Plant Cell Reports
Plant Cell Reports (1994) 1 4 : 5 0 - 5 4
,9 Springer-Verlag 1994
Peanut agglutinin from callus and cell suspension cultures of Arachis hypogaea L. Icy D'Silva * and Sunil Kumar Podder Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India * Present address: Department o f Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Received 8 June 1993/Revised version received 12 May 1994 - Communicatedby J. Widholm
Abbreviations: BA, benzyladenine; 2,4-D, 2,4dichlorophenoxyacetic acid; EDTA, ethylenediaminetetraacetic acid; HAU(s), haemagglutination unit(s); IEF, isoelectric focusing; KN, kinetin; LS, Linsmaler and Skoog (1965) medium; Mm, medium promoting minimum growth of cells; Mx, medium promoting maximum growth of cells; NAA, naphthalene-l-acetic acid; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonytfluoride; PNA, peanut agglutinin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SHAA, specific haemagglutination activity; TCA, trichloroacetic acid
storage of carbohydrates (Barondes 1981; Etzler 1986; Chrispeels and Raikhel 1991) have been postulated for agglutinins in planta. However, no conclusive evidence has been presented for any of them (Etzler 1986). Agglutinins have played an increasingly important role in blood typing, in cell separation and identification, as carriers of chemotherapeutic agents and in the study of cell membranes (Lis and Sharon 1986a, 1986b). Among the several lectins known, PNA is by far the most popular, especially for cell separation and identificaton (Lis and Sharon 1986b). The only abundant source of PNA, so far, has been the seed cotyledons. Plant tissue and cell culture could not only be an alternate continuous source of lectins but could also be a useful and important model system to study the regulation of biosynthesis of PNA. A few reports on the presence of agglutinins (Del CampiUo et al. 1981; James et al. 1985; Malek-Hedayet et al. 1987; Sato et al. 1993) in tissue cultures have appeared. Earlier work from our laboratory has shown that ribosome-inactivating proteins and agglufinins were synthesized in callus and ceil suspension cultures of R. communis L. and A. precatorius L. (D'Silva et al. 1994). The purpose of the present study was to ascertain whether PNA could be continuously synthesized in callus and cell suspension cultures of A. hypogaea, to study the effect of growth regulators on the synthesis of PNA in vitro and to purify and characterize the lectin obtained from cultures.
Introduction
Materials and methods
Agglutinins have been isolated from seeds (Pratt et al. 1990; Datta et al. 1991) and tissues (Borrebaeck 1984) of a variety of plants. Several physiological functions, viz. involvement in defence mechanisms, cell recognition, as well as transport, immobilization and
Seeds of A. hypogaea L. vat. JL-24 were obtained from the National Seed Corporation, Bangalore. Growth regulators, EDTA, PMSF, BSA, lactose, SDS and standard molecular weight markers were from Sigma Chemical Co. (SL Louis, USA). Sepharose 4B, PhastGel-IEF and IEF standard markers were purchased from Pharmaeia (Uppsala, Sweden). [3H]leucine was supplied by BARC (Bombay, India). All other
Summary. Synthesis of peanut agglutinin was induced in callus and cell suspension cultures of cotyledons of peanut (Arachis hypogaea L.). The lectin was synthesised in cultures through several passages, Biosynthesis of peanut agglutinin was regulated by the type and concentration of exogenous growth regulators and was positively correlated to the growth of the cultures, indicating that the agglutinin may have a role to play during cell growth. Movement of agglutinin from the cells into the medium not only facilitated easy isolation of the lectin but also provided a clue that it may probably serve as a defence molecule. The synthesized lectin purified from culture, was found to be biologically active, and was found to be comparable with the lectin from seeds, in terms of its electrophoretic mobility.
Correspondence to: I. D'Silva
51 chemicals were of analytical grade. All experiments were repeated three times.
Callus and cell suspension cultures. Seeds of A. hypogaea L. var. JL24 were dehulled, surface-sterillzed with 0.1% mercuric chloride for 10 rain and rinsed in double-distilled water, three times. Cotyledons were inoculated to 100 ml liquid or agar-solidified LS (Linsmaler and Skoog 1965) media in 250-ml flasks. The media were supplemented with 58 mM sucrose, 0.1 mM thiamine-HC1, 1.1 la2VI uscorbic acid and various concentrations of growth regulators viz. ABA, 2,4-D, BA, KN and NAA; singly, and in combination. The media were adjusted to pH5.9. The cultures were incubated at 28 + 2 ~ under a 16-h photoperiod with a photon flux density of 10 to 15 I.Ixnol m -2 s -1 produced from cool white daylight fluorescent tubes. Liquid cultures were continuously agitated on an orbital shaker at 100 rpm. The calli and suspensions were transferred to fresh medium every three weeks. Calli and suspensions were subcultured three times before evaluating the effect of growth regulators on lectin levels. Fresh weight of callus was determined every four days, over a period of four weeks, to determine the growth of cultures.
Regulation of the levels of PNA. The influence of M x and M m on the levels of PNA in callus oaltures was determined as shown later. To confirm the consistency of lectin production, the lectin content of callus cultures, over five subcultures on M x, was determined.
Biosynthesis of PNA in cells. Ten days after the third subculture, [3H]leucine (0.5 mCi/35 ml medium) was added to the cell suspensions of A. hypogaea. The cultures were agitated at 100 rpm and samples were drawn every 48 h for 12 d. PNA was isolated from the cells and the medium. The proteins were precipitated with a final concentration of 10% TCA, kept on ice for 2 h, and centrifuged at 3 , 1 2 4 x g for 20min. The pellet was washed with 10%TCA. The pellet obtained after three cycles of washing was dissolved in 1 N sodium hydroxide and then transferred to methoxyethanol:toluene (1:1, v/v) containing 0.05% 1,4 bis[5-phenyl-2-oxazolyl]-benzene and 0.5% diphenyloxazole. Radioactivity was measured on a LKB 1211Racbeta liquid scinfiUation counter.
Purification of PNAfrom cultures. Callus (100 g fresh weight) was hocnogenized in a Waringblendor with 150 ml extraction buffer consisting of 100 mM phosphate (pH 7.2), 0.15 M sodium chloride, 0.1 mM EDTA and 0.1 mM PMSF. The mixture was stirred for 4 h and centrifuged at 27,200 x g for 30 min. The supematant was passed through cheese cloth and dialyzed against 10 mM PBS. The dialyzed sample was loaded on a Lactamyl-Sepharose 4B high-affinity matrix (Hegde et al. 1991) column (2x 15 em) equilibrated with PBS, and with a flow rate of 25 rnllh. Unbound protein was washed out until the A280 decreased to 0.05. The bound protein was eluted and collected as 2.5 ml fractions with 50 ml of 0.4 M lactose in PBS. The fractions were pooled and dialyzed against several changes of double-distilled water. The protein was o lyopl~ized and stored at -20 C. All operations were carried out at 4 C. Cells (100 g fresh weight) from suspension cultures were separated from the liquid media (2.5 1)by passing the suspensions through a Whatman No. 1 filter paper placed in a Buchner funnel. The cells were washed with extraction buffer. qlae washed cells and medium were individually processed. The cells were sonieated in extraction buffer (1:1.5, v/v). The mixture was stirred for 4 h , centrifuged at 27,200x g for 30rain and processed further as described for callus. The medium was dialyzed against 10 mM PBS and centrifuged at 27,200 x g for 30 min. The supernatant was passed through cheese cloth and loaded onto a Laetamyl-Sepharose 4B affinity matrix. The remaining procedures were carried out as described for callus.
Biochemical determinations. SDS-PAGE of the purified lectin from culture and seeds was carried out according to the method of Laemmli (1970) on a 7% running gel. The gel was stained with Coomassie brilliant blue. The molecular weight of the proteins was determined by comparing the eleetrophoretie mobilities with those of standard molecular weight marker proteins. IEF of proteins was carried out on PhastSystem (Pharmacia). PhastGel varying from pH 3 to 9 was used. The gel was stained with Coomassie brilliant blue. The range of pI was estimated by using standard IEF markers. Protein concentrations were determined by the method of Lowry et al. (1951) with BSA as the standard. Seed leetin of A. hypogaea was isolated as described by Singh (1989) using a high-affinity Lactamyl-Sepharose matrix (Hegde et al. 1991) and was electrophoresed along with the lectin obtained from cultures.
Biological activity of PNA from cultures. Hacmagglutination assay was carried out of lectin
in
plastic microtiter plates. A 100 Ixl quantity
sample was serially
diluted with 100 }2.1 of PBS and
mixed with 100p.1 of 4% (v/v) rabbit erythrocytes n PBS. Specific haemagglutination activity (SHAA) o f t he lectin is given in terms of HAU(s) mg-lprotein. One HAU is defined as the minimum amount of protein required for 100% agglutination at 4 ~ at the end of 2h.
Results and discussion
Regulation of levels of lectin in culture Optimal growth of callus and liquid cultures of A. hypogaea was obtained on LS medium supplemented with 10.7 t.tM NAA, 2.3 ~tM KN and 1.9 ~tM ABA. The addition of ABA promoted a slight increase in the level of lectin. ABA has been reported to promote lectin synthesis during the in vitro development of embryos of wheat (Morris et al. 1986) and rice (Stinissen et al. 1984). The synthesis of PNA was dependent on the type and concentration of exogenous growth regulators (Table 1). A maximum of 12.4 mg of PNA was obtained from 100 g fresh weight of callus. Table 1 gives the composition of Mx and Mm. Transfer of caUus back to Mx resulted in increased levels of lectin. Fig.1 demonstrates the effect of change of exogenous growth regulators on the accumulation of PNA. Accumulation of PNA was maximum during the exponential phase of growth of the cultures and declined during the stationary phase (Fig. 2). Though the lag phase of growth extended over 8 d after transfer to fresh medium, increase in the levels of PNA was observed from the fourth day. The levels of PNA continued to increase up to the twentieth day and declined thereafter. There was a direct positive correlation between the growth of cultures and levels of PNA indicating that PNA may have a role to play during cell expansion. Such a direct correlation and hormonal regulation of lectin biosynthesis has been demonstrated in Phaseolus vulgaris cultures (Borrebaeck and Linsefors 1985).
52 The PNA content of 100 g of seeds is 80 mg. Seeds are relatively dry in comparison to cultures and have a much higher protein content. Synthesis of PNA from cultures, through several passages, remained relatively constant (12 rag/100 g fresh weight of callus) thus making in vitro culture attractive as an alternate source of lectins.
r- l
120 "~ 100
0
~ Table 1. Effect of growth regulators on callus and lecfin production in callus cultures of A. hypogaea on LS medium supplemented with 58 m M sucrose, 0.1 m M thiamine-HC1 and 1.1 p.M ascorbie acid at the
60
40 u.
f~ o
20
end of the fourth subculture.
00~ Growth regulators (btM) 2,4-D
KN
BA
NAA
2.3
-] 2.2
_
10.7 10.7 10.7
2.3 2.3
PNA b
callus a
(rag/100 g fresh weight of callus)
+ + + ++ +++ +++
0 0.5 0 1.2 11.0 12.4
ABA
2.3
[-
Amount of
1.9}
aRating of callus growth: - none, + low, ++ moderate, +++ high, ++++ profuse. bAverage of 3 rephcations. SE calculated on the original values did not exceed + 2%. [ ] =Mm {} = M x
4
8
0
12 16 20 Time ( d )
24
28
Fig. 2. Relation between the growth of cells ( 9 and levels of PNA (o) in callus cultures of A. hypogaea grown on Mx. Samples for analysis were drawn every 4 days, over a period of 28 d.
Biosynthesis of PNA in cells [3H]leucine incorporation studies confirmed the synthesis of PNA in A. hypogaea cells as well as the movement of PNA into the medium. There was a linear increase of lectin synthesis for 10 d, after the tenth day following transfer to fresh medium (Fig. 3).
~'12
:
Mx
~-F
Mm
~'-
Mx
)I
"~o
t0
E U
/
10F
0
0
9-~
-
8
8 %"~ .= .
6
.~
"~
J
~o 4,
/
J
f------" ' -
.c_
,t-
o~ o 4 o E
2:
a.
4
2
9
0 0
3
6
9 12 15 Time (weeks)
' 2
4
6 8 Time (d)
10
12
2
~
o
18
Fig. 1. Influence of M x and M m media on the levels of PNA in callus cultures of A. hypogaea. The cultures were transferred to fresh medium every 3 weeks over aperiod of 18 weeks and samples were taken every 3 weeks.
Fig. 3. Incorporation of [3H]leueine in PNA in cells (e) and medium (o) of cell suspension cultures of A. hypogaea. [3H]leucine (0.5 m C i / 3 5 ml medium) was added to the cell suspension culture 10 d after the third subculture. The cultures were agitated on an orbital shaker at 100 rpm. The dialyzed sample obtained from the cells/medium was passed through a Lactamyl-Sepharose 4B high-affinity matrix column to get [3H]leucine incorporated PNA.
53 Significant amount of PNA was isolated and purified from cultures. Movement of PNA into the medium indicates that the cells are competent to synthesize the lectin, but are unable to retain large amounts of it. The lectin probably lacks a specific sorting determinant, that would in a normal plant cell be responsible for targeting the lectin to the vacuoles, as demonslrated in barley (Bednarek et al. 1990). PNA probably serves as a defence molecule. Movement of agglutinin into the culture medium has been reported in case of soybean (Malek-Hedayat et al. 1987) and winged bean (Meimeth et al. 1982).
molecular weight of PNA was found to be 27,000 (Fig. 4). The IEF profile of the lectin from culture exhibited a range of charged species with pls from 5.4 to 6.55 (Fig. 5), identical to that of seed lectin, studies of which have shown that the isoforms of the lectin are due to several genes.
Purification of PNA from cultures PNA was purified from A. hypogaea callus cultures using a high-affinity matrix as described in "Materials and Methods". The steps involved in the purification have been listed in Table 2. A single peak of the galactose-binding lectin was obtained from cultures of A. hypogaea on a Lactamyl-Sepharose 4B matrix. 12 mg PNA was obtained from 100 g of callus of A. hypogaea on elution with 0.4 M lactose in PBS (Table 2). The Table 2. Pudfication of PNA from 100 g callus of A. hypogaea
Step
Total protein (mg)
Protein yield (%)
Crude extract Centrifugation (Supematant) Dialysis Affinity Flow-through Lactose-eluted
1500 312
100 20.8
95
6.3
73 12
4.9 0.8
SHAA (HAUs mg-1 protein)
Fig. 4. SDS-PAGE pmJSle of PNA from seeds and cultures of A. hypogaea. M, standard markers; lane 1, seed PNA; lane 2, suspension culture PNA. The standard markers were BSA (66 kD), bovine carbonic anhydrase (29 kD), bovine trypsinogen (24 kD) and soybean trypsin inhibitor (20.1 kD).
48
level of PNA in cells and that secreted into the culture medium at the end of 3 weeks after the fourth subculture was 4 mg from 100 g of cells and 14 mg from 2.5 1 of medium of cell suspension culture. The yield of lectin from cell suspension cultures was higher than that from callus cultures since the lectin from suspension cultures could be recovered not only from the cells but also from the liquid medium. The same could not be recovered from agar-solidified media. Purification of PNA from the culture medium was easier as this involved fewer number of steps as described in "Materials and Methods".
Biochemical characteristics of PNA from cultures The purified PNA from the cultures comigrated with that from seeds on a SDS-polyacrylamide gel. The subunit
Fig. 5. IEF gel profile of PNA from seeds and cultures of A. hypogaea. M, standard markers; lane 1, seed PNA; lane 2, suspension culture PNA. The standard markers were phyeocyanin (4.65), IMaetoglobulin B (5.1), bovine carbonic anhydrase (6.0), human carbonic arthydrase (6.5) equine myoglobin (7.0), human haemaglobin A (7.1), human haemaglobin C (/.5), lentil lectins (7.8, 8.8, 8.2) and eytoehrome e
(9.6).
54
The SDS-PAGE profiles and IEF profiles of the agglutinin from callus cultures and cell suspension cultures (cells and medium) were identical (Fig. 4 and 5).
Biological activity of PNA from cultures The lectin obtained from cultures was biologically active. The SHAA ofPNAfrom cells and medium of suspension cultures was 15 and 78 HAUs mg -1 protein respectively. Seed PNA had a SHAA of 98 HAUs mg -1 protein.
Conclusion PNA was synthesized in cultures through several passages. The synthesis was regulated by exogenously supplied growth regulators and was positively correlated with the growth of the cells. Movement of the agglutinin from the cells into the medium facilitated easy isolation of the lectin. The lectin synthesized in culture was found to be biologically active and comparable to the seed lectin. In vitro culture could not only be used as an alternate source of PNA but also provided a model system to study the regulation of the biosynthesis of PNA through exogenously supplied growth regulators, thus providing a clue to their physiological functions.
Acknowledgments. IDS thanks the Deparlment of Bioteclmology, Government of India, for a post-doctoral fellowship. Thanks are due to Dr. L. D'Souza for the gift of membrane filters. Sincere thanks to Prof. C.S. Valdyanathan for providing the cell culture facilities.
References Barondes SH (1981) Armu. Rev. Bioehem. 50:207-23
Bednarek SY, WiLkins TA, DombrowskiJE, Raildad NV (1990) The Plant Cell 2:1145-1155 Borrebaeck CAK (1984) Planta 161:223-228 Borrebaeck CAK, Linsefors L (1985) Plant Physiol. 79:659-662 Chrispeels MJ, Raildael NV (1991) The Plant Ceil 3:1-9 DattaPK, FigueroaMODCR, LajoloFM (1991) Plant Physiol. 97: 856-862 Dd Campiilo E, Howard J, Shannon LM (1981) Z. Pflanzenphysiol. 104:97-102 D'Silva I, Vaidyanathan CS, Podder SK (1994) Plant Science (in press) EtzlerME (1986) In: LienerIE, SharonN, GoldsteinIJ (eds) The Lectins. Properties, Functions and Applications in Biology and Medicine. Academic Press Inc, Florida, pp 371-435 Hegde R, Maiti TK, Podder SK (1991) Anal. Bioehem. "194:101-109 James DW Jr., Ghosh M, Etzler ME (1985) Plant Physiol. 77:630-634 Laemmli UK (1970) Nature 227:680-685 Linsmaier EM and Skoog F (1965) Physiol. Plant. 18:100-126 Lis H, Sharon N (1986a) In: LienerIE, Sharon N, GoldsteinIJ (eds) The Lectins. Properties, Functions, and Applications in Biology and Medicine. Academic Press Inc, Florida, pp 265-291 Lis H, Sharon N (1986b) In: IAener IE, Sharon N, Goldstein IJ (eds) The Lectins. Properties, Functions, and Applications in Biology and Medicine. Academic Preess Inc, Florida, pp 293-370 Lowry OH, Rosebrough NJ, Farr AL, Randall ILl (1951) J. Biol Chem. 193:265-275 Malek-Hedayat S, Meiners SA, Metcalf TN I~, Schindler M, Wang J-L, Ho S (1987) J. Biol. Chem. 262:7825-7830 Meimeth T, Thanh Van TK, Marcottee JL, Trinn TH, Clarke AE (1982) Plant Physiol. 70:579-584 Morris PC, Maddoek SE, Jones MGK, Bowles DJ (1986)Plant Ceil Reports 5:460-463 Pratt RC, Singh NK, Shade RE, Murdoek LL, Bressan RA (1990) Plant Physiol. 93:1453-1459 Sato A, Barcellos GBS, Riedel EC, Cameiro JA, Carlini CR, Esquibel MA (1993) Plant Cell Reports 12:233-236 Singh PLK (1989) In: Lectin-induced lymphocyte activation : role of ganglioside in the initial recognition event. Thesis, lISt, Bangalore, pp 114 Stinissen HM, Peumans WJ, De Langhe E (1984) Plant Cell Reports 3:55-59