Planta

Planta (1982)156:108-116

9 Springer-Verlag 1982

Fractionation and characterization of cellular membranes from root tips of garden cress (Lepidium sativum L.) Thomas J. Buckhout, Liane Heyder-Caspers, and Andreas Sievers * Botanisches Institut der Universit/it, Venusbergweg 22, D-5300 Bonn 1, Federal Republic of Germany

Abstract. The first step in the gravitropic reaction chain, i.e. perception, is known to occur in the statenchyma of the root cap. Because of the importance of the root tip in graviperception, a procedure has been developed to isolate root tips from garden cress (Lepidium sativum L.). The root tip fraction contains the tissues of the root cap plus the lower half of the meristem zone, but is clearly separated from the tissues of the elongation zone, the zone of gravitropic response. Membranes from the root tip and root base fractions have been centrifuged on sucrose density gradients and the marker enzyme profiles analyzed. These results show that the marker enzyme profiles for vacuoles, dictyosomes, mitochondria, and plasma membranes are similar in the root tip or root base fractions. The endoplasmic reticulum (ER) has a shoulder of cytochrome c reductase activity at a density of 1.16 g cm-3 which is distinct from the other enzyme activities and is only observed in root tip preparations. The specific enzyme activity for ER, cytochrome c reductase, was enriched in root tip membranes 1.7 fold. This latter increase is interpreted as at least in part an increased ER content in the root tip.

Key words: Endoplasmic reticulum - Gravitropism - Lepidium - Membrane fractionation Root (membranes). Introduction

In the root the cells responsible for sensing the gravity force (statocytes) are located in the root * To whom correspondence should be addressed Abbreviations." ASG=6-acyl-steryl glucoside; ER=endoplasmic reticulum; IDP = inosine-5'-diphosphate; INT = 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazoliumchloride; P M = plasma membrane; SG = steryl glucoside

0032-0935/82/0156/0108/$01.80

cap (Sievers and Volkmann 1972; Barlow 1974; Juniper 1976; Volkmann and Sievers 1979; Jackson and Barlow 1981). The structure of these cells has been extensively studied, especially in Lepidium sativum L. (Iversen 1969; Sievers and Volkmann 1972, 1977a, 1977b; Volkmann 1974; Sievers et al. 1976). The structural polarity of the statocytes is not gravity induced but genetically determined, indicating a structural-functional relationship (Sievers et al. 1976). Although little is currently known concerning the mechanism of gravity perception and signal transmission, there are observations that might indicate important components in this process. For example, both gravitropic and agravitropic lateral roots contain sedimentable amyloplasts; however, only gravitropic lateral roots contain an underlying endoplasmic reticulum (ER) complex (Schmitz 1977). Or, when the starch in the amyloplasts is artificially depleted following hormone treatment, the roots no longer respond to gravity (Iversen 1969). Even short physical contact between the amyloplasts of the statenchyma and the distal ER has been suggested to be sufficient for graviperception (Hensel and Sievers 1981). In addition, the unique function of the statocyte ER may be related to the unique structure as indicated by the high capacity of the ER to bind Concanavalin A (Schneider and Sievers 1981). More recent results involving electro-physiological measurements in Lepidium roots (Behrens et al. 1982) show a symmetrical pattern of external current flow with current leaving the elongation zone and entering the root cap and meristem zone. After 90 ~ rotation to the horizontal an apparent reversal of current flow occurs. Current leaves the root cap on the upper surface of the root but enters the root cap on the lower surface. The authors interpret these results in terms of altered ion trans-

T.J. Buckhout et al. : Lepidium root tip membranes

port across the plasma membrane, which may be involved in graviperception. Thus, there are indications that membranes of the ER and the plasma membrane and the interaction of the amyloplasts with the ER may be important in the early reactions of the perception process. We have developed a procedure for isolation of root tips from Lepidium and report here an ultrastructural comparison of isolated and control root tips, the qualitative distribution of membranes on sucrose density gradients based on marker enzyme analysis and the quantitation of membrane content based on marker enzyme activity. Materials and methods Plant material. Seeds of Lepidium sativum L. cv Krause (ca. 400 ml dry volume) were soaked for 30 rain in tap water and spread evenly on wire mesh racks one layer thick. The seeds were grown in the dark at 24+__2~ C for 34 h. The roots protruding through the wire mesh were harvested and the cut roots stored in ice-cold 50 mM sucrose solution until needed (maximum storage 2 h). Roots grown for 34 h had an average, in situ length of 7.5 _+1.2 mm.

Isolation of root tips. Roots were collected by filtration through hylon cloth and chopped in 50 m M sucrose with a mechanical chopper similar to that described by Cunningham et al. (1966). The root material was chopped for 3 rain at approximately 240 cycles per rain, washed in 50 mM sucrose and mixed with 200 ml 20% Percoll (Pharmacia Fine, Uppsala, Sweden) solution in 50 m M sucrose and stood on ice for 5 min. The tissue separated at unit gravity into an interface (root base fraction) and a pellet fraction. The underlying pellet fraction was recovered and subsequently mixed with 40% Percotl and stood on ice as before. The resulting pellet was recovered and designated the root tip fraction.

Homogenization and centrifugation. Root material was homogenized with a mechanical razor blade chopper for 4 rain at approximately 400 cycles per min in 50 m M Tris(hydroxymethyl)aminornethane-HCL (Tris), pH 8.0, at 20 ~ C, 50 m M KCI, 14 m M mercaptoethanol, 1 mM (ethylene-bis(oxyethylenenitrile))tetraacetic acid (0.1 m M in the presence of MgC12), + 3 mM MgCI2, and 0.4 M sucrose. The ratio of tissue fresh weight to buffer volume was 1 : 2. The homogenate was filtered through a single layer of nylon cloth, centrifuged at 4,000 rpm for 15 min (HB 4 Sorvatl rotor) and chromatographed through a Sepharose CL-2B gel filtration column (Pharmacia Fine, Uppsala, Sweden). The column (2.10 cm) was equilibrated and eluted with homogenization buffer at a flow rate of 85 ml h-1. Fractions of 1 ml were collected and void-volume fractions pooled and used for subsequent membrane fractionation. Voidvolume fractions were cleanly separated from soluble, retained fractions as judged by carotenoid measurements (data not shown). The pooled void-volume fractions were layered onto a continuous, 1 . 5 - 0 . 4 M (1.6-0.4 M in high-Mg 2+ gradients) sucrose gradient and centrifuged for 4 h at 25,000 rpm (Spinco SW 27 rotor). All sucrose solutions were prepared in homogenization buffer minus mercaptoethanol. Gradients were fractionated from the bottom into 0.5 ml fractions and either analyzed immediately or frozen at - 2 0 ~ C until needed with the maximum storage time being 48 h.

109

Enzyme assays. ATPase and IDPase activity was determined by the measurement of released inorganic phosphate, using the modified method of Scherer and Morr~ (1978). Fractions to be tested for ATPase activity were chromatographed through Sephadex G50 columns (bed volume 2 ml) equilibrated with 0.1 M 2-(N-morpholino)ethanesulfonic acid-Tris buffer, pH 5.5, at 20 ~ C, plus 50 mM sucrose. Void-volume fractions were collected and 50 gl samples were taken for the ATPase assay. The assay was run in the above buffer plus 5 m M MgC12 plus or minus 50 m M KC1 in a total volume of 100 ~tl. The assay was started by the addition of 10 gl 30 m M ATP and continued for 30 rain at 30 ~ C. The reaction was terminated with 400 gl 10% trichloroacetic acid (TCA) and inorganic phosphate was measured. Non-specific ATP-phosphatase activity was taken as the enzyme activity in the absence of MgCI> IDPase activity was assayed plus or minus 0.1% Triton X-100, according to Bowles and Kauss (1976), in a total volume of 100 gl containing 50 gl enzyme fraction. Cytochrome c reductase was assayed in the presence of 22 gg ml-1 antimycin A essentially by the method of Hodges and Leonard (1972). The final assay volume was 500 gl, the reaction was run at room temperature, and n o n - N A D H reduction of cytochrome c was determined as the enzyme activity in the absence of added N A D H . Succinate-p-iodonitrotetrazolium violet reductase was measured in a total volume of 500 gl, containing 100 gl membrane fraction, according to Pennington (1961). The assay for c~-mannosidase was performed by the method of Boller and Kende (1979) as modified by Scherer (1981). Sterol-UDP-glucose transferase was assayed by a modification of the method of Hartmann-Bouillon et al. (1979). Membrane fractions (60 gl) were incubated for 3 rain at 30 ~ C with 20 gl 0.3 M Tris-HC1, pH 8.0, containing 0.8% Triton X-100 (w/v) and 1 mM mercaptoethanol and the reaction started with the addition of 20 gl 1.7 mM UDP-glucose containing 0.35 kBq UDP-[14C]glucose (Amersham Buchler, Braunschweig, FRG). The assay was continued for 30 rain and stopped by boiling for 1 rain. The isolation of steryl glucoside (SG) and 6-acylsteryl glucoside (ASG) was conducted as described by Quantin et al. (1980). Glucan synthetases were assayed by the procedure of Jesaitis et al. (1977). Glucan synthetase I was assayed at 30 ~ C for 30 min and glucan synthetase II at 30 ~ C for 45 min. The glucan synthetase II activity was linear for at least 1 h. Fraction density was determined by accurately weighing 100 ~tl samples, or refractometrically. Both methods gave identical results. Protein was determined following TCA precipitation by the method of Lowry et al. (1951) with bovine serum albumin as a standard.

Microscopy. For electron microscopy, roots or root tips were fixed in K M n O 4 (2%) at room temperature and subsequent dehydration and embedding were performed as described by Sievers and Volkmann (1972). Root tip and root base fractions to be analyzed by light microscopy were stained with iodophenol to visualize the statenchyma in the calyptra. Fraction purity was determined by counting the number of intact root tips under low-power magnification. The data is expressed as percent root tip tissue and represents the results of three determinations from each of four separate preparations. Root length was determined by measuring photographically enlarged root tips to the nearest 0.1 mm.

Results

The isolation and characterization of root tip fractions. The root fractionation procedure as de-

110

T.J. Buckhout et al. : Lepidium root tip membranes

Fig. l. Light micrographs of root fractious from Lepidium sativum L. Lepidium roots were fractionated as described in Materials and methods. The tissues were stained with iodophenol to visualize the starch-containing statenchyma. The root separated into two fractions designated root base fraction (a) and root tip fraction (b). Bar equals 1 mm

scribed in Materials and methods yields essentially two fractions of material. The first fraction is composed of the root base which consists of material extending 7.5 mm basal to the root tip but is almost devoid of calyptra and meristem tissue and designated root base fraction (Fig. 1 a). The second fraction, identified by iodophenol-stained amyloplasts in the statenchyma, contained 8 5 + 2 % structurally intact root tips of average length 0.54_+0.15 m m and was designated as the root tip fraction (Fig. I b). The resulting root tip preparation has been subjected to electron microscopic analysis to determine the effect of isolation on the ultrastructure of the statenchyma. Root tips isolated in the absence or presence of 100 or 250 m M sucrose contained statocytes with a reduced quantity of ER, vesiculated ER, and distorted cell polarity (data not shown). These artifacts were not observed with roots isolated in 50 m M sucrose. The strict polarity found in root statenchyma (Sievers and Volkmann 1972) is also found in the isolated root tips (Fig. 2a). That the amyloplasts are located directly above the distal ER and not randomly distributed, reflects the tip-first sedimentation of the root tips through the Percoll gradient during isolation. The ER is preserved as shortened cisternae (Fig. 2b) lacking the parallel structure found in control cells (Fig. 2c).

Membrane fractionation. An extensive study of marker enzyme distribution on sucrose density gradients has been conducted in Lepidium. Figures 3-8 are each the result of an analysis of a single sucrose gradient. The mitochondria as measured by the enzyme succinate-2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT)-re-

ductase equilibrate at a buoyant density of 1.181 g cm -3, regardless of whether the membranes are isolated from root tip (Figs. 3a, 4a) or root base fractions (Figs. 5 a, 6 a) or whether the membranes were homogenized in low-Mg 2+ (Figs. 3 a, 5 a) or high-Mg 2+ (Figs. 4a, 6a). The profile of INT-reductase activity was similar to that for cytochrome c oxidase (data not shown); however, the INTreductase activity was reported because of the nonlinear oxidase kinetics in Lepidium. The 0~-mannosidase activity has been used as a marker enzyme for vacuolar membranes (Boller and Kende 1979; Scherer 1981). In the absence of M g 2+, activity is found as a main peak at a density of approximately 1.125g cm -a with a prominent shoulder at 1.094 g c m - 3 (Figs. 3 b, 5 b), or with the addition of Mg 2+ as two peaks, one at 1.107g cm -3 and a second at 1.140g cm -3 (Figs. 4b, 6 b). Antimycin A-insensitive cytochrome c reductase equilibrates at a density of 1.124 g cm - 3 (Figs. 3 a, 5 a). In the presence of Mg 2 +, the peak of reductase activity is found at 1.178 g cm -3 (Figs. 4a, 6a). The activity profile for inosine-5"-diphosphatase (IDPase) is a single-peaked profile at a buoyant density of 1.132 (Figs. 3a, 5a) or in the presence of Mg 2+ 1.138 g cm -3. The activity of a second Golgi marker, glucan synthetase I (Ray et al. 1969; Morr6 et al. 1977), has been compared to the IDPase activity profile (Fig. 7). In the presence of 0.1% Triton, IDPase was stimulated greater than 4 fold. Both with or without Triton, the activity peak occurred at a density of 1.130 g cm - a. The profiles of the two enzymes were identical. The ATPase profile occurred as two clearly resolved peaks of activity (Figs. 3 b-6 b, 7, 8). In low-

T.J. Buckhout et al. : Lepidiurn root tip membranes

111

Fig. 2a-e. Electron micrographs of statocytes from the isolated root tip fraction (a and b) and a control root (e). The characteristic polarity found in statocytes is present in the isolated tips (a) with ER preserved as shortened cisternae (arrow, b) lacking the strictly parallel structure found in control cells (e, photograph from W. Hensel). The ER can be associated with lipid droplets and microbodies in isolated root tip and control statocytes. Fixation: KMnO4. A =amyloplast; ER=endoplasmic reticulum; L = lipid droplet; Mb = microbody ; N = nucleus. Bar equals 1 gm

Mg 2+ gradients, the low-density peak occurred at approximately 1.10 g c m - 3 and in high-Mg 2 + gradients at 1.13 g cm -3. The second peak of activity occurred at a density of 1.145 in low- or 1.152 g c m - 3 in high-Mg 2§ gradients. The relative distribution of enzyme activity between the two peaks was somewhat variable. Glucan synthetase II and sterol transferase often used as PM markers both

occurred as a single peak of activity at a density of ca. 1.140 g cm -3 (Fig. 8). In this same gradient, the K§ ATPase showed a prominent peak of activity at 1.140 g cm -3 and a second, minor peak at 1.10 g cm -3 (Fig. 8). There was no indication of a second peak in the profile of glucan synthetase II or sterol transferase, regardless of whether the steryl glucoside (SG) or the subse-

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