Modifications of Etioplasts in Cotyledons during Prolonged ... - ECOBIO

Plant Physiol. (1994)106: 1555-1565

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings’ Identification of Etiolation-Related Plastidial Aminopeptidase Activities Abdelhak El Amrani, Ivan Couée*, Jean-PierreCarde, Jean-PierreCaudillère, and Philippe Raymond Station de Physiologie Végétale, lnstitut National de la Recherche Agronomique, BP 81, 33883 Villenave d’Ornon Cedex, France (A.E.A., I.C., J.-P.C., P.R.); and Université Bordeaux 1, Laboratoire de Physiologie Cellulaire, Végétale, Unité associée Centre National de la Recherche 568, 33405 Talence Cedex, France (J.-P.C.)

provide a continuous and sufficient supply of carbon and nitrogen for seed germination and early development until breaking to light and development of the photosynthetic apparatus (Mullet, 1988). Thus, the control by carbon and nitrogen availability of etioplast development and of the conversion of etioplasts into chloroplasts has received little attention. Furthermore, the situations that have generally been considered are Glc excess and nitrogen limitation (Avelange et al., 1990). However, the supply of carbon during early development can become limiting when germinationtakes place in difficult seedbed conditions with delayed breaking to light. We have previously shown that, in the case of sugar beet (Beta vulgaris L.) seedlings grown under optimum conditions in the dark at 2OoC, SUC,glutelin, and lipid reserves were rapidly depleted and Glc levels in hypocotyls and cotyledons declined as early as 6 to 8 d after imbibition, whereas Arg and Asn were accumulated, thus indicating sufficient nitrogen supply (El Amrani et al., 1992). Indeed, cotyledons, which are the initial site of heterotrophy-autotrophy transition, since the gemination of sugar beet seeds is epigeal, were shown to be in a situation of Glc limitation, with a decrease of the total adenine nucleotide pool (El Amrani et al., 1994), and an increase of ammonium-scavenging amino acids such as Asn and Arg (El Amrani et al., 1992), thus suggesting, in accordance with previous studies of Glc starvation (James et al., 1993), that proteolysis was activated. Furthermore, it has been shown that lipid degradation and fatty acid /3-oxidation could be activated by Glc starvation (Dieuaide et al., 1992). These processes, occumng after the depletion of reserves, would thus affect structural proteins and lipids. The onset of Glc limitation between d 6 and d 8 after imbibition (El Amrani et al., 1992, 1994) coincided with a decline of greening capacity in cotyledons as measured by the rate of Chl synthesis under exposure to light (El Amrani et al., 1994). Burke and Oliver (1993), studying optimal thermal environments for seedling establishment during epigeal germination, have also related poor Chl accumulation

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris 1.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greeningcapacity. However, this situation of efficient greening capacity was shortlived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.

The transition from heterotrophy to autotrophy is a critical event during the early growth of seedlings (Burke and Oliver, 1993). This requires the differentiation of proplastids into the much larger chloroplasts containing the thylakoid membranes. Many steps of chloroplast development can occur in the absence of light, thus leading to etioplast formation, especially in monocotyledonous plants. In contrast, in dicotyledonous plants, leaf and chloroplast development is greatly inhibited in the absence of light (Mullet, 1988). In green algae, this chloroplast development is also controlled by nutritional factors such as oxygen concentration and carbon and nitrogen availability (Mullet, 1988). In the case of higher plants, however, it is often assumed that seed reserves This work was partly funded by the Institut Technique Français de la Betterave Industrielle. * Corresponding- author; fax 33-56843245.

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Abbreviations:AA-NH-Nap, L-aminoacyl-2-naphthylamide; Nap, 2-naphthylamine;6PGDH, gluconate-6-phosphate dehydrogenase. ~. . -

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with low soluble sugar content in cucumber cotyledons, The structural damage resulting from Glc h i t a t i o n could have affected a number of cellular targets. We have focused our study on plastids. The biogenetic development of chloroplasts from proplastids or etioplasts requires the accumulation of proteins through nuclear gene expression, subsequent protein import, and plastidial gene expression (Dahlin and Cline, 1991) with a regulation at the transcriptional and posttranscriptional levels (Klein and Mullet, 1986). In parallel, degradation of specific proplastidial, or etioplastic, proteins such as NADPH-Pchlide oxidoreductase and other prolamellar body proteins must take place (Dahlin and Cline, 1991). Furthermore, carotenoid and Chl synthesis depends on the activity of plastidial enzymes. Any of these mechanisms could function improperly during prolonged etiolation either through the lack of energy substrate when the conversion is initiated by light or through intrinsic damage. In the case of sugar beet, we showed the detrimental effects of prolonged dark growth, such as the loss of the greening capacity, not to be fully compensated by the exogenous addition of Glc (El Amrani et al., 1994). Therefore, we studied purified plastids from cotyledons at different stages of etiolation to identify at what level structural damage may occur. We thus show that etioplast development in the dark results in significant protein loss and in activation of aminopeptidase activities that are inactivated by light and by Glc. These biochemical data are compared with ultrastructural studies showing an alteration of intemal membranes in etioplasts. These etiolation-related aminopeptidase activities may thus play a role in bringing etioplasts to a stage of development at which they are no longer convertible to chloroplasts. MATERIALS A N D METHODS Plant Material

Seeds of sugar beet (Beta vulgaris L., cv Véga) were sown in perlite-vermiculite (50-50%, w/w) and watered with a mineral nutrient solution for neutrophilic plants as previously described (El Amrani et al., 1994). Germination and growth were carried out in the dark at 2OOC. Complete germination as determined by the emergence of the radicle was obtained in 4 d (El Amrani et al., 1992). In some experiments, plantlets were then exposed to light at 400 pmol photon m-’s-’ from a metal halide lamp (HQI-TS 400W/D; Osram, Paris, France). Cotyledons were excised under a dim green light at different stages of etiolation, which was usually carried out for 4, 6, 8, 10, and 12 d, and immediately used for the isolation of plastids or exposed to light. In this latter case, 30 pairs of excised cotyledons were placed in flasks containing 20 mL of the mineral nutrient solution described by Saglio and Pradet (1980) complemented with 50 m~ Mes, pH 6.2. Exposure to light (400 pmol photon m-’s-l) was carried out at 2OOC. The solution was changed every 8 h, and air was bubbled continuously in the flasks throughout the experiment. Isolation of Plastids from Cotyledons

Approximately 100 g of sugar beet cotyledonswere excised and immediately placed in ice-cold water under a dim green

Plant Physiol. Vol. 106, 1994

light. All of the subsequent isolation steps were carried out at O to 4OC. After 10 min, the water was drained and replaced with 200 mL of medium A consisting of 50 m~ Tris-HCI, pH 7.6, 330 m~ sorbitol, 2 m~ EDTA, 2 m~ MgC1, 1%(w/v) soluble PVP, 1O m~ ascorbic acid, and 5 m~ Cys. The material was homogenized in this medium in a Waring blender. The blender was set to full speed for 3 s. The homogenate was then filtered through two layers of 45-pm mesh nylon cloth and one layer of Miracloth (Calbiochem Corp., La Jolla, CA). The remaining solid material was re-homogenized using the same procedure. This was repeated once more. The three filtrates were pooled. All of the following cenírifugations were carried out in swinging buckets in a Beclanan JS-7.5 (Beckman Instruments, Geneva, Switzerland) rotor. The pooled filtrates were centrifuged at 3008 for 2’ min. The supematant was then centrifuged for 10 min at 24408. The resulting pellet was gently resuspended in 100 mL of washing medium, which consisted of medium A minus sclluble PVP, ascorbic acid, and Cys. This suspension was recentrifuged for 8 min at 1900g. Two milliliters of washing medium were added to each pellet. These crude etioplasts were further purified either on a preformed 5 to 80% (v/v) Percoll gradient (Pharmacia, Uppsala, Sweden) (Liitke-Brinkhaus and Kleinig, 1987) or on a step gradient consisting of an upper layer containing 8 mL of 35% (v/v) Percoll (Pharmaci(3,Uppsala, Sweden) and a lower layer containing 6 mL of 70% (v/v) Percoll (Baumgartner et al., 1989). Percoll gradients were made in washing medium and centrifuged at 12OOg for 10 min. The lower yellow band containing the intact etioplasts was removed, diluted with 10 volumes of washing medium, and sedimented at 19008 for 8 min. Step gradients were used in all of the experiments described in the figures and tables. Plastids were observed with a phase contrast lens in a Nikon photomicroscope. The number of plastids per v l h m e unit was determined by visual counting on a hematocytometer. Analysis of Proteins, Carotenoids, and Chi

Protein was determined by the method of Brad.Ford (1976) using 7-globulin as the standard. SDS-PAGE malysis of proteins was carried out in a Mighty Small I1 (Hoefer, San Francisco, CA) electrophoresis unit with gradient ,gels of 9 to 15% acrylamide in the presence of 0.1% (w/v) SDS, essentially as described by O’Farrell (1975). Carotertoids were determined as described by Jeffrey et al. (1974). Total Chl was extracted and measured as described previously (El Amrani et al., 1994). Enzyme Assays

Cyt c oxidase (EC 1.9.3.1), isocitrate lyase (EC 4.1.3.1), and alcohol dehydrogenase (EC 1.1.1.2) were used as markers of mitochondria, glyoxysomes, and the cytosol, respectively, and assayed as described by Rafael (1987), De Bellis and Nishimura (1991), and Bergmeyer (1974), respectively. 6PGDH (EC 1.1.1.44) activity was measured as described by Simcox et al. (1977). The assay of 6PGDH in the absence and in the presence of 0.025% (w/v) Triton X-100 was used to determine the immediate and latent, menibrane-enclosed, activities as described by Joumet and Douce (1985).

Effects of Prolonged Dark Growth on Etioplasts Aminopeptidase activity was assayed using AA-NH-Nap substrates (Sigma) in a reaction medium containing 50 m potassium phosphate buffer, pH 7.5, 0.5 m AA-NH-Nap, and 10 pL of enzyme in a final volume of 1 mL at 25OC. The production of Nap was monitored spectrofluorimetrically at 410 nm in a Hitachi F 2000 spectrofluorimeter with an excitation wavelength of 350 nm. Aminopeptidase activity was calculated from the slope of the recorder trace in terms of Nap produced from the substrate using the standard curve relating fluorescence intensity to Nap concentration (Sarath et al., 1989). '251-Caseinewas prepared and used to determine endopeptidase activity according to the method of Sarath et al. (1989).This activity was assayed in 50 m Tris-HCl buffer, pH 7.5, containing 20 m KC1 in the absence of exogenous ATP. Native Cel Electrophoresis of Aminopeptidases

Native gel electrophoresis was camed out essentially as described by Orr et al. (1972) in a Mighty Small I1 (Hoefer) electrophoresis unit. Slab gels of 10% (w/v) acrylamide (1.5 mm thick) were polymerized with N,N,N',N'-tetramethylethylenediamine. Electrophoresis was carried out at pH 8.3 with a 40-mA current per gel at 4OC during 4 h. Aminopeptidase activity was revealed as described by Thayer et al. (1988). The gels were incubated in 0.5 m AA-NH-Nap in 12.5 m potassium phosphate buffer, pH 6.4, during 30 min and then in 1 M sodium acetate buffer, pH 4.2, containing 0.1% Fast Garnet GBC (Sigma) and 5% Brij 35 (Sigma) for approximately 10 min until the insoluble azo dye of Nap (Wagner, 1986) appeared as red bands. After severa1 washings with 7% acetic acid, gels were photographed.

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lation of plastids as described above except that homogenization was carried out with a mortar and pestle. The plastidial fractions were frozen in liquid nitrogen and kept at -8OOC until the measurement of radloactivity. Aliquots of plastidial fractions were precipitated with 10% (w/v) TCA and 1 m Met in the presence of 0.05 mg mL-' BSA and heated at 100°C for 15 min. The cooled precipitate was collected on a glass fiber filter (Whatman GF/C) and washed with 5% (w/ w) TCA, 1 m Met, and then 70% ethanol. The filter was humidified with 150 pL of water, and proteins were solubilized by incubation at 55OC for 2 h in the presence of 1 mL of protosol (Dupont, Boston, MA). The radioactivity was measured in ACS (Amersham, UK) scintillation liquid.

EM Plastid preparations were fixed with 2.5% (v/v) glutaraldehyde in 100 m sodium phosphate buffer, pH 7.2, containing 330 m sorbitol, for 1.5 h at 4OC. After thorough washing with the phosphate buffer, the plastids were pelleted (1900g, 10 min) and postfixed with 1%(w/v) osmium tetroxide in the same medium for 2 h at 4OC. These pellets were then treated with 1%(w/v) tannic acid in phosphate buffer, pH 7.2, for 1 h at 20OC. The pellets were dehydrated with ethanol and propylene oxide and embedded in Epon. Cotyledons were processed according to the same procedure, except that sorbitol was not included in the fixation buffers. Ultrathin sections were collected on uncoated 600-mesh HT grids (Gilder, Grantham, UK), stained with uranyl and lead, and then observed with a Philips CMlO electron microscope. RESULTS Plastid Development and Capacity of Carotenoid Synthesis

Western Dot Blot Analysis

The large subunit of Rubisco was quantitated by westem dot blot with the monoclonal antibody 7-7 obtained and described by Meyer et al. (1991). Westem dot blot analysis was camed out using protein corresponding to 2 x 106 etioplasts bound to nitrocellulose on each spot. The nitrocellulose was blocked with 1% BSA in Tris-buffered saline buffer consisting of 50 m Tris-HC1, pH 7.6, containing 0.2 M NaCl. The blot was visualized through the action of an alkaline phosphatase-conjugated antibody developed in goat (Sigma). The color was developed using 0.165 mg mL-' 5bromo-4-chloro-3-indolyl phosphate and 0.330 mg mL-' nitroblue tetrazolium. In Vivo Labeling of Proteins

Seeds of sugar beet were surface sterilized with commercial hypochlorite and rinsed with sterile 0.01 M HCl and sterile water. Sterilized seeds were then germinated in the dark under axenic conditions on perlite-vermiculite soaked with the mineral nutrient solution for neutrophilic plants. Batches of 50 cotyledons were excised with sterile blades under a dim green light and incubated with 150 PCi of ~ - [ ~ ~ S l M (>1000 et Ci "01-I) in 5 mL of sterile water during 3 h. The labeling was ended by washing the cotyledons with 100 m Met. The cotyledons were then immediately homogenized for the iso-

In situ EM observations of etiolated cotyledons (Fig. 1, a and b) showed the transformation of proplastids at d 4 into etioplasts at d 6 with the development of the prolamellar body. The dry embryo of sugar beet does not contain amyloplasts (E1 Amrani et al., 1992). However, the presence of starch in proplastids and etioplasts (Fig. 1, a and b) was consistent with the increase of starch content in the embryo from d 2 to d 6, in parallel with the mobilization of starch from the perisperm (E1 Amrani et al., 1992). The amount of carotenoid per cotyledon (Fig. 2) and the greening capacity, as determined by the rate of production of Chl (E1 Amrani et al., 1994) or by that of carotenoids (Fig. 2) after illumination, increased during proplastid-etioplast transition. After d 6, whereas the amount of carotenoid per cotyledon remained constant, the rate of carotenoid synthesis upon illumination, whether in the absence or in the presence of exogenous Glc, started to decrease (Fig. 2), in strict accordance with the decrease in the rate of Chl synthesis (E1 Amrani et al., 1994). Thus, the profile of carotenoid accumulation and synthesis confirmed the previously described (E1 Amrani et al., 1994) irreversible decrease of greening and the effects of Glc limitation. During this period of loss of greening capacity, etioplasts were found to contain more and more osmiophilic globuli, most of them being in the prolamellar body or close to the prothylakoid membranes (Fig. 1, c and d). In addition, some alterations of plastid membranes occurred. The mem-

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Figure 1. Electron micrographs of plastids in cotyledons of dark-grown sugar beet plantlets. Cells in cotyledons from plantlets that had grown for 4 d (a), 6 d (b), or 12 d (c) in the dark were observed with a X25,000 magnification. The views show the ultrastructure of plastids in these cells with typical ultrastructures of proplastid at d 4 and etioplast at d 6 and the development of osmiophilic globuli at d 12 (d, with a X40,000 magnification). Mitochondria (m), peroxisomes (px), starch (s), prolamellar body (pb), prothylakoids (pt), and plastoglobuli (pg) are indicated. Bar = 0.5 nm.

Effects of Prolonged Dark Growth on Etioplasts

v)

c O V W

-r

6 -

5 -

c O

o

.-= õ

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w Exposure t o light O

4

6

8

10

12

Time of growth (d] Figure 2. Effects of light on the carotenoid content of cotyledons from dark-grown sugar beet plantlets. Excised cotyledons from 4-, 6-, 8-, IO-, and 12-d dark-grown plantlets were incubated as described in “Materialsand Methods” in the presence of 0.1 M Clc or 0.1 M mannitol (control)and exposed to 400 pmol photon m-* s-’ for O, 4, 8, 12, or 24 h. Carotenoids were extracted and measured as described in “Materialsand Methods.” Results are t h e means (f SE) of at least three measurements from one typical experiment.

brane lamellae became fuzzy and contained small, dense aggregates in some places. Furthermore, although some large etioplasts could be observed as early as d 6, the proportion of large etioplasts increased from d 6 to d 12. Figure ICshows an example of an enlarged and distended etioplast at d 12 of dark growth. Purification of Plastids from Sugar Beet Cotyledons

Carotenoids were used as a marker of plastids (Journet and Douce, 1985). The method of purification was worked out from a number of published methods using low (