Characterization of the major proteins from Vitis vinifera seeds

Plant Scienc~ 62 (1989)73-81 Elsevier Scientific Publishers Ireland Ltd.

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CHARACTERIZATION OF THE MAJOR PROTEINS FROM V I T I S V I N I F E R A SEEDS

ELISABETTA GIANAZZA°, GIULIANO TEDESCOb, PIERLUIGI VILLAe, ATTILIO SCIENZA c, GIOVANNI CARGNELLO ~, PIER GIORGIO RIGHETTI° and ANNA OSNAGHI" ~Facolt~ di Farmacia Dipartimento di Scienze e Tecnologie Biomediche, bFacolt~ di Scienze, Dipartimento di Biologi~ Sezione di Botanica Sistematic~ ~Facolt~ di Agrari~ Iatituto di Coltit~zioni Arboree, via Celoria ~ 1-20133 Milano and ~Istituto Sperimentale per la Viticultur~ SOP, Corso Eiz~. udi 60, I-1~100A sti (Italy/ (Received November 15th, 1988) (Revision received February 3rd, 1989) (Accepted February 3rd, 1989)

The major protein from the endosperm of Vitis ~in(fera cv. Chardonnay is a globulin, homogeneous by size (M ~ 400 kD after PAGE) and highly heterogeneous by charge, 23 bands being resolved by IEF, with pI values 4.8-5 for the major components, and up to pH 7 for the minor ones. The native structure is assembled from non-covalently bound subunits, with M 65 kD, which in turn are composed of disulfide-bridged peptides, M 19-- 21 kD and 3 8 - 44 kD. The focusing pattern of the denatured protein includes 15 acidic (pI values = 4.25-4.8) and two alkaline (M = 26 kD, pI = 6.8-6.9) com.vonents. None of the major bands contains sugar or lipid moieties. Several enzymatic activities are detected: esterase, phosphogiucomutase, acid phosphatase, peroxydase, malic, alcohol and a little lactic dehydrogenase; no protease inhibitors can be identified. The quali~luantitative variability among proteins extracted from individual seeds amounts to approx. 10%; only large samples, including20-- 30 seeds, are thus likely to be representative of the genetic set-up of a given Vitis clone. Key words: endosperm; enzyme; immobilized pH gradient; protein; systematics; Vitis

Introduction Chemiosystematics has been widely applied to staple crops, such as cereals, legumes and potatoes [1-5]. In Mediterranean countries grape-growing ranks as a major farming activity. Yet, the current systematics of the Vitis genus still rests on classical ampelographic work [6,7] and the newer biochemical techniques haven't attained routine application in vine selection and breeding (see, however Refs. 8 - 1 3 for grape juice components, Ref. 14 for leaves, Ref. 15 for pollen). In the chemical approach to systematics, pollen grains and seeds have long been recognized as the most suitable material. In fact they correspond to a well defined step in the vegetable cycle (in contrast to a continuum of transient growth states for other parts of the plant, leaves for instance) and over and over their composition has been shown to be species-spe-

cific and invariant under different growing • conditions [16]. As for Vitis vinifera, our group has detailed that pollen wall proteins are indeed clone-specific and independent from: (i) the time of sample collection, (ii) the stock on which the scion is grafted and (iii) the location where the vine is grown [17,18]. In the present account we report on chemicophysical parameters (charge, mass, subunit structure) and on some biological activities (enzymes, protease inhibitors) of the endosperm proteins from V. vinifera seeds. We have also investigated to which extent the protein pattern varies (i) among individual seeds from an inbred population and (ii) upon outbreeding. A parallel study is under way on the low-molecular mass components (mostly pigments) of the fruit. Together, the current investigations are meant to establish a data base for the selection of relevant traits to a revised systematics within the genus Vitis.

0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

74 Material and methods

Samples Vine grapes were obtained by controlled breeding of V. vinifera cv. Chardonnay either with autologous (C x C) or with heterologous pollen (from the cultivars: Durella, C x D; Mars, C x M; Petit Arvine, C x P; Rous, C x R). Putative C × C was also collected from vineyards where Chardonnay was the main cultivar being grown. Samples from vintage 1987 were stored at - 20 °C. Upon thawing, the seeds were freed from the pulp and washed extensively in distilled water (empty kernels float and could thus be discarded). The kernels were then crushed with tweezers, and endosperms dissected from testa and most perisperm.

Protein extraction Seed proteins were classified according to their solubility by a sequential extraction procedure, devised as a simplified Landry and Moureaux protocol [19]: 0.5 M NaCI, followed by 0.5 M NaC] + H2COs/Na (pH 10) + 25 mM DTT, then by H2COs/Na (pH 10) + 25 mM DTT + 0.5% SDS. For routine analysis by isoelectric focusing, the seed endosperms, ground with mortar and pestle, were extracted for 1 h at 4 °C with 10 vol. of 0.2 M glycine [20] and centrifuged 2 × 20 rain at 10 000 and at 12 000 rev./ rain with a JA20 rotor in a centrifuge Beckman J2-21 (Palo Alto, Calif.). At each step the floating layer of lipids was carefully discarded. For very small samples the tissue-to-liquid ratio was increased to 1 : 2 0 and the 250 /d-tubes were spinned for 10 rain in a BioItalia mod 3223 centrifuge.

Electrophoretic techniques Isoelectricfocnsing. Vine seed proteins were fractionated by charge with isoelectric focusing (IEF) on mixed bed CA-Immobiline gels [ 2 1 23]. Immobilized pH gradients (IPGs) [24], covering non-linearly the pH range 4--10 [25] and with an acrylamide matrix T% = 4 C% = 4, were polymerized according to standard procedures [26], washed in 1% glycerol and

dried. To favor the solubility of globulin components around their isoelectric point (pI), the slabs were reswollen in 2% carrier ampholytes (0.5°/0 Pharmalyte 3--10, 0.3°/0, 4--6.5, 0.5°/0 Ampholine 3.5--10, 0.4°/0 4 - 7, 0.3°/0 4 - 6, from Pharmacia-LKB Biotechnology, Uppsala, Sweden). The samples were always loaded near the cathode, as anodic applications resulted in extensive protein precipitation. The run was overnight at 1.5 W-- 500 V (for a slab 110 × 240 x 0.5 ram) followed by I h at 5 W-- 1,300 V. In a few experiments, the gel was also made to contain 8M urea and the samples were reduced with 2°/0 2-mercaptoethanol (2-SH). The electrophoretic equipment was from LKB. Proteins were stained according to Righetti and Drysdale [27] or to Merril et al. [28].

SDS-electrophoresis Proteins were separated according to size by electrophoresis in presence of SDS on a polyacrylamide gradient T% = 7.5--17.5 with the discontinuous buffer system of Laemmli [29]. The electrophoretic chamber was from BioRad (Richmond, Cal.). Proteins were stained in 0.3% Coomassie Blue R250.

Two-dimensional (2-D)mapping The subunit composition of proteins was investigated by sequential fractionation either through native-denaturing IEF or through IEF--SDS-electrophoresis. In the former case, a gel strip from a standard run was equilibrated for 5 rain in 6 M u r e a - 0 . 5 % 2-SH, and overlaid at right angle to a urea-containing gel. Otherwise, strips from either native or denaturing IEF were equilibrated for 10 rain in 3% S D S - 20/0 2-SH, then embedded on a SDS-slab gel, as previously described [30].

Spe cific stains Glycoproteins and lipoproteins. Glycoproteins were detected by the periodic acid-Shiff procedure of Zacharius et al. [31] as well as by the Alcian Blue staining of Wardi and Michos [32]; for lipoproteins, the Sudan Black solution by Prat et al. [33] was tried.

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Zymograms The following enzyme activities were tested: esterase (EST, [34]); phosphogiucomutase (PGM, [35]); acid (AcP, [36]) and alkaline (ALP, [37]) phosphatases; malic (MDH, [38]), lactic (LDH, [39]) and alcohol (ADH, [40]) dehydrogenases; amylase (AMY, [41]); peroxydase (POD, [42]). Except for the latter, the reaction mixture was poured as 1.5-ram thick agarose layer (1% agarose H, gelling temperature -- 37°C, from LKB), overlaid to the IEF slab and incubated for 1--3 h at 37°C. POD stain was developed by shaking gel strips in 10 mL substrate solution. Anti-proteases were tested according to Uriel and Berges [43] and to Erlanger et al. [44].

Results Protein extraction When attempting to process the whole seed through protein extraction, interfering material is released from the wooden testa, which makes the resulting solution unsuitable for electrophoretic fractionation. Extracts from which the oily layer had been discarded after centrifugation and whole supernatants (remixed and homogenized) give the same banding pattern, but upon storage lipids might easily undergo peroxydation. To fit the requirements of IEF, a medium of high dielectric constant and negligible ionic strength was selected, i.e. isoelectric giycine. In order to check whether

Fig. 1. M evaluation for the native storage protein f~om Vitis vinifera endosperm, and its subunits. (A) Disc electrophoresis on a polyaerylamide gradient T% = 4-- 10 of the extract with 0.5 M NaCI; approximate M scale by reference to standard proteins. (B) SDS-electrophoresis on a polyarrylamide gradient T% = 7.5-- 17.5 of 0.2 M glyeine and 0.5 M NaCI extracts without reducing agents; M, scale by reference to standard proteins. (C) SDS~leetrophoresis as above on glycine, NaCI, H2CO/Na + DTT and SDS extracts; all samples were added with 2% 2-SH.

76

!

pH

!

-8

A

-7

!

-6

|

-5

Fig. 2. Molecular parameters for the vine storage protein subunits. (A) Native IEF on a non-linear 4-- I0 gradient (pattern at the top) followed by IEF under dissociating conditions (urea + 2-SH) at right angle. (B) Native IEF followed by SDS-electrophoresis. (C) IEF in urea, sample added with 2-SH, followed by SDS~lectrophoresis. pI and M scales as marked. Sample: C × C.

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such an extract would be representative of the seed protein content, we exposed the endosperm meal to solvents of increasing solubilizing power: neutral, with high ionic strength; alkaline and containing a reducing agent; added with an ionic surfactant. The four solutions are compared in Fig. 1C by SDS-electrophoresis. Except for some minor low M component, identical patterns are obtained with glycine and with buffer extracts, while just a few more bands are stained in the SDS track.

Molecular parameters Figures 1B and 1C also compare, in the case of NaC1- and glycine~xtracts, the SDS banding patterns obtained with and without 2-SH. In the latter condition, two components are resolved, a major band, M = 65 kD and a minor

one, M = 28 kD. Upon reduction, the larger assembly splits into a number of subunits, their M-values ranging between 19--21 and 38--44 kD. On pore-gradient electrophoresis (PAGE; T% ffi 4 - 1 0 ) the undenatured protein runs as a band with Mr ~ 400 kD (Fig. 1A). Isoelectric focusing in native conditions resolves approx. 25 protein bands (Fig. 2B, top); the pI values of the major cluster are in the range 4 . 8 - 5 , while a number of components are found up to pH 7. Upon partial denaturation new bands appear, with pI approx. 6.2 when the samples are added with 2-SH and at pH 5 . 5 - 6 when run in 8 M urea (not shown). The combined effect of a reducing and of a chaotropic agent generates the fully denatured pattern (Fig. 2C, top), featuring: 2 distinct alkaline bands, pI 6.8-6.9, and a number of minor corn-

Fig. 3. Specific stains for the vine endosperm proteins. From left to right: CBB, Coomassie stain; Ag, silver nitrate stain; PAS, glyeoprotein stain, then zymograms for: EST, esterase; PGM, phosphoglucomutase; ACP, acid phosphatase; MDH, malie dehydrogenase; LDH, lactic dehydrogenase; ADH, alcohol dehydrogenase; POD, perox/dase. Sample: C × C. pH scale (from computer simulation} as marked.

78 ponents between pH 6 and 7; a major cluster of 10 bands with pI 4.4-4.8 and 5 acidic subunits, pI 4.25- 4.30. The quaternary structure of the endosperm proteins was further investigated by 2-D electrophoresis. Figure 2A shows the outcome of the sequence: native-denaturing IEF. Some lateral spreading (caused by differential conductivity) in the transfer from lst- to 2nd-d reduces the overall resolution; it is clear however that little selectivity exists of subunit composition among the native assemblies. The same impression comes from the sequence: native IEF-SDS-electrophoresis (Fig. 2B). In fact, the horizontal streaks in the 2-D pattern imply subunits with identical M into native aggregates of different pI. The individual peptides are resolved in a pIM plane with the denaturing IEF--SDS~lectrophoresis sequence of Fig. 2C. For the major spots, a relationship seems to exist between pI and M . The most acidic components form a cloud of tiny spots, loosely ordered in vertical arrays; the alkaline bands have a M of 25 kD. By comparing Fig. 2B with 2C, only the spot with pI = 5.4, M = 10 kD, is found in the same position, and may be assumed to represent an individual protein as opposed to a subunit from some complex structure. The first two lanes of Fig. 3 compare the conventional Coomassie protein stain with Merril's silvering procedure [28], the number of resolved bands being virtually the same in either case. This finding, confirmed by staining a 2-D map with silver nitrate, shows that the relative concentrations of the major and of the minor protein components from vine endosperm are far apart, and that the former have little affinity for silver (the amount of protein per lane in this experiment is twice as large as for Coomassie stain). This feature in turn implies an odd aminoacid composition, with little or no lysine and/or cystine-methiom ine (C. Merril, oral presentation at "Electrophoresis '88", Copenhagen, July 4 - 7). The third lane of Fig. 3 shows a gel stained for glycoproteins. One prominent PAS-positive band is shown, with pI = 6.5, stained, albeit

with reduced contrast, also by Alcian Blue, and a fainter band, pI 5.5. (In SDS-electrophoresis, however, the only stainable zone hardly entered the running gel, apparent M = 300 kD, and couldn't thus be associated to any of the protein components.) No lipid moiety was detected by Sudan Black.

Biological activities The last 7 lanes of Fig. 3 form a composite from distinct zymograms. Esterase gives a number of positive bands, with a cluster between pH 7 and 8 and two distinct isozymes at pH 5.9 and 6.5. The two major components of phosphoglucomutase focus at pH 5.3 and 5.7, while the bands positive for acid phosphatase are grouped between pH 4.7 and 5.3. No alkaline phosphatase as well as no amylase activity could be observed. Malic dehydrogenase isozymes are centered between pH 4.4 and 5, with additional components at pI 5.3--5.5. Lactic dehydrogenase scores very low activity, and a single band is detected at pH 6, while for alcohol dehydrogenase the major component focuses at pH 6.2 and two minor bands are at pH 6 and 6.8. Finally, the most active peroxydase isozyme is resolved at pH 5.4. Up to 60 ~l of glycine extract couldn't inhibit the proteolytic activity of 0.4 mg/ml trypsin. Pattern variability When comparing the protein pattern of C × CseedswithC × D,C x M,C × P a n d C × R, both in the native and in the denatured state, the same number of bands is resolved, although the relative intensities may vary to a significant extent. A similar picture comes from their SDS-banding pattern (not shown). The level of enzymatic activity also varies among the 5 samples, however the pI of the positive bands is alike in most cases. The only exception is C × M, in which some more active components are stained for AcP and PGM (not shown). The IEF protein profile of the seeds from individual grapes in comparison with the extract from a large number of kernels either obtained by artificial pollination or from naturally pollinated fruits in a homogeneous cul-

79

ture, shows a degree of variability between 10O/oand 20% (not shown). Discussion The major endosperm protein from 17. vini. fera seeds is extracted by NaCI and precipitated upon dialysis against distilled water, and therefore exhibits the solubility properties of a globulin. Peptides of approx. 20 and 40 kD are linked by disulfide bridges to form 65 kD subunits, which then assemble in a quaternary structure, 9400 kD. Both the constituent peptides and the resulting assembly are highly heterogeneous by charge, 15 isoelectric components are resolved in u r e a - 2 - S H and as many as 23 bands in native conditions. The molecular mass of individual peptides also varies over about 2 - 5 kD around the average values given above (this is especially evident from the 2-D pattern of Fig. 2C). On the contrary, both the unreduced subunit and the native structure appear quite homogeneous by size (compare sections A - C of Fig. 1). Heterodispersion is common finding for storage seed proteins. These are usually encoded by multiple gene copies, whose codominant expression speeds up the accumulation of aleurone grains in the seed endosperm; divergence by mutation may easily arise within such gene families. Selective pressure on the other hand is likely to be loose. The function storage proteins play is merely the accumulation of aminoacids to be metabolized upon germination, and a large number of structures might fulfill this role. The only requirements seem to be that the storage granules are efficiently packed, which entails some type of balance between their hydrophilie and hydrophobic regions, and that the target sequences for the proteases in the germinating seed are preserved [45]. The presence of disulfide bridges and mostly the peculiar contrast between the much larger heterogeneity in size at the level of the fully dissociated peptides than for the larger molecular structures might suggest that the primary synthetic product of the vine seed is a polypep-

tide ;;,65 kb long, which is then cleaved into two unequal halves. The evolution of alternative splitting sites few kD apart might explain the complex peptide map of the protein. This hypothesis may be supported by the finding of some 10% 65-kD component in the SDS pattern even after thiol reduction (Fig. 1C). A non-negligible amount of variation among individual seeds has been observed, which is reduced but not abolished when the specimen is obtained by artificial pollination. A simple explanation to this finding might come from an unequal post-translational processing. The proposal of a proteolytic splitting might be instrumental also in this respect. The data above also mean that a large number of grapes are required to compose a representative sample for a given Vitis clone (N = 2 0 - 30 seeds). Acknowledgements Work supported in part by grants from Ministero Pubblica Istruzione, Roma. We thank Dr. Ib S#ndergaard, University of Copenhagen, for help with quantitative gel evaluation, Prof. Mario Orsenigo, University of Milano, for critically reading the manuscript and Mr. R. Cavatorta for assistance in photographic reproduction. References 1

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