Plant Physiol. (1986) 80, 1-6 0032-0889/86/80/0001/06/$0 1.

00/0

Lectins in Castor Bean Seedlings'

Received for publication April 10, 1985 and in revised form August 29, 1985

SUZANNE M. HARLEY2 AND HARRY BEEVERS*

Biology Department, University ofCalifornia, Santa Cruz, California 95064

ABSTRACT The amounts of the two lectins (ricin and Ricinus communis agglutinin) in tissues of castor bean seedlings were followed during germination and early growth. For measurement, lectins in extracts were separately eluted from Sepharose columns; an antibody to the agglutinin was also used to detect the lectins by immunodiffusion. The endosperm of the dry seed contains 3.5 mg total lectin (5.6% of the total seed protein), which declines by 50% by day 4 and more rapidly thereafter as the tissue is completely consumed. The cotyledons of the dry seed also contain lectins but the amounts are less than 1% of those in the endosperm, and, as in the endosperm, they are constituents of the albumin fraction of the isolated protein bodies. No lectins were detected in the green cotyledons of 10-day seedlings that had been exposed to light from day 5. The embryonic axes of 2-day seedlings contained very small amounts of lectins but they were not detectable in the aerial parts of seedlings grown for 3 weeks or in cells from endosperm grown in tissue culture. The ability of proteinases and glycosidases (isolated from endosperm of 4-day seedlings) to hydrolyze the lectins was examined. No hydrolysis of the two lectins was observed, but the subunits, separated by reduction with 2-mercaptoethanol, were hydrolyzed slowly by a proteinase and some release of mannose was observed in the presence of the glycosidases. Ricin was converted to its subunits by cysteine and an enzyme in an endosperm extract accelerated chain separation by glutathione.

matrix and the globulin proteins of the crystalloid. The lectins are components of the matrix proteins (44, 46) and are the only glycoproteins detectable in the protein bodies (8, 44, 46). Agglutinating activity has been detected in extracts of primary axes and cotyledons (27). Protein bodies have been observed in electron micrographs of castor bean cotyledons (I. J. Mettler, personal communication), and crystalloids have been isolated from cotyledons of 2-d-old seedlings (41). We report here the purification of ricin and RCA, from certain tissues of castor bean seedlings at various stages of growth using the technique developed for purifying these lectins from seeds by Nicolson et al. (25) and attempts to degrade ricin using hydrolases from the endosperm tissue.
MATERIALS AND METHODS

Plant Materials. Castor bean seeds (Ricinus communis L. cv

Hale) were soaked overnight in running tap water, sown in moist vermiculite, and germinated in the dark at 3OC and 85% RH. The time of planting was taken as d 0. Endosperm tissue was collected from seedlings at daily intervals. To obtain green cotyledons and leaves, 5-d-old seedlings were selected, the endosperms removed, and the seedlings replanted in vermiculite with the cotyledons exposed. The plants were placed in a growth chamber at 30C and grown with a 12-h light/12-h dark cycle, I with light at 110 E/m2 s. For comparison, similar seedlings were returned to the dark. The plants were watered daily with Hoagland solution. Lyophilized cells from an 18-d suspension culture, derived from castor bean endosperm, were kindly provided by Dr. R. R. Theimer. Determination of Total Endosperm Protein. Endosperm tissue was extracted with 1 N NaOH, using 2 ml per endosperm, by grinding in a mortar and pestle with sand. The homogenate was centrifuged at 20,000g for 15 min. The supernatant was decanted through two layers of Miracloth and the pellet re-extracted with 1 N NaOH. One ml of extract was mixed with 1 ml of 1 N HCI and 1 ml of 20% (w/v) TCA and held on ice for 5 to 10 min. The precipitated protein was pelleted and the pellet dissolved in 0.5 N NaOH. An appropriate dilution was assayed for protein (18), using BSA as the standard. The protein content of other extracts was assayed in a similar manner. Quantification of the Lectin Levels. All operations were conducted at 4C. Twenty seedlings of known age were harvested and their endosperms extracted with 25 to 40 ml 0.1 M Na phosphate (pH 7.2), 0.15 M NaCl, 3 mM NaN3 by grinding in a mortar and pestle with sand. The extract was centrifuged at 20,000g for 15 min, the supernatant decanted through two layers of Miracloth, and the pellet re-extracted. The combined supernatant was brought to 65% saturation with (NH4)2SO4. A crude lectin fraction precipitated and was collected by centrifuging at 20,000g for 15 min. The (NH4)2SO4 pellet was suspended in PBS; 10 mm Na phosphate (pH 7.2), 150 mM NaCl, 3 mM NaN3, dialyzed against PBS, and loaded onto a Sepharose 4B-200 (Sigma) column equilibrated with PBS. The column was washed with PBS to remove contaminating proteins. Ricin was eluted

The two castor bean lectins, ricin and RCA1,,3 are glycoproteins with mol wt of 60,000 and 120,000, respectively (25). Ricin is composed of two subunits linked by a single disulfide bond, an A-chain which is a potent inhibitor of protein synthesis by 80S ribosones, and a carbohydrate binding B-chain (30). RCA,, a strong hemagglutinin, is a tetramer composed of two A'-chains and two B'-chains. Each A'-chain is joined to a B'-chain by a single disulfide bond (31), two such heterodimers being held together by noncovalent forces (3). Ricin and RCA, are serologically related, although ricin has certain antigenic determinants that RCA, lacks (34). The lectins are localized in the protein bodies of the castor bean endosperm (44, 46). The protein bodies isolated from dry seeds contain two protein fractions, the albumin proteins of the
'Supported by National Science Foundation Grant PCM 78-19575 from the United States National Science Foundation. 2 Predoctoral fellowship from the National Science Foundation. Present address: Biology Department, University of California, Los Angeles, CA 90024. 3Abbreviations: RCA,, Ricinus communis agglutinin; PBS, phosphate buffered saline: Hb, hemoglobin; CPase, carboxypeptidase; LeuNA, Lleucyl-,B-napthylamide; ProNA, L-prolyl-,3-naphthylamide; BANA, a-N-

benzoyl-DL-arginine-,f-naphthylamide; ,@-HexNAc'ase, fl-N-acetylhexosaminidase; a-Man'ase, a-mannosidase.

HARLEY AND BEEVERS

Plant Physiol. Vol. 80, 1986

with 7.5 mM N-acetylgalactosamine in PBS; RCA, was eluted with 50 mm galactose in PBS (25). The fractions containing ricin and RCA, were pooled separately and the concentrations of lectins determined from the A280 extinction coefficients (32). To estimate the overall effectiveness of the purification and assay procedure an extract from twenty 4-d endosperms was divided into two equal portions and 5.0 mg ricin (roughly half of that already present) was added to one of them. Both portions were carried separately through the complete procedure and eluted from Sepharose columns. The sample spiked with ricin was then found to contain 4.35 mg more ricin than the control. The final figures for the amount of lectin per endosperm were therefore corrected for overall losses (13%) during the purification procedure. For assays on cotyledons, 12.5 g cotyledons were extracted in 36 ml PBS and subjected to the same procedure. Preparation of the Lectins and their Subunits for Enzymic Digestion. The purification steps were conducted at 4C. Ricin and RCA1 were purified from extracts of dry castor bean seeds by elution from Sepharose 4B-200 with N-acetylgalactosamine and galactose, respectively (25). The A- and B-chains of ricin were separated on DEAE-cellulose (Whatman DE-52) as described (29), except that 0.5% (v/v) 2-mercaptoethanol was included in all of the buffers and the B-chain was eluted stepwise with 0.1 M NaCl. The RCA1 subunits were separated on DEAEcellulose (Whatman DE-52) by the method of Saltvedt (39), except for the inclusion of 0.5% (v/v) 2-mercaptoethanol in all of the buffers. The concentrations of ricin, RCAI, and the ricin A- and B-chains were determined using the A280 extinction coefficients (32). The concentrations of the RCA1 A- and Bchains were measured by the method of Lowry et al. (18), using BSA as the standard. Preparation of Hydrolases from Castor Bean Endosperm. ProNAase, CPase, HB-ase, and a mixture of LeuNAase/ BANAase were prepared as previously described (45). ,BHexNAc'ase and a-Man'ase were prepared as described in (12) and used in combination. Hydrolysis of the Lectins. One ml of lectin or subunit, 6 to 8 mg/ml, was mixed with 0.1 ml of hydrolase and incubated at 25C. The three alkaline proteases were used in 0.1 M Na phosphate (pH 7.0), 3 mm NaN3, and the other proteases and the glycosidases in 0.1 M Na citrate (pH 4.5), 3 mM NaN3. At intervals of 20 to 25 h, an aliquot of reaction mixture was removed and the reaction stopped by the addition of 0.5 volume of 20% (w/v) TCA. The protein that precipitated was pelleted by centrifugation. Aliquots of the supematant were assayed for amino acids (16), nonamino sugars (4), or amino sugars (38), using glycine, mannose, and N-acetylglucosamine, respectively, as standards. Preparation of Thiol:Protein Disufide Reductase. All steps except the heat treatment were conducted at 4C. Endosperm tissue from 4-d-old castor bean seedlings, 36 g, was extracted with 70 ml of 0.1 M Na phosphate (pH 7.5), 2 mM EDTA, 3 mM NaN3 by homogenizing in a blender for 1 min. The homogenate was centrifuged at 20,000g for 15 min, and the supematant decanted through two layers of Miracloth. The supernatant, 80 ml, was placed in a 250-ml Erlenmeyer flask, brought to 50C in a water bath (2.5 min), and held at 50C for 12.5 min. The flask was cooled to 6C by plunging it into ice (3 min). The cooled solution was centrifuged (20,000g, 15 min) and the supernatant dialyzed against 50 mM Na phosphate (pH 7.8), to remove small metabolites that might interfere with the reductase assay. Thiol:protein reductase was assayed at 25C by monitoring the formation of oxidized glutathione, using insulin as the disulfide

with acetone, dried, and extracted twice with buffer (2% PVP, 150 mm NaCl, 10 mm DTT, 50 mm Na phosphate, pH 7.3), using 2 ml of buffer per g of tissue. The extracts were centrifuged at 16,000g for 15 min. Extracts of dried cells from tissue culture and fresh roots, hypocotyls, and embryo axes were prepared in a similar manner. Immunodiffusion. Ouchterlony double diffusion plates were prepared on microscope slides, using 3 ml of 1% Noble agar (Difco) in PBS per slide (24). Galactose, 0.1 M, was included in the agar to prevent any interaction between the lectins and the carbohydrate chains of the IgG (14). To avoid any interference from other serum proteins, IgG was purified from serum on DEAE-cellulose (40). Antiserum to RCA1 prepared in rabbits was the kind gift of Dr. L. M. Shannon. The concentration of the purified IgG which also reacts with ricin was adjusted to 1 mg/ml in PBS. For the double diffusion tests, 10 to 25 Al of concentrated extract was challenged with 10 ,l of IgG. There was never a reaction of castor bean extracts with IgG purified from nonimmune serum. Protein Body Isolation. Protein bodies were isolated from the endosperm and cotyledons of dry castor bean seeds using the nonaqueous glycerol method (44). Endosperms were homogenized in a blender, while the cotyledons were ground in a mortar and pestle with sand. The albumins were solubilized with 10 mm Tris-HCl (pH 7.5), and the globulins with 0.1 M Tris-HCl (pH 7.5) 1% SDS. SDS/PAGE. SDS/PAGE was performed in slab gels according to O'Farrell (28). The gels were fixed in 12% (w/v) TCA for 30 min, followed by 60 min in methanol:water:acetic acid, 9:9:2. Proteins were stained with either Coomassie brilliant blue R (28) or silver (20). Glycoproteins were stained by the thymol/ sulfuric acid method (37). Hemagglutination Assay. A 2% suspension of red blood cells in PBS was incubated with an equal volume (25 Ml) of sample in PBS in a plastic Petri dish at room temperature (14). After 60 min, the cells were examined for agglutination. Trypsinized, glutaraldehyde-fixed human type A cells (Sigma) were used. In Vitro Translation. A poly-U directed in vitro translation system was prepared using wheat germ and used as previously reported (1 1). Prior to assaying for protein synthesis, 30 Ml of ribosomes were incubated at 30C in the presence or absence of 40 Mg of ricin, with either 3.2 mm DTT, 3.2 mM cysteine, or no reductant. The total volume was 110 Ml. After 30 min, the other translation components were added, bringing the final volume 100 to 0.4 ml and the ricin concentration to Ig/ml. The amount of ["4C]phenylalanine incorporated into TCA precipitable products in 20 min was determined. RESULTS Lectin Breakdown in Endosperm Tissue. Figure 1 shows the changes in protein, dry weight, and fresh weight of the endosperm during germination and early growth. The amount of protein per endosperm declines gradually until d 3, after which the rate of decline increases. The dry weight remains high until d 4, and then declines rapidly. The fresh weight increases to d 6, especially from d 3 to 6, but falls as the endosperm becomes visibly
senescent. The amounts of ricin and RCA1 are essentially equal at all stages (Fig. 2C). The changes in total lectin content of the endosperm during the 7-d period are shown in Figure 2. Figure 2C shows that the amount of lectin per endosperm decreases after d 1. The dry seed contains 3.5 mg of lectin, and approximately 50% of it is still present at d 4. From d 4 there is a more rapid decline in lectin levels. In the dry seed, the lectins constitute 5.6% of the total seed protein (Fig. 2A). The increase in lectin per total endosperm protein seen around d 5 (Fig. 2A) is not due to synthesis ofricin and RCAI, as the lectins decline continuously

containing protein (23).

Extraction of Lectins from other Tissue. For cotyledons and leaves, from older seedlings, 0.5 to 2 g tissue was ground in 25 ml acetone and centrifuged. The pellet was washed thoroughly

LECTINS IN CASTOR BEAN SEEDLINGS

E
c

0.07
Z
0

0.06
0.05
0.04

a0 CL

E
0

C E

._E J
c -J

0.03

E
.

0.02 0
ci

15

0
'5
10
,
._

0 CI
o .N

0
J
.C
N

021 E

10

c:1

>

dry 0 1 seed

2 3 4 5 6 7

U01

Seedling age,days

ci 0

FIG. 1. Changes in the protein content, dry wt, and fresh wt of the castor beam endosperm during germination.

-J

0 2.0
1.5 1.0

on a per endosperm basis (Fig. 2C). The proportion of lectin to other proteins increases around d 5 because the lectins are degraded more slowly than the bulk protein. A delay in lectin degradation, compared to the decline of other storage proteins, has also been observed by Gifford et al. (8). The amounts of lectins shown in Figure 2 are those measured after specific elution of ricin and RCA1 from Sepharose 4B, using the A280 extinction coefficients (32). Closely similar values for ricin and RCA, at all stages were obtained when the lectins were measured by the protein assay of Lowry et al. (18). Enzymes which May Function in Lectin Breakdown. Since the lectins comprise over 5% of the endosperm protein and are broken down during germination, enzymes that could possibly hydrolyze the lectins were examined. Tully and Beevers (45) characterized five proteases from the endosperm of germinating castor beans. The proteases were named for the substrates used to detect them. ProNAase, LeuNAase, and BANAase have alkaline pH optima, and CPase and Hb-ase have acidic optima. Ion exchange chromatography can be used to yield fractions enriched for ProNAase, LeuNAase/BANAase, and Hb-ase, and CPase activities (45). Ricin, RCAI, and the isolated subunits were incubated separately with each of the four protease fractions under optimal pH conditions. No release of amino acids from intact ricin and RCA, could be detected after 83 h. When the lectin subunits were incubated under similar conditions only the Hb-ase was able to release amino acids from the lectin subunits. The rates of release, shown in Table I, are 4 to 5% that of the hydrolysis of hemoglobin, the substrate used to detect Hb-ase. When the lectins and subunits were incubated for 83 h with a mixture of the three alkaline proteases, no amino acid release could be detected. When the pH was then lowered to 4.5 with citric acid and the two acidic proteases added, a more rapid release of amino acids was seen than when the Hb-ase was used alone (Table I). Again, only the separated subunits were hydrolyzed, not the intact lectins ricin and RCA1. The activities of the acidic proteases are highest late in germination (45), when the most rapid decline in lectin was seen (Fig. 2C). It was perhaps not unexpected that the castor bean proteases failed to degrade intact ricin and RCA, since ricin is notorious for its resistance to proteolytic cleavage (24). It is not hydrolyzed by trypsin (32), chymotrypsin (32), or pepsin (5). However, the separated A- and B-chains are digested by trypsin and other proteases (30, 32). The sensitivity of the subunits to proteolysis

0.
0

EF

E 0 2.0 0 E
'.5
0

Ow

w
C

0 10 C

1.0

Lii
01

.2_

cE

0.5
0
Dry

E
0
1
2 3 4 5 6 7

Seed

Seedling Age, Days

FIG. 2. Changes in the amount of lectin (ricin + RCA,) in the castor bean endosperm during germination. At all times during germination, the amount of ricin (@) was equal to the amount of RCA, (0) as shown in (c).

Table I. Release ofAmino Acids during Protease Digestion of the Subunits of the Castor Bean Lectins Amino Acid Release Substrate Hb-ase + CPase, after

Hb-ase
Ricin A-chain Ricin B-chain RCA A-chain RCA B-chain 17.8 8.9 5.3 9.4

LeuNAase, ProNAase, BANAase nmol/h

22.6 10.9 7.0 11.5

is probably due to the conformational changes the subunits undergo upon reduction of the disulfide bond holding them together (3, 33). There are a number of ways the disulfide bond linking the A- and B-chains could be reduced in vivo. Glutathione could reduce disulfide bonds. In vitro, the amount of glutathione oxidized by reaction with the lectin can be determined by measuring NADPH consumption in the presence of glutathione reductase. Some reduction occurred in the absence of extract, but the rate was increased in the presence of enzyme extract (Table II). Thus, the castor bean endosperm appears to contain an enzyme reducing lectin at the expense of glutathione.

HARLEY AND BEEVERS

Plant Physiol. Vol. 80, 1986

Table II. Reduction of Castor Bean Lectins by Glutathione Each assay contained 25 mg/ml lectin, 3.3 mM glutathione, 0.2 mM NADPH, 0.5 unit glutathione reductase, 0.1 ml extract of endosperm tissue of 4-d-old castor bean seedlings. NADPH Consumed Lectin -Extract +Extract nmol/min Ricin 9.32 12.9a RCA 8.04 20.3a aThese figures have been corrected for NADPH oxidation in the absence of added lectin, which was less than 5% of the corrected rates. Table III. Effect of Reducing Agents on the Ability of Ricin to Inhibit Protein Synthesis in a Cell-free Translation System Preparedfrom Wheat Germ Protein synthesis was determined as the amount of ['4C]phenylalanine incorporated into protein in 20 min. Incorporation of [14C] -Phe(CPM) Reductant Inhibition -Ricin +Ricin dpm % None 9,886 8,149 18 DTT 9,896 2,076 79 Cysteine 7,120 62 2,724

Lectins in Other Tissues. Protein bodies were isolated from the cotyledons of dry castor bean seeds, by the nonaqueous method used to isolate protein bodies from the endosperm (44). The protein bodies from cotyledons resembled those from endosperm (44) under light microscopy using phase optics. Both types of protein bodies can be differentially extracted to give albumin and globulin protein fractions. Figure 3 shows the SDS/ PAGE profile of the albumins and globulins of protein bodies from cotyledons and endosperm. Differences in the protein profiles can be seen in the lower mol wt globulins (Fig. 3A). Two proteins present in the endosperm indicated by dots to the right of the bands in lane 4 are not observed in the cotyledon and cotyledons have an albumin band (dot to the right of lane 5) that is missing from the endosperm. After reduction by 2-mercaptoethanol, the endosperm and the cotyledon albumins each have a band that the other lacks (Fig. 3B, lanes 3 and 5). Bands which correspond to ricin and RCA, in the absence of 2-mercaptoethanol can be seen in the albumin proteins from both cotyledons

1 2 3 4 5 6 78

thioredoxin (42). Cysteine could also reduce the disulfide bond linking the lectin A- and B-chains. Free ricin A-chain is a more effective inhibitor of cell-free protein synthesis than the entire ricin molecule (30). If a reductant such as 2-mercaptoethanol or DTT is included in the translation system, A-chain is liberated (30). The data in Table III show that cysteine was effective in generating free Achain. In the absence of reductant, protein synthesis was inhibited 18% by ricin. In the presence of DTT or cysteine, inhibition was increased 3- to 4-fold due to the release of A-chain. Because ricin and RCA, are glycoproteins (32), glycosidases which could hydrolyze the oligosaccharide chains (15) were also studied. The oligosaccharide chains of the lectins are asparagine linked and composed of two N-acetylglucosamine residues and four to seven mannose residues (30). Binding studies with concanavalin A have shown that the mannoses are terminal and/or 2-0-linked (30). The oligosaccharide structures have not been determined, but from their composition it can be concluded that they are of the 'simple' or 'high-mannose' type chains, which contain only N-acetylglucosamine and mannose. As such, the mannose linkages are probably a and the N-acetylglucosamine are ft. When ricin, RCA1, or the lectin subunits were incubated with a mixture of f3-HexNAc'ase and a-Man'ase, an extremely slow release of nonamino sugars could be detected from the subunits, but not from the whole lectins. Approximately 0.5 nmol of mannose equivalent was released per h. This was 0. 1 % of the rate of hydrolysis of p-nitrophenyl-a-mannoside. No release of amino sugars was detected. As with the proteases, the activities of the glycosidases are highest late in germination (12) when the most rapid decline in lectin levels was seen (Fig. 2C).

However, while there is only one interchain disulfide bond, the B chain of ricin contains four interchain disulfides (31). We do not know which bonds were reduced in our system. Glutathionerequiring thiol:protein disulfide oxidoreductases have been purified from bovine liver and rat liver and were capable of reducing the interchain disulfide bond found in ricin (1). A protein disulfide reductase that uses NADPH or NADH has been described from peas (13). NADPH-dependent thioredoxin reductase from wheat will reduce insulin or ribonuclease in the presence of

1 23 45 67 8

aim~~~~*-

~~~~~~~.

-w.

FIG. 3. SDS/PAGE of protein fractions of cotyledon and endosperm protein bodies from dry castor bean seeds. The gel was a linear gradient of 6 to 20% acrylamide, stained with Coomassie brilliant blue R. The proteins in (A) were not reduced with 2-mercaptoethanol, while those in (B) were reduced. A, lanes 1 and 7, ricin; lanes 2 and 8, RCA1; lane 3, endosperm albumins; lane 4, endosperm globulins; lane 5, cotyledon albumins; lane 6, cotyledon globulins. B, lane 1, ricin; lane 2, RCA1; lane 3, endosperm albumins; lane 4, endosperm globulins; lane 5, cotyledon albumins; lane 6, cotyledons globulins; lanes 7 and 8, low and high mol wt standards (Bio-Rad), the mol wt from bottom to top being 14,300, 21,000, 30,000. 43,000, 68,000, 94,000, and 130,000.

LECTINS

5
also
stained

and endosperm (Fig. 3). The protein bands which correspond the lectins (Fig. 3A) and the lectin subunits (Fig. 3B)

When an extract of 4-d-old cotyledons was pharose 4B, a protein peak was detectable
was

positively for glycoprotein.

separated

on

column. There are species of ricin which do not bind Sepharose (17, 21). Nevertheless, it is quite clear that the endosperm cells maintained in tissue culture do not contain the high of lectins present in seed endosperm (Fig. 2).
to
level

when

used to elute the column (Fig. 4). When examined by SDS/PAGE, bands which corresponded and RCA1 were detected. Furthermore, the proteins lactose formed precipitin bands when challenged fusion tests with anti-RCA, IgG, but not IgG purified nonimmune serum. The fractions eluted by agglutinated red blood cells. Therefore, the cotyledons seeds contain both ricin and RCA1, but the than those found in the endosperm of the seedling (Table
this
material
to

0.1 M

lactose

DISCUSSION
The seeds of
organs of
many plant species contain specific
or

lectins

and

eluted

by

the amounts of seed lectins

in

double

serologically

related molecules in

plants

grown

from

some of

these

seeds

have been

lactose

also

of the
are

castor

levels

much

same

IV).

begins

to antibody RCA,

are exposed to light, Chl synthesis within 1 d and as the cotyledons become photosynthetic organs the levels of ricin and RCA, drop. The immunoreaction

When 5-d-old seedlings

was

measured by immunoassays. Legumes such as Phaseolus vulgaris (2), Psophocarpus tetragonolobus (u9), Glycine max (36), and have been examined for distribution of Dolichos biflorus seed lectins and their fate during germination and early growth. The of the has been especially useful for studying lectins in the Gramineae such as wheat, where the

(43)

sensitivity

immunoassays

agglutinain is present in very low amounts (1 gg/seed compared lectin to 1-4 mg/seed in legumes [22]). These plants contain
one

barely detectable
at 9 d and
extracts

in concentrated

or

collection

of

isolectins.

extracts

of greening cotyledons

absent
of

tests were still positive using

by d 10. Ouchterlony cotyledons

from

Castor
ricin and

bean, the object of the present study, has

RCA1,

two

lectins,
deter-

for extracts of cotyledons and leaves from or leaves from castor bean plants grown greenhouse conditions.

seedlings kept

in the dark.

114-d

which although

sharing
it is not

many

antigenic

Double diffusion

tests

were

negative

minants, are quite distinct. Thus,

the
amounts

possible

to determine

of each

3-week-old for
4

lectin in
on

an

extract or mixture

with the

seedlings

months

ricin and RCA,. A 10-ml Sepharose column ricin and RCA, from 1.2 g of embryo 2-d seedlings. Protein bands that coincided with ricin RCA, PAGE were isolated from hypocotyls of seedlings. 15 g of tissue gave 1 ml of concentrate which required sensitive silver stain (20) for visualization 0.1 analyzed by SDS/PAGE. Extracts of 4-d were devoid of Sepharose binding material gave negative
was used
to

Embryo axes from 2-d-old

seedlings

contain

low

amounts

antibody to RCA1. However, the two lectins can be purified and the basis of their different carboseparated from each other hydrate binding specificities (25) in stepwise elution from Se-

purify

pharose 4B.
lectins
mg of
are

The

castor bean
have

seed contains 1.73 mg of ricin and

axes from
and

1.78 mg of RCA1.

During germination

and early growth the two

of 74

depleted simultaneously

and in the endosperm

to

on

SDS/

4-d

However,
the

seedlings

the levels

RCAl.

declined

0.053 mg ricin and 0.056

when

roots

from

seedlings

In the endosperm of the dry seed both lectins are present the protein bodies, where they form part of the water matrix proteins (44, 46). Protein bodies are also present
in

in

soluble
the

and

Ouchterlony tests. An extract of 1.5 g dry weight of endosperm grown tissue culture contained a small amount material, but no detectable ricin or RCA, could Sepharose 4B with lactose. The same weight germinating seeds would contain 10.2 mg 6.2 mg at d 7 (Fig. 2), amounts that would give large noprecipitate and easily purified on a small (20-40 ml) Sepharose
cells

cotyledons, where again ricin and RCA1

fraction.
sperm

Cotyledons contain

part of the albumin much less lectins than the endoare

of

immunoreactive

be

eluted

(Table IV) and as the content drops to undetectable d-old seedlings

cotyledons turn green levels. The embryo

the lectin
from 2-

axes

of

low
were

amounts

of lectin

and hypocotyls from 4-d-old seedlings contain of lectin. From each of these tissues the lectins
by Nicolson
et

at

purified using the technique developed

and shown

aL

(25).
Some lectins from

same technique
I

example, and root

I

root

CS
4c
0.

~.5kC 0
0

~~~~~~0.040.02

hemagglutinin
trast,
the

lectins from Arachis hypogaea (35),

a-galactosidase from immunoreactive materials

legume tissues have been purified by the to be identical to the seed lectins; for lectin from Onobrychis vicitfolia (10) hypocotyl
and
a

hypocotyl

leaves of Dolichos (43)

II
30

230

260

Psophocarpus (39),roots are not identical to the seed lectins. During germination and early growth

roots,

Vigna radiata (9). In confound in pods, stems and stems and derived callus from of G. max (6) and Pisum sativum (7)
of castor bean the lectins

FIG. 4. Purification of ricin and RCA, cotyledons of germinating castor beans by Sepharoseaffinity raphy.
from
an

4-d-old
chromatog-

endosperm are degraded most rapidly after the bulk of the (Fig. 3; 8). This is in contrast storage protein has been to where the lectins of the (43) and Glycine cotyledons are mobilized simultaneously with the other storage reserves. The small amounts of lectins in the young cotyledons

in the

depleted

Dolichos

(36)

of castor bean disappear

as

Table IV. Lectin Quantities in the Cotyledons

4-d-old Castor
Bean

and

Endosperm of

photosynthetic.
seedlings
decline
50%

Lectins

found

the seedling becomes established and in the organs of most young

Seedlings

Endosperm Cotyledon
mg lectin/g fresh wt mg lectin/g dry wt mg lectin/seedling

E:C

Cotyledon, %
of Endosperm
5.4

germination (43).

to undetectable levels within 2 to 3 weeks of However, 344d-old seedlings of wheat contain of the agglutinin present in ungerminated grains, with the

2.4
6.8
1.7

0.13
0.24

.8.5
28.3

highest levels in actively growing regions (22). In an attempt to elucidate the breakdown of the lectins in the
substrates for proteases and endosperm, they were tested carbohydrases isolated from the tissue, where they are contained in the vacuole f(26).None of the enzymes able to break down
as
was

0.005

340

lectin, % of total
protein
4.1
0.15
27.3

the native lectins, but

some release of amino acids

was observed

HARLEY AND BEEVERS

Plant Physiol. Vol. 80, 1986

when the SH-proteinase Hbase was added to the separated A and B chains of both ricin and RCA,. Evidence was obtained that glutathione could reduce the S-S bonds of the lectins to bring about chain separation and the reaction was stimulated by an enzyme in the endosperm extract. Cysteine also brought about chain separation. A slow release of mannose from the subunits was observed when the glycosidases were added. From what is known in other systems, it seems likely that both protein and carbohydrate moieties would be more readily hydrolyzed if they were first cleaved by a peptide:N-glycosidase (15), but no such activity could be detected with crude extracts or the carbohydrases isolated from the endosperm tissue ( 12).
Acknowledgments-We thank Dr. L. M. Shannon, University of CaliforniaRiverside for his gift of antiserum to RCA,, and Dr. R. R. Theimer, Botanisches Institut Munchen for supplying dried cells of endosperm from suspension culture.
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