Plant Physiol. (1980) 65, 600-6040032-0889/80/65/0600/05/$00.50/0

Endoplasmic Reticulum of Mung Bean CotyledonsACCUMULATION DURING SEED MATURATION AND CATABOLISM DURING SEEDLING GROWTH1

Received for publication September 12, 1979 and in revised form November 13, 1979

NEIL R. GILKES AND MAARTEN J. CHRISPEELS2Department of Biology, C-016, University of California, San Diego, La Jolla, California 92093

ABSTRACT

Homogenates of mung bean cotyledons were subjected to equilibriumdensity centrifugation on linear sucrose gradients and the positions of thevarious organelies determined by assay of marker enzymes. Measurementof phospholipid distribution on such gradients showed that the major peakof phospholipid at a density of 1.11 to 1.13 grams per cubic centimetercoincided with the position of the endoplasmic reticulum (ER), confirmingultrastructural evidence that storage parenchyma cells are rich in ER.Germination and seedling growth were accompanied by a rapid decline inER-associated phospholipid but a marked increase in the ER markerenzyme NADH cytochrome c reductase. Similar experiments with devel-oping seeds indicated that the amount of ER-associated phospholipidincreases during cotyledon expansion reaching a maximum during seedmaturation. There was no subsequent decline during seed desiccation,instead ER-associated phospholipid levels were maintained in the dry seeduntil germination when catabolism was initiated 12 to 24 hours after thestart of imbibition. This timing indicates that the observed ER breakdownis not an expression of the overall senescence of the cotyledons, but mayrepresent the dismantling of the extensive rough ER used for reserveprotein synthesis during cotyledon development.

Previous work aimed at elucidating the control mechanismsinvolved in the catabolism ofreserve proteins in legume cotyledonshas shown that in mung beans mobilization is dependent on thede novo synthesis of the proteinase vicilin peptidohydrolase (8, 9);immunocytochemical evidence shows that the enzyme is presentin cytoplasmic organelles before it accumulates in the proteinbodies (2). We have postulated that the RER may be the site ofsynthesis and/or transport of this proteinase, and are currentlyexamining changes in the ER which occur during germinationand seedling growth. We are studying synthesis, catabolism, mor-phological changes and enzyme content of the ER and seek torelate our findings to the process of reserve protein mobilization.

Biochemical observations (14) indicated that cotyledons arecapable of phospholipid synthesis early during germination andthat these new phospholipids become associated with the ER.However, phospholipid catabolism exceeds synthesis resulting ina net phospholipid decline in the cotyledons. We now report thatthis decline is a reflection of the rapid decrease in the amount ofER in the cotyledons, possibly the extensive ER which functionedduring seed maturation in the synthesis of reserve protein. Prelim-inary morphological observations (13) show that seedling growthis accompanied by a decline in the amount of tubular ER in thecotyledons.

1 Supported by grants from the National Science Foundation (MetabolicBiology).

2 To whom requests for reprints should be addressed.

MATERIALS AND METHODS

Plant Material. Seeds of Vigna radiata L. Wilczek, cv. Berkenwere purchased from W. Atlee Burpee Co., Riverside, Cal. Forseed germination studies, seeds were germinated and grown in thedark as previously described (9). For seed development studiesseeds were planted in a soil mixture (5) and grown in a greenhouseunder natural light conditions in La Jolla during June and July.Flowers were tagged when they appeared, to facilitate later sam-pling of the pods.

Homogenization and Sucrose Density Centrifugation. Cotyle-dons were harvested at appropriate times, testas and axes removed,and rinsed in water. Routinely, 12 cotyledons were homogenizedwith a mortar and pestle in 2.6 ml ice-cold 12% (w/w) sucrosecontaining 50 mM Tris-HCl (pH 7.5), 1 mm EDTA, and 0.1 or 3.0mM MgCl2. The homogenate was centrifuged at 500g, 2 min toremove starch, nuclei, and cell wall debris. The supernatant wasloaded on a linear sucrose gradient (14-50o or 14-65% w/wsucrose in the above medium) and centrifuged at 150,000g, 3 h, 4C, using a SW41 rotor in a Beckman L3-50 ultracentrifuge (Beck-man Instruments, Palo Alto, Cal.). Gradients were formed andfractionated using a Buchler Auto Densi-flow IIC gradient appa-ratus (Buchler Instruments, Fort Lee, N.J.).

Preparation of Protein Body Limiting Membranes. Thirty 3-day-old cotyledons were gently homogenized (to reduce proteinbody rupture) with a mortar and pestle in 7.5 ml 14% sucrose, 50mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.1 mMiMgCl2. Thehomogenate was filtered through two layers of Miracloth andloaded on a linear sucrose gradient (16-65%, w/w, in the abovemedium), and centrifuged at 95,000g for 1 h using a SW 27.1rotor. The turbid band at 1.28-1.30 g cm-3 was removed with apasteur pipette and diluted with water to a final concentration of12% w/w sucrose. This material was vortexed for 3 min andloaded on a second gradient (16-65%, w/w, sucrose, 10 mM Tris[pH 7.5]) and centrifuged 150,000g, 1 h using a SW 41 rotor.Enzyme Assays. p-Nitrophenyl-a-D-mannosidase as assayed as

previously described (9). NADH Cyt c reductase and inosinediphosphatase (IDPase) were assayed as described by Bowles andKauss (6). Phosphoryl choline:glyceride transferase was deter-mined by the method of Johnson and Kende (16). Preparation ofreduced Cyt c and assay of Cyt oxidase was by the method ofSottocasa et al. (24). Catalase was determined as described byBeers and Sizer (3).Other assays. Lipids were extracted from gradient fractions as

described by Folch et aL (12) and assayed for phosphorus afterdigestion in 72% HC104 by the method of Bartlett (1). Chlorophyllwas estimated from its absorption at 665 nm after extraction with2:1 (v/v) chloroform-methanol.

RESULTS

The appearance of a linear 14-65% (w/w) sucrose gradient aftercentrifuging a 5OOg supernatant fraction of a cotyledon homoge-nate is shown in Figure IA. The distribution of organelles on such

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FIG. 1. A: appearance of a sucrose gradient (14-65%, w/w, 50 mM Tris-HCI [pH 7.51, 1 mm EDTA, 0.1 mM MgCl2) produced by centrifugation ofa 5OOg supernatant fraction of a cotyledon homogenate (2 days of seedlinggrowth). Notice the double band at a density of 1.18 g-cm-3. B: particulatematerial generated by disruption of the band at 1.29 g.cm3 (intact proteinbodies) and recentrifugation on a sucrose gradient (14-65%, w/w, 10 mMTris-HCl [pH 7.5]). Densities were determined by sampling with a pasteurpipette.

a gradient was investigated by determination of various markerenzyme activities (Fig. 2). NADH Cyt c reductase, a marker forthe ER (23), shows a major peak of activity at 1.11-1.13 g.cCm3and a smaller peak at 1.18-1.19 gcm3. Cyt oxidase, specificallyassociated with the inner mitochondrial membrane (23), alsoshows a peak at 1.18-1.19 g* cm-3, whereas catalase, characteristicof intact microbodies (4), peaks at 1.21-1.23 g.cm3 with a smallerpeak of soluble (nonsedimentable) activity which presumablyindicates partial rupture of these organelles. IDPase, an enzyme

reported to be characteristic of the Golgi apparatus, though notexclusively (for a discussion, see ref. 23), shows a major peak ofsoluble activity while no clearly defined sedimentable activity isevident. Activity and distribution of this enzyme showed essen-tially no change during storage at 4 C over 6 days (data notshown).An additional class of organelle, the protein body, is abundant

in legume cotyledons. In mung bean cotyledons it has been shownto contain a number of hydrolytic activities (25) which are suffi-ciently specific to be used as marker enzymes for the intactorganelle. The distribution of one such enzyme, p-nitrophenyl-a-D-mannosidase, shows a small peak of sedimentable activity at1.28-1.29 g.cm3 on sucrose gradients, consistent with the equilib-rium density previously reported for mung bean protein bodies(11), while the bulk of activity is seen at the top of the gradientindicating large scale rupture and release ofprotein body contents.No enzyme activities characteristic of the limiting membrane ofthe protein body have been reported at this time. The question oflocation of these protein body "ghosts" on sucrose gradients hastherefore been approached differently. The turbid band at 1.29 g.cm-3 on a sucrose gradient (Fig. IA), representing protein bodiesstill intact after homogenization (Fig. 2), was removed and lysisinduced by dilution and vortexing. This material when centrifuged

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FIG. 2. Distribution of phospholipid (nmol) and organelle marker en-zyme activities (NADH Cyt c reductase and Cyt oxidase, AOD&o/min;IDPase, nmol Pi released/h; catalase AOD,.o/min; and a-D-mannosidase,AOD4,0/h), on a sucrose gradient (14-50%, w/w, 50 mM Tris-HCl [pH7.51, 1 mm EDTA, 0.1 mm MgCl2) following centrifugation of a 500gsupernatant fraction of a cotyledon homogenate (2 days of seedlinggrowth). All activities are expressed on a per cotyledon basis.

on a second sucrose gradient was found to generate a diffusemembrane band at 1.17-1.19 g.cm3 (Fig. 1B). This suggests thatthe material visible on normal gradients at this density contains,in addition to mitochondria (see above), the limiting membranesofprotein bodies ruptured during homogenization. Indeed, carefulexamination of this region of the gradient indicates the presenceof two distinct membrane bands (Fig. lA).The distribution of phospholipids on gradients (Fig. IA) shows

a major peak at 1.11-1.13 g.cm3 with smaller peaks at 1.17-1.19and 1.28-1.29 g.cm3. The coincident distribution of the majorpeak with that of NADH Cyt c reductase suggests that the ERaccounts for the bulk of the phospholipid in the cotyledon cells.This view is supported by the results shown in Figure 3. With 1mM EDTA and 0.1 mm MgCl2 present in the homogenization andgradient media, NADH Cyt c reductase and phospholipid arecoincident at a density of 1.12-1.13 g.cm3; also coincident is thepeak of phosphoryl choline:glyceride transferase, an exclusive ERmarker in castor bean endosperm (18). Increasing the concentra-tion of Mg2+ to 3.0 mm results in the "shift" in density of both theenzyme activities and the major phospholipid peak to a density of1.16 gcmFigure 4 documents the distribution of phospholipid on sucrose

gradients during seedling growth; although continuing to be as-sociated with the ER, the level of phospholipid in this major peakundergoes a marked decline. By contrast, the level ofphospholipidat _1.19 g-cm-3 remains relatively constant. Figure 4 also illus-trates a large (6-fold) increase in the level of NADH Cyt creductase during the first 3 days of germination; this high levelsubsequently declines as the cotyledons senesce. Experiments inwhich homogenate fractions from 1- and 3-day-old cotyledonswere mixed indicate that the increased enzyme activity during

Plant Physiol. Vol. 65, 1980 601

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FIG. 3. Effect of the presence of 0.1 (top and bottom left) or 3.0 mMMgC12 (top and bottom right) in the homogenization and gradient mediaon the distribution of phospholipid and ER marker enzyme activities on

sucrose gradients (14-50%, w/w, 50 mm Tris-HCl [pH 7.51, 1 mo EDTA).Gradients were produced by centrifugation of 5OOg supernatant fractionsof cotyledon homogenates (30 h of seedling growth). Phosphoryl choline:glyceride transferase activity is expressed as pmol product/h cotyledon;phospholipid and NADH Cyt c reducase as in Figure 2.

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FRACTION NUMBERFIG. 4. Distribution of phospholipid and NADH Cyt c reductase on

sucrose gradients (14-50%o, w/w, 50 mm Tris-HCl [pH 7.5], 1 mm EDTA,0.1 mM MgCl2) following centrifugation of 5OOg supernatant fractions ofcotyledon homogenates (1, 3, and 5 days of seedling growth). Phospholipidand NADH Cyt c reductase are expressed as in Figure 2.

seedling growth does not result from the disappearance of inhibit-ing factors (data not shown).

In order to relate these ER-associated events seen during seed-ling growth to the process of cotyledon maturation, organelleshave also been fractionated from developing cotyledons (Fig. 5).The ER, as determined by NADH Cyt c reductase, equilibrates ata similar density to the ER derived from cotyledons duringgermination. Also detectable were a peak of Chl and a peak of

FRACTION NUMBERFIG. 5. A: distribution of phospholipid, Chl and organelle marker

enzyme activities on a sucrose gradient (14-50%o, w/w, sucrose, 50 mm Tris[pH 7.5], 1 mm EDTA, 0.1 mM MgCl2) after centrifugation of a 5OOgsupematant fraction from a homogenate ofdeveloping cotyledons (18 daysafter flowering). B: Effect of increasing MgCl2 concentration in homoge-nization and gradient media to 3.0 mm on distribution of NADH Cyt c

reductase and IDPase. Chl is expressed as OD6U5/cotyledon; other activitiesas in Figure 2.

Cyt oxidase activity at 1.15 and 1.18 g cm-3, respectively. Incontrast to the situation in cotyledons during seedling growth, aportion of the IDPase activity is seen to be sedimentable at adensity of 1.12 g cm-3. This activity, as in developing pea coty-ledons (21), shows no significant change during storage of thegradient fractions at 4 C for 6 days (data not shown). A shift inthe density of NADH Cyt c reductase is again observed onincreasing the concentration of Mg2+ in the homogenization andgradient media, while no such shift is seen in the density ofparticulate IDPase activity. The significance of the large peak ofsoluble Cyt c reductase activity is not known; it can be separatedfrom the particulate activity by chromatography of the homoge-nate on a Sepharose 4B column, is not NADH dependent, andshows kinetics which distinguish it from the particulate activity(data not shown).

Figure 6 documents the amounts of ER-associated phospholipidin the cotyledons at various stages of seed development andgermination. The dry weight:fresh weight ratio is included as anindex of seed maturity. As seeds accumulate dry matter duringdevelopment, ER-associated phospholipid shows a linear increase(Fig. 6a). During maturation and desiccation (30 days after flow-ering) the amount of phospholipid levels off and remains at thislevel in the dry seed (35-40 days after flowering). During germi-nation and seedling growth the rapid catabolism of ER phospho-lipid is observed (Fig. 6b). The high level of NADH Cyt creductase activity expressed during seedling growth far exceedsthe activity detectable during seed development which is equiva-lent to that seen in the newly imbibed cotyledons.

DISCUSSION

Organelles derived from mung bean cotyledons at various stagesof seed development and seedling growth have been resolved onsucrose gradients and shown to exhibit sedimentation properties

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ER CATABOLISM IN MUNG BEAN COTYLEDONS

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FIG. 6. Levels of ER-associated phospholipid (nmol) and NADH Cytc reductase (AOD55o/min) during seed development, maturation and seed-ling growth. Cotyledons of appropnate ages were homogenized and the5OOg supernatnat fractions resolved on sucrose gradients (14-50%o, w/w,sucrose, 50 mM Tris-HCI [pH 7.5], 1 mM EDTA, 0.1 InM MgCl2). Gradientfractions corresponding to the bulk of the ER (23-36%, w/w, sucrose) were

pooled and assayed. Phospholipid and reductase are expressed as in Figure2.

typical of organelles from a variety of plant tissues (23). By thecriteria of: (a) coincidence with established ER marker enzyme

activities; and (b) sedimentation behavior in the presence of highand low Mg2+ concentrations, we have identified the major phos-pholipid component of the cotyledons as the ER.The criterion of "Mg2+ shift" (for a discussion, see ref. 23) is

validated by the absence of an effect on non-ER components. Noinfluence of Mg2+ concentration was observed on the peak den-sities of Cyt oxidase (Fig. 3), catalase (data not shown), or sedi-mentable IDPase activity from developing cotyledons (Fig. 2).The bulk of the microsomal phospholipid equilibrates at a densityof 1.16 g* cm 3 in the presence of 3.0 mm Mg2+ (which causes ER-associated ribosomes to be retained) while a trail of phospholipidinto the lower density region of the gradient suggests the presence

of a heterogeneous population of relatively smooth material whichmay represent smooth ER, Golgi cistemae and transitional forms.The majority of the protein bodies are broken during the

homogenization procedure employed in these experiments. Evi-dence is presented which indicates that the protein body limitingmembrane equilibrates at a density of 1.18-1.19 g.cm-3 when theprotein body contents are released. This rather high equilibriumdensity may be characteristic of membranes of this origin; studieson castor bean endosperm indicate equilibrium densities of 1.15(27) and 1.22 (20) g cm-' for this membrane, depending on theisolation procedure employed. In the present study protein bodymembranes do not appear to be resolved well from mitochondria,thus the relative contributions of these two organelles to thephospholipid peak seen at 1.18-1.19 g cm-' is not known.Our previous studies (14) have shown that seedling growth is

accompanied by a decline in the levels of phospholipids in thecotyledons. Evidence presented here attributes this decline to acatabolism of the phospholipids of the ER, confirming a similarobservation for Phaseolus vulgaris (19). The significance of thisobservation has been clarified by an examination of ER phospho-lipid levels during seed development. The data in Figure 6 suggeststhat the ER which functions during seed development is notcatabolized during desiccation; instead it is preserved in the dryseed until the onset of germination, at which point rapid break-down is initiated. The timing of the initiation of this breakdownindicates that it is not a reflection of over-all cotyledon senescence

as suggested by McKersie and Thompson (19). We hypothesizethat it may represent the dismantling of the extensive rough ER

which functions in reserve protein biosynthesis during cotyledondevelopment.These biochemical data are in agreement with morphological

descriptions of the ER in legume cotyledons. Early work showedthat cotyledon expansion is accompanied by an increase in theamount of rough ER cisternae (7, 22). A recent study of Viciafaba (15) using thick sections to visualize the ER shows it toconsist of an interconnecting array of cisternal and tubular ele-ments in developing cotyledons. During the period of storageprotein deposition there is an apparent proliferation of these formsfollowed by a conversion of the cisternae to a reticular configu-ration during seed desiccation. The tubular and cisternal forms ofER are also present in the cotyledons of mung beans imbibed for24 h. Seedling growth is accompanied by an apparent increase inER cisternae and a decrease in the extensive tubular form (Harrisand Chrispeels, in preparation). This contrasts with the situationin other seeds; in castor bean endosperm (17, 26) and wheataleurone tissue (10) there is very little ER at the beginning ofgermination but it increases markedly during early seedlinggrowth. We postulate that the observed decline in ER-associatedphospholipid in mung bean cotyledons during seedling growth isa reflection of this decrease in the tubular form of ER.The presence of phospholipid synthesizing enzymes in the

cotyledons (14), their association with the ER (Fig. 3) and theaccumulation of labeled phospholipid precursors by the ER duringgermination (14) suggest that, in addition, the ER present in themature seed functions as a basis for the synthesis of additional ERcomponents during seedling growth. The large increase in NADHCyt c reductase may be a reflection of this process. The extensiveER breakdown concurrent with this synthesis would result in theobserved net decline in ER-associated phospholipids in the coty-ledons. The observed decline in ER-associated phospholipidsoccurring at the same time as the increase in NADH-Cyt creductase indicates that the amount of this enzyme is not alwaysa good measure for the amount of ER.How such simultaneous ER catabolism and synthesis is regu-

lated in the cotyledons remains to be elucidated. Recent evidenceindicates that the protein bodies contain phospholipase D andacid phosphatase, enzymes capable of degrading membrane-as-sociated phospholipids. We have suggested that this organelleconstitutes a lytic compartment where membrane material maybe sequestered by a process involving autophagy (25).

LITERATURE CITED

1. BARTLETT, GR 1953 Phosphorus assay in column chromatography. J Biol Chem234: 466-468

2. BAUMGARTNER B, KT TOKUYASU, MJ CHRISPEELS 1978 Localization of vicilinpeptidohydrolase in the cotyledons of mung bean seedlings by immunofluo-rescence microscopy. J Cell Biol 79: 10-19

3. BEERS RF, IW SIZER 1952 A spectrophotometric method for measuring thebreakdown of hydrogen peroxide by catalase. J Biol Chem 195: 133

4. BEEVERS H 1979 Microbodies in higher plants. Annu Rev Plant Physiol 30: 159-193

5. BOLLINI R, MJ CHRISPEELS 1978 Characterization and subcellular localization ofvicilin and phytohemaglotinin, the two major reserve proteins of Phaseolusvulgaris L. Planta 142: 291-298

6. BOWLEs DJ, H. KAuss 1976 Characterization, enzymic and lectin properties ofisolated membranes from Phaseolus aureus. Biochim Biophys Acta 443: 360-374

7. BRIARTY LG 1973 Stereology in seed development studies: some preliminarywork. Caryologica 25: Suppl 289-301

8. CHRISPEELS MJ, B BAUMGARTNER, N HARRIS 1976 Regulation of reserve proteinmetabolism in the cotyledons of mung bean seedlings. Proc Nat Acad Sci USA73: 3168-3172

9. CHRISPEELS MJ, D BOULTER 1975 Control of storage protein metabolism in thecotyledons of germinating mung beans: role of endopeptidase. Plant Physiol55: 1031-1037

10. COLBORNE AJ, G MORRIs DL LAIDMAN 1976 The formation of endoplasmicreticulum in the aleurone cells of germinating wheat: an ultrastructural study.J Exp Bot 27: 759-767

11. ERICSON MC, MJ CHRISPEELS 1973 Isolation and characterization of glucosa-mine-containing storage glycoproteins from the cotyledons of Phaseolus aureusPlant Physiol 52:' 8-104

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604 GILKES AND CHRISPEELS12. FOLCH J, M. LEEs, GH STANLEY 1957 A simple method for the isolation and

purification of total lipids from animal tissue. J Biol Chem 226: 497-50913. GILKEs NR, N HARs, MJ CmusPELs 1978 Transformation of the endoplasmic

reticulum in the cotyledons of mung bean seedlings. Plant Physiol. 61: S- 1514. GisKFS NR, EM HERMAN, MJ CHRISPEELS 1979 Rapid degradation and limited

synthesis of phospholipids in the cotyledons of mung bean seedlings. PlantPhysiol 64: 38-42

15. HARRIs N 1979 Endoplasmic reticulum in developing seeds of Viciafaba. Planta146: 63-69

16. JOHNSON KD, H KENDE 1971 The hormonal control of lecithin synthesis inbarley aleurone cells: regulation of the CDP-choline pathway by gibberellin.Proc Nat Acad Sci USA 68: 2674-2677

17. LoRD JM 1978 Evidence that a proliferation of endoplasmic reticulum precedesthe formation of glyoxysomes and mitochondria in germinating castor beanendosperm. J Exp Bot 29: 13-23

18. LoRD JM, T KAGAWA, TS MooRE, H BEEVERS 1973 Endoplasmic reticulum asthe site of lecithin formation in castor bean endosperm. J Cell Biol 57: 659-667

19. McKERsIE BD, JE THoMPSON 1975 Cytoplasmic membrane senescence in beancotyledons. Phytochemistry 14: 1485-1491

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20. METTLER U, H BEEVERS 1979 Isolation and characterization of the protein bodymembrane of castor beans. Plant Physiol 64: 506-511

21. NAGAHASHI J, L BEEVERS 1978 Subcellular localization of glycosyl transferaseinvolved in glycoprotein biosynthesis in the cotyledons of Pisum sativum L.Plant Physiol 61: 451-459

22. OPIK H 1968 Development of the cotyledon cell structure in ripening Phaseolusvulgaris seeds. J Exp Bot 19: 64-76

23. QUAIL PH 1979 Plant cell fractionation. Annu Rev Plant Physiol 30: 425-48424. SOTTOCASA GL, B. KUYLENSTIERNA, L. ERNSTER, A BERGSTRAND 1967 An

electron-transport system associated with the outer membrane of liver mito-chondria. J Cell Biol 32: 415-438

25. VAN DER WILDEN W, EM HERMAN, MJ CHRISPEELS 1980 Protein bodies ofmungbean cotyledons as autographic organelles. Proc Nat Acad Sci USA. In press

26. VIGIL EL 1970 Cytochemical and developmental changes in microbodies (glyox-ysomes) and related organelles of castor bean endosperm. J Cell Biol 46: 435-454

27. YOULE RJ, AHC HUANG 1976 Protein bodies from the endosperm of castorbean. Subfractionation, protein components, lectins, and changes during ger-mination. Plant Physiol 58: 703-709

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