele. Newbury D E, Joy D C, Echlin P, Fiori C E and Goldstein J I 1986 Advanced Scanning Electron Microscopy and X-Ray Microanalysis. Plenum Press, New York and London. 454 p. Perumalla C J, Peterson C A and Enstone D E 1990 A survey of an- Acknowledgements giosperm species to detect hypodermal casparian bands. I. Roots with uniseriate hypodermis and epidermis. Bot. J. Linn. Soc. 103, 93–112. The authors thank Prof Dr Gerd Bicker for critical Peterson C A 1987 The exodermal casparian band of onion roots reading the manuscript and his comments on language blocks the apoplastic movement of sulfate ions. J. Exp. Bot. 38, and style. The valuable help of the staff from the In- 2068–2081. stitute of Biometry, School of Veterinary Medicine Peterson C A 1988 Exodermal casparian bands: their significance for ion uptake by roots. Physiol. Plant. 72, 204–208. Hannover is gratefully acknowledged. This work was Peterson C A 1989 Significance of the exodermis in root function. supported by the Deutsche Forschungsgemeinschaft In: Structural and functional aspects of transport in roots. Ed. (Schwerpunktprogramm “Apoplast”; Ste 312/ 9–2). B.C. Loughman, 0. Gasparikováˇ and J. Kolek. pp. 35–40. Kluwer Academic Publishers, Dordrecht. 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