An Analytical Microscopical Study on the Role of the Exodermis in Apoplastic Rb+(K+) Transport in Barley Roots
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Plant and Soil 207: 209–218, 1999. 209 © 1999 Kluwer Academic Publishers. Printed in the Netherlands. An analytical microscopical study on the role of the exodermis in apoplastic RbC(KC) 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 demon- strated 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 RbC as a tracer for KC in a short-time application on selected regions of intact roots from intact plants. After subsequent quench-freezing with liquid propane the distribution of KC and RbC 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 vis- ible 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 % KC, 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 KC and KC being externally applied C Rb was used as a tracer. The validity of this method Cryo-preparation has already been confirmed by Kochian and Lucas C (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 respect- ively (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.