characterization of 2s and 7s storage proteins in embryos

s

ELSEVIER Plant Science 122 (1997) 141-151

Characterization of 2s and 7s storage proteins in embryos of oil palm

Fabienne Morcillo*, FrCdtrique Aberlenc-Bertossi, Pierre Trouslot, Serge Hamon, Yves Duval

ORSTOMICIRAD-CP, Laboratoire de Resources Gkn&tiques et AmPlioration des Piantes Tropicales, BP 5045, 34032 Montpellier cedex 1, France

. Received 25 March 1996; revised 6 November 1996; accepted 12 November 1996

Abstract

Storage proteins that accumulated during oil palm embryo development were extracted, purified and characterized. Only water- and low-salt-soluble proteins, with respective sedimentation coefficients of 2S and 7S, were detected in mature embryos. After purification by gel filtration, the various protein classes identified were characterized by electrophoresis and amino acid composition analysis. The 2S proteins comprise polypeptides of 22 kD and 19 kD, which are acidic (PI< 6) and basic (PI> 9) respectively. The 7s proteins predominate and are heterogeneous oligomers (Mr of 156 and 201 kD), comprising a polypeptide triplet of Mr between 45 and 65 kD with no disulphide bonds. Their amino acid composition is broadly similar to those of the 7S proteins of other monocotyledon embryos, but differs from those of the legume 7S vicilins. Histological examinations and electrophoresis showed that the 2S and 7S proteins appeared at the third month after fertilization, and no qualitative changes were detected up to the sixth month of embryo development. The characterization of the embryo storage proteins of oil palm is discussed with reference to legume, cereal and palm seed storage proteins. This study will enable further investigation of storage protein synthesis during somatic embryogenesis. 0 1997 Elsevier Science Ireland Ltd. All rights reserved

Keywordx Elaeis guineensis Jacq.; Embryo maturation; Monocotyledon; Albumins; Globulins .

L Abbreviations: DW, dry weight; DEAE, diethylamine ethyl; 1. Introduction

ND, non-denaturing; IEF, isoelectrophoretic focusing; ME, #I-mercaptoethanol; Mr, molecular mass; PAGE, polyacry- Oil palm (EZueis guineensis Jacq.) is cultivated Iamide gel electrophoresis; PI, isoelectric point; PMSF, phenyl- methylsulphonyl fluoride; SDS, sodium dodecyl sulphate; % T,

for its high-yield oil production. As this is an

g of monomer-acrylamide and N, N methylenebisacry- outcrossing species, individual palms are highly

Iamide-per 100 ml gel mixture before polymerization (w/v). heterozygous and vary widely in yield, oil quality, * Corresponding author. and disease resistance. Nevertheless, improve-

OI67-9452/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved PIISO168-9452(96)04555-4

142 F. Morcillo et al. / Plant Science 122 (1997) 141-151

ments in vegetative propagation through somatic embryogenesis hold promise for the cloning of high-yielding palms [l -41. However, the plantlets resulting from the somatic embryos are less vigo- rous than plantlets resulting from zygotic embryos placed on the same germination medium. This poor vigour may be due to incomplete or non- maturation of the somatic embryos [5].

Redenbaugh et al. have suggested [6] that storage proteins could be relevant markers in the assessment of the maturation of somatic embryos and hence of the quality of the resulting plantlets. Plant regeneration protocols have been improved through the characterization of the storage proteins and the control of their synthesis during the maturation of somatic embryos in numerous species [7-lo].

There is little information available about seed storage proteins of palm species. Ultrastructural studies have revealed protein crystalloids in the protein bodies of endosperm cells from Washing- tonia ( Washingtonia firifera Wendl.) and coconut palms (Cocos nucifera L.) [11,12]. Sjogren and Spychalski [13] have noted in the endosperm of coconut palm a salt-soluble protein termed ‘cocosin’, which has been characterized as an 11s globulin [14]. In addition, polypeptides in total protein extracts have been shown to be recognized by antibodies to 7s and 11s soybean globulins

Crystalloid proteins have not been detected in the embryos of Washingtonia palm and date palm (Phoenix dactylifera L.) [12], but both species the electrophoretic patterns of total proteins appear to be almost the same in the endosperm and embryo [11,15]. Findings for oil palm are consis- tent with those obtained for other palm species. Crystalloid proteins have been found to be specifi- cally localized in the protein bodies of the endosperm cells [16], whilst the major polypeptide compositions of proteins were shown to be similar in the endosperm and embryo [17]. Unlike other crop plants such as cereals [18] and legumes [19], which have been extensively studied, palm embryo storage proteins have not previously been characterized.

In this paper, we present the classification of the major proteins of oil palm embryos, according

1121.

I

to their solubility, sedimentation coefficients and electrophoretic patterns. The description of the major storage proteins, which have been proposed as indicators of embryo maturation [20], will be useful for the study of oil palm somatic embryogenesis.

2. Materials and methods

2.1. Plant material

Embryos were extracted from seeds collected from various oil palm dura x pisifera progeny. Mature seeds from three hybrids identified as A (PO 3781D x P 0 4096P), B (PO 2903D x P 0 4096P) and C (PO 3223D x PO 2758P) were sup- plied by the Station de Recherche sur le Palmier à Huile (SRPH) of Pobe, Benin, after artificial dry- ing (9% DW). Immature seeds from hybrid B were harvested 2.5, 3, 4 and 6 months after fertilization at IDEFOR/DPO La Mé Station, Côte d’lvoire.

:

.

2.2. Seed germination

Dry seeds of hybrid B were heat-treated (40°C for 80 days) to break down the coat-imposed dormancy [2l]. To induce germination, 200 seeds were soaked in water to achieve a moisture content of 21.5% DW and then placed in plastic bags and incubated at 25°C in the dark. Plantlets were collected as soon as they emerged from the germ pore, around 8 days later.

2.3. Extraction and estimation of proteins

Excised embryos, or plantlets, were immedi- ately stored in liquid nitrogen until needed. Stor- age proteins were fractionated by means of sequential extractions from batches of 100-200 embryos or plantlets. The first extraction was performed with K-phosphate buffer (25 mM, pH7) containing NaCl (0.2, 0.5, 1 and 2 M), leupeptin (10 pM) (L2023, Sigma) and PMSF (1 mM) (Sigma), with continuous stirring for 30 min at 4°C. The first supernatant was collected after centrifugation (27 O00 x g for 30 min at 4°C). The

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F. Morcillo et al. 1 Plant Science 122 (1997) 141-151 143

remaining pellet was resuspended in 55% iso- propanol (v/v) (30 min at 4°C) and the second supernatant was collected after centrifugation, as described above. A third extraction was performed on the residue, following the same proto- col, with a solution of sodium merystate (1.2 M). The third supernatant was collected. Each opera- tion was repeated two to four times.

The first supernatant was dialyzed against distilled water for 20 h at 4°C. Water-insoluble proteins were separated from the soluble fraction by centrifugation at 27000 x g , 4"C, for 1 h and were resuspended in an NaCl solution (0.2, 0.5, 1 or 2 M). The second and third supernatants were dialyzed against water as described previously. Insoluble proteins were resuspended in iso- propanol and sodium merystate solution respectively.

Protein content was estimated by the Micro- Kjeldahl [22] and Bradford methods [23]. The Micro-Kjeldahl method was only used for the quantitative analysis of the different classes of extracted proteins. Proteins were acid-treated (36 NH,SO,, 340°C) and their NH$ is content mea- sured using the Nessler reagent (Merck, reference solution A 901 1 and reference solution B 9012). A solution of ammonium sulphate (1 mM nitrogen) was used as a standard. Protein content was estimated using conversion factor of 6.25, and ex- pressed as mg/g DW. Dry weight was estimated from batches of 30 embryos, after dehydration for 14 h at 100°C. All experiments were carried out in triplicate. The Bradford method was used for a rapid estimation of proteins before qualitative analysis.

2.4. Determination of sedimentation coeflcients I

Sedimentation coefficients of the solubilized proteins were estimated on sucrose gradients. Each sample (0.5 ml) was applied to the top of a linear gradient composed of 10.5 ml of 5-30% (w/w) sucrose containing 0.2 M NaCl and 0.7% (v/v) /J-mercaptoethanol (ME) and was cen- trifuged at 40000 x g for 24 h using a SW 41 rotor (Beckman). The gradient was fractionated into 0.5 ml aliquots and the protein contents were estimated according to Bradford (1976). Cy-

-

tochrome c (2.5 S) and alcohol dehydrogenase (7.2 S) were used as markers for sedimentation values.

2.5. Protein puriJication and electrophoretic characterization

The major storage proteins were purified by gel filtration on a Sephacryl S300 (Pharmacia) column (1 m x 1 cm) equilibrated with 0.2 M NaCl, K-phosphate buffer (25 mM, pH7). Elution was carried out with the same buffer at a flow rate of 60 ml/h. All fractions containing major proteins with the same patterns in SDS-PAGE were pooled and dialyzed against distilled water for electrophoretic characterization.

In order to determine the molecular mass of major proteins, non-denaturing gel electrophoresis (ND-PAGE) was performed according to Sigma Technical Bulletin MKR-137. Protein samples, of 50-80 p g , were loaded into wells and electrophoresed (30 mA per gel, for 1 h and 15 min). Gels were stained with Coomassie brilliant blue. Molecular masses of native proteins were determined from the slopes of Ferguson plots [24] using electrophoretic mobilities of standards (a- lactalbumin, chicken egg albumin, bovine serum albumin and urease) on a set of gels with various polyacrylamide concentrations (4.5- 12% T).

Denaturing gel electrophoresis (SDS-PAGE) was performed to determine subunit masses for major proteins in 12.5% T polyacrylamide gels according to Laemmli [25]. Each well was loaded with 27 pg of proteins diluted in sample buffer and heated to 95°C for 5 min. For the preparation of reduced polypeptides, ß -mercaptoethanol was included in the sample buffer at a final concentra- tion of 5% (v/v). Gels were run at 30 mA per gel for 1 h and 15 min, then stained with Coomassie brilliant blue after precipitation of proteins with 10% (v/v) trichloroacetic acid. Electrophoretic patterns were compared with the mobilities of the following protein markers: phosphorylase-b (92 kD), bovine serum albumin (66 kD), ovalbumin (45 kD), carbonic . anhydrase (3 1 kD), trypsin inhibitor (21 kD) and lysozyme (14 kD).

IEF gel electrophoresis was performed according to the method of OFarrel[26]. Tube gels (5%

144 F. Morcillo et QI. /Plant Science 122 (1997) 141-151

T) were focused for 15 min at 200 V, 60 min at 300 V, overnight at 400 V, and 60 min at 800 V. Isoelectric points were determined as described by Pharmacia Biotech (1 1-B-045-07).

After protein separation in the first dimension (ND-PAGEy SDS-PAGE + ME or IEF), the gels (or gel pieces) were incubated in 2% SDS, 10% glycerol, 0.5 M Tris-HC1 @H 6.8), and electrophoresed in a second dimension (SDS-PAGE). Gels were stained with Coomassie brilliant blue.

2.6. Amino acid analysis

Purified fractions of proteins were vacuum hydrolysed with 6 N HCl for 24 h at 110°C and separated on a Beckman 420 HPLC. Amino acids were analysed after reaction with orthophthaldi- aldehyde (OPA) [27]. For cysteine analysis, protein samples were hydrolysed with dimethyl sulphoxide (DMSO) before separation. Proline was detected after derivatization of amino acids with 9-fluorenylmethyloxycarbonyl chloride (FMOC) according to Einarsson [28]. The analysis was carried out in triplicate using samples of 150 embryos.

I

I 3. Results

3.1. Extraction of proteins and determination of their sedimentation values

Solubility analyses on proteins extracted from mature oil palm embryos indicated that most proteins were water- and salt-soluble. Proteins content did not differ significantly between the three studied hybrids (F(2,17) = 2.97 and 0.19, respectively, a > 0.05). The mean values were 98 mg/g DW for water-soluble proteins and 151 mg/ g DW .for salt-soluble proteins. Only traces of alcohol- and acid-soluble proteins were detected.

Differential extractions, with a range of salt concentrations (0.2-2 M), showed that major proteins were rendered soluble in low-salt buffer (0.2 M NaCl) after two successive extractions. The water- and salt-soluble extracts were resolved by sucrose gradient centrifugation into 2s and 7s proteins respectively.

3.2. Electrophoretic characterization of the 2S and 7s proteins

SDS PAGE analysis of oil palm embryo 2s and 7s fractions is shown in Fig. 1. The proteins shown had been previously purified by gel filtration on a Sephacryl S300 column. Gel patterns indicated the removal of the protein contaminants as compared with the crude extract (Fig. 1A and B). The major polypetides of these two fractions appeared at the third month after fertilization and no qualitative changes were detected between the third and sixth months of embryo development (Fig. 1C). Proteins represented by the main bands of both classes disappeared quickly at the begin- ning of seedling growth.

The structures of the 2s and 7s proteins were studied by 1D- and 2D-electrophoresis. In the first dimension of ND-PAGE/SDS-PAGE, the 2S . proteins were resolved into five major native proteins (Fig. 2A) of low molecular masses (Fer- guson plots). The polypeptide composition of these proteins was determined in the second dimension under denaturing conditions (Fig. 2A). Of the five proteins, four consisted of major polypeptides of 22 kD. In one, the pattern indicated that the native protein may comprise two polypeptide types: 22 kD and 19 kD. 2D-electrophoresis (SDS-PAGE + ME/SDS-PAGE - ME) yielded similar patterns under reducing and non-reducing conditions (Fig. 2B) and indicated that the polypeptides identified were not linked by disulphide bonds.

Electrophoretic patterns (Fig. 2C) indicated that the 7s proteins consisted of two major native proteins (156 and 201 kD) which comprised three similar polypeptides (Mr 45-65 kD) and a few minor polypeptides of about 25 and 40 kD. In spite of their different masses, the major native proteins were similar oligomeric proteins of heterogeneous size. Addition of the subunit molecular masses and comparison with the molecular masses of the native proteins suggested that the salt-soluble fraction was composed of trimeric or tetrameric proteins. The non-reduced and reduced 7s proteins gave similar electrophoretic patterns, indicating that these proteins do not contain disulphide-linked polypeptides (data not shown).

.

3

F. Morcillo et al. /Plant Science 122 (1997) 141-151

M CI 1 2s M CL 7s 4 :

: I . - . ? .

cc.-

145

sg - 2s 7s

1 2 3 1 2 3

9 7 6 6

4 5

3 1

2 1

C Fig. I. Electrophoretic analysis of water-soluble (2s) and salt-soluble (7s) purified proteins from developing oil palm embryos (Coomassie brilliant blue). SDS-PAGE (12% T) of gel filtration fractions containing 2s (A) and 7s (B) proteins. M, molecular weight standards; C, crude protein extract of water-soluble (CI) and salt-soluble proteins (C2) before separation on gel filtration. SDS-PAGE (12% T) of 28 and 7s proteins from oil palm embryos at 3 (lane l), 4 (lane 2) and 6 (lane 3) months after fertilization (C). Sg, soluble proteins extracted from seedling.

The isoelectric point of polypeptides was studied by IEF gel electrophoresis and 2D-electrophoresis (IEF/SDS-PAGE). The 2s proteins consisted of a series of basic and acidic isomers (Fig. 3A). 2D-electrophoresis showed that the

polypeptides of 22 and 19 kD were composed of acidic (PX -= 6) and basic (PI > 9) isomers, respectively (Fig. 3B). In contrast, the major polypeptides of the 7s proteins were slightly basic @I w 8) (Fig. 3A).

146

22 .,

F. Morcillo et al. PImr Scieiiw 122 (1997) 141-151

A -=+ /

a i 6 6

45

31

21

14

Fig. 2. Electrophoretic characterization of water-soluble (2s) and salt-soluble proteins (7s) from mature oil palm embryos (Coomassic brilliant blue). A, Two-dimensional analysis of 3s protcins. First dimension, ND-PAGE (7% T}. Second dimension, SDS-PAGE (1 2% T) under non-reducing conditions. B, Two-dimensional analysis of 2s proteins. First dimcnsion, SDS-PAGE (12% T) under non-reducing conditions ( - ME). Second dimension, SDS-PAGE under reducing conditions ( f ME). C, Two-dimensional analysis of 7s proteins. Fir$ dimension, ND-PAGE (7% T). Second dimension, SDS-PAGE (12% T) under non-reducing conditions;

1

i'

F. Morcilla et cil. Pbrzt Scierice 122 (IY97) 141-151 147 '- .

I

J

. I I

J

I

'

,,

9 1 6 6

46

31

21

14

Fig. 2.

3.3. Anzino acid composirio?z of 2s atid 7s piareins

The amino acid compositions of the purifìed 2s and 7s proteins from oil palm embryos are shown in Table 1. In oil palm, high contents of AsplAsn and Glu/Gln (totalling 36% and 27% for the 2s and 7s proteins, respectively), Arg (16 and 11%) and Gly (9 and 8%) were detected in both protein fractions. The 2s proteins were characterized by their high Cys content, which was four to five times greater than in 7s proteins.

4. Discussion '

In the work reported here, the only proteins stored in oil palm embryos were water- and low- salt-soluble. According to the dekitions of Os- born [29], they may be classified as albumins and globulins, respectively. Amongst the various water-soluble protejns , found in dicotyledon species, the abundant 2s albumins, some of which arecrich in sulphur-containing &o acids, are considered to be storage proteins [30-321.

In the mature oil palm embryo, water-soluble proteins represented 40% of extracted proteins.

They were composed of five major proteins characterized by undeteinined low molecular masses. The abundance, sedimentation coefficients, and level of sulphur-containing amino acids observed indicates the presence of albumin storage proteins in the oil palm embryos. The albumins of oil palm embryos comprised polypeptides with several charged forms which probably reflect the se- quence heterogeneity observed with multigene families. This feature was reported by Higgins [33] as a specific feature of storage proteins. Unlike the albumins described in numerous legume seeds, the albuniin storage proteins of oil palm embryos have no disulphide bonds [32].

Globulins have been more extensively described than albumins. There are two types, 7s (vicilin- type) and 11s (legumin-type), distinguishable in size and sugar content [19,31]. They are mainly found in legume seeds, although 7s globulins are the major storage protein in monocotyledon embryos [18,34]. In oil palm embryos, globulins of the 7s type predominate. No other major proteins (including 11s proteins) were found, in spite of the range of salt concentrations used and the addition of detergent (data not shown). 7s vicilins are often trimers of similar subunits (Mr 50-70 kD depending on the species) held together by

148 F. Morcilla er al, Plant Scieiice 122 (1997) 141-151

B

I . . ' . . 7s

t I I I I l I 1 t I I I 10.3 9.2 8.0 6.9 5.9 4.7

PH I Fig. 3. Isoelectric point of water-soluble (2s) and salt-soluble proteins (7s) from mature oil palm embryos (Coomassie brilliant blue).

A, IEF of 2s and 7s proteins. The pH gradient was determined from a separate gel without added proteins; B, two-dimensional analysis of 2s proteins. First dimension, IEF. Second dimension, SDS-PAGE (12% T) under non-reducing conditions.

non-covalent bonds. However, after the assembly of the 7s subunits, they may sometimes be prote- olysed or glycolysed [35], which could explain the two sizes observed in the native proteins from oil palm embryos. Some legume 7s proteins do not

undergo post-translational cleavage, examples including soybean ß-conglycinin and phaseolin from Phaseohs uulgaris. The latter consist of polypeptides which have molecular masses in the range 40-76 kD [19], similar to the 48-65 kB

F. Morcillo el al. 1 Plant Science 122 (1997) 141-151 149

1

range of ehe major subunits of oil palm globulins. The SDS-PAGE patterns of oil palm 7s globulins appear comparable to those of bean phaseolin (Mr 45-5 kD range, described by Koenig et al. [36]) and cereal globulin (50-60 kD depending on the species [34]). Amino acid composition analysis reveals greater similarity with 7s globulins from cereal embryos than with the corresponding 7s legume proteins [34], and in particular Asp/Asn and Leu contents were typically lower. In contrast Pro, Gly and Ala contents were higher.

Relatively few differences were observed between the electrophoretic patterns of embryo proteins from oil palm and those from other palm species [11,15]. For example, in the 45-66 kD range there were three major common protein bands in oil and date palm, whilst only one of these bands was evident in Washingtonia electrophoretic patterns. In the corresponding

1

L *

Table 1 Amino acid composition (% composition) of 2s and 7s proteins fractions from oil palm embryos. The analysis was carried out in triplicate fon samples of 150 embryos

4

2s 7s

Mean S.D. Mean S.D.

J

Asxa 1.0 0.07 Glxb 29.3 0.53 Ser 4.1 0.24 His 1.3 0.04 GlY 9.0 0.15 Thr 3.0 0.11 Arg 15.8 0.72 Ala , 3.9 0.34 Tyr 1.3 0.32 Met 0.7 0.02 Val 2.4 0.05 Phe 1.3 0.13 Ile 1.4 0.18 Leu 3.7 0.10 LYS 5.8 0.22 C y s 4.9 0.30 Pro 4.9 0.53

10.1 0.17 17.3 0.38 6.1 0.09 3.4 0.13 8.4 0.40 3.5 0.15

10.6 0.25 6.5 0.01 1.7 0.13 0.7 0.05 6.0 0.17 4.3 0.03 3.4 0.02 6.9 0.01 4.5 0.23 1.1 0.16 5.3 0.16

a Asx, aspartate and asparagine. Glx, glutamate and glutamine.

IEF gels, the charges of the major polypeptides were not identical: the 45-66 kD polypeptides were more basic in oil palm than in date and Washingtonia palm @I ranging between 6.2 and 7.3). In addition, the basic polypeptides around 19 kD in oil palm embryos were not present in date palm. In general, marked similarities were observed between the protein profiles of oil palm embryos and these seen with endosperm and/or embryos of date palm, Washington palm and coconut palm [12,15]. In all four species, the major polypeptides had molecular masses around 55 kD, and between 16 and 22 kD. The differences observed in the patterns of the reduced and non-reduced proteins extracted from oil palm were not seen with date and Washing- tonia embryos. In all the analysed palms, there were no proteins containing disulphide-bonded subunits. The similarities described collectively suggest that homologous classes of storage proteins occur in each of these palm species.

Following the characterization of storage proteins in mature oil palm embryos, we investi- gated these proteins during embryo development. The fruits of oil palm ripen over a period of six months after fertilization. A morphological study indicated that the embryos had finished growing in size about 2.5 months after fertilization [17]. In the work reported here, we observed storage protein deposition as of the third month after fertilization. The lack of change ín protein content (data not shown), and the similarities of electrophoretic patterns at 3 and 6 months after fertilization, suggest that storage proteins required for germination are available from this period onwards, and could explain why seeds extracted from immature fruit are able to germinate [37].

Our study describes the first characterization and purification of storage proteins of the oil palm embryo, in which 2s albumins and 7s globulins have been identified. Merkle et al. [20] have proposed storage proteins as being markers of embryo maturation. The 7s globulins, which predominate both in oil palm embryos and in many other monocotyledon embryos might therefore potentially serve as maturation markers in the study of somatic embryogenesis.

I50 F. Morcillo el al. / Plant Science 122 (1997) 141-151

Acknowledgements ,

We thank Dr Prevot and Dr Rival for their kind help and Mr Gallois for his participation in this work. We also thank Dr N. Djegui, Director of the Oil Palm Research Station of Pobe and Dr B. Nouy, Dr Kouame, Director of IDEFOR/ DPO La Mé Station, and Dr Durand-Gasselin for the supply of seeds. The assistance of Alain Rival in the preparation of the illustrations is gratefully acknowledged. We thank James Tregear for cor- rection on the english manuscrit.

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